!

ABSTRACT!

JAYARAMAN,(PRIYA(ANURADHA.(Innate(Immune(Responses(to(Infection(by(the(Pathogenic(Spirochetes(Leptospira*interrogans(and(Borrelia*burgdorferi.((Under(the(direction(of(Dr.(Frank(Scholle).((

Leptospira*interrogans(and(Borrelia*burgdorferi(cause(zoonotic(and(vectorIborne(

diseases(that(pose(an(agricultural(and(veterinary(public(health(threat,(respectively.(This(

body(of(work(addresses(the(early(innate(immune(responses(to(infection(using(a(mouse(

model,(which(is(relevant(to(the(transmission(cycle(of(infection.(In(chapter(1,(an(overview(of(

innate(responses(to(infection(are(described.(Cellular(and(humoral(responses(play(an(

important(role(in(early(inflammatory(responses(and(clearance(or(persistence(of(infection.(

Intracellular(signaling,(a(mechanism(that(results(in(cytokine(production(and(affects(later(

immune(responses,(is(also(described.(These(immune(responses(are(also(explained(in(the(

context(of(infection(by(spirochetes.(In(addition(to(this,(the(importance(of(studying(pathogenic(

spirochetes(is(addressed(in(this(chapter.(In(chapter(2,(the(contributing(role(of(the(adaptor(

protein(TRIF(to(innate(responses(during(leptospiral(infection(is(investigated.(Mice(lacking(

TRIF(exhibit(a(dysregulated(cytokine(response(and(an(altered(humoral(response(to(

leptospiral(infection.((In(chapter(3,(the(role(of(TLR(signaling(in(response(to(B.*burgdorferi(

and(RNA(from(B.*burgdorferi(is(explored,(and(a(mechanism(for(Type(I(IFN(induction(

mediated(by(TLR7(is(addressed.(Finally,(Chapter(4(highlights(the(main(points(in(this(

dissertation,(while(discussing(possible(future(directions(that(seek(to(elucidate(critical(roles(

for(TLRIsignaling,(adaptor(proteins,(and(impact(on(immune(responses(during(infection(by(

spirochetes.((((

( (

(

(

(

(

(

(

(

(

(

(

(

©(Copyright(2015(by(Priya(Anuradha(Jayaraman(

All(Rights(Reserved

Innate(Immune(Responses(to(Infection(by(the(Pathogenic(Spirochetes(Leptospira*interrogans(and(Borrelia*burgdorferi(

(

(

by(Priya(Anuradha(Jayaraman(

(

(

A(dissertation(submitted(to(the(Graduate(Faculty(of(North(Carolina(State(University(in(partial(fulfillment(of(the((

requirements(for(the(degree(of(Doctor(of(Philosophy(

(

(

Microbiology(

(

(

Raleigh,(North(Carolina(

2015(

(

APPROVED(BY:(

(

(

_______________________________( ( _______________________________(Dr.(Frank(Scholle( ( ( ( ( Dr.(Michael(L.(Sikes(Committee(Chair(((_______________________________( ( _______________________________(Dr.(Jonathan(W.(Olson( ( ( ( Dr.(Charles(S.(Apperson(

ii(((

(

DEDICATION!

I(would(like(to(dedicate(this(dissertation(to(my(family(and(friends,(who(have(always(

supported(me.((

(

((((((((((((( ((

iii(((

(

BIOGRAPHY!

Priya(was(born(in(Upstate(NY(and(went(to(Binghamton(University((State(University(of(New(

York),(where(she(earned(a(B.A.(in(Spanish(with(a(double(minor(in(Chemistry(and(

Anthropology.(After(graduating(from(SUNY(Binghamton(in(2007,(she(worked(as(a(laboratory(

technician(at(the(New(York(State(Department(of(Health(before(enrolling(in(the(School(of(

Public(Health(at(SUNY(Albany.(During(this(time,(she(became(more(interested(in(the(fields(of(

Microbiology(and(Immunology(and(moved(to(North(Carolina(after(graduating(with(an(MPH(

degree.(At(NC(State,(she(has(continued(to(explore(mechanisms(of(innate(immunity(that(

resulted(in(chronic(inflammation(after(infection(by(spirochetes.(In(addition(to(her(

microbiology(pursuits,(she(was(a(part(of(the(a(cappella(group(Ladies(in(Red(during(her(first(

two(years(at(NC(State,(while(continuing(to(participate(in(Binghamton(University(a(cappella(

alumni(events(and(recording(two(live(albums(and(one(studio(album(with(her(acaIbuddies.(

Her(love(for(both(music(and(microbiology(are(well(known(to(her(friends(and(family,(and(her(

dedication(to(microbiology(research(has(led(to(the(written(work(you(are(about(to(read.((

iv(((

(

ACKNOWLEDGMENTS!

I(would(like(to(thank(several(people(for(helping(me(along(my(journey.(I(would(first(like(to(

thank(my(current(advisor,(Dr.(Frank(Scholle(for(taking(me(into(his(lab(and(helping(me(round(

out(a(research(project(that(is(fairly(substantial(and(has(turned(into(prettyI(well(rounded(story.(

I(would(also(like(to(thank(Dr.(Scholle(and(my(committee(members(Dr.(Michael(Sikes,(Dr.(

Jonathan(Olson,(and(Dr.(Charles((Apperson,(for(pushing(me(to(be(a(better(writer,(speaker(

and(allIaround(more(critical(thinker(in(design(and(implementation(of(experiments.(I(am(very(

grateful(for(that.(I(would(like(to(thank(Dr.(Jennifer(Miller(for(her(insight(when(I(was(in(her(lab(

conducting(research(on(bacteria(that(cause(chronic(inflammatory(disease.(It(was(because(of(

this(research(that(I(became(very(interested(in(causative(factors(of(chronic(inflammation.(I’d(

like(to(extend(a(thankIyou(to(my(former(labmates(and(colleaguesb(Alex,(Amy,(Breanna,(

Heather,(Karissa,(Mehnaz,(Michael,(and(Sylvia.(Thank(you(to(the(Scholle(lab(members(

Farah(and(Lindsey,(who(have(been(my(adoptive(lab(family(and(provided(helpful(insight(into(

experiments(and(planning(weddings.(Lindsey,(you’re(my(twinsie,(thanks(for(joining(me(in(

teaching,(singing(along(to(musicals(or(working(in(lab(to(the(best(musical(disney(radio(station(

ever.(Thanks(to(all(of(the(Microbiology(faculty,(staff(and(students(for(your(guidance(during(

my(time(here.(On(a(personal(note(I(want(to(thank(both(of(my(A(cappella(families(for(letting(

me(be(a(part(of(an(incredible(experience.(My(family,(who(has(supported(me(and(all(of(my(

choices(that(led(me(here.(Mom(and(Nimi,(you’re(wonderful.(Lucy,(thanks(for(putting(up(with(

the(13+(hour(drives.(You(truly(are(a(oneIcat(wonder.(And(finally,(Matt.(Words(cannot(

express(how(thankful(I(am(for(your(support(during(these(last(fourIplus(years(or(how(much(I(

love(you.(Thank(you(so(much.((

(

(

v((((

(

TABLE!OF!CONTENTS!

List!of!Tables!............................................................................................................!vi!!!List!of!Figures!..........................................................................................................!vii!!Chapter!1:!Literature!Review!...................................................................................!1!!

References!...................................................................................................!47!!Chapter!2:!The!adaptor!molecule!Trif!contributes!to!murine!host!defense!during!Leptospiral!infection!..................................................................................!78!!!

Abstract!........................................................................................................!79!Keywords.....................................................................................................!!79!Introduction!..................................................................................................!80!Materials!and!methods!................................................................................!83!Results!..........................................................................................................!89!Discussion....................................................................................................!97!References!.................................................................................................!104!Ethical!statement!.......................................................................................!111!Acknowledgements!...................................................................................!111!Figure!Legends!and!Figures!!....................................................................!112!!

Chapter!3:!Murine!TLR7!dependent!ISG!and!cytokine!responses!to!B.#burgdorferi!and!B.#burgdorferi!RNA!...................................................................!120((!

Abstract!......................................................................................................!120!Introduction!................................................................................................!121!Materials!and!methods!..............................................................................!123!Results!........................................................................................................!129!Discussion..................................................................................................!133!Figures!........................................................................................................!137!References!.................................................................................................!143!

!

Chapter!4:!Discussion!.........................................................................................!150!!

References!.................................................................................................!155!(

(

vi(((

(

LIST!OF!TABLES!

Chapter!1:!Literature!Review:!Innate!immune!responses!to!infection!by!Spirochetes!!

Table!1.!TLR’s!and!immune!responses!to!Leptospiral!and!and!Borrelial!infection.!Table(outlining(the(different(TLRs,(localization,(and(recognition(of(L.*interrogans*and*B.*burgdorferi,(if(previously(described.(References(are(listed(in(the(far(right(column(of(the(table.(

..................................................................................................................................!27!!

Table!2.!RLRs,!NLRs!and!immune!responses!to!Leptospiral!and!Borrelial!infection.!Table(outlining(additional(PRRs(including(RLRs,(NLRs(and(CIType(Lectins,(localization,(and(recognition(of(L.*interrogans(and(B.*burgdorferi,(if(previously(described.(References(are(listed(in(the(far(right(column(of(the(table.(

..................................................................................................................................!40!!!Chapter!2:!The!adaptor!molecule!Trif!contributes!to!murine!host!defense!during!Leptospiral!infection!

Supplemental!Table!1.!Functional!observation!of!leptospiral!infection!in!bladders!.!(Bladders(were(removed(from(mice(postIeuthanasia(and(incubated(in(5mL(EMJH(as(described(in(methods(for(10(days.(10uL(of(cultured(medium(was(observed(on(a(slide(via(Dark(Field(microscopy(for(motile(Leptospires.(*One(mouse(died(at(4(d.p.i.(

................................................................................................................................!117!

Chapter!3:!Murine!TLR7!dependent!ISG!and!cytokine!responses!to!B.#burgdorferi!and!B.#burgdorferi#RNA!

Table!1.!RNA!from!B.#burgdorferi#is!predicted!to!bind!TLR7.!!Sequences(of(Mouse(PRR’s((by(Uniprot(ID)(and(whole(genome(sequences(of(B31(and(N40(were(analyzed(for(RNAIProtein(Interactions(using(the(RPIISeq(program((53,(54).((RF,(SVM)>0.5(indicates(a(positive(predicted(binding(probability(between(Bb(RNA(and(the(PRR(in(the(left(column.(!

................................................................................................................................!142!

vii(((

(

LIST!OF!FIGURES!

Chapter!1:!Literature!Review:!Innate!immune!responses!to!infection!by!Spirochetes!!

Figure!1.!Outcomes!of!leptospiral!infection.!(Comparison(between(acute(or(chronic(infection(as(indicative(of(biphasic(disease.(Reproduced(and(modified(from((38).(

....................................................................................................................................!4!!!

Figure!2.!Innate!responses!to!borrelial!infection.!Diagram(depicting(the(tickImammal(interface(and(cellular(immune(responses(near(site(of(tick(bite,(including(macrophage(activation(and(initiation(of(the(adaptive(immune(response.(Reproduced(and(modified(from((56).(

..................................................................................................................................!11!

Figure!3.!Cellular!responses!and!cytokine!production!during!infection!by!L.#interrogans!and!B.#burgdorferi.!Simplified(schematic(of(immune(responses(to(leptospiral(and(borrelial(infection.((1)(L.*interrogans(are(able(to(evade(neutrophil((Nφ)(responses,(but*B.*burgdorferi*are(recognized(and(killed(by(neutrophils,(resulting(in(proIinflammatory(cytokine(production(and(Dendritic(Cell((DC)(activation.((2)(Recognition(of(both(L.*interrogans*and(B.*burgdorferi(by(Natural(Killer((NK)(cells(also(results(in(proIinflammatory(cytokine(production(and(T(cell(activation((3).((Monocytes((Mo)(also(recognize(Pathogen(Associated(Molecular(Patterns((PAMPs)(from(L.*interrogans*and(B.*burgdorferi*via(Pathogen(Recognition(Receptor((PRR)(signaling,(and(can(undergo(maturation(to(become(macrophages((Mφ)((4)(or(DCs((5).(Activation(of(these(cell(types(ultimately(results(in(initiation(of(adaptive(immune(responses.((6)(L.*interrogans*is(also(able(to(evade(complement(without(recognition(by(AntiILeptospira*IgM.!

..................................................................................................................................#12##!Figure!4.!PRRVPAMP!recognition!during!leptospiral!and!borrelial!infection.!Simplified(schematic(showing(recognition(of(Borrelia((blue)(or(Leptospira((purple)(by(TLRs(or(NLRs((Orange)(and(recruitment(of(adaptors((yellow)(that(result(in(downstream(signaling(and(induction(of(proIinflammatory(cytokines(or(Type(I(Interferon((IFN)(or(Interferon(Stimulated(Genes((ISG).(Adapted(from((4,(50,(56,(137,(139,(207I210)(

..................................................................................................................................!26!!!

viii(((

(

Chapter!2:!The!adaptor!molecule!Trif!contributes!to!murine!host!defense!during!Leptospiral!infection!!

Figure!1.(Leptospiral!infection!and!changes!in!weight!of!infected!mice.(WT(and(Trif5/5(mice(were(i.p.(injected(with(2x(108(bacteria/mouse(and(weighed(daily((A).(Changes(in(weight(over(time(are(described(in(methods(and(averaged(across(multiple(experiments.(Kinetics(of(leptospiral(load(in(kidneys((B),(lungs((C),(and(blood((D)(as(measured(by(copies(of(L.*interrogans(16s(rRNA(per(1000(copies(of(betaIactin(using(qRTIPCR.(*p<0.05,(***p<0.005.(ND(=(Not(detected.(................................................................................................................................!111!!

Figure!2.!Induction!of!Type!I!and!II!IFN!and!ISG!in!WT!and!Trif3/3!infected!mice.!(ISG((Ifit1,*Oasl2,*Gbp2)(Transcripts(from(kidneys((A),(blood((B)(and(lungs((C)(were(measured(using(qRTIPCR(in(addition(to(levels(of(Ifnγ(and(Cxcl10(transcripts(from(kidneys((D),(blood((E)(and(lungs((F)(and(normalized(to(copies(per(1000(copies(of(beta(actin,(prior(to(foldIchange(calculations(as(described(in(methods.(IFNγ(in(soluble(extracts(from(kidneys(of(mice(at(1(d.p.i.((G)(were(determined(by(cytokine(sandwich(ELISAs.(Levels(of(Irf3(in(the(kidneys,(blood(and(lungs,(were(measured(using(qRTIPCR(as(described(above.((Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01,(***p<0.005.

................................................................................................................................!112!

Figure!3.!ProVinflammatory!cytokine!induction!in!kidneys!of!WT!and!Trif3/3!infected!mice.!!ProIinflammatory(transcripts(from(kidneys((A,C,(E,(G)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(copies(of(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Levels(of((B)(TNFα,((D)(ILI6((F)(ILI1β,((and((H)(ILI12p70(in(soluble(extracts(from(kidneys(of(mice(at(1(d.p.i.(were(determined(by(cytokine(sandwich(ELISAs.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(................................................................................................................................!113##!Figure!4.!ProVinflammatory!cytokine!induction!in!blood!of!WT!and!Trif3/3!infected!mice.!ProIinflammatory(transcripts(from(blood(at(1(d.p.i.((A)(and(3(d.p.i.((B)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(copies(of(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Levels(of(ILI6(in(sera(of(mice(at(1(d.p.i.((C)(and(3(d.p.i.((D)(were(determined(by(cytokine(sandwich(ELISA.(

ix(((

(

Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(!

................................................................................................................................!114!!!

Figure!5.!Leptospiral!specific!IgM!and!IgG!responses!between!WT!and!Trif3/3!Mice.!Levels(of(leptospiralIspecific(IgM((at(3,(7,(and(14(d.p.i.((A,(B,(C),(leptospiralIspecific(IgG(at(7(and(14(d.p.i.((D,(E)(were(quantified(by(ELISA(as(described(in(Methods.(OD(490(measurements(of(leptospiralIspecific((IgG2c((F)(,(IgG1((G,(H)(and(IgA((I)(were(determined(by(ELISA(as(described(in(Methods.(Data(are(depicted(as(Mean(+/I(SEM(averaged(across(multiple(independent(experiments.(*p>0.05,(**p<0.01,(***p<0.005.!

................................................................................................................................!115!!!

Figure!6.!Diagram!of!Leptospiral!pathogenesis!in!WT!and!TrifV/V!mice.!!(A)(Bacterial(burden(in(the(kidneys((16s)(,(and(Weight(change(during(the(course(of(leptospiral(infection(in(WT(and(TrifI/I(mice((B)(Immune(response(for(WT(and(TrifI/I((mice((by(AntiILeptospira(IgM((ALIIgM)(TNF(in(the(kidneys(as(a(marker(for(renal(inflammation(in(WT(and(TrifI/I(mice.(

................................................................................................................................!116!!!

Supplemental!Figure!1.!Creatinine!levels!in!serum!of!WT!and!Trif3/3!infected!mice.Creatinine(was(measured(in(serum(of(mice(at(3(d.p.i.((A)(using(a(kit(from(Cayman(Chem(following(the(manufacturers(instructions.(Serum(Nitrite(levels(were(measured(in(mice(at(7(d.p.i.((B)(and(14(d.p.i.((C)(as(described(in(methods.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.!................................................................................................................................!118!!!

Supplemental!Figure!2.!ProVinflammatory!cytokine!induction!in!lungs!of!WT!and!Trif3/3!infected!mice.(ProIinflammatory(transcripts(from(lungs(at(1(d.p.i.((A),(3(d.p.i.((B),(and(7(d.p.i.((C)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(((................................................................................................................................!119!!!

Chapter!3:!Murine!TLR7!dependent!ISG!and!cytokine!responses!to!B.!burgdorferi!and!B.!burgdorferi!RNA!

x((((

(

Figure!1.!infection!of!B.#burgdorferi#in!WT!and!Tlr73/3!mice.!Mice(were(i.d.(injected(with(BSKIII(or(B.*burgdorferi*(Bb)(for(1(or(4(weeks(as(described(in(methods.((A)(Quantification(of(RecA(in(joints(as(a(marker(of(bacterial(burden(and(detection(of(host(response(in(serum(by(AntiIBorrelia(IgG(ELISA((B)(or(AntiIBorrelia(IgM(ELISA((C).(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(multiple(experiments.(*p<0.05,(***p<0.005(

................................................................................................................................!137!!

Figure!2.!ISG!Induction!in!WT!and!Tlr73/3!mice!at!1!week!post!infection.!ISG(transcript(expression(was(measured(in(the(joints(of(WT((filled(bars)(and(Tlr75/5(gray(bars)(mice(by(qRTIPCR(as(described(in(methods.(Dashed(line(at(2(indicates(a(threshold(for(fold(induction(over(uninfected.(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(2(independent(experiments.((

................................................................................................................................!137!!

Figure!3.!ISG!induction!in!BMDMs!of!WT!and!Tlr73/3!mice.!(BMDMs(were(stimulated(with(RPMI(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(2ug,(or(Imiquimod((R848)(at(1ug(as(described(in(methods(for(6(hours.(ISG(mRNA(expression(was(measured(using(qRTIPCR(and(fold(change(was(calculated(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(2(independent(experiments.((

................................................................................................................................!138!!

Figure!4.!Cytokine!production!in!culture!supernatants!from!BMDMs!of!WT!and!TLR73/3!mice.!BMDMs(were(stimulated(with(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(1ug,(or(Imiquimod((R848)(at(1ug(for(4(or(24(hours(as(described(in(methods.(Cytokine(production(was(measured(by(sandwich(ELISA(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.((

................................................................................................................................!139!

Figure!5.!ISG!induction!in!BMDMs!of!WT!and!Myd883/3!mice.!(BMDMs(were(stimulated(with(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(2ug,(or(Imiquimod((R848)(at(1ug(as(described(in(methods(for(6(hours.(ISG(induction(was(measured(using(qRTIPCR(and(fold(change(was(calculated(as(previously(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.(

................................................................................................................................!140!

Figure!6.!ILV6!production!in!WT!and!Myd883/3!BMDMs.!BMDMs(were(stimulated(with(media,(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(1ug,(or(the(TLR7(agonist(Imiquimod((R848)(at(1ug(for(24(hours(as(described(in(

xi(((

(

methods.(Cytokine(production(was(measured(by(sandwich(ELISA(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.(

................................................................................................................................!141

1(

Chapter!1!

1.1!Innate!immune!responses!to!infection!by!Spirochetes!

Borrelia*burgdorferi(and(Leptospira*interrogans(are(two(pathogenic(

spirochetes(that(can(cause(chronic(infection(in(humans(and(animals.(Innate(immune(

responses(have(been(extensively(studied(using(animal(models(and(in*vitro(studies.(

Innate(immunity(to(infection(by(these(spirochetes(is(comprised(of(humoral(and(

cellular(responses(that(make(up(the(innate(and(adaptive(branches(of(immune(

responses.(Cellular(responses(can(include(mechanisms(of(cell(mediated(pathogen(

destruction,(whereas(humoral(responses(in(innate(immunity(include(cytokine(and(

chemokine(production(and(complement.(In(this(review,(the(importance(of(

understanding(immune(responses(in(regards(to(two(pathogenic(spirochetes,(L.*

interrogans*and*B.*burgdorferi,(is(addressed,(including(model(organisms(of(infection.(

Innate(immune(responses(to(infection(by(L.*interrogans*and*B.*burgdorferi(are(also(

described,(including(cellular(and(humoral(responses,(and(intracellular(signaling.((

!

1.1.1!Leptospira#interrogans!and!Leptospirosis!

The(neglected(tropical(disease(Leptospirosis(is(caused(by(members(of(the(

leptospira(genus,(including(Leptospira*interrogans((1I4).(Leptospires(are(aerobic,(as(

the(bacteria(can(freely(live(in(the(soil(and(water,(surviving(in(alkaline(environments(

for(months(at(a(time((1,(3).(The(leptospiral(genome(consists(of(two(circular(

chromosomes(with(genes(that(code(for(several(virulence(factors(including(

2(

lipoproteins,(lipopolysaccharide((LPS)(and(several(genes(involved(in(LPS(

biosynthesis((5).(Several(studies(have(provided(insight(into(key(differences(in(

genomes(of(pathogenic(leptospires,(and(highlighted(the(importance(of(LPS(during(

infection(and(shown(that(expression(of(these(genes(differs(by(host(model(of(infection(

(5I7).(Mutants(have(been(generated(to(identify(genes(that(might(be(implicated(in(

infection(and(transmission((8I12).((

Leptospires(are(transmitted(by(contact(between(infected(animal(fluids(or(

contaminated(water(and(broken(skin(via(cuts(and(abrasions((1I3).((Rats(are(

asymptomatic(reservoir(carriers,(as(are(some(species(of(mice((13I15).(Humans(are(

considered(incidental(hosts(based(on(the(transmission(cycle((1,(3).(Patients(and(

animals(infected(with(pathogenic(Leptospira*spp.(exhibit(a(variable(range(of(

symptoms.(Symptoms(of(acute(infection(begin(with(headache,(fever,(myalgia,(and(

extend(to(jaundice,(kidney(and(liver(failure,(pulmonary(hemorrhaging,(and(death(

(Figure!1)((2,(16,(17).(Severe(symptoms(and(resulting(fatal(cases(have(been(

documented(as(Weil’s(Disease,(and(additional(cases(of(uveitis(in(horses(and(carditis(

in(humans(have(also(been(reported((2,(16I18).(Cases(of(Leptospirosis(have(been(

reported(since(at(least(the(beginning(of(the(early(20th(century((19),(in(addition(to(

epidemiological(surveys((19I23).(Leptospirosis(is(prevalent(in(the(tropics((22I25)(and(

outbreaks(are(frequently(reported(worldwide((17,(21,(26I28).(There(is(currently(no(

approved(vaccine(against(pathogenic(Leptospira*spp.(but(research(is(in(progress(to(

design(a(safe((and(effective(vaccine(for(domestic(animals((29).!(

3(

Because(symptoms(are(variable(and(are(similar(to(many(other(symptoms(

especially(for(cases(occurring(in(the(tropics,(early(and(accurate(diagnosis(and(

treatment(is(imperative(for(bacterial(clearance(and(resolution(of(infection.((Methods(

of(detecting(Leptospira*spp.((have(been(documented((21,(30,(31).(The(microscopic(

agglutination(test((MAT)(has(been(regarded(as(one(of(the(standard(assays(to(

diagnose(leptospiral(infection,(but(ELISAs(are(also(used(to(detect(a(host(response(to(

leptospirosis(by(quantifying(the(levels(of(immunoglobulin((Ig)(specific(to(leptospires(

in(patient(serum,(and(several(variations(of(the(assay(have(been(developed(for(

detection(of(leptospires((17,(21,(32,(33).(Quantitative(PCR(for(detection(of(the(

Leptospira(16s(rRNA(subunit(was(developed(in(the(early(90’s(and(is(still(used(today(

(34I36).(Because(early(detection(is(critical(for(treatment(of(disease,(rapid(diagnostic(

methods(are(also(being(developed(and(evaluated(for(sensitivity(and(specificity((30,(

37).(Leptospirosis(is(treatable(with(penicillin(if(detected(early,(but(administration(of(

antibiotics(like(penicillin(at(a(later(stage(of(infection(is(ineffective((2,(10).(It(is(

imperative(that(the(immune(response(to(leptospiral(infection(is(elucidated(because(of(

the(prevalence(of(leptospirosis,(variability(in(symptoms,(establishment(of(chronic(

infection(in(human(and(rodent(reservoir(hosts,(and(need(to(develop(more(efficient(

diagnostic(assays(and(effective(vaccines.(

(

4(

(

Figure!2.!Outcomes!of!leptospiral!infection.!(Comparison(between(acute(or(chronic(infection(as(indicative(of(biphasic(disease.(Reproduced(and(modified(from((38).(

(

1.1.2!Animal!models!of!Leptospiral!infection!

Leptospirosis(has(been(studied(using(many(different(animal(models.(Rats(are(

asymptomatic(reservoir(hosts,(due(to(chronic(carriage(and(shedding(in(the(kidneys(

(6,(13,(14).((Hamsters(and(guinea(pigs(die(rapidly(upon(intraperitoneal(injection(with(

leptospires((39I42).(Recently(the(route(of(inoculation(was(investigated(in(hamsters,(

because(although(intraIperitoneal(injection(is(the(conventional(method(used,(it(does(

not(mimic(the(natural(route(of(infection((42).(Subcutaneous(injection(of(L.*interrogans*

serovar(sv.)(Copenhageni(strain(st.)(Fiocruz(L1I130(yielded(similar(results(to(

intraperitoneal(injection,(but(intradermal(injection(of(hamsters(did(not(induce(a(

5(

systemic(response((42).(Outcomes(due(to(alternate(inoculation(route(in(other(animal(

models,(including(rats(and(mice,(have(not(been(determined.((

C3H/HeJ(mice(die(within(7(days(after(injection(with(leptospires,(but(C3H/HeN(

mice(survive(infection((43I45).(This(is(likely(due(to(a(mutation(resulting(in(the(

absence(of(a(functional(TLR4(in(C3H/HeJ(mice((43I45).(A(recent(animal(model(has(

been(developed(using(10Iweek(old(C3H/HeJ(mice(showing(inflammation(in(the(

kidney,(but(not(in(the(liver(or(lung(by(upIregulation(of(proIinflammatory(mRNA(

transcripts,(and(an(increase(IgG1(antibody(production((46).((Balb/c(mice(show(mild(

symptoms,(and(remain(infected(with(chronic(carriage(of(leptospires,(but(C57BL/6(

mice(show(moderate(symptoms(of(infection(and(renal(fibrosis(during(chronic(

leptospiral(infection((45,(47,(48).(Because(C57BL/6(mice(show(acute(and(chronic(

symptoms(of(leptospiral(infection,(they(have(been(used(as(an(animal(model(to(

elucidate(mechanisms(of(host(defense(and(leptospiral(pathogenesis((48I51).(

Ultimately,(the(genetic(variation(between(strains(and(outcomes(during(infection(are(

characteristics(that(make(mice(a(useful(animal(model(to(dissect(mechanisms(of(

acute(and(chronic(infection.((

(

1.2.1!Borrelia#burgdorferi!and!Lyme!Disease!

Lyme(disease((LD)(was(first(reported(in(Connecticut(in(the(1980’s((7,(52I54).((

The(causative(agent(is(the(pathogenic(spirochete(Borrelia*burgdorferi((7,(52I54).(B.*

burgdorferi*is(a(gram(negative(microaerophile(containing(one(linear(chromosome(

6(

and(several(linear(and(circular(plasmids(varying(in(composition(by(isolate((55,(56).(

The(genome(sequence(of(B.*burgdorferi*B31(has(been(previously(reported((55,(56),(

and(is(comprised(of(one(linear(chromosome(approximately(910(kb(in(length(and(at(

least(17(linear(and(circular(plasmids(containing(genes(that(code(for(outer(surface(

proteins,(adhesins,(response(regulators,(and(proteins(that(enable(immune(evasion.(

(55,(56).(The(linear(plasmid(lp28(encodes(the(vls(locus,(enabling(antigenic(variation(

and(subsequent(evasion(of(humoral(responses((56I58).(Some(of(the(outerIsurface(

proteins((Osp)(expressed(from(linear(or(circular(plasmids(mediate(infection(and(

dissemination(in(ticks(and(mammals(as(part(of(the(enzootic(cycle.(The(circular(

plasmid(cp26(contains(the(gene(coding(for(OspC,(a(surface(lipoprotein(required(for(

dissemination(in(mice(and(temperature(regulation((55,(56).(OspC(is(required(for(

establishment(of(the(initial(infection(in(mice((59,(60).(The(expression(of(OspC,(which(

peaks(during(initial(infection(in(mice,(is(inversely(correlated(with(the(expression(of(

linear(plasmid(lp54(encoding(gene(ospA.(OspA(is(required(for(colonization(in(ticks,(

by(binding(the(receptor(TROSPA(in(the(midgut(after(transmission((61I65).((

B.*burgdorferi*is(transmitted(primarily(by(infected(Ixodes*spp(ticks.(Ixodes*

scapularis(is(found(on(the(east(coast(and(Ixodes*pacificus(is(found(on(the(west(coast(

(56,(65I68).(Infected(ticks(will(“quest”(to(find(a(mammalian(host(and,(once(on(the(

skin,(create(a(hypostome,(subsequently(penetrating(the(dermis(and(attaching(to(the(

skin((56,(65I68).(Tick(salivary(proteins(have(immunomodulatory(properties(that(

impact(responses(in(animals,(and(recently(have(been(considered(as(targets(for(

7(

vaccines(and(drugs(to(facilitate(the(immune(response(in(animals(against(bacteria(

(56,(65I68).(As(seen(in(leptospiral(infection,(wild(mammals(are(transmission(hosts(

and(humans(are(incidental(hosts(in(the(transmission(cycle(of(B.*burgdorferi*(2,(56).((

After(a(tick(bite,(B.*burgdorferi*will(replicate(and(disseminate(rapidly(from(the(

site(of(infection,(establishing(infection(at(other(sites((69).(Symptoms(of(infection(can(

begin(with(a(bull’sIeye(rash(at(the(origin(of(the(tick(bite(site,(known(as(Erythema(

migrans((EM)((69).(Asymptomatic(cases(have(also(been(reported(but(EM(is(a(critical(

feature(of(clinical(manifestation(at(1I2(weeks(after(a(tick(bite(that(leads(to(accurate(

diagnosis(of(this(disease((69).(Other(lesions(may(also(form(where(dissemination(of(

spirochetes(occur,(but(these(are(smaller(and(don’t(represent(the(characterized(bullsI

eye(target(appearance((69).(Additional(symptoms(include(fever,(fatigue(and(

headaches,(but(failure(to(treat(infection(with(antibiotics(results(in(an(extension(of(the(

acute(symptoms(to(Lyme(Arthritis((69).((

Serological(methods(of(diagnosing(B.*burgdorferi*infection(have(been(the(

conventional(approach(and(are(more(accurate(compared(to(more(direct(methods(like(

PCR(and(culturing(bacteria((70I73).(Concerns(over(direct(techniques(have(been(

previously(addressed(by(the(Centers(for(Disease(Control((CDC)(due(to(an(elevated(

number(of(false(positive(results(from(enrichment(steps(of(cultured(specimens(from(

patients((74).(The(current(methods(of(diagnosing(LD(are(serological(assessment(of(

exposure(using(FDA(approved(antigenIspecific(ELISAs(and(a(followIup(with(western(

blots((70I73).(LD(can(be(treated(with(administration(of(tetracycline((doxycycline)(for(

8(

14(days,(or(penicillin(if(patients(have(a(history(of(adverse(responses(to(the(family(of(

tetracyclines.((Despite(treatment,(humoral(immunity(to(B.*burgdorferi*is(short(lived(

and(no(vaccine(is(currently(available(on(the(market(for(humans.(Initial(studies(in(

mice(showed(that(immunization(with(recombinant(OspA(provided(a(protective(

response(after(challenge(with(B.*burgdorferi*by(tick(feeding((75).(Based(on(these(

studies,(a(vaccine(containing(recombinant(OspA(was(developed(and(passed(preI

clinical(and(clinical(trials((75).(Due(to(its(low(efficacy(in(producing(a(sustained(

immune(response,(the(vaccine(was(taken(off(the(market((75).(Additional(vaccine(

candidates(are(being(developed,(but(since(humans(are(not(primary(hosts,(and(the(

exposure(is(based(on(environmental(interactions,(prevention(of(tick(interactions(are(

important(to(take(into(consideration.((

(

1.2.2!Animal!models!of!infection!by!B.#burgdorferi!

B.*burgdorferi*pathogenesis(has(been(studied(in(mice,(rats(and(hamsters,(but(mice(

are(the(primary(animal(model(to(elucidate(mechanisms(of(inflammation(and(arthritis(

(76I81).(Genetic(differences(between(mice(strains(have(been(used(to(dissect(

mechanisms(of(varied(host(responses(that(result(in(moderate(to(severe(phenotypes(

(82I86).(C3H/HeJ(mice(show(severe(symptoms,(including(increased(ankle(joint(

swelling(during(the(peak(of(arthritis(severity,(while(C57BL/6J(mice(show(a(more(

moderate(phenotype((82I86).((The(differences(in(these(phenotypes(have(been(

9(

explained(by(differences(in(induction(of(IFN(and(ISG,(which(is(an(additional(topic(in(

this(chapter((82I86).((

(2.1!Cellular!responses!in!innate!immunity!to!infection!by!Leptospira!and!

Borrelia#

2.1.1!Neutrophils!

Neutrophils(are(the(earliest(responders(to(the(site(of(infection(in(the(innate(

immune(response(to(bacteria(and(fungi((87I89).(Neutrophils(develop(from(

hematopoietic(stem(cells((HSC)(in(the(bone(marrow,(and(are(bound(to(stromal(cells(

expressing(CXCL12(with(a(CXCR4(receptor((87I89).(CXCR2(mediates(neutrophil(

release,(resulting(in(migration(en*masse(to(the(site(of(infection(and(multiple(

mechanisms(that(result(in(inflammation,(activation(of(other(cell(types,(and(control(of(

infection((87I89).(Neutrophils(are(a(critical(component(to(innate(immune(responses,(

as(patients(with(lower(counts(are(more(susceptible(to(disease((87,(88).(Mechanisms(

include(degranulation(of(microbicidal(compounds,(reactive(oxygen(species((ROS)(

and(reactive(nitrogen(species((RNS),(secretion(of(cytokines(and(chemokines(that(

recruit(other(cells(to(the(site(of(infection,(phagocytosis(of(bacteria(or(fungi,(and(finally(

release(of(neutrophils(extracellular(traps((NETs)(prior(to(cell(death,(or(apoptosis.((

Depending(on(the(disease,(neutrophils(have(beneficial(and(damaging(

functions(that(can(heal(or(damage(tissue(because(they(can(destroy(nucleated(and(

nonInucleated(tissue(via(degranulation(and(release(of(microbicidal(compounds((87I

89).(Microbicidal(compounds(include(serine(proteases,(cathepsins,(myeloperoxidase(

10(

(MPO),(matrixmetalloproteases((MMP)((87I89).(Upon(recognition(of(bacteria(or(

fungi,(neutrophils(will(undergo(degranulation,(leading(to(selfIdestruction(and(

destruction(of(bacteria((87I89).(In(humans,(neutrophils(express(all(TollIlike(

Receptors((TLRs)(except(for(TLR3(and(7(and(MyD88(is(the(predominant(adaptor(via(

TLR4(that(leads(to(production(of(proIinflammatory(cytokines,(including(ILI6,(ILI1β,(

TNFα,(and(IFNγ((87,(88).(NodILikeIReceptor((NLR)(pathways,(including(that(of(

NLPR3(and(subsequent(inflammasome(activation(also(have(been(described((87,(

88).(Secretion(of(proIinflammatory(cytokines(and(chemokines(results(in(recruitment(

of(antigen(presenting(cells((APCs)(including(dendritic(cells((DCs)(and(monocytes.(

The(surface(molecule(CD11b,(expressed(on(neutrophils(will(bind(DCISIGN(and(

activate(DCs(to(produce(ILI23(and(ILI17((89).(The(cytokines(and(chemokines(

produced(also(affect(macrophage(polarization(to(different(types,(and(activation(of(B(

and(T(cells(by(BILymphocyte(Stimulator((BLyS)(or(IFNγ,(respectively((88,(89).(NETs(

are(comprised(of(heterochromatin(and(antimicrobial(proteins(that(are(exocytosed(

from(neutrophils(and(trap(and(lyse(bacteria(and(fungi((87I89).((

(

11(

(

Figure!2.!Innate!responses!to!borrelial!infection.!Diagram(depicting(the(tickImammal(interface(and(cellular(immune(responses(near(site(of(tick(bite,(including(macrophage(activation(and(initiation(of(the(adaptive(immune(response.(Reproduced(and(modified(from((56).(

(

(

12(

((Figure!3.!Cellular!responses!and!cytokine!production!during!infection!by!L.#interrogans!and!B.#burgdorferi.!Simplified(schematic(of(immune(responses(to(leptospiral(and(borrelial(infection.((1)(L.*interrogans(are(able(to(evade(neutrophil((Nφ)(responses,(but*B.*burgdorferi*are(recognized(and(killed(by(neutrophils,(resulting(in(proIinflammatory(cytokine(production(and(Dendritic(Cell((DC)(activation.((2)(Recognition(of(both(L.*interrogans*and(B.*burgdorferi(by(Natural(Killer((NK)(cells(also(results(in(proIinflammatory(cytokine(production(and(T(cell(activation((3).((Monocytes((Mo)(also(recognize(Pathogen(Associated(Molecular(Patterns((PAMPs)(from(L.*interrogans*and(B.*burgdorferi*via(Pathogen(Recognition(Receptor((PRR)(signaling,(and(can(undergo(maturation(to(become(macrophages((Mφ)((4)(or(DCs((5).(Activation(of(these(cell(types(ultimately(results(in(initiation(of(adaptive(immune(responses.((6)(L.*interrogans*is(also(able(to(evade(complement(without(recognition(by(AntiILeptospira*IgM.!

(

(

The(roles(of(neutrophils(have(been(studied(in(the(innate(immune(response(to(

infection(by(both(L.*interrogans*and(B.*burgdorferi*(Figure!2,!Figure!3).(CoIculturing(

13(

normal(human(serum(with(nonpathogenic(L.*biflexa(resulted(in(nearly(complete(lysis(

of(all(bacteria,(but(not(with(L.*interrogans*(90),(suggesting(that(pathogenic(

leptospires(were(not(sensitive(to(killing(by(polymorphonuclear(cells((PMNs).(

However,(incubation(of(Human(Umbilical(Vein(Endothelial(Cells((HUVECs)(with(the(

pathogenic(spirochete(L.*interrogans*sv.(icterohemorrhagiae(st.Teramo,(in(a(time(

and(dose(dependent(manner,(followed(by(addition(of(PMNs(from(whole(blood(

resulted(in(increased(adhesion(of(neutrophils(to(endothelial(cells((91).(Incubation(of(

PMNs(with(B.*hermsii(leads(to(bacterial(killing((92)(and(increased(expression(of(ILI8(

in(humans,(or(KC(in(mice,(as(well(as(increased(migration(and(adhesion(to(

endothelial(cells(during(bacterial(infection((91,(93I95).(CoIculturing(B.*burgdorferi,(

HUVECs(and(isolated(PMNs(showed(increased(adhesion(of(neutrophils,(but(

antibodyIblocking(of(ILI8(with(HUVECs(resulted(in(reduced(neutrophil(migration((94,(

96).((

(2.1.2!Natural!Killer!Cells!

Natural(Killer((NK)(cells(also(appear(early(during(the(innate(immune(response(

to(infection(by(bacteria(and(viruses((97I99).(NK(cells(are(a(subset(of(innate(lymphoid(

cells(named(for(their(cytotoxic(abilities,(and(are(located(in(the(lymph(nodes(and(

spleen((97I99).(Two(populations(of(distinct(functions(have(been(identified(in(humans(

based(on(expression(of(the(surface(markers(CD56(and(CD16((97I99).(In(humans,(

CD56hiCD16lo(expressing(NK(cells(have(cytotoxic(functions,(killing(target(cells(that(

have(been(infected(with(virus(due(to(absence(of(MHC(Class(I(expression((97I99).((

14(

CD56loCD16hi(expressing(NK(cells(are(identified(by(additional(surface(markers,(

including(CXCR3,(and(primarily(secrete(proIinflammatory(cytokines,(including(IFNγ,(

TNFα,(and(GMICSF((97I99).((

Depending(on(the(cytokines(produced,(nonIcytotoxic(functions(of(NK(cells(

also(include(directing(T(cell(polarization(towards(TIhelper((TH)(cell(subtypes,(

including(a(TH1(phenotype(by(IFNγ(or(a(TH17(phenotype(by(ILI22,(and(inducing(

maturation(of(DCs((97I99).((Mice(lack(a(CD56(homologue,(but(their(NK(cells(contain(

the(markers(CD27(and(MAC1((97I99).(CD27hiMac1lo(expressing(murine(NK(cells(

have(cytotoxic(functions(and(produce(IFNγ,(whereas(CD27loMac1hi(expressing(NK(

cells(predominantly(produce(cytokines.((NK(cells(are(activated(by(the(IFN(and(proI

inflammatory(cytokines(including(ILI1(and(IFNγ(induced(ILI12,(ILI15,(and(ILI18((97I

99).((

NK(cells(do(not(play(a(role(in(pathology(of(arthritis(development(in(mice(

infected(with(B.*burgdorferi,(but(still(contribute(to(immune(responses(and(

inflammation((99I106).(NK(cell(functions(in(response(to(B.*burgdorferi(have(been(

studied(in*vitro,*in(mice,(in(whole(blood(and(serum(from(patients(and(healthy(controls(

(105).(Activation(of(IFNγ(producing(NK(cells(by(B.*burgdorferi*sonicate,(and(purified(

surface(proteins(OspA(and(OspB(has(been(identified(in(mice((102,(107).(NK(cell(

activation(and(IFNγ(production(was(also(assessed(in(susceptible(and(resistant(

mouse(strains,(including(susceptible(C3H/HeJ(mice,(resistant(Balb/c(mice,(C57BL/6(

mice,(DBA2/J,(and(a(congenic(NK(depleted(strain((102).((Results(of(this(study(

15(

showed(that(NK(cells(were(the(primary(source(of(IFNγ(production,(which(is(elevated(

in(susceptible(C3H/HeJ(mice(but(not(mice(with(a(resistant(phenotype((102).(

Cytotoxic(function(determined(by(4HIChromium(release(was(higher(in(YACI1(murine(

lymphoma(cells(treated(with(culture(supernatants(of(C3H(popliteal(lymph(node(

cultures((102).(These(studies(collectively(show(that(NK(cells(are(important(in(the(

innate(immune(response(to(B.*burgdorferi.(

While(NK(cells(have(been(implicated(in(host(defense(against(B.*burgdorferi,(

fewer(studies(have(shown(NK(involvement(during(leptospiral(infection.(NK(cells(were(

not(enhanced(in(activity(after(treatment(of(YACI1(cells(with(LPS(from(L.*interrogans*

sv.(Copenhageni(or(L.*interrogans*sv.(hebdomadis(even(though(splenocytes(from(

Balb/c(mice(showed(a(higher(mitogenic(response(after(stimulation(with(either(strain(

(108).(Additional(studies(showed(an(elevation(of(CXCR3,(CXCL10,(and(IFNγ(in(

serum(of(patients(with(severe(leptospirosis(compared(to(healthy(controls((109I111).(

A(dose(dependent(induction(of(IFNγ,(ILI12,(and(TNFα(was(detected(in(culture(

supernatants(of(peripheral(blood(mononuclear(cells((PBMCs)(from(patients(with(

leptospirosis(and(PBMCs(from(healthy(controls(after(treatment(with(L.*interrogans,(

as(well(as(proliferation(of(PBMCs,(suggesting(involvement(of(NK(cells(in(

inflammation(during(infection((Figure!3)((112,(113).(Treatment(of(PBMCs(for(6(

hours,(followed(by(antibiotic(treatment(and(no(addition(of(cytokines(led(to(an(

increase(in(γδ+T(cells(and(CD56hiCD16hiCD3I(expressing(NK(cells((112,(113).(Similar(

increases(in(TH1(and(NK(responses(have(been(documented(in(cattle(after(

16(

vaccination(and(challenge(with(L.*borgpetersenii(sv.(Hardjo(st.(203((114).(Additional(

studies(are(required(to(determine(the(function(of(NK(cells(and(other(innate(lymphoid(

cells(in(the(innate(responses(to(leptospiral(infection(of(mice.((

(

2.1.3!Monocytes!and!Macrophages!

Mononuclear(phagocytes(are(present(in(several(tissues(near(epithelia(and(

can(be(further(classified(into(monocytes,(macrophages(and(dendritic(cells((115,(

116).(Both(monocytes(and(macrophages(are(involved(in(varied(cellular(responses(to(

microbial(infection((115,(116).(Monocytes(and(macrophages(are(extensively(involved(

in(innate(immune(responses(to(infection(by(bacteria(and(viruses((115I117).(

Monocytes(develop(in(the(bone(marrow(from(hematopoietic(stem(cells(and(myeloid(

progenitors,(and(can(differentiate(into(macrophages(or(dendritic(cells(depending(on(

the(pathogen(involved(and(the(mechanism(of(infection((115I117).(Several(tissueI

resident(groups(of(macrophages(have(been(identified,(including(alveolar(

macrophages(in(lungs,(Kupffer(cells(in(livers,(and(microglia(in(the(brain,(and(the(

resulting(pathologies(from(inflammation(vary(by(disease((118).(Alveolar(

macrophages(from(infected(hamsters(have(been(studied(during(leptospiral(infection(

(119),(where(pulmonary(dysfunction,(enhanced(nitric(oxide(production,(and(

enhanced(lymphocyte(production(in(infected(hamsters(have(been(reported((119).((

Macrophages(and(dendritic(cells(can(sense(extracellular(pathogens(via(pathogen(

17(

recognition(receptors((PRRs)(present(on(the(plasma(membrane(or(in(the(endosome(

(118,(120I124).((

Sensing(extracellular(or(intracellular(pathogens(via(pathogen(associated(

molecular(patterns((PAMPs)(results(in(activation(of(macrophages(and(subsequent(

polarization(towards(either(the(M1(or(M2(subtype((118,(120I124).(Macrophage(

polarization(has(been(extensively(studied(in(response(to(infection(by(bacteria(and(

viruses((118,(120I124).(M1(macrophages(are(activated(by(pathogens(or(

inflammatory(cytokines,(and(genes(that(code(for(proIinflammatory(cytokines(ILI6,(ILI

12,(TNFα,(and(ILI1β(are(upregulated((124).(M2(macrophages(can(be(divided(into(

three(subgroups(based(on(the(cytokines(produced,(and(are(involved(in(wound(

healing((124).(M1(and(M2(macrophages(can(also(skew(adaptive(immune(responses(

towards(different(functional(subsets((118,(120).(M1(macrophages(direct(T(cell(

differentiation(towards(a(Th1(or(TH17(subtype(associated(with(bacterial(killing,(

inflammation(and(host(defense.(M2(macrophages(are(more(closely(associated(with(

wound(healing,(tissue(repair,(and(bacterial(persistence((118,(120).((

Macrophages(are(an(important(cell(type(involved(in(the(response(to(

leptospiral(infection.(L.*interrogans*st.(Lai(and(L.*interrogans(strain(Luo(adhere(to(the(

surface(of(murine(and(human(macrophages(from(10(to(40(minutes(in(culture,(with(a(

preference(for(primary(macrophages(compared(to(THPI1(human(monocyteIlike(cells(

or(J774.1(mouse(macrophageIlike(cells((125).(In(human(and(mouse(macrophages,(

leptospires(are(phagocytosed,(but(intracellular(replication(and(an(exacerbated(host(

18(

response(has(been(found(in(human(macrophages(compared(to(murine(

macrophages((125I129).(These(results(correlate(with(mice(being(an(established(

resistant(reservoir(host(in(the(leptospiral(transmission(cycle((Figure!1,!Figure!3).(

Leptospires(induce(activation(of(J774.1(cells(in(a(timeIand(dose(dependent(manner,(

activating(multiple(caspases,(including(caspaseI8((126I128).(Activation(of(CaspaseI

8(during(infection(of(J774.1(cells(by(L.*interrogans,(but(not(avirulent(L.*biflexa(

induces(apoptosis((126I128).(Similar(results(have(also(been(shown(in(mouse(

peritoneal(macrophages((MPM)((126I128).(MPMs(cannot(phagocytose(leptospires(

without(an(antigen(specific(antibody(response((129).(Recent(studies(have(used(

arrays(to(determine(the(innate(responses(in(mouse(and(human(macrophages(during(

leptospiral(infection,(supporting(previous(work(showing(an(upregulation(of(apoptotic(

genes(that(code(for(CaspaseI8(and(FasIassociating(protein(like(death(domain(

(FADD),(and(highlighting(the(differences(in(outcomes(between(primary(human(blood(

mononuclear(cells((HBM)(and(MPMs((129).((

The(roles(of(macrophages(during(infection(by(B.*burgdorferi(have(also(been(

studied((Figure!2)((78,(130I135).(B.*burgdorferi(attaches(to(the(surface(of(

macrophages(and(the(bacteria(are(phagocytosed(during(infection,(leading(to(

degradation(in(the(lysosome(and(recognition(of(PAMPs(including(lipoprotein(and(

flagellin(by(PRRs((134,(136,(137).!Immune(signaling(in(macrophages(during(murine(

bone(marrow(derived(macrophages((BMDMs)(have(been(used(for(in*vitro(studies(to(

elucidate(mechanisms(of(innate(immune(signaling(responses(to(infection(by(B.*

19(

burgdorferi((83,(138I140).(Human(PBMCs(and(BMDMs(from(wildItype((WT)(and(

various(knockout(strains(have(been(used(to(identify(molecular(mechanisms(of(

immune(responses(to(B.*burgdorferi(pathogenesis((83,(138I140).(!

(

2.1.4!Dendritic!Cells!

Dendritic(cells((DCs)(are(antigenIpresenting(cells((APC)(that(are(generally(

thought(to(bridge(the(gap(between(innate(and(adaptive(immunity((116).(Like(many(

aforementioned(cell(types,(DCs(originally(develop(in(the(bone(marrow(from(HSCs(

and(can(differentiate(from(both(lymphoid(and(myeloid(progenitor(cells((116).(Like(

macrophages,(DCs(develop(from(monocytes(into(subtypes(and(have(been(identified(

by(surface(markers,(including(Ly6C(in(mice,(where(Ly6Chigh(CX3CR1low,(CCR2+(and(

CD62L+(expression(is(found(in(one(subtype(and(Ly6Clow(populations(can(be(

differentiated(with(type(2(cytokines(including(ILI4(and(GMCSF((116).(DCs(were(

initially(characterized(as(a(derivative(of(lymphoid(progenitors,(and(subsets(that(are(

either(CD8+CD4+(or(CD8ICD4I(are(found(in(the(spleen,(thymus,(lymph(nodes((116).(

Unlike(residentItissue(DCs,(lymphoid(DCs(do(not(migrate,(but(the(diverse(array(of(

markers(and(tissue(specific(actions(enable(DCs(to(become(effective(APCs.((

Multiple(serovars(of(L.*interrogans*contain(surface(carbohydrates(that(are(

recognized(by(the(CItype(Lectin(DCISIGN(on(DCs((141,(142).(Recognition(of(L.*

interrogans*by(PBMC(derived(DCs(from(multiple(donors(led(to(increased(expression(

of(CD83(and(CD86,(markers(of(DC(maturation,(and(production(of(cytokines(IL12p70,(

20(

ILI10(and(TNFα((141,(142).(The(roles(of(DCs(and(T(cells(have(been(separately(

identified(during(leptospiral(pathogenesis,(but(an(understanding(of(the(crosstalk(

between(both(DCs(and(T(cells(may(be(an(important(factor(in(designing(an(effective(

vaccine(against(leptospirosis,(which(has(not(yet(been(successful((141,(142).((

The(function(of(DCs(during(B.*burgdorferi(pathogenesis(have(been(studied(

using(PBMC(derived(DCs(to(investigate(innate(immune(responses(to(B.*burgdorferi(

infection((Figure!2,!Figure!3)((68,(143).(Using(PBMC(derived(DCs,(it(was(

determined(that(Human(TLR9(played(a(critical(role(in(the(inflammatory(response(to(

B.*burgdorferi*infection(and(induction(of(Type(I(IFN((144).(B.*burgdorferi(

internalization(by(phagocytosis(results(in(antigen(presentation(on(MHC(II(and(

subsequent(activation(of(mixed(TH1/TH2(subset(of(CD4+(T(helper(cells((68,(143).(

TH1(cells(are(often(associated(with(inflammation,(while(TH2(cells(are(associated(

with(helminth(infection(and(wound(healing((68,(143).((

(

2.2!Humoral!responses!in!immunity!to!Leptospira!and!Borrelia#

2.2.1!Complement!

The(complement(pathways(have(been(extensively(characterized(and(play(a(

critical(role(in(several(immune(responses(against(bacteria((145).(Complement(

recognizes(debris,(foreign(agents(including(bacteria,(regulates(cellular(responses(

during(infection,(and(lyses(bacteria(through(multiple(pathways((145).((The(classical(

pathway(is(very(well(characterized(and(is(often(defined(by(an(antigenIantibody(

21(

interaction(that(is(recognized(by(complement(factor(C1pq,(which(is(cleaved(to(bind(

the(antibodyIantigen(formed(complex((145).(This(initiates(a(cascade(that(cleaves(

additional(factors(to(form(a(complex(bound(and(create(pores(in(the(target(cells(that(

results(in(lysis(of(that(cell((145).(The(alternate(pathway(is(a(feedback(regulation(of(

C3bBb(cleavage(to(form(C3b(subunits.(Finally,(the(mannoseIbindingIlectin((MBL)(

pathway(is(initiated(when(MBL(binds(to(carbohydrates(found(on(surfaces(of(

pathogens((145,(146).(In(addition(to(target(cell(lysis,(complement(plays(a(role(in(

opsonization(and(agglutination,(which(prevent(infection(by(targeting(infectious(

agents(for(lysis(and(initiate(the(adaptive(immune(response((145,(146).(((

Complement(mediated(responses(against(L.*interrogans*and(B.*burgdorferi(

have(been(studied((147I151).(Leptospiral(evasion(of(complement(was(originally(

determined(by(culturing(pathogenic(or(nonpathogenic(leptospires(with(serum,(

resulting(in(lysis(of(L.*biflexa(but(not(L.*interrogans*(152).(Indeed,*pathogenic(

leptospires(secrete(multiple(virulence(factors(that(competitively(bind(factors(of(the(

complement(pathways((152I154).(Secreted(proteases(in(culture(supernatants(from(

serovars(of(pathogenic(leptospires(were(shown(to(inhibit(complement(activity(by(

cleaving(complement(factor(C3b,(inhibiting(C4b,(and(binding(factor(H((152I154).(

Leptospiral(adhesins(are(also(involved(in(evasion(of(complement,(as(proteins(like(

LigB(and(Lsa30(bind(C4b(in(order(to(inhibit(complement(activity((155,(156).((

Like(pathogenic(species(of(Leptospira,(B.*burgdorferi*has(mechanisms(to(

evade(complement(by(acquiring(complement(factor(H(and(inhibiting(C3b(activity(

22(

(157I163).(Complement(regulators(acquiring(surface(proteins((CRASPs)(on(the(

outer(surface(of(B.*burgdorferi(can(bind(factor(H,(a(regulator(of(C3b(activity(in(both(

the(classical(and(alternative(pathways((162I166).(In(addition(to(this,(tick(salivary(

proteins(have(immunomodulatory(properties(during(transmission,(and(can(inhibit(

initiation(of(classical(and(alternative(complement(pathways((157,(167).((

Other(complement(components(have(also(been(studied(in(murine(host(

defense(against(borrelial(infection.(Mice(lacking(CD21(and(CD35((Complement(

receptor(1(and(2)(are(unable(to(produce(AntiIBorrelia(Ig(responses,(but(bacterial(

burden(and(arthritis(severity(do(not(differ(from(WT(C57BL/6(mice((168).(C3I/I(mice(

have(a(higher(bacterial(burden(and(a(lower(immune(response,(supporting(previous(

studies(that(highlight(the(importance(of(C3(in(host(defense((161,(169).(C5I/I(mice(

immunized(with(serum(containing(antiIBorrelia(antibodies(are(protected(from(

challenge(with(B.*burgdorferi*(170).(Complement(activity(aids(cellular(responses(and(

lysis(of(B.*burgdorferi(during(host(defense((151,(171,(172).((

!

!

2.2.2!Antibody!responses!to!Leptospiral!and!Borrelial!infection!

Antibodies(play(a(central(role(in(the(immune(response(and(are(critical(for(

clearance(of(microbial(infections,(in(addition(to(other(mechanisms(of(host(defense(

and(responses.(Five(classes(of(Immunoglobulins((Ig)(have(been(characterizedb(IgD,(

23(

IgM,(IgG,(IgE,(and(IgA,(and(many(of(these(antibodies(can(be(further(subdivided(into(

subclasses(associated(with(responses(by(cell(types.((

IgD(is(often(coIexpressed(with(IgM(on(mature(naïve(B(cells(prior(to(class(

switching((173,(174).(IgM(is(produced(by(B(cells(and(has(multifunctional(roles(in(the(

response(to(microbial(infection,(including(initiation(of(the(complement(cascade,(lysis(

of(bacteria(or(infected(cells,(and(agglutination((175).(B(cell(responses(and(IgM(

production(during(leptospiral(and(borrelial(infection(are(initiated(by(TLR(signaling(and(

are(dependent(on(the(adaptor(proteins(MyD88(and(Trif,(which(will(be(discussed(later(

in(this(chapter((4,(50,(176I180).((

IgM(to(IgG(class(switching(is(associated(with(the(onset(of(adaptive(immune(

responses,(where(IgG(is(also(produced(by(B(cells(and(can(be(identified(by(subclass(

based(on(the(TH(cell(subset(that(is(present(during(infection((181I185).(IgG1(is(

associated(with(a(TH2(response(and(production(of(ILI4,(ILI5,(ILI10(and(ILI13,(and(

has(been(identified(as(the(predominant(subclass(in(serum(of(patients(with(a(late(

stage(of(Lyme(Disease((181I185).!IgG2a(in(Balb/c(mice,(and(IgG2c(in(C57BL/6(mice(

are(associated(with(a(TH1(response,(and(production(of(cytokines(ILI6,(IFNγ(and(

TNF((186,(187).((Other(subclasses(of(IgG,(including(IgG3(and(IgG4(are(also(found(in(

mice(and(humans(to(a(lesser(extent((182).(IgG(subclass(antibodies(have(been(

identified(as(potential(diagnostic(markers(during(leptospiral(infection,(but(their(

contribution(to(host(defense(against(pathogenic(leptospires(has(not(yet(been(

determined((188).!

24(

(IgE(is(produced(after(class(switching(from(plasma(cells(and(is(recognized(by(

FcεRI(or(FcεRII(receptors(on(mast(cells,(dendritic(cells,(and(basophils(in(response(to(

allergic(diseases(and(parasitic(infections((189,(190).((The(role(of(IgE(in(leptospiral(

pathogenesis(is(not(known,(but(has(been(investigated(during(infection(of(B.*

burgdorferi(in(mice(and(from(patients(diagnosed(with(Lyme(disease((191)((192)(and(

antiIBorrelia(IgE(has(been(detected(in(patients(with(persistent(Lyme(Disease((191).(

IgA(is(found(throughout(the(mucosal(surfaces,(and(is(produced(more(than(any(

of(the(other(Ig(subtypes((193I195).((IgA(production(occurs(after(DC(recognition(of(

sIgA(bound(to(bacteria(or(M(cells(expressing(the(CIType(Lectin(receptor(DectinI1,(

which(lead(to(activation(of(CD4+(T(cells((193I195),(Secretory(IgA((sIgA)(is(produced(

in(response(to(microbiota(lining(the(mucosal(surfaces,(forming(a(dimer(between(both(

of(its(subtypes(and(neutralizing(toxins(and(secreted(proteins(from(pathogenic(

bacteria(after(induction(within(Peyer’s(patches(in(gutIassociatedIlymphoid(tissue(

(GALT)((193I195).((Bacterial(and(viral(infections(are(cleared(by(neutralizing(sIgA(

during(the(early(immune(responses((193I195).((

Leptospiral(specific(IgA(has(been(identified(in(diagnostic(assays(as(a(marker(

of(early(infection(in(humans,(with(progressively(decreasing(levels(over(the(course(of(

6(months((196,(197).(Additional(studies(have(also(shown(a(correlation(between(IgA(

deposition(in(tissues(from(guinea(pigs(and(patients(with(severe(leptospirosis(and(

pulmonary(hemorrhaging((152).(The(role(of(IgA(in(the(immune(responses(to(

leptospirosis(during(early(infection(in(mice(has(not(been(established,(but(may(be(an(

25(

important(factor(due(to(the(association(of(IgA(deposits(with(with(kidney(nephropathy(

and(persistent(colonization(of(leptospires(in(the(kidneys(152).((IgA(has(also(been(

detected(during(infection(by(B.*burgdorferi*,(but(not(in(response(to(membrane(blebs(

(198I200).((In(addition(to(this,(IgA(detection(in(response(to(antigens(has(been(used(

in(development(of(vaccine(candidates(against(B.*burgdorferi((151,(200I203).(((

!

2.3!Intracellular!responses!to!leptospiral!and!borrelial!infection!

Cellular(and(humoral(responses(to(Leptospira(or(Borrelia(have(also(provided(

mechanistic(insight(into(innate(immune(signaling(during(infection((2,(4,(56).(

Intracellular(signaling(is(initiated(by(the(recognition(of(PAMPs(by(PRRs((204,(205).(

As(previously(mentioned,!PAMPs(include(Nucleic(Acids,(Lipoproteins,(LPS,(Flagellin(

and(other(molecules(that(are(recognized(by(NodILikeIReceptors((NLRs),(RigIlikeI

Receptors((RLRs),(TLRs(and(other(receptors(on(the(plasma(membrane(or(in(the(

cytoplasm((204,(205).(TLRs(are(a(critical(component(of(the(innate(immune(response(

(204,(205)(and(their(roles(have(been(studied(in(the(context(of(bacterial,(viral,(fungal,(

and(parasitic(infection((204,(205).((Toll(receptors(were(originally(identified(in(

Drosophila,(and(have(since(been(studied(extensively(in(mammals,(including(humans(

and(mice((204,(205).(There(are(13(TLRs(that(have(been(identified(in(humans(and(12(

in(mice((206).(TLRs(can(be(grouped(by(location(within(a(cell,(recognized(PAMP,(and(

outcome(depending(on(the(signaling(pathway(that(is(initiated((Table!1,!Figure!4)(

(206).(!

26(

(((

(

Figure!4.!PRRVPAMP!recognition!during!leptospiral!and!borrelial!infection.!Simplified(schematic(showing(recognition(of(Borrelia((blue)(or(Leptospira((purple)(by(TLRs(or(NLRs((Orange)(and(recruitment(of(adaptors((yellow)(that(result(in(downstream(signaling(and(induction(of(proIinflammatory(cytokines(or(Type(I(Interferon((IFN)(or(Interferon(Stimulated(Genes((ISG).(Adapted(from((4,(50,(56,(137,(139,(207I210)(

(

(

(

(

(

((

27(

Table!1.!TLR’s!and!immune!responses!to!Leptospiral!and!and!Borrelial!infection.!Table(outlining(the(different(TLRs,(localization,(and(recognition(of(L.*interrogans*and*B.*burgdorferi,(if(previously(described.(References(are(listed(in(the(far(right(column(of(the(table.(

TLR!( PAMP!( Localization!( Recognizes!Leptospira!or!Borrelia?!(

References!(

TLR1/2/6/10*(( Lipotechoic(Acid((

Plasma(Membrane((

Leptospira*spp.,(B.*burgdorferi*((

(4,(50,(62,(211I218).(

TLR3(( dsRNA(( Endosome(( Unknown(( (137)(

TLR4(( LPS(( Plasma(Membrane((

Leptospira*spp.((

(43I45,(50,(219I221).(

TLR5(( Flagellin(( Plasma(Membrane((

B.*burgdorferi(( (26,(222,(223)(

TLR7/8(( ssRNA(( Endosome(( B.*burgdorferi(( (56,(137,(139,(208,(209)(

TLR9(( CpG(DNA(( Endosome(( B.*burgdorferi(( (139,(144,(223).(

TLR13(( Bacterial(23s(rRNA((

Endosome(( Unknown(( (224,(225)(

*TLR10(is(not(found(in(mice(and(has(only(been(studied(during(B.*burgdorferi*infection((

((2.3.1!TLR!signaling!during!infection!by!spirochetes!

2.3.1.1!Lipoprotein!sensing!TLRs!

TLR2(is(a(plasmaImembraneIbound(TLR(that(forms(heterodimers(with(either(TLR1(

or(TLR6((226,(227).((Upon(recognition(of(lipoproteins(or(lipotechoic(acids,(the(

adaptor(molecules(Myeloid(Differentiation(Factor(88((MyD88)(and(TIRIdomainI

28(

containing(adapterIinducing(interferonIβ((TRIF)(are(recruited(downstream(of(TLR2,(

leading(to(NFIκB(mediated(cytokine(induction(or(production(of(Type(I(Interferon,(

respectively((204I206).(It(is(only(recently(that(TRIF(has(been(implicated(in(

recruitment(to(TLR2((228I230).((TLR2(is(involved(in(innate(immune(responses(to(

spirochetes((Table!1,!Figure!3)((62,(211I218).((

TLR2(conferred(a(protective(response(to(mice(infected(with(the(relapsing(

fever(causing(spirochete(Borrelia*hermsii((217),(and(is(required(for(cytokine(

production(during(infection(of(human(monocytes(by(pathogenic(spirochete(Borrelia*

garinii*(231).(Recognition(of(B.*burgdorferi*sonicate(and(subsequent(NFIκB(mediated(

proIinflammatory(cytokine(production(had(originally(demonstrated(a(role(for(TLR2(

during(B.*burgdorferi*pathogenesis((211).(Additional(studies(determined(that(TLR2/6(

heterodimers(recognize(outer(surface(proteins,(including(OspA(lipoprotein((62,(212,(

232).((In(addition(to(this,(multiple(tissues(from(Tlr2I/I(mice(infected(with(B.*burgdorferi*

had(a(higher(burden(at(two(and(four(weeks(post(infection,(in(addition(to(severe(

swelling(of(the(ankle(joints(as(a(marker(of(arthritis(and(inflammation((233I235).((

Interestingly,(human(TLR2,(but(not(murine(TLR2(is(required(for(innate(

immune(responses(to(leptospiral(LPS(during(infection(by(L.*interrogans*(50,(213,(

220).((Tlr2I/I(mice(infected(with(L.*interrogans*survive(infection(and(show(mild(to(

moderate(symptoms(during(pathogenesis,(but(studies(in(human(monocyteI

macrophageIlike(THPI1(cells(show(an(induction(of(TNF(via(TLR2(and(CD14(with(

leptospiral(LPS,(which(may(be(due(to(the(unique(and(varied(structure(of(LPS(in(

29(

pathogenic(species(of(leptospires((50,(213,(220).(TLR2(is(one(of(multiple(surface(

TLRs(that(have(been(implicated(in(responses(to(infection(by(spirochetes,(but(other(

plasmaImembrane(bound(TLRs(are(involved(as(well,(including(TLR4.((

The(role(of(plasmaImembrane(bound(TLR4(in(response(to(microbial(infection(

has(been(extensively(characterized((205,(227,(236).(Early(studies(showed(that(TLR4(

recognizes(Lipopolysaccharide((LPS),(a(major(virulence(factor(in(gramInegative(

bacteria((49,(237,(238).((Recognition(of(LPS(by(TLR4((involves(the(recruitment(of(

MD2(and(the(physical(interaction(of(TLR4(and(LPS(239I241).(Signaling(downstream(

of(TLR4(is(mediated(by(multiple(adaptor(molecules,(including(MyD88(and(TRIF((242,(

243).(When(MyD88(is(recruited(to(TLR4,(downstream(signaling(involves(the(

activation(of(kinases(by(phosphorylation!(Table!1,!Figure!4)!and(activation(of(the(

transcription(factors(NFIκB,(and(MAPK((243).(Activation(of(transcription(factors(like(

NFIκB(will(result(in(their(translocation(to(the(nucleus(and(initiation(of(proI

inflammatory(cytokine(gene(expression.(When(TRIF(is(recruited(to(TLR4,(the(

resulting(downstream(pathway(leads(to(activation(of(the(transcription(factor(IRF3(and(

subsequent(expression(of(Type(I(Interferons((IFN)(and(Interferon(Stimulated(Genes(

(ISG).((

Unlike(L.*interrogans,(B.*burgdorferi(does(not(have(LPS,(but(its(genome(has(

several(genes(that(code(for(lipoproteins((55,(244).(Lipoproteins(from(B.*burgdorferi*

are(recognized(by(TLR2((245I247).(LPS(is(an(important(virulence(factor(of(L.*

interrogans*that(induces(innate(immune(responses(to(infection(by(inducing(TNF(via(

30(

TLR2(and(CD14(recognition((213).((Previous(studies(showed(that(attenuated(strains(

of(Leptospira(with(mutated(LPS(were(not(able(to(cause(lethal(infection(of(hamsters(

(219,(248).(Hamsters(infected(with(the(virulent(L.*interrogans*sv.(Manilae(st.(L495(

died(7(days(post(infection(and(bacteria(could(be(isolated(from(the(blood(and(tissues(

(219,(248).(However,(hamsters(infected(with(an(attenuated(mutant(showed(no(signs(

of(illness(and(bacteria(could(not(be(cultured(from(blood(and(tissues((219,(248).((

Additional(studies(revealed(an(association(with(changes(in(the(regulation(of(OI

antigen(expression(on(LPS(and(resulting(acute(or(chronic(infections(in(guinea(pigs(

and(rats,(respectively((43,(249).((These(and(other(studies(established(Leptospira*

LPS(as(an(important(virulence(factor(due(to(differential(recognition(and(resulting(

responses(via(TLR4(in(animal(models((43,(219I221).(Overall,(TLR2(and(TLR4(play(a(

critical(role(in(the(response(to(infection(by(spirochetes,(but(lipoproteins(are(not(the(

only(ligands(sensed(by(host(TLRs((139).((

(

2.3.1.2!Recognition!of!RNA!by!spirochetes!via!Nucleic!Acid!sensing!TLRs!

Nucleic(acid(sensing(TLRs(are(located(in(the(endosome((205).(It(is(generally(

thought(that(TLR3(recognizes(doubleIstranded(RNA((dsRNA)(produced(by(viruses(

(205).((Indeed,(TLR3(is(extensively(involved(in(antiIviral(signaling(responses((250I

253).!However,(TLR3(has(recently(also(been(implicated(in(immune(signaling(

responses(to(bacterial(infection((254,(255).(TRIF(is(the(only(adaptor(protein(for(

TLR3,(where(downstream(signaling(leads(to(production(of(Type(I(IFN(and(ISGs(via(

31(

dimerization(and(translocation(of(the(transcription(factor(IRF3((205,(236).(TLR7(and(

TLR8(are(also(located(in(the(endosome,(but(recognize(singleIstranded(RNA((205,(

236).((The(roles(of(TLR7(and(TLR8(in(humans(and(mice(have(been(studied(in(the(

context(of(bacterial,(viral(and(fungal(infection((205,(236).(The(role(of(TLR7/8(in(

humans(has(been(studied(in(response(to(infection(by(B.*burgdorferi*(208,(256)(but(it(

is(not(known(whether(RNA(from(L.*interrogans*induces(an(immune(response.(RNA(

isolated(from(B.*burgdorferi*induces(Type(I(IFN(and(NFIkB(dependent(cytokines(ILI

6,(ILI10,(and(IFNγ(in(human(PBMCs(via(TLR7,(and(TLR8(recognizes(RNA(from(B.*

burgdorferi*in(the(phagosome(of(PBMCs((Table!1,!Figure!4)((208,(256).(TLR13(was(

recently(identified(as(a(bacterial(rRNA(sensor((224,(225,(257I259).(Transfection(of(

RNA(from(lactic(acid(bacteria(resulted(in(increased(MyD88(dependent(signaling(

(260).(In(addition(to(this,(shRNA(knock(down(of(TLR13(in(RAW264.7(cells(led(to(

abrogation(of(RNA(mediated(ILI1β(expression,(but(not(R848(mediated(ILI1β(

expression((224,(225).(TLR13(specifically(recognizes(23s(rRNA(from(bacteria((224,(

225).(TLR13(is(predicted(to(recognize(RNA(from(B.(burgdorferi(and(will(be(discussed(

in(Chapter(3.(While(the(endosomal(TLR3,(TLR7/8(and(TLR13(recognize(different(

types(of(RNA,(TLR9(is(the(only(TLR(that(recognizes(DNA,(specifically(CpG(DNA(

from(bacteria((261).(TLR9(has(not(been(studied(in(the(context(of(leptospiral(infection,(

but(its(role(during(infection(by(B.*burgdorferi*has(been(explored((139,(144,(223).(

TLR9(is(not(required(for(phagocytosis(of(B.*burgdorferi*or(proIinflammatory(cytokine(

32(

production,(but(it(is(required(for(an(effective(Type(I(IFN(response(in(PBMCs(and(not(

mouse(BMDMs((139,(144,(223).((

(

2.3.1.3!TLR5!

TLR5(is(a(plasmaImembrane(bound(TLR(that(recognizes(bacterial(flagellin,(

resulting(in(proIinflammatory(cytokine(expression((262I264).(TLR5(is(not(required(for(

phagocytosis(of(B.*burgdorferi*in(murine(macrophageIlike(RAW(264.7(cells,(but(is(

required(for(induction(of(proIinflammatory(cytokines,(including(ILI6,(ILI1β,(and(IFNγ(

(Table!1,!Figure!4)((223).(Induction(of(proIinflammatory(cytokines(in(human(

monocytes(or(THPI1(monocyteIlike(cells(is(not(mediated(by(TLR5(during(infection(by(

B.*burgdorferi,(which(may(also(highlight(differences(in(rodent(and(human(host(

responses((26,(222,(223).(TLR5(has(not(been(studied(in(the(context(of(leptospiral(

infection(in(mice,(but(TLR5(expression(in(blood(from(human(donors(was(elevated(

after(infection(with(L.*interrogans,(in(addition(to(TLR2(and(TLR4,(and(blocking(TLR5(

with(monoclonal(antibodies(resulted(in(reduced(ILI6(and(TNFα(production(during(

leptospiral(infection((26).(Additional(studies(are(required(to(elucidate(cooperative(

signaling(mechanisms(between(TLR5(and(other(plasmaImembrane(bound(TLRs(

during(infection(by(both(B.*burgdorferi*and(L.*interrogans.(Because(mice(can(act(as(

reservoir(hosts(for(either(pathogenic(spirochete,(studies(with(Tlr55/5(mice(may(also(

provide(insight(into(this(intracellular(response(and(highlight(additional(differences(

between(mouse(and(human(immune(responses.((

33(

2.3.1.4!TLR10!

TLR10(was(identified(in(humans(by(rapid(amplification(of(cDNA(ends((RACE)(

from(splenic,(thymic,(lymph(node(and(lung(tissue(using(primers(spanning(a(novel(

region(in(the(human(genome(sequence((265).((Sequence(similarity(also(most(closely(

matched(human(TLR1(and(TLR6,(which(suggested(a(potential(role(for(human(TLR10(

in(the(immune(response((265).(TLR10(is(not(functional(in(mice(because(it(appears(in(

the(genome(sequence(as(a(pseudogene,(and(this(has(hindered(advances(in(

elucidating(signaling(pathways(downstream(of(ligandIreceptor(interactions((265I

268).(This(TLR(is(expressed(primarily(on(B(cells(and(plasmacytoid(DCs((266,(268,(

269)(and(can(homodimerize(or(heterodimerize(with(other(TLRs,(including(TLR(1(and(

6((266,(268,(269).(Recently,(a(role(for(TLR10(as(an(antiIinflammatory(recognition(

receptor(was(determined((270I272).(Recent(studies(showed(that(antibody(blocked(

TLR10(in(human(PBMCs(resulted(in(increased(proIinflammatory(cytokine(production(

after(stimulation(with(the(TLR2(agonist(PAMCYS(or(B.*burgdorferi*(272).(The(role(of(

TLR10(during(Leptospiral(infection(has(yet(to(be(studied.((

(

2.3.2!Adaptor!Molecules!of!TLR!signaling!

MyD88(and(TRIF(are(both(located(in(the(cytosol(of(a(cell,(and(will(interact(with(

TLRs(depending(on(the(PAMP(that(is(recognized((Table!1,!Figure!3)!(236).(MyD88(

is(associated(with(all(TLRs(except(for(TLR3,(which(exclusively(signals(using(TRIF(

(205,(236).(MyD88(has(a(Toll/ILI1R((TIR)(domain(that(is(present(in(the(cytosol(and(

34(

interacts(with(TIR(domains(of(TLRs,(including(TLR2,(TLR4,(TLR5,(TLR7(and(TLR9(

(273).(Recruitment(of(MyD88(to(TLRs(based(on(the(specificity(of(TIR(domains(in(

cytosolic(regions(of(TLRs(has(been(demonstrated(by(identifying(mutations(that(

change(specificity(of(MyD88(to(TRIF(recruitment(during(the(immune(response((204,(

273,(274).(In(addition(to(TLR(signaling,(MyD88(and(TRIF(are(also(associated(with(

other(signaling(pathways.(MyD88(is(also(an(adaptor(to(ILI1R,(and(is(involved(in(ILI1(

family(signaling((275,(276).((Like(TLRs,(ILI1R(contains(a(cytoplasmic(TIR(domain,(

which(recruits(MyD88,(resulting(in(proIinflammatory(cytokine(induction,(specifically(

from(the(ILI1(family((277).(((

TRIF(is(another(adaptor(molecule(that(is(generally(associated(with(production(

of(interferons((Table!1,!Figure!4)!(273).(TRIF(is(associated(with(TLR2,(TLR3,(and(

TLR4(in(response(to(microbial(infection((205,(236).(It(is(generally(thought(that(TRIFI

dependent(signaling(via(multiple(TLRs(leads(to(the(activation(of(IRFs(and(

subsequent(expression(of(ISGs((229,(254,(278,(279).(However,(recent(studies(have(

shown(an(association(between(TRIF(and(NLRP3(in(a(nonIcanonical(inflammasome(

based(mechanism((280).(The(induction(of(Type(I(IFN(has(been(shown(to(regulate(

the(inflammasome(in(a(TRIF(dependent(manner((280).(Additional(mechanisms(

between(TRIF(and(caspases(during(the(course(of(infection(have(also(been(studied(

(280I284).(TRIF(is(not(only(required(for(CaspaseI11(dependent(activation(in(

response(to(bacterial(infection((280),(but(CaspaseI1(cleaves(TRIF(in(order(to(

downregulate(autophagy(in(response(to(infection(by(Pseudomonas*aeruginosa(

35(

(284).(There(are(other(models(of(infection(that(have(demonstrated(an(inflammatory(

response(independent(of(the(TRIFIIFN(pathway(but(involving(CaspaseI11((281).(

Production(of(other(proIinflammatory(cytokines,(including(RANTES(and(CXCL10(are(

TLR4(and(TRIFIdependent((285I287).(MyD88(and(TRIF(have(also(been(studied(in(

the(context(of(the(adaptive(response(to(infection.(Myd885/5(and(Trif5/5(Bone(Marrow(

Derived(Dendritic(Cells((BMDCs)(had(reduced(levels(of(the(proIinflammatory(

cytokines(ILI6(and(TNF,(and(lower(IFNy(production(in(a(DCIT(cell(coIculture(system,(

suggesting(that(both(adaptor(molecules(contribute(to(a(TIcell(response(during(

Campylobacter*jejuni(pathogenesis((288).(The(relationship(between(MyD88,(Trif(and(

the(TH1(subset(of(CD4+(T(cells(has(also(been(explored(in(other(models(of(bacterial(

pathogenesis((254,(289,(290).((B(cell(production(is(reduced(in(Myd885/5(and(Trif5/5(

mice,(and(IgG1(production,(a(hallmark(of(the(TH2(response,(is(reduced(in(Trif5/5(mice(

but(not(Myd885/5(mice((291).(Impaired(Ig(production(and(class(switching(has(been(

documented(in(Trif5/5(mice(but(not(in(the(context(of(leptospiral(infection((279,(291I

293).((MyD88(and(TRIF(also(contribute(to(IgE(production(in(B(cells(in(response(to(

allergens,(but(the(TLR4ITRIF(pathway(mediates(class(switching(to(IgG((293,(294).((

MyD88(is(essential(for(an(effective(immune(response(in(mice(to(B.*burgdorferi*

and*L.*interrogans*(Figure!4)((50,(295I297).(Bacterial(burden(in(joints,(hearts(and(

ears(of(mice(detected(as(copies(of(RecA(are(significantly(higher(in(Myd885/5(mice(

compared(to(C57BL/6(mice((295I297).(Myd885/5(mice(infected(with(L.*interrogans*sv.(

Copenhageni(died(within(5(days(due(to(an(elevated(inflammatory(response(and(a(

36(

lower(B(cell(response((50).(TRIF(is(not(required(for(Type(I(IFN(induction(during(

infection(of(BMDMs(by(B.*burgdorferi,(but(contributes(to(inflammatory(responses(via(

TLR2((139,(230).(In(addition(to(this,(both(inflammatory(and(Type(I(IFN(responses(are(

ablated(in(the(absence(of(both(MyD88(and(TRIF,(highlighting(potential(synergistic(

effects(during(the(immune(response(to(infection(by(B.*burgdorferi*(139,(230).(

Synergistic(effects(of(both(MyD88(and(TRIF(have(been(reported(elsewhere((236,(

274,(289,(292,(298I301).((The(role(of(TRIF(during(leptospiral(infection(is(a(topic(of(

this(dissertation.(

!

2.3.3!Consequences!of!TLR!signaling!

( Downstream(signaling(of(TLRs(and(adaptor(molecules(leads(to(dimerization(

of(transcription(factors(to(for(Interferon(regulatory(factors((IRFs)(or(NFIκB((205,(236).(

Dimerization(of(these(factors(leads(to(translocation(in(the(nucleus(and(subsequent(

transcription(of(genes(coding(for(cytokines(and(chemokines((Table!1,!Figure!4)(

(205,(236).(NFIκB(mediated(cytokines(are(often(a(result(of(MyD88(signaling((205,(

236).(ILI6(is(a(proIinflammatory(cytokine(produced(by(neutrophils,(TH17(helper(T(

cells,(macrophages,(and(DCs((Figure!3)((205,(236).(ILI12(is(produced(by(activated(

macrophages(and(TH1(T(helper(cells(and(has(a(common(subunit(to(IL23((205,(236).(

TNFα(is(a(proIinflammatory(cytokine(produced(by(multiple(cell(types(and(recruits(

TH1(helper(T(cells(and(activates(M1(macrophage(polarization((205,(236).(ILI4(and(

ILI10(are(Type(2(cytokines(that(are(produced(by(M2(macrophages(and(TH2(cells(

37(

(205,(236).(ILI1β(and(IL18(are(members(of(the(IL1(family(that(are(produced(in(

macrophages((205,(236).(Production(of(these(cytokines(due(to(signaling(pathways(

leads(to(recruitment,(migration,(activation(and(maturation(of(multiple(cell(types,(

leading(to(the(cellular(responses(previously(described(in(this(chapter.(

(

2.3.4!Interferons!and!ISG!

Interferons(are(a(class(of(cytokines(that(are(produced(by(all(cell(types(in(

response(to(microbial(infections((205,(236).(Type(I(IFN(is(primarily(comprised(of(

IFNα(or(IFNβ,(and(its(role(in(immune(responses(to(viral(and(bacterial(pathogenesis(

has(been(extensively(characterized((302).((IFNα(is(constitutively(expressed(at(low(

levels,(while(IFNβ(is(produced(in(response(to(viral(or(bacterial(infection((302).(IFNβ(

production(is(also(closely(associated(with(regulation(of(IFNγ(via(STAT1,(and(is(also(

implicated(in(B(cell(enhancement,(proliferation(and(survival((303,(304).((IFNβ(has(

multiple(roles(in(bacterial(infection((305).((Previous(work(showed(detection(of(serum(

IFNα(in(mice(injected(with(IFNβ,(but(not(detection(of(serum(IFNα(in(mice(injected(

with(IFNβ,(demonstrating(regulation(of(IFNα(by(IFNβ((306).((

Type(I(IFN(has(been(studied(in(response(to(infection(of(Borrelia(but(not(

Leptospira((140,(307).((Results(from(a(microarray(based(study(showed(an(elevation(

in(Type(I(IFN(and(ISG(in(a(mouse(model(of(severe(arthritis((C3H/HeJ)(compared(to(a(

moderate(phenotype((C57BL/6)((83,(84).(Additional(studies(in(mice(that(were(treated(

with(a(blocking(antibody(to(IFNα,(or(congenic(C57Bl/6(mice(with(the(C3H/H3J(B.*

38(

burgdorferi–associated(locus(1((Bbaa1)(allele,(showed(that(arthritis(was(reduced(in(

joints(of(mice(with(an(ablated(Type(I(interferon(response((140).(The(Type(I(IFN(

response,(measured(by(IFNβ(production,(was(also(induced(by(multiple(components(

of(B.*burgdorferi,(including(nucleic(acids,(lipoproteins,(and(currently(unidentified(

secreted(molecules(in(B.*burgdorferi*cultured(supernatants((139).((

Type(II(IFN(is(primarily(IFNγ(and(is(implicated(in(the(immune(response(to(

Leptospira(and(Borrelia((47,(109,(308I310).(IFNγ(is(expressed(by(multiple(cell(types(

including(T(cells(and(NK(cells.(Its(class(of(ISGs(includes(the(Type(II(inducible(

chemokines(CXCL10,(which(is(produced(by(CD8(T(cells(and(NK(cells,(and(is(

recognized(by(CXCR3,(a(receptor(expressed(primarily(in(NK(cells((111).(IFNγ(and(

IFNγIproducing(cells(have(been(studied(in(murine(and(human(responses(to(infection(

by(B.*burgdorferi*(83,(308,(311,(312).(Ankle(joint(swelling(and(histopathological(

analysis(of(arthritis(severity(were(elevated(in(C3H/(Ifnγ5/5(mice(compared(to(the(WT(

C3H/HeJ(mice((308).(An(increase(in(Type(I(and(II(IFN(expression(was(also(detected(

by(microarray(and(qRTIPCR(in(joints(of(C3H(mice(compared(to(C57BL/6(mice((83,(

84).(This(increase(in(interferon(production(correlated(with(a(significant(increase(in(

inflammation(and(arthritis(severity(as(well((83,(84).(The(role(of(IFNγ(during(

leptospiral(infection(has(been(investigated(in(humans(and(mice((47,(50,(111).(DoseI

response(experiments(with((C57BL/6/(Ifnγ5/5(mice(showed(survival(through(28(days(

after(intraIperitoneal(infection(even(at(high(doses(of(2x108(leptospires((and(reduced(

inflammation(based(on(histopathological(analysis(of(kidneys((47).(TLR2(and(TLR4(

39(

dependent(expression(of(IFNγ(in(B(cells(and(T(cells(has(been(identified(in(mice,(after(

infection(with(L.*interrogans(sv.(Copenhageni(st.(Fiocruz(L1I130((50).(Plasma(levels(

of(IFNγ(are(elevated(in(patients(with(leptospirosis(compared(to(healthy(controls,(as(

are(CXCL10,(and(TIcell(producing(granzyme(A(and(B(in(patients(diagnosed(with(

leptospirosis((109,(111).((

Type(III(IFN(is(a(recent(addition(to(the(class(of(Interferons(and(has(been(

implicated(in(the(antiviral(response((313).((There(are(4(subtypes(of(IFNλ(that(have(

been(documented((313).(Type(III(IFN(has(recently(been(implicated(in(immune(

responses(against(viruses(and(bacteria((208,(209,(314).(Multiple(viruses,(including(

Dengue,(Hepatitis(C(virus,(Sendai(virus(activate(Type(III(IFN(signaling(using(RLRs,(

and(recent(studies(have(shown(a(peroxisomal(mitochondrial(antiviral(signaling(

protein((MAVS)(and(JAKISTAT1(dependent(mechanism(of(activation.(This(

mechanism(is(also(found(in(response(to(infection(by(the(intracellular(bacteria(Listeria*

monocytogenes((208,(209,(314).((Stimulation(of(human(PBMCs(with(RNA(from(B.*

burgdorferi*or(live(B.*burgdorferi*induced(expression(of(ILI28((IFNL3)((208,(209,(

314).(Type(III(IFN(responses(to(B.*burgdorferi*infection(in(mice(have(not(been(

explored(and(the(function(of(Type(III(IFN(in(response(to(leptospiral(infection(has(not(

yet(been(addressed.((

(

(

(

40(

Table!2.!RLRs,!NLRs!and!immune!responses!to!Leptospiral!and!Borrelial!infection.!Table(outlining(additional(PRRs(including(RLRs,(NLRs(and(CIType(Lectins,(localization,(and(recognition(of(L.*interrogans(and(B.*burgdorferi,(if(previously(described.(References(are(listed(in(the(far(right(column(of(the(table.(

PRR!Family!(

PAMP!( Localization!( Recognizes!Leptospira!or!Borrelia?!(

References!(

NLRPs!(1V14)!(

( ( ( (

NLRP1( Muramyl(Dipeptide(( Cytosol(( Unknown(( (315,(316).(

NLRP3( Intracellular(bacteria,(bacterial(RNA,(ATP,(DangerIassociated(molecular(patterns((DAMPs)((

Cytosol(( Leptospira*spp.,((

(4,(51,(207).(

NLRC’s!( ( Cytosol(( ( (

NOD1,2( (NIacetylglucosamine,((NIacetylmuramic(acid((

Cytosol(( B.*burgdorferi((

(137,(210,(317,(318)(

NLRC3( (regulates(STING)(( Cytosol(( Unknown(( (315,(316).(

NLRC4( Flagellin(( Cytosol(( Unknown(( (315,(316).(

AIM2( DNA(( Cytosol(( B.*burgdorferi((

(139)(

STING( DNA(( Cytosol(( B.*burgdorferi((

(139)(

RLRs!( ( ( ( (

RIGII,(MDA5((

5’ppp(dsRNA(of(varying(lengths((

Cytosol(( Unknown(( (319I322).(

(

41(

2.3.5!NodVLikeVReceptors!

NLRs(are(cytoplasmic(PRRs(that(are(involved(in(inflammation,(and(are(

structurally(characterized(by(an(aminoIterminal(domain,(a(centrally(located(

nucleotideIbinding(domain((NBD),(and(a(carboxyIterminal(domain(containing(

leucineIrich(repeats((LRR)((315,(316).(NucleotideIbinding(oligomerization(domainI

containing(protein(1(and(2((NOD1(and(NOD2)(are(associated(with(inflammatory(

responses,(while(other(NLRs(including(NLRP1,(NLRP3,(and(NLRC4(are(associated(

with(inflammasome(activation((Table!2)((315,(316).((NLRP3(mediated(activation(of(

the(inflammasome(has(been(characterized,(and(can(be(triggered(by(multiple(stimuli,(

including(ROS,(changes(in(intracellular(potassium(content,(and(lysosome(

degradation((315,(316).((NLR(signaling(goes(through(the(adaptors(ApoptosisI

associated(SpeckIlike(protein(containing(a(CARD((ASC)(and(a(SerineIthreonine(

protein(Kinase(with(a(caspase(activation(and(Recruitment(domain((RICK),(leading(to(

activation(of(the(inflammasome.(The(inflammasome(produces(a(highly(inflammatory(

response(that(results(in(caspase(activation(and(a(triggered(form(of(cell(death(called(

pyroptosis((315,(316).((

ProIinflammatory(cytokine(production(is(reduced(in(cells(from(Rick5/5(mice(but(

not(Nod15/5(mice,(and(PBMCs(from(volunteers(that(had(reduced(NOD2(function(had(

a(lower(proIinflammatory(cytokine(response,(including(reduced(ILI6(and(ILI8(during(

B.*burgdorferi*infection((137,(210,(317,(318).(Similar(results(were(also(shown(in(

BMDMs(from(Nod25/5(mice(that(were(stimulated(with(B.*burgdorferi,(leading(to(lower(

42(

proIinflammatory(cytokine(production(compared(to(WT(BMDMs(in(addition(to(

reduced(IFNα(and(CXCL10((137,(210,(317,(318).(Mice(lacking(NOD2(also(had(lower(

bacterial(load(in(the(hearts(and(bladders,(and(exacerbated(arthritis(and(carditis(

based(on(histopathological(analysis,(suggesting(that(NOD2(might(be(contributing(to(

infection,(but(suppressing(the(arthritis(and(carditis(phenotype((137,(210,(317,(318).(

Il1r5/5(mice(show(reduced(inflammation(and(joint(swelling(after(injection(with(B.*

burgdorferi,(but(production(of(ILI1β(is(mediated(by(TLR2(and(MyD88(instead(of(

NOD1(or(NOD2((137,(210,(317,(318).(In(addition(to(this,(AscI/I(and(RickI/I(mice(

showed(reduced(joint(swelling(after(injection(with(B.*burgdorferi,(but(Nlrp3I/I(mice(has(

similar(levels(of(inflammation(compared(to(WT,(highlighting(multiple(mechanisms(of(

PRRs(in(the(innate(immune(response(to(borrelial(infection((137,(210,(317,(318).(

NLRP3(is(the(most(characterized(NLR,(and(has(been(implicated(in(the(

immune(response(to(leptospiral(infection,(but(it(is(not(implicated(in(chronic(fibrosis(

(Table!2)((4,(51,(207).(Leptospiral(LPS(upregulates(ILI1β(in(a(TLR2/4/MyD88(and(a(

TLR4/TRIF(dependent(manner,(but(downregulation(of(Na/KIATPase(in(mouse(

BMDMs(activated(NLRP3,(suggesting(a(potential(role(for(the(inflammasome(during(

leptospiral(infection((4,(51,(207).(Renal(fibrosis,(a(marker(of(chronic(leptospiral(

infection,(is(not(NLR(dependent(as(Nlrp35/5(mice,*Nod1/25/5(mice(and(Caspase515/5(

mice(show(similar(characteristics(of(fibrosis(by(histopathological(analysis(during(

chronic(leptospiral(infection((4,(51,(207).((Additional(studies(would(be(useful(to(

assess(the(contribution(of(NLRs(to(acute(leptospiral(infection(in(mice(and(tools(for(

43(

genetic(manipulation(of(leptospires(need(to(be(developed(in(order(to(generate(

mutants(that(can(identify(what(virulence(factors(contribute(to(this(immune(response.(

(

2.3.6!RigVlikeVReceptors!

RLRs(have(been(implicated(in(antiviral(responses((Table!2)((319I322).(RLRs(

are(known(for(inducing(Type(I(and(III(IFN(responses(and(are(structurally(

characterized(by(a(caspase(recruitment(domain(on(the(N(terminus,(a(DExD/HIbox(

Helicase,(and(a(CIterminal(domain(that(recognizes(double(stranded(RNA((319I322).(

Two(characterized(RLRs(are(Retinoic(Acid(Inducible(Gene(I((RigII)(and((MDA5),(

both(of(which(recognize(5’pppIdsRNA(of(short(or(long(lengths(of(nucleotides,(

respectively((319I322).(The(adaptor(to(both(receptors(is(MAVS,(which(is(located(on(

the(mitochondrial(outer(membrane(and(peroxisomes(and(is(a(target(for(viral(evasion(

of(innate(immune(responses((319I322).(Recently(an(RLR(dependent(mechanism(of(

Type(III(IFN(induction(has(been(implicated(in(the(immune(response(to(infection(by(L.*

monocytogenes((314).(Potential(roles(for(RLRs(in(the(immune(response(to(B.*

burgdorferi*have(been(speculated,(because(it(is(generally(thought(that(B.*burgdorferi*

is(phagocytosed,(and(RNA(is(recognized(by(PRRs(in(the(endosome((208,(209,(323).(

The(potential(recognition(of(RNA(by(RLRs(and(other(cytosolic(PRRs(is(currently(

unknown,(and(additional(studies(are(required(to(determine(the(role(of(RLR’s(and(

MAVS(during(spirochete(infection.(LPS(is(the(major(PAMP(for(L.*interrogans,(and(

44(

RNA(has(not(been(identified(as(a(ligand(during(leptospiral(infection,(so(the(roles(of(

RLRs(are(unknown(here(as(well.((

!

2.3.7!Other!PRRs!

Herein(TLRs(NLRs(and(RLRs(have(been(described(by(location,(function,(and(

contribution(to(immune(responses(during(leptospiral(and(borrelial(infection.(However,(

there(are(other(PRRs(that(play(a(role(in(host(responses.(CItype(lectins(are(a(large(

family(of(carbohydrateIbinding(receptors,(although(binding(of(CItype(Lectins(to(

proteins(and(other(compounds(has(been(previously(reported((324).(This(family(of(

PRRs(has(expanded(over(time(to(include(seventeen(subtypes,(highlighting(the(

diverse(responses(associated(with(PAMP(recognition((324).(These(plasma(

membrane(bound(receptors(are(located(on(a(variety(of(cell(types,(including(DCs,(NK(

cells,(macrophages,(monocytes,(epithelial(cells,(and(endothelial(cells((324).(The(CI

type(Lectin(DCISIGN(expressed(on(DCs(plays(a(role(in(microbial(infection(and(

recognition(of(viruses(and(bacteria,(including(L.*interrogans(and(B.*burgdorferi((142,(

325,(326).((

Glycoproteins(from(leptospires(have(been(implicated(in(DC(maturation((327).(

Recognition(of(mannose(components(in(leptospires(by(expressed(DCISIGN(results(

in(increased(expression(of(DC(maturation(surface(markers(CD86(and(CD83,(as(well(

as(the(production(of(ILI12,(TNFα,(and(ILI10((142,(325,(326).(Interestingly,(variation(

in(cytokine(production(was(identified(as(a(result(of(infection(depending(on(virulence(

45(

of(leptospiral(species((142,(325,(326).(Analysis(of(monocytic(and(plasmacytoid(DCs(

from(blister(fluid((BF)(in(erythema(migrans((EM)(lesions(treated(with(borrelial(

lipoproteins(showed(an(increase(in(expression(of(CD11c(and((CD86,(both(of(which(

are(surface(markers(in(mature(DCs((142,(325,(326).(Similar(increases(in(ILI12,(

TNFα,(and(ILI10,(as(seen(with(leptospiral(infection,(were(also(reported(in(BF(from(

EM(lesions(in(patients(with(Lyme(disease((142,(325,(326).((

Other(CItype(lectins(have(also(been(studied(in(immune(responses(to(borrelial(

infection((328,(329).(PBMCs(with(antibodyIblocked(DectinI1(or(DectinI2(did(not(

exhibit(differences(in(cytokine(production(compared(to(regular(PBMCs((329).(Similar(

results(were(obtained(in(murine(peritoneal(macrophages(lacking(DectinI1(or(DectinI

2,(as(well(as(histopathological(analysis(of(knockout(mice(during(borrelial(infection(

(329).(Additional(studies(are(required(to(assess(the(roles(of(other(CItype(lectins(

during(infection(by(spirochetes,(including(markers(present(on(inflammationI

associated(cell(types(like(NK(cells((324).((

(

3.1!Summary!

Here(we(have(summarized(innate(immune(responses(to(leptospiral(and(borrelial(

infection(by(describing(cellular(responses,(complement(as(a(humoral(response,(and(

intracellular(signaling.(The(importance(of(studying(both(L.*interrogans*and(B.*

burgdorferi*has(been(addressed(by(a(description(of(the(disease(etiology,(

epidemiology(and(animal(models(used(to(study(infection.(In(Chapter(2,(the(innate(

46(

immune(response(to(leptospiral(infection(will(be(investigated(by(assessing(the(

contribution(of(the(adaptor(TRIF(to(infection(in(intracellular(signaling(pathways.(In(

Chapter(3,(the(innate(immune(response(to(borrelial(infection(is(addressed(by(

determining(the(roles(of(TLR7(and(MyD88(in(recognition(and(cytokine(production(in(

response(to(B.*burgdorferi*and(RNA(isolated(from(B.*burgdorferi.(Chapter(4(will(

highlight(the(main(points(of(the(previous(chapters,(identify(future(directions,(and(

address(the(impact(of(both(studies(in(this(dissertation.((

(

(

(

(

(

(

(

(

(

(

(

(

(

(

47(

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50(

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44.( Viriyakosol(S,(Matthias(MA,(Swancutt(MA,(Kirkland(TN,(Vinetz(JM.(TollIlike(receptor(4(protects(against(lethal(Leptospira(interrogans(serovar(icterohaemorrhagiae(infection(and(contributes(to(in(vivo(control(of(leptospiral(burden.(Infection(and(immunity.(2006(Febb74(2):887I95.(

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51(

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52(

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66.( Marques(AR.(Lyme(disease:(a(review.(Curr(Allergy(Asthma(Rep.(2010(Janb10(1):13I20.(

67.( Brinkerhoff(RJ,(Gilliam(WF,(Gaines(D.(Lyme(disease,(Virginia,(USA,(2000I2011.(Emerg(Infect(Dis.(2014(Octb20(10):1661I8.(

53(

68.( Mason(LM,(Veerman(CC,(Geijtenbeek(TB,(Hovius(JW.(Menage(a(trois:(Borrelia,(dendritic(cells,(and(tick(saliva(interactions.(Trends(Parasitol.(2014(Febb30(2):95I103.(

69.( Bockenstedt(LK,(Wormser(GP.(Review:(unraveling(Lyme(disease.(Arthritis(Rheumatol.(2014(Sepb66(9):2313I23.(

70.( Kopnitsky(MJ.(The(evolution(of(Borrelia(serology(tests.(MLO(Med(Lab(Obs.(2014(Junb46(6):24I5.(

71.( Stillman(BA,(Monn(M,(Liu(J,(Thatcher(B,(Foster(P,(Andrews(B,(et(al.(Performance(of(a(commercially(available(inIclinic(ELISA(for(detection(of(antibodies(against(Anaplasma(phagocytophilum,(Anaplasma(platys,(Borrelia(burgdorferi,(Ehrlichia(canis,(and(Ehrlichia(ewingii(and(Dirofilaria(immitis(antigen(in(dogs.(J(Am(Vet(Med(Assoc.(2014(Jul(1b245(1):80I6.(

72.( Arvikar(SL,(Steere(AC.(Diagnosis(and(Treatment(of(Lyme(Arthritis.(Infect(Dis(Clin(North(Am.(2015(Junb29(2):269I80.(

73.( Marques(AR.(Laboratory(Diagnosis(of(Lyme(Disease:(Advances(and(Challenges.(Infect(Dis(Clin(North(Am.(2015(Junb29(2):295I307.(

74.( Nelson(C,(Hojvat(S,(Johnson(B,(Petersen(J,(Schriefer(M,(Beard(CB,(et(al.(Concerns(regarding(a(new(culture(method(for(Borrelia(burgdorferi(not(approved(for(the(diagnosis(of(Lyme(disease.(MMWR(Morb(Mortal(Wkly(Rep.(2014(Apr(18b63(15):333.(

75.( GomesISolecki(M.(Blocking(pathogen(transmission(at(the(source:(reservoir(targeted(OspAIbased(vaccines(against(Borrelia(burgdorferi.(Frontiers(in(cellular(and(infection(microbiology.(2014b4:136.(

76.( Benach(JL,(Coleman(JL,(GarciaIMonco(JC,(Deponte(PC.(Biological(activity(of(Borrelia(burgdorferi(antigens.(Ann(N(Y(Acad(Sci.(1988b539:115I25.(

77.( Schmitz(JL,(Lovrich(SD,(Callister(SM,(Schell(RF.(Depletion(of(complement(and(effects(on(passive(transfer(of(resistance(to(infection(with(Borrelia(burgdorferi.(Infection(and(immunity.(1991(Octb59(10):3815I8.(

78.( Du(Chateau(BK,(England(DM,(Callister(SM,(Lim(LC,(Lovrich(SD,(Schell(RF.(Macrophages(exposed(to(Borrelia(burgdorferi(induce(Lyme(arthritis(in(hamsters.(Infection(and(immunity.(1996(Julb64(7):2540I7.(

79.( Barthold(SW,(Moody(KD,(Terwilliger(GA,(Jacoby(RO,(Steere(AC.(An(animal(model(for(Lyme(arthritis.(Ann(N(Y(Acad(Sci.(1988b539:264I73.(

54(

80.( Moody(KD,(Barthold(SW.(Relative(infectivity(of(Borrelia(burgdorferi(in(Lewis(rats(by(various(routes(of(inoculation.(Am(J(Trop(Med(Hyg.(1991(Febb44(2):135I9.(

81.( Akins(DR,(Bourell(KW,(Caimano(MJ,(Norgard(MV,(Radolf(JD.(A(new(animal(model(for(studying(Lyme(disease(spirochetes(in(a(mammalian(hostIadapted(state.(J(Clin(Invest.(1998(May(15b101(10):2240I50.(

82.( Weis(JJ,(McCracken(BA,(Ma(Y,(Fairbairn(D,(Roper(RJ,(Morrison(TB,(et(al.(Identification(of(quantitative(trait(loci(governing(arthritis(severity(and(humoral(responses(in(the(murine(model(of(Lyme(disease.(Journal(of(immunology((Baltimore,(Md(:(1950).(1999(Jan(15b162(2):948I56.(

83.( Crandall(H,(Dunn(DM,(Ma(Y,(Wooten(RM,(Zachary(JF,(Weis(JH,(et(al.(Gene(expression(profiling(reveals(unique(pathways(associated(with(differential(severity(of(lyme(arthritis.(Journal(of(immunology((Baltimore,(Md(:(1950).(2006(Dec(1b177(11):7930I42.(

84.( Miller(JC,(Ma(Y,(Crandall(H,(Wang(X,(Weis(JJ.(Gene(expression(profiling(provides(insights(into(the(pathways(involved(in(inflammatory(arthritis(development:(murine(model(of(Lyme(disease.(Exp(Mol(Pathol.(2008(Augb85(1):20I7.(

85.( Ma(Y,(Miller(JC,(Crandall(H,(Larsen(ET,(Dunn(DM,(Weiss(RB,(et(al.(IntervalIspecific(congenic(lines(reveal(quantitative(trait(Loci(with(penetrant(lyme(arthritis(phenotypes(on(chromosomes(5,(11,(and(12.(Infection(and(immunity.(2009(Augb77(8):3302I11.(

86.( Ma(Y,(Bramwell(KK,(Lochhead(RB,(Paquette(JK,(Zachary(JF,(Weis(JH,(et(al.(Borrelia(burgdorferi(arthritisIassociated(locus(Bbaa1(regulates(Lyme(arthritis(and(K/BxN(serum(transfer(arthritis(through(intrinsic(control(of(type(I(IFN(production.(Journal(of(immunology((Baltimore,(Md(:(1950).(2014(Dec(15b193(12):6050I60.(

87.( Kruger(P,(Saffarzadeh(M,(Weber(AN,(Rieber(N,(Radsak(M,(von(Bernuth(H,(et(al.(Neutrophils:(Between(host(defence,(immune(modulation,(and(tissue(injury.(PLoS(pathogens.(2015(Marb11(3):e1004651.(

88.( Bardoel(BW,(Kenny(EF,(Sollberger(G,(Zychlinsky(A.(The(balancing(act(of(neutrophils.(Cell(host(&(microbe.(2014(May(14b15(5):526I36.(

89.( Nathan(C.(Neutrophils(and(immunity:(challenges(and(opportunities.(Nature(reviews(Immunology.(2006(Marb6(3):173I82.(

90.( Wang(B,(Sullivan(J,(Sullivan(GW,(Mandell(GL.(Interaction(of(leptospires(with(human(polymorphonuclear(neutrophils.(Infection(and(immunity.(1984(Mayb44(2):459I64.(

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91.( Dobrina(A,(Nardon(E,(Vecile(E,(Cinco(M,(Patriarca(P.(Leptospira(icterohemorrhagiae(and(leptospire(peptidolgycans(induce(endothelial(cell(adhesiveness(for(polymorphonuclear(leukocytes.(Infection(and(immunity.(1995(Augb63(8):2995I9.(

92.( Butler(T,(Aikawa(M,(HabteIMichael(A,(Wallace(C.(Phagocytosis(of(Borrelia(recurrentis(by(blood(polymorphonuclear(leukocytes(is(enhanced(by(antibiotic(treatment.(Infection(and(immunity.(1980(Junb28(3):1009I13.(

93.( Benach(JL,(Habicht(GS,(Gocinski(BL,(Coleman(JL.(Phagocytic(cell(responses(to(in(vivo(and(in(vitro(exposure(to(the(Lyme(disease(spirochete.(Yale(J(Biol(Med.(1984(JulIAugb57(4):599I605.(

94.( Burns(MJ,(Sellati(TJ,(Teng(EI,(Furie(MB.(Production(of(interleukinI8((ILI8)(by(cultured(endothelial(cells(in(response(to(Borrelia(burgdorferi(occurs(independently(of(secreted([corrected](ILI1(and(tumor(necrosis(factor(alpha(and(is(required(for(subsequent(transendothelial(migration(of(neutrophils.(Infection(and(immunity.(1997(Aprb65(4):1217I22.(

95.( Hung(CC,(Chang(CT,(Chen(KH,(Tian(YC,(Wu(MS,(Pan(MJ,(et(al.(Upregulation(of(chemokine(CXCL1/KC(by(leptospiral(membrane(lipoprotein(preparation(in(renal(tubule(epithelial(cells.(Kidney(international.(2006(Mayb69(10):1814I22.(

96.( Sellati(TJ,(Burns(MJ,(Ficazzola(MA,(Furie(MB.(Borrelia(burgdorferi(upregulates(expression(of(adhesion(molecules(on(endothelial(cells(and(promotes(transendothelial(migration(of(neutrophils(in(vitro.(Infection(and(immunity.(1995(Novb63(11):4439I47.(

97.( Cella(M,(Miller(H,(Song(C.(Beyond(NK(cells:(the(expanding(universe(of(innate(lymphoid(cells.(Front(Immunol.(2014b5:282.(

98.( Cooper(MA,(Colonna(M,(Yokoyama(WM.(Hidden(talents(of(natural(killers:(NK(cells(in(innate(and(adaptive(immunity.(EMBO(Rep.(2009(Octb10(10):1103I10.(

99.( Strowig(T,(Brilot(F,(Munz(C.(Noncytotoxic(functions(of(NK(cells:(direct(pathogen(restriction(and(assistance(to(adaptive(immunity.(Journal(of(immunology((Baltimore,(Md(:(1950).(2008(Jun(15b180(12):7785I91.(

100.( Dattwyler(RJ,(Thomas(JA,(Benach(JL,(Golightly(MG.(Cellular(immune(response(in(Lyme(disease:(the(response(to(mitogens,(live(Borrelia(burgdorferi,(NK(cell(function(and(lymphocyte(subsets.(Zentralbl(Bakteriol(Mikrobiol(Hyg(A.(1986(Decb263(1I2):151I9.(

56(

101.( Golightly(M,(Thomas(J,(Volkman(D,(Dattwyler(R.(Modulation(of(natural(killer(cell(activity(by(Borrelia(burgdorferi.(Ann(N(Y(Acad(Sci.(1988b539:103I11.(

102.( Brown(CR,(Reiner(SL.(Activation(of(natural(killer(cells(in(arthritisIsusceptible(but(not(arthritisIresistant(mouse(strains(following(Borrelia(burgdorferi(infection.(Infection(and(immunity.(1998(Novb66(11):5208I14.(

103.( Wingender(G,(Kronenberg(M.(Invariant(natural(killer(cells(in(the(response(to(bacteria:(the(advent(of(specific(antigens.(Future(Microbiol.(2006(Octb1(3):325I40.(

104.( Miyagi(T,(Gil(MP,(Wang(X,(Louten(J,(Chu(WM,(Biron(CA.(High(basal(STAT4(balanced(by(STAT1(induction(to(control(type(1(interferon(effects(in(natural(killer(cells.(J(Exp(Med.(2007(Oct(1b204(10):2383I96.(

105.( Katchar(K,(Drouin(EE,(Steere(AC.(Natural(killer(cells(and(natural(killer(T(cells(in(Lyme(arthritis.(Arthritis(Res(Ther.(2013b15(6):R183.(

106.( Bordon(Y.(Natural(killer(T(cells:(Lyme(scaled(back.(Nature(reviews(Immunology.(2014(Octb14(10):648I9.(

107.( Ma(Y,(Seiler(KP,(Tai(KF,(Yang(L,(Woods(M,(Weis(JJ.(Outer(surface(lipoproteins(of(Borrelia(burgdorferi(stimulate(nitric(oxide(production(by(the(cytokineIinducible(pathway.(Infection(and(immunity.(1994(Sepb62(9):3663I71.(

108.( Isogai(E,(Isogai(H,(Fujii(N,(Oguma(K.(Biological(effects(of(leptospiral(lipopolysaccharide(to(mouse(B,(T(and(NK(cells.(Nihon(Juigaku(Zasshi.(1990(Octb52(5):923I30.(

109.( De(Fost(M,(Chierakul(W,(Limpaiboon(R,(Dondorp(A,(White(NJ,(van(Der(Poll(T.(Release(of(granzymes(and(chemokines(in(Thai(patients(with(leptospirosis.(Clin(Microbiol(Infect.(2007(Aprb13(4):433I6.(

110.( de(Fost(M,(Hartskeerl(RA,(Groenendijk(MR,(van(der(Poll(T.(Interleukin(12(in(part(regulates(gamma(interferon(release(in(human(whole(blood(stimulated(with(Leptospira(interrogans.(Clin(Diagn(Lab(Immunol.(2003(Marb10(2):332I5.(

111.( Chierakul(W,(de(Fost(M,(Suputtamongkol(Y,(Limpaiboon(R,(Dondorp(A,(White(NJ,(et(al.(Differential(expression(of(interferonIgamma(and(interferonIgammaIinducing(cytokines(in(Thai(patients(with(scrub(typhus(or(leptospirosis.(Clin(Immunol.(2004(Novb113(2):140I4.(

112.( Klimpel(GR,(Matthias(MA,(Vinetz(JM.(Leptospira(interrogans(activation(of(human(peripheral(blood(mononuclear(cells:(preferential(expansion(of(TCR(gamma(

57(

delta+(T(cells(vs(TCR(alpha(beta+(T(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2003(Aug(1b171(3):1447I55.(

113.( Tuero(I,(Vinetz(JM,(Klimpel(GR.(Lack(of(demonstrable(memory(T(cell(responses(in(humans(who(have(spontaneously(recovered(from(leptospirosis(in(the(Peruvian(Amazon.(The(Journal(of(infectious(diseases.(2010(Feb(1b201(3):420I7.(

114.( Zuerner(RL,(Alt(DP,(Palmer(MV,(Thacker(TC,(Olsen(SC.(A(Leptospira(borgpetersenii(serovar(Hardjo(vaccine(induces(a(Th1(response,(activates(NK(cells,(and(reduces(renal(colonization.(Clin(Vaccine(Immunol.(2011(Aprb18(4):684I91.(

115.( Guilliams(M,(Ginhoux(F,(Jakubzick(C,(Naik(SH,(Onai(N,(Schraml(BU,(et(al.(Dendritic(cells,(monocytes(and(macrophages:(a(unified(nomenclature(based(on(ontogeny.(Nature(reviews(Immunology.(2014b14(8):571I8.(

116.( Kushwah(R,(Hu(J.(Complexity(of(dendritic(cell(subsets(and(their(function(in(the(host(immune(system.(Immunology.(2011(Augb133(4):409I19.(

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118.( Wynn(TA,(Chawla(A,(Pollard(JW.(Macrophage(biology(in(development,(homeostasis(and(disease.(Nature.(2013(Apr(25b496(7446):445I55.(

119.( Marinho(M,(OliveiraIJúnior(IS,(Perri(SHV,(Peiró(JR,(Pavanelli(TF,(Salomão(R.(Response(activity(of(alveolar(macrophages(in(pulmonary(dysfunction(caused(by(Leptospira(infection.(Journal(of(Venomous(Animals(and(Toxins(including(Tropical(Diseases.(2008b14:58I70.(

120.( Mills(CD,(Lenz(LL,(Ley(K.(Macrophages(at(the(fork(in(the(road(to(health(or(disease.(Front(Immunol.(2015b6:59.(

121.( Sica(A,(Mantovani(A.(Macrophage(plasticity(and(polarization:(in(vivo(veritas.(J(Clin(Invest.(2012(Marb122(3):787I95.(

122.( Mills(CD.(M1(and(M2(Macrophages:(Oracles(of(Health(and(Disease.(Crit(Rev(Immunol.(2012b32(6):463I88.(

123.( Mege(JL,(Mehraj(V,(Capo(C.(Macrophage(polarization(and(bacterial(infections.(Curr(Opin(Infect(Dis.(2011(Junb24(3):230I4.(

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58(

125.( Li(S,(Ojcius(DM,(Liao(S,(Li(L,(Xue(F,(Dong(H,(et(al.(Replication(or(death:(distinct(fates(of(pathogenic(Leptospira(strain(Lai(within(macrophages(of(human(or(mouse(origin.(Innate(Immun.(2010(Aprb16(2):80I92.(

126.( Jin(D,(Ojcius(DM,(Sun(D,(Dong(H,(Luo(Y,(Mao(Y,(et(al.(Leptospira(interrogans(induces(apoptosis(in(macrophages(via(caspaseI8I(and(caspaseI3Idependent(pathways.(Infection(and(immunity.(2009(Febb77(2):799I809.(

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128.( Merien(F,(Baranton(G,(Perolat(P.(Invasion(of(Vero(cells(and(induction(of(apoptosis(in(macrophages(by(pathogenic(Leptospira(interrogans(are(correlated(with(virulence.(Infection(and(immunity.(1997(Febb65(2):729I38.(

129.( Xue(F,(Zhao(X,(Yang(Y,(Zhao(J,(Cao(Y,(Hong(C,(et(al.(Responses(of(murine(and(human(macrophages(to(leptospiral(infection:(a(study(using(comparative(array(analysis.(PLoS(neglected(tropical(diseases.(2013b7(10):e2477.(

130.( Strle(K,(Drouin(EE,(Shen(S,(El(Khoury(J,(McHugh(G,(RuzicISabljic(E,(et(al.(Borrelia(burgdorferi(stimulates(macrophages(to(secrete(higher(levels(of(cytokines(and(chemokines(than(Borrelia(afzelii(or(Borrelia(garinii.(The(Journal(of(infectious(diseases.(2009(Dec(15b200(12):1936I43.(

131.( Linder(S,(Heimerl(C,(Fingerle(V,(Aepfelbacher(M,(Wilske(B.(Coiling(phagocytosis(of(Borrelia(burgdorferi(by(primary(human(macrophages(is(controlled(by(CDC42Hs(and(Rac1(and(involves(recruitment(of(WiskottIAldrich(syndrome(protein(and(Arp2/3(complex.(Infection(and(immunity.(2001(Marb69(3):1739I46.(

132.( Cinco(M,(Cini(B,(Murgia(R,(Presani(G,(Prodan(M,(Perticarari(S.(Evidence(of(involvement(of(the(mannose(receptor(in(adhesion(of(Borrelia(burgdorferi(to(monocyte/macrophages.(Infection(and(immunity.(2001(Aprb69(4):2743I7.(

133.( DuChateau(BK,(Munson(EL,(England(DM,(Lovrich(SD,(Callister(SM,(Jensen(JR,(et(al.(Macrophages(interact(with(enriched(populations(of(distinct(T(lymphocyte(subsets(for(the(induction(of(severe(destructive(Lyme(arthritis.(J(Leukoc(Biol.(1999(Febb65(2):162I70.(

134.( Montgomery(RR,(Malawista(SE.(Entry(of(Borrelia(burgdorferi(into(macrophages(is(endIon(and(leads(to(degradation(in(lysosomes.(Infection(and(immunity.(1996(Julb64(7):2867I72.(

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135.( Georgilis(K,(Steere(AC,(Klempner(MS.(Infectivity(of(Borrelia(burgdorferi(correlates(with(resistance(to(elimination(by(phagocytic(cells.(The(Journal(of(infectious(diseases.(1991(Janb163(1):150I5.(

136.( Montgomery(RR,(Nathanson(MH,(Malawista(SE.(The(fate(of(Borrelia(burgdorferi,(the(agent(for(Lyme(disease,(in(mouse(macrophages.(Destruction,(survival,(recovery.(Journal(of(immunology((Baltimore,(Md(:(1950).(1993(Feb(1b150(3):909I15.(

137.( PetnickiIOcwieja(T,(Kern(A.(Mechanisms(of(Borrelia(burgdorferi(internalization(and(intracellular(innate(immune(signaling.(Frontiers(in(cellular(and(infection(microbiology.(2014b4:175.(

138.( Lochhead(RB,(Ma(Y,(Zachary(JF,(Baltimore(D,(Zhao(JL,(Weis(JH,(et(al.(MicroRNAI146a(provides(feedback(regulation(of(lyme(arthritis(but(not(carditis(during(infection(with(Borrelia(burgdorferi.(PLoS(pathogens.(2014(Junb10(6):e1004212.(

139.( Miller(JC,(MaylorIHagen(H,(Ma(Y,(Weis(JH,(Weis(JJ.(The(Lyme(disease(spirochete(Borrelia(burgdorferi(utilizes(multiple(ligands,(including(RNA,(for(interferon(regulatory(factor(3Idependent(induction(of(type(I(interferonIresponsive(genes.(Infection(and(immunity.(2010(Julb78(7):3144I53.(

140.( Miller(JC,(Ma(Y,(Bian(J,(Sheehan(KC,(Zachary(JF,(Weis(JH,(et(al.(A(critical(role(for(type(I(IFN(in(arthritis(development(following(Borrelia(burgdorferi(infection(of(mice.(Journal(of(immunology((Baltimore,(Md(:(1950).(2008(Dec(15b181(12):8492I503.(

141.( Faisal(SM,(Chen(JW,(McDonough(SP,(Chang(CF,(Teng(CH,(Chang(YF.(Immunostimulatory(and(antigen(delivery(properties(of(liposomes(made(up(of(total(polar(lipids(from(nonIpathogenic(bacteria(leads(to(efficient(induction(of(both(innate(and(adaptive(immune(responses.(Vaccine.(2011(Mar(16b29(13):2381I91.(

142.( Gaudart(N,(Ekpo(P,(Pattanapanyasat(K,(van(Kooyk(Y,(Engering(A.(Leptospira(interrogans(is(recognized(through(DCISIGN(and(induces(maturation(and(cytokine(production(by(human(dendritic(cells.(FEMS(Immunol(Med(Microbiol.(2008(Augb53(3):359I67.(

143.( Divan(A,(Budd(RC,(Tobin(RP,(NewellIRogers(MK.(gammadelta(T(Cells(and(dendritic(cells(in(refractory(Lyme(arthritis.(J(Leukoc(Biol.(2015(Aprb97(4):653I63.(

144.( Petzke(MM,(Brooks(A,(Krupna(MA,(Mordue(D,(Schwartz(I.(Recognition(of(Borrelia(burgdorferi,(the(Lyme(disease(spirochete,(by(TLR7(and(TLR9(induces(a(type(I(IFN(response(by(human(immune(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2009(Oct(15b183(8):5279I92.(

60(

145.( Ricklin(D,(Hajishengallis(G,(Yang(K,(Lambris(JD.(Complement:(a(key(system(for(immune(surveillance(and(homeostasis.(Nature(immunology.(2010(Sepb11(9):785I97.(

146.( Trouw(LA,(Daha(MR.(Role(of(complement(in(innate(immunity(and(host(defense.(Immunology(Letters.(2011(7//b138(1):35I7.(

147.( SahagunIRuiz(A,(Breda(LC,(Castiblanco(Valencia(MM,(Elias(WP,(MuntheIFog(L,(Garred(P,(et(al.(Studies(of(the(binding(of(ficolinI2(and(ficolinI3(from(the(complement(lectin(pathway(to(Leptospira(biflexa,(Pasteurella(pneumotropica(and(Diarrheagenic(Escherichia(coli.(Immunobiology.(2015(Octb220(10):1177I85.(

148.( Vieira(ML,(Atzingen(MV,(Oliveira(R,(Mendes(RS,(Domingos(RF,(Vasconcellos(SA,(et(al.(Plasminogen(binding(proteins(and(plasmin(generation(on(the(surface(of(Leptospira(spp.:(the(contribution(to(the(bacteriaIhost(interactions.(J(Biomed(Biotechnol.(2012b2012:758513.(

149.( Kraiczy(P,(Skerka(C,(Kirschfink(M,(Zipfel(PF,(Brade(V.(Mechanism(of(complement(resistance(of(pathogenic(Borrelia(burgdorferi(isolates.(Int(Immunopharmacol.(2001(Marb1(3):393I401.(

150.( Patarakul(K,(Cole(MF,(Hughes(CA.(Complement(resistance(in(Borrelia(burgdorferi(strain(297:(outer(membrane(proteins(prevent(MAC(formation(at(lysis(susceptible(sites.(Microb(Pathog.(1999(Julb27(1):25I41.(

151.( Nowling(JM,(Philipp(MT.(Killing(of(Borrelia(burgdorferi(by(antibody(elicited(by(OspA(vaccine(is(inefficient(in(the(absence(of(complement.(Infection(and(immunity.(1999(Janb67(1):443I5.(

152.( Fraga(TR,(Barbosa(AS,(Isaac(L.(Leptospirosis:(aspects(of(innate(immunity,(immunopathogenesis(and(immune(evasion(from(the(complement(system.(Scand(J(Immunol.(2011(Mayb73(5):408I19.(

153.( da(Silva(LB,(Miragaia(Ldos(S,(Breda(LC,(Abe(CM,(Schmidt(MC,(Moro(AM,(et(al.(Pathogenic(Leptospira(species(acquire(factor(H(and(vitronectin(via(the(surface(protein(LcpA.(Infection(and(immunity.(2015(Marb83(3):888I97.(

154.( Fraga(TR,(Courrol(Ddos(S,(CastiblancoIValencia(MM,(Hirata(IY,(Vasconcellos(SA,(Juliano(L,(et(al.(Immune(evasion(by(pathogenic(Leptospira(strains:(the(secretion(of(proteases(that(directly(cleave(complement(proteins.(The(Journal(of(infectious(diseases.(2014(Marb209(6):876I86.(

61(

155.( Choy(HA.(Multiple(activities(of(LigB(potentiate(virulence(of(Leptospira(interrogans:(inhibition(of(alternative(and(classical(pathways(of(complement.(PloS(one.(2012b7(7):e41566.(

156.( Souza(NM,(Vieira(ML,(Alves(IJ,(de(Morais(ZM,(Vasconcellos(SA,(Nascimento(AL.(Lsa30,(a(novel(adhesin(of(Leptospira(interrogans(binds(human(plasminogen(and(the(complement(regulator(C4bp.(Microb(Pathog.(2012(Sepb53(3I4):125I34.(

157.( de(Taeye(SW,(Kreuk(L,(van(Dam(AP,(Hovius(JW,(Schuijt(TJ.(Complement(evasion(by(Borrelia(burgdorferi:(it(takes(three(to(tango.(Trends(Parasitol.(2013(Marb29(3):119I28.(

158.( Shoemaker(RC,(Giclas(PC,(Crowder(C,(House(D,(Glovsky(MM.(Complement(split(products(C3a(and(C4a(are(early(markers(of(acute(lyme(disease(in(tick(bite(patients(in(the(United(States.(Int(Arch(Allergy(Immunol.(2008b146(3):255I61.(

159.( Bykowski(T,(Woodman(ME,(Cooley(AE,(Brissette(CA,(Wallich(R,(Brade(V,(et(al.(Borrelia(burgdorferi(complement(regulatorIacquiring(surface(proteins((BbCRASPs):(Expression(patterns(during(the(mammalItick(infection(cycle.(Int(J(Med(Microbiol.(2008(Sep(1b298(Suppl(1:249I56.(

160.( Garcia(RC,(Murgia(R,(Cinco(M.(Complement(receptor(3(binds(the(Borrelia(burgdorferi(outer(surface(proteins(OspA(and(OspB(in(an(iC3bIindependent(manner.(Infection(and(immunity.(2005(Sepb73(9):6138I42.(

161.( Lawrenz(MB,(Wooten(RM,(Zachary(JF,(Drouin(SM,(Weis(JJ,(Wetsel(RA,(et(al.(Effect(of(complement(component(C3(deficiency(on(experimental(Lyme(borreliosis(in(mice.(Infection(and(immunity.(2003(Augb71(8):4432I40.(

162.( Kraiczy(P,(Hellwage(J,(Skerka(C,(Kirschfink(M,(Brade(V,(Zipfel(PF,(et(al.(Immune(evasion(of(Borrelia(burgdorferi:(mapping(of(a(complementIinhibitor(factor(HIbinding(site(of(BbCRASPI3,(a(novel(member(of(the(Erp(protein(family.(Eur(J(Immunol.(2003(Marb33(3):697I707.(

163.( Alitalo(A,(Meri(T,(Lankinen(H,(Seppala(I,(Lahdenne(P,(Hefty(PS,(et(al.(Complement(inhibitor(factor(H(binding(to(Lyme(disease(spirochetes(is(mediated(by(inducible(expression(of(multiple(plasmidIencoded(outer(surface(protein(E(paralogs.(Journal(of(immunology((Baltimore,(Md(:(1950).(2002(Oct(1b169(7):3847I53.(

164.( Kraiczy(P,(Hartmann(K,(Hellwage(J,(Skerka(C,(Kirschfink(M,(Brade(V,(et(al.(Immunological(characterization(of(the(complement(regulator(factor(HIbinding(CRASP(and(Erp(proteins(of(Borrelia(burgdorferi.(Int(J(Med(Microbiol.(2004(Aprb293(Suppl(37:152I7.(

62(

165.( Stevenson(B,(ElIHage(N,(Hines(MA,(Miller(JC,(Babb(K.(Differential(binding(of(host(complement(inhibitor(factor(H(by(Borrelia(burgdorferi(Erp(surface(proteins:(a(possible(mechanism(underlying(the(expansive(host(range(of(Lyme(disease(spirochetes.(Infection(and(immunity.(2002(Febb70(2):491I7.(

166.( Alitalo(A,(Meri(T,(Ramo(L,(Jokiranta(TS,(Heikkila(T,(Seppala(IJ,(et(al.(Complement(evasion(by(Borrelia(burgdorferi:(serumIresistant(strains(promote(C3b(inactivation.(Infection(and(immunity.(2001(Junb69(6):3685I91.(

167.( Kraiczy(P,(Stevenson(B.(Complement(regulatorIacquiring(surface(proteins(of(Borrelia(burgdorferi:(Structure,(function(and(regulation(of(gene(expression.(Ticks(Tick(Borne(Dis.(2013(Febb4(1I2):26I34.(

168.( Jacobson(AC,(Ma(Y,(Zachary(JF,(Weis(JJ,(Weis(JH.(Mice(lacking(CD21(and(CD35(proteins(mount(effective(immune(responses(against(Borrelia(burgdorferi(infection.(Infection(and(immunity.(2007(Aprb75(4):2075I8.(

169.( van(Burgel(ND,(Balmus(NC,(Fikrig(E,(van(Dam(AP.(Infectivity(of(Borrelia(burgdorferi(sensu(lato(is(unaltered(in(C3Ideficient(mice.(Ticks(Tick(Borne(Dis.(2011(Marb2(1):20I6.(

170.( Bockenstedt(LK,(Barthold(S,(Deponte(K,(Marcantonio(N,(Kantor(FS.(Borrelia(burgdorferi(infection(and(immunity(in(mice(deficient(in(the(fifth(component(of(complement.(Infection(and(immunity.(1993(Mayb61(5):2104I7.(

171.( Sole(M,(Bantar(C,(Indest(K,(Gu(Y,(Ramamoorthy(R,(Coughlin(R,(et(al.(Borrelia(burgdorferi(escape(mutants(that(survive(in(the(presence(of(antiserum(to(the(OspA(vaccine(are(killed(when(complement(is(also(present.(Infection(and(immunity.(1998(Junb66(6):2540I6.(

172.( Suhonen(J,(Hartiala(K,(TuominenIGustafsson(H,(Viljanen(MK.(Borrelia(burgdorferiIIinduced(oxidative(burst,(calcium(mobilization,(and(phagocytosis(of(human(neutrophils(are(complement(dependent.(The(Journal(of(infectious(diseases.(2000(Janb181(1):195I202.(

173.( Edholm(ES,(Bengten(E,(Wilson(M.(Insights(into(the(function(of(IgD.(Dev(Comp(Immunol.(2011(Decb35(12):1309I16.(

174.( Preud'homme(JL,(Petit(I,(Barra(A,(Morel(F,(Lecron(JC,(Lelievre(E.(Structural(and(functional(properties(of(membrane(and(secreted(IgD.(Mol(Immunol.(2000(Octb37(15):871I87.(

63(

175.( Chen(K,(Xu(W,(Wilson(M,(He(B,(Miller(NW,(Bengten(E,(et(al.(Immunoglobulin(D(enhances(immune(surveillance(by(activating(antimicrobial,(proinflammatory(and(B(cellIstimulating(programs(in(basophils.(Nature(immunology.(2009(Augb10(8):889I98.(

176.( Elsner(RA,(Hastey(CJ,(Olsen(KJ,(Baumgarth(N.(Suppression(of(LongILived(Humoral(Immunity(Following(Borrelia(burgdorferi(Infection.(PLoS(pathogens.(2015(Julb11(7):e1004976.(

177.( Hastey(CJ,(Ochoa(J,(Olsen(KJ,(Barthold(SW,(Baumgarth(N.(MyD88I(and(TRIFIindependent(induction(of(type(I(interferon(drives(naive(B(cell(accumulation(but(not(loss(of(lymph(node(architecture(in(Lyme(disease.(Infection(and(immunity.(2014(Aprb82(4):1548I58.(

178.( Hastey(CJ,(Elsner(RA,(Barthold(SW,(Baumgarth(N.(Delays(and(diversions(mark(the(development(of(B(cell(responses(to(Borrelia(burgdorferi(infection.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Jun(1b188(11):5612I22.(

179.( Adler(B,(Murphy(AM,(Locarnini(SA,(Faine(S.(Detection(of(specific(antiIleptospiral(immunoglobulins(M(and(G(in(human(serum(by(solidIphase(enzymeIlinked(immunosorbent(assay.(J(Clin(Microbiol.(1980(Mayb11(5):452I7.(

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181.( Panelius(J,(Seppala(I,(Granlund(H,(Nyman(D,(Wahlberg(P.(Evaluation(of(treatment(responses(in(late(Lyme(borreliosis(on(the(basis(of(antibody(decrease(during(the(followIup(period.(Eur(J(Clin(Microbiol(Infect(Dis.(1999(Sepb18(9):621I9.(

182.( Widhe(M,(Ekerfelt(C,(Forsberg(P,(Bergstrom(S,(Ernerudh(J.(IgG(subclasses(in(Lyme(borreliosis:(a(study(of(specific(IgG(subclass(distribution(in(an(interferonIgammaIpredominated(disease.(Scand(J(Immunol.(1998(Junb47(6):575I81.(

183.( Seppala(IJ,(Kroneld(R,(Schauman(K,(Forsen(KO,(Lassenius(R.(Diagnosis(of(Lyme(borreliosis:(nonIspecific(serological(reactions(with(Borrelia(burgdorferi(sonicate(antigen(caused(by(IgG2(antibodies.(J(Med(Microbiol.(1994(Aprb40(4):293I302.(

184.( Mathiesen(T,(von(Holst(H,(Fredrikson(S,(Wirsen(G,(Hederstedt(B,(Norrby(E,(et(al.(Total,(antiIviral,(and(antiImyelin(IgG(subclass(reactivity(in(inflammatory(diseases(of(the(central(nervous(system.(J(Neurol.(1989(Mayb236(4):238I42.(

185.( Olsson(I,(Hammarstrom(L,(Smith(CI,(Hovmark(A,(Asbrink(E.(IgG(subclasses(of(specific(antibodies(in(Ixodes(ricinusIborne(borreliosis.(Clin(Exp(Immunol.(1987(Sepb69(3):618I23.(

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186.( Xia(J,(Winkelmann(ER,(Gorder(SR,(Mason(PW,(Milligan(GN.(TLR3I(and(MyD88IDependent(Signaling(Differentially(Influences(the(Development(of(West(Nile(VirusISpecific(B(Cell(Responses(in(Mice(following(Immunization(with(RepliVAX(WN,(a(SingleICycle(Flavivirus(Vaccine(Candidate.(Journal(of(Virology.(2013b87(22):12090I101.(

187.( Martin(RM,(Brady(JL,(Lew(AM.(The(need(for(IgG2c(specific(antiserum(when(isotyping(antibodies(from(C57BL/6(and(NOD(mice.(Journal(of(Immunological(Methods.(1998b212(2):187I92.(

188.( Oliveira(TR,(Longhi(MT,(de(Morais(ZM,(Romero(EC,(Blanco(RM,(Kirchgatter(K,(et(al.(Evaluation(of(Leptospiral(Recombinant(Antigens(MPL17(and(MPL21(for(Serological(Diagnosis(of(Leptospirosis(by(EnzymeILinked(Immunosorbent(Assays.(Clinical(and(Vaccine(Immunology(:(CVI.(2008b15(11):1715I22.(

189.( He(JS,(Narayanan(S,(Subramaniam(S,(Ho(WQ,(Lafaille(JJ,(Curotto(de(Lafaille(MA.(Biology(of(IgE(production:(IgE(cell(differentiation(and(the(memory(of(IgE(responses.(Curr(Top(Microbiol(Immunol.(2015b388:1I19.(

190.( Gould(HJ,(Ramadani(F.(IgE(responses(in(mouse(and(man(and(the(persistence(of(IgE(memory.(Trends(Immunol.(2015(Janb36(1):40I8.(

191.( Bluth(MH,(Robin(J,(Ruditsky(M,(Norowitz(KB,(Chice(S,(Pytlak(E,(et(al.(IgE(antiIBorrelia(burgdorferi(components((p18,(p31,(p34,(p41,(p45,(p60)(and(increased(blood(CD8+CD60+(T(cells(in(children(with(Lyme(disease.(Scand(J(Immunol.(2007(Aprb65(4):376I82.(

192.( Benach(JL,(Gruber(BL,(Coleman(JL,(Habicht(GS,(Golightly(MG.(An(IgE(response(to(spirochete(antigen(in(patients(with(Lyme(disease.(Zentralbl(Bakteriol(Mikrobiol(Hyg(A.(1986(Decb263(1I2):127I32.(

193.( Gutzeit(C,(Magri(G,(Cerutti(A.(Intestinal(IgA(production(and(its(role(in(hostImicrobe(interaction.(Immunol(Rev.(2014(Julb260(1):76I85.(

194.( Gommerman(JL,(Rojas(OL,(Fritz(JH.(ReIthinking(the(functions(of(IgA(+)(plasma(cells.(Gut(Microbes.(2014b5(5):652I62.(

195.( Macpherson(AJ,(McCoy(KD,(Johansen(FE,(Brandtzaeg(P.(The(immune(geography(of(IgA(induction(and(function.(Mucosal(Immunol.(2008(Janb1(1):11I22.(

196.( da(Silva(MV,(Nakamura(PM,(Camargo(ED,(Batista(L,(Vaz(AJ,(Romero(EC,(et(al.(Immunodiagnosis(of(human(leptospirosis(by(dotIELISA(for(the(detection(of(IgM,(IgG,(and(IgA(antibodies.(Am(J(Trop(Med(Hyg.(1997(Junb56(6):650I5.(

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197.( da(Silva(MV,(Camargo(ED.([An(immunoenzyme(test((ELISA)(for(the(detection(of(circulating(classIIgA(antibodies(in(human(leptospirosis].(Rev(Inst(Med(Trop(Sao(Paulo.(1992(MayIJunb34(3):239I42.(

198.( Baig(S,(Olsson(T,(Hansen(K,(Link(H.(AntiIBorrelia(burgdorferi(antibody(response(over(the(course(of(Lyme(neuroborreliosis.(Infection(and(immunity.(1991(Marb59(3):1050I6.(

199.( Baig(S,(Olsson(T,(Link(H.(Predominance(of(Borrelia(burgdorferi(specific(B(cells(in(cerebrospinal(fluid(in(neuroborreliosis.(Lancet.(1989(Jul(8b2(8654):71I4.(

200.( Whitmire(WM,(Garon(CF.(Specific(and(nonspecific(responses(of(murine(B(cells(to(membrane(blebs(of(Borrelia(burgdorferi.(Infection(and(immunity.(1993(Aprb61(4):1460I7.(

201.( Cacciapuoti(B,(Ciarrocchi(S,(Ciceroni(L.(The(complementIkilling(of(Borrelia(burgdorferi.(Target(antigens(and(sensitizing(antibodies.(Zentralbl(Bakteriol.(1998(Julb288(1):121I9.(

202.( Kramer(MD,(Wallich(R,(Simon(MM.(The(outer(surface(protein(A((OspA)(of(Borrelia(burgdorferi:(a(vaccine(candidate(and(bioactive(mediator.(Infection.(1996(MarIAprb24(2):190I4.(

203.( Radolf(JD,(Goldberg(MS,(Bourell(K,(Baker(SI,(Jones(JD,(Norgard(MV.(Characterization(of(outer(membranes(isolated(from(Borrelia(burgdorferi,(the(Lyme(disease(spirochete.(Infection(and(immunity.(1995(Junb63(6):2154I63.(

204.( Bryant(CE,(Symmons(M,(Gay(NJ.(TollIlike(receptor(signalling(through(macromolecular(protein(complexes.(Mol(Immunol.(2015(Febb63(2):162I5.(

205.( Khoo(JJ,(Forster(S,(Mansell(A.(TollIlike(receptors(as(interferonIregulated(genes(and(their(role(in(disease.(J(Interferon(Cytokine(Res.(2011(Janb31(1):13I25.(

206.( Kawasaki(T,(Kawai(T.(TollIlike(receptor(signaling(pathways.(Front(Immunol.(2014b5:461.(

207.( LacroixILamande(S,(d'Andon(MF,(Michel(E,(Ratet(G,(Philpott(DJ,(Girardin(SE,(et(al.(Downregulation(of(the(Na/KIATPase(pump(by(leptospiral(glycolipoprotein(activates(the(NLRP3(inflammasome.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Mar(15b188(6):2805I14.(

208.( Love(AC,(Schwartz(I,(Petzke(MM.(Borrelia(burgdorferi(RNA(induces(type(I(and(III(interferons(via(TollIlike(receptor(7(and(contributes(to(production(of(NFIkappaBIdependent(cytokines.(Infection(and(immunity.(2014(Junb82(6):2405I16.(

66(

209.( KrupnaIGaylord(MA,(Liveris(D,(Love(AC,(Wormser(GP,(Schwartz(I,(Petzke(MM.(Induction(of(type(I(and(type(III(interferons(by(Borrelia(burgdorferi(correlates(with(pathogenesis(and(requires(linear(plasmid(36.(PloS(one.(2014b9(6):e100174.(

210.( Oosting(M,(Buffen(K,(Malireddi(SR,(Sturm(P,(Verschueren(I,(Koenders(MI,(et(al.(Murine(Borrelia(arthritis(is(highly(dependent(on(ASC(and(caspaseI1,(but(independent(of(NLRP3.(Arthritis(Res(Ther.(2012b14(6):R247.(

211.( Hirschfeld(M,(Kirschning(CJ,(Schwandner(R,(Wesche(H,(Weis(JH,(Wooten(RM,(et(al.(Cutting(edge:(inflammatory(signaling(by(Borrelia(burgdorferi(lipoproteins(is(mediated(by(tollIlike(receptor(2.(Journal(of(immunology((Baltimore,(Md(:(1950).(1999(Sep(1b163(5):2382I6.(

212.( Lien(E,(Sellati(TJ,(Yoshimura(A,(Flo(TH,(Rawadi(G,(Finberg(RW,(et(al.(TollIlike(receptor(2(functions(as(a(pattern(recognition(receptor(for(diverse(bacterial(products.(J(Biol(Chem.(1999(Nov(19b274(47):33419I25.(

213.( Werts(C,(Tapping(RI,(Mathison(JC,(Chuang(TH,(Kravchenko(V,(Saint(Girons(I,(et(al.(Leptospiral(lipopolysaccharide(activates(cells(through(a(TLR2Idependent(mechanism.(Nature(immunology.(2001(Aprb2(4):346I52.(

214.( Ruby(J,(Rehani(K,(Martin(M.(Treponema(denticola(activates(mitogenIactivated(protein(kinase(signal(pathways(through(TollIlike(receptor(2.(Infection(and(immunity.(2007(Decb75(12):5763I8.(

215.( Nussbaum(G,(BenIAdi(S,(Genzler(T,(Sela(M,(Rosen(G.(Involvement(of(TollIlike(receptors(2(and(4(in(the(innate(immune(response(to(Treponema(denticola(and(its(outer(sheath(components.(Infection(and(immunity.(2009(Sepb77(9):3939I47.(

216.( Hartiala(P,(Hytonen(J,(Yrjanainen(H,(Honkinen(M,(Terho(P,(Soderstrom(M,(et(al.(TLR2(utilization(of(Borrelia(does(not(induce(p38I(and(IFNIbeta(autocrine(loopIdependent(expression(of(CD38,(resulting(in(poor(migration(and(weak(ILI12(secretion(of(dendritic(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2010(May(15b184(10):5732I42.(

217.( Dickinson(GS,(Piccone(H,(Sun(G,(Lien(E,(Gatto(L,(Alugupalli(KR.(TollIlike(receptor(2(deficiency(results(in(impaired(antibody(responses(and(septic(shock(during(Borrelia(hermsii(infection.(Infection(and(immunity.(2010(Novb78(11):4579I88.(

218.( Shin(JE,(Choi(Y.(Treponema(denticola(suppresses(expression(of(human(betaIdefensinI2(in(gingival(epithelial(cells(through(inhibition(of(TNFalpha(production(and(TLR2(activation.(Mol(Cells.(2010(Aprb29(4):407I12.(

67(

219.( Murray(GL,(Morel(V,(Cerqueira(GM,(Croda(J,(Srikram(A,(Henry(R,(et(al.(GenomeIwide(transposon(mutagenesis(in(pathogenic(Leptospira(species.(Infection(and(immunity.(2009(Febb77(2):810I6.(

220.( Nahori(MA,(FournieIAmazouz(E,(QueIGewirth(NS,(Balloy(V,(Chignard(M,(Raetz(CR,(et(al.(Differential(TLR(recognition(of(leptospiral(lipid(A(and(lipopolysaccharide(in(murine(and(human(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2005(Nov(1b175(9):6022I31.(

221.( Nascimento(AL,(Ko(AI,(Martins(EA,(MonteiroIVitorello(CB,(Ho(PL,(Haake(DA,(et(al.(Comparative(genomics(of(two(Leptospira(interrogans(serovars(reveals(novel(insights(into(physiology(and(pathogenesis.(J(Bacteriol.(2004(Aprb186(7):2164I72.(

222.( Dennis(VA,(Dixit(S,(O'Brien(SM,(Alvarez(X,(Pahar(B,(Philipp(MT.(Live(Borrelia(burgdorferi(spirochetes(elicit(inflammatory(mediators(from(human(monocytes(via(the(TollIlike(receptor(signaling(pathway.(Infection(and(immunity.(2009(Marb77(3):1238I45.(

223.( Shin(OS,(Isberg(RR,(Akira(S,(Uematsu(S,(Behera(AK,(Hu(LT.(Distinct(roles(for(MyD88(and(TollIlike(receptors(2,(5,(and(9(in(phagocytosis(of(Borrelia(burgdorferi(and(cytokine(induction.(Infection(and(immunity.(2008(Junb76(6):2341I51.(

224.( Hidmark(A,(von(Saint(Paul(A,(Dalpke(AH.(Cutting(edge:(TLR13(is(a(receptor(for(bacterial(RNA.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Sep(15b189(6):2717I21.(

225.( Li(XD,(Chen(ZJ.(Sequence(specific(detection(of(bacterial(23S(ribosomal(RNA(by(TLR13.(Elife.(2012b1:e00102.(

226.( Kawai(T,(Akira(S.(The(role(of(patternIrecognition(receptors(in(innate(immunity:(update(on(TollIlike(receptors.(Nature(immunology.(2010(Mayb11(5):373I84.(

227.( Kawai(T,(Akira(S.(TollIlike(receptors(and(their(crosstalk(with(other(innate(receptors(in(infection(and(immunity.(Immunity.(2011(May(27b34(5):637I50.(

228.( Sheedy(FJ,(O'Neill(LA.(The(Troll(in(Toll:(Mal(and(Tram(as(bridges(for(TLR2(and(TLR4(signaling.(J(Leukoc(Biol.(2007(Augb82(2):196I203.(

229.( Aubry(C,(Corr(SC,(Wienerroither(S,(Goulard(C,(Jones(R,(Jamieson(AM,(et(al.(Both(TLR2(and(TRIF(contribute(to(interferonIbeta(production(during(Listeria(infection.(PloS(one.(2012b7(3):e33299.(

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230.( PetnickiIOcwieja(T,(Chung(E,(Acosta(DI,(Ramos(LT,(Shin(OS,(Ghosh(S,(et(al.(TRIF(mediates(TollIlike(receptor(2Idependent(inflammatory(responses(to(Borrelia(burgdorferi.(Infection(and(immunity.(2013(Febb81(2):402I10.(

231.( Rupprecht(TA,(Kirschning(CJ,(Popp(B,(Kastenbauer(S,(Fingerle(V,(Pfister(HW,(et(al.(Borrelia(garinii(induces(CXCL13(production(in(human(monocytes(through(TollIlike(receptor(2.(Infection(and(immunity.(2007(Sepb75(9):4351I6.(

232.( Bulut(Y,(Faure(E,(Thomas(L,(Equils(O,(Arditi(M.(Cooperation(of(TollIlike(receptor(2(and(6(for(cellular(activation(by(soluble(tuberculosis(factor(and(Borrelia(burgdorferi(outer(surface(protein(A(lipoprotein:(role(of(TollIinteracting(protein(and(ILI1(receptor(signaling(molecules(in(TollIlike(receptor(2(signaling.(Journal(of(immunology((Baltimore,(Md(:(1950).(2001(Jul(15b167(2):987I94.(

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240.( Dziarski(R,(Gupta(D.(Role(of(MDI2(in(TLR2I(and(TLR4Imediated(recognition(of(GramInegative(and(GramIpositive(bacteria(and(activation(of(chemokine(genes.(J(Endotoxin(Res.(2000b6(5):401I5.(

241.( Poltorak(A,(RicciardiICastagnoli(P,(Citterio(S,(Beutler(B.(Physical(contact(between(lipopolysaccharide(and(tollIlike(receptor(4(revealed(by(genetic(complementation.(Proc(Natl(Acad(Sci(U(S(A.(2000(Feb(29b97(5):2163I7.(

242.( Kawai(T,(Takeuchi(O,(Fujita(T,(Inoue(J,(Muhlradt(PF,(Sato(S,(et(al.(Lipopolysaccharide(stimulates(the(MyD88Iindependent(pathway(and(results(in(activation(of(IFNIregulatory(factor(3(and(the(expression(of(a(subset(of(lipopolysaccharideIinducible(genes.(Journal(of(immunology((Baltimore,(Md(:(1950).(2001(Nov(15b167(10):5887I94.(

243.( Tan(Y,(Kagan(JC.(A(crossIdisciplinary(perspective(on(the(innate(immune(responses(to(bacterial(lipopolysaccharide.(Mol(Cell.(2014(Apr(24b54(2):212I23.(

244.( Casjens(SR,(Mongodin(EF,(Qiu(WG,(Luft(BJ,(Schutzer(SE,(Gilcrease(EB,(et(al.(Genome(stability(of(Lyme(disease(spirochetes:(comparative(genomics(of(Borrelia(burgdorferi(plasmids.(PloS(one.(2012b7(3):e33280.(

245.( Radolf(JD,(Norgard(MV,(Brandt(ME,(Isaacs(RD,(Thompson(PA,(Beutler(B.(Lipoproteins(of(Borrelia(burgdorferi(and(Treponema(pallidum(activate(cachectin/tumor(necrosis(factor(synthesis.(Analysis(using(a(CAT(reporter(construct.(Journal(of(immunology((Baltimore,(Md(:(1950).(1991(Sep(15b147(6):1968I74.(

246.( Cox(DL,(Akins(DR,(Bourell(KW,(Lahdenne(P,(Norgard(MV,(Radolf(JD.(Limited(surface(exposure(of(Borrelia(burgdorferi(outer(surface(lipoproteins.(Proc(Natl(Acad(Sci(U(S(A.(1996(Jul(23b93(15):7973I8.(

247.( Sellati(TJ,(Bouis(DA,(Kitchens(RL,(Darveau(RP,(Pugin(J,(Ulevitch(RJ,(et(al.(Treponema(pallidum(and(Borrelia(burgdorferi(lipoproteins(and(synthetic(lipopeptides(activate(monocytic(cells(via(a(CD14Idependent(pathway(distinct(from(that(used(by(lipopolysaccharide.(Journal(of(immunology((Baltimore,(Md(:(1950).(1998(Jun(1b160(11):5455I64.(

248.( Murray(GL,(Lo(M,(Bulach(DM,(Srikram(A,(Seemann(T,(Quinsey(NS,(et(al.(Evaluation(of(238(antigens(of(Leptospira(borgpetersenii(serovar(Hardjo(for(protection(against(kidney(colonisation.(Vaccine.(2013(Jan(7b31(3):495I9.(

249.( Nally(JE,(Chow(E,(Fishbein(MC,(Blanco(DR,(Lovett(MA.(Changes(in(lipopolysaccharide(O(antigen(distinguish(acute(versus(chronic(Leptospira(interrogans(infections.(Infection(and(immunity.(2005(Junb73(6):3251I60.(

70(

250.( Alexopoulou(L,(Holt(AC,(Medzhitov(R,(Flavell(RA.(Recognition(of(doubleIstranded(RNA(and(activation(of(NFIkappaB(by(TollIlike(receptor(3.(Nature.(2001(Oct(18b413(6857):732I8.(

251.( Doyle(S,(Vaidya(S,(O'Connell(R,(Dadgostar(H,(Dempsey(P,(Wu(T,(et(al.(IRF3(mediates(a(TLR3/TLR4Ispecific(antiviral(gene(program.(Immunity.(2002(Sepb17(3):251I63.(

252.( Wietek(C,(Miggin(SM,(Jefferies(CA,(O'Neill(LA.(Interferon(regulatory(factorI3Imediated(activation(of(the(interferonIsensitive(response(element(by(TollIlike(receptor((TLR)(4(but(not(TLR3(requires(the(p65(subunit(of(NFIkappa.(J(Biol(Chem.(2003(Dec(19b278(51):50923I31.(

253.( Yamamoto(M,(Sato(S,(Hemmi(H,(Hoshino(K,(Kaisho(T,(Sanjo(H,(et(al.(Role(of(adaptor(TRIF(in(the(MyD88Iindependent(tollIlike(receptor(signaling(pathway.(Science.(2003(Aug(1b301(5633):640I3.(

254.( Derbigny(WA,(Johnson(RM,(Toomey(KS,(Ofner(S,(Jayarapu(K.(The(Chlamydia(muridarumIinduced(IFNIbeta(response(is(TLR3Idependent(in(murine(oviduct(epithelial(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2010(Dec(1b185(11):6689I97.(

255.( Derbigny(WA,(Shobe(LR,(Kamran(JC,(Toomey(KS,(Ofner(S.(Identifying(a(role(for(TollIlike(receptor(3(in(the(innate(immune(response(to(Chlamydia(muridarum(infection(in(murine(oviduct(epithelial(cells.(Infection(and(immunity.(2012(Janb80(1):254I65.(

256.( Cervantes(JL,(La(Vake(CJ,(Weinerman(B,(Luu(S,(O'Connell(C,(Verardi(PH,(et(al.(Human(TLR8(is(activated(upon(recognition(of(Borrelia(burgdorferi(RNA(in(the(phagosome(of(human(monocytes.(J(Leukoc(Biol.(2013(Decb94(6):1231I41.(

257.( Fieber(C,(Janos(M,(Koestler(T,(Gratz(N,(Li(XD,(Castiglia(V,(et(al.(Innate(immune(response(to(Streptococcus(pyogenes(depends(on(the(combined(activation(of(TLR13(and(TLR2.(PloS(one.(2015b10(3):e0119727.(

258.( Oldenburg(M,(Kruger(A,(Ferstl(R,(Kaufmann(A,(Nees(G,(Sigmund(A,(et(al.(TLR13(recognizes(bacterial(23S(rRNA(devoid(of(erythromycin(resistanceIforming(modification.(Science.(2012(Aug(31b337(6098):1111I5.(

259.( Signorino(G,(Mohammadi(N,(Patane(F,(Buscetta(M,(Venza(M,(Venza(I,(et(al.(Role(of(TollIlike(receptor(13(in(innate(immune(recognition(of(group(B(streptococci.(Infection(and(immunity.(2014(Decb82(12):5013I22.(

71(

260.( Eigenbrod(T,(Franchi(L,(MunozIPlanillo(R,(Kirschning(CJ,(Freudenberg(MA,(Nunez(G,(et(al.(Bacterial(RNA(mediates(activation(of(caspaseI1(and(ILI1beta(release(independently(of(TLRs(3,(7,(9(and(TRIF(but(is(dependent(on(UNC93B.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Jul(1b189(1):328I36.(

261.( Hemmi(H,(Takeuchi(O,(Kawai(T,(Kaisho(T,(Sato(S,(Sanjo(H,(et(al.(A(TollIlike(receptor(recognizes(bacterial(DNA.(Nature.(2000(Dec(7b408(6813):740I5.(

262.( Gewirtz(AT,(Navas(TA,(Lyons(S,(Godowski(PJ,(Madara(JL.(Cutting(edge:(bacterial(flagellin(activates(basolaterally(expressed(TLR5(to(induce(epithelial(proinflammatory(gene(expression.(Journal(of(immunology((Baltimore,(Md(:(1950).(2001(Aug(15b167(4):1882I5.(

263.( Hayashi(F,(Smith(KD,(Ozinsky(A,(Hawn(TR,(Yi(EC,(Goodlett(DR,(et(al.(The(innate(immune(response(to(bacterial(flagellin(is(mediated(by(TollIlike(receptor(5.(Nature.(2001(Apr(26b410(6832):1099I103.(

264.( Morris(AE,(Liggitt(HD,(Hawn(TR,(Skerrett(SJ.(Role(of(TollIlike(receptor(5(in(the(innate(immune(response(to(acute(P.(aeruginosa(pneumonia.(Am(J(Physiol(Lung(Cell(Mol(Physiol.(2009(Decb297(6):L1112I9.(

265.( Chuang(T,(Ulevitch(RJ.(Identification(of(hTLR10:(a(novel(human(TollIlike(receptor(preferentially(expressed(in(immune(cells.(Biochim(Biophys(Acta.(2001(Mar(19b1518(1I2):157I61.(

266.( Hasan(U,(Chaffois(C,(Gaillard(C,(Saulnier(V,(Merck(E,(Tancredi(S,(et(al.(Human(TLR10(is(a(functional(receptor,(expressed(by(B(cells(and(plasmacytoid(dendritic(cells,(which(activates(gene(transcription(through(MyD88.(Journal(of(immunology((Baltimore,(Md(:(1950).(2005(Mar(1b174(5):2942I50.(

267.( Govindaraj(RG,(Manavalan(B,(Lee(G,(Choi(S.(Molecular(modelingIbased(evaluation(of(hTLR10(and(identification(of(potential(ligands(in(TollIlike(receptor(signaling.(PloS(one.(2010b5(9):e12713.(

268.( Guan(Y,(Ranoa(DR,(Jiang(S,(Mutha(SK,(Li(X,(Baudry(J,(et(al.(Human(TLRs(10(and(1(share(common(mechanisms(of(innate(immune(sensing(but(not(signaling.(Journal(of(immunology((Baltimore,(Md(:(1950).(2010(May(1b184(9):5094I103.(

269.( Kim(D,(Kim(YJ,(Koh(HS,(Jang(TY,(Park(HE,(Kim(JY.(Reactive(oxygen(species(enhance(TLR10(expression(in(the(human(monocytic(cell(line(THPI1.(Int(J(Mol(Sci.(2010b11(10):3769I82.(

270.( Regan(T,(Nally(K,(Carmody(R,(Houston(A,(Shanahan(F,(Macsharry(J,(et(al.(Identification(of(TLR10(as(a(key(mediator(of(the(inflammatory(response(to(Listeria(

72(

monocytogenes(in(intestinal(epithelial(cells(and(macrophages.(Journal(of(immunology((Baltimore,(Md(:(1950).(2013(Dec(15b191(12):6084I92.(

271.( Lee(SM,(Kok(KH,(Jaume(M,(Cheung(TK,(Yip(TF,(Lai(JC,(et(al.(TollIlike(receptor(10(is(involved(in(induction(of(innate(immune(responses(to(influenza(virus(infection.(Proc(Natl(Acad(Sci(U(S(A.(2014(Mar(11b111(10):3793I8.(

272.( Oosting(M,(Cheng(SC,(Bolscher(JM,(VesteringIStenger(R,(Plantinga(TS,(Verschueren(IC,(et(al.(Human(TLR10(is(an(antiIinflammatory(patternIrecognition(receptor.(Proc(Natl(Acad(Sci(U(S(A.(2014(Oct(21b111(42):E4478I84.(

273.( Gay(NJ,(Symmons(MF,(Gangloff(M,(Bryant(CE.(Assembly(and(localization(of(TollIlike(receptor(signalling(complexes.(Nature(reviews(Immunology.(2014(Augb14(8):546I58.(

274.( Verstak(B,(Arnot(CJ,(Gay(NJ.(An(alanineItoIproline(mutation(in(the(BBIloop(of(TLR3(Toll/ILI1R(domain(switches(signalling(adaptor(specificity(from(TRIF(to(MyD88.(Journal(of(immunology((Baltimore,(Md(:(1950).(2013(Dec(15b191(12):6101I9.(

275.( Dinarello(CA.(InterleukinI1(in(the(pathogenesis(and(treatment(of(inflammatory(diseases.(Blood.(2011(Apr(7b117(14):3720I32.(

276.( Cohen(P.(The(TLR(and(ILI1(signalling(network(at(a(glance.(J(Cell(Sci.(2014(Jun(1b127(Pt(11):2383I90.(

277.( Garlanda(C,(Dinarello(CA,(Mantovani(A.(The(interleukinI1(family:(back(to(the(future.(Immunity.(2013(Dec(12b39(6):1003I18.(

278.( Choi(YJ,(Im(E,(Chung(HK,(Pothoulakis(C,(Rhee(SH.(TRIF(mediates(TollIlike(receptor(5Iinduced(signaling(in(intestinal(epithelial(cells.(J(Biol(Chem.(2010(Nov(26b285(48):37570I8.(

279.( Kolb(JP,(Casella(CR,(SenGupta(S,(Chilton(PM,(Mitchell(TC.(Type(I(interferon(signaling(contributes(to(the(bias(that(TollIlike(receptor(4(exhibits(for(signaling(mediated(by(the(adaptor(protein(TRIF.(Sci(Signal.(2014b7(351):ra108.(

280.( Rathinam(VA,(Vanaja(SK,(Waggoner(L,(Sokolovska(A,(Becker(C,(Stuart(LM,(et(al.(TRIF(licenses(caspaseI11Idependent(NLRP3(inflammasome(activation(by(gramInegative(bacteria.(Cell.(2012(Aug(3b150(3):606I19.(

281.( Casson(CN,(Copenhaver(AM,(Zwack(EE,(Nguyen(HT,(Strowig(T,(Javdan(B,(et(al.(CaspaseI11(activation(in(response(to(bacterial(secretion(systems(that(access(the(host(cytosol.(PLoS(pathogens.(2013b9(6):e1003400.(

73(

282.( Gunther(C,(Buchen(B,(He(GW,(Hornef(M,(Torow(N,(Neumann(H,(et(al.(CaspaseI8(controls(the(gut(response(to(microbial(challenges(by(TnfIalphaIdependent(and(independent(pathways.(Gut.(2015(Aprb64(4):601I10.(

283.( Gurung(P,(Malireddi(RK,(Anand(PK,(Demon(D,(Vande(Walle(L,(Liu(Z,(et(al.(Toll(or(interleukinI1(receptor((TIR)(domainIcontaining(adaptor(inducing(interferonIbeta((TRIF)Imediated(caspaseI11(protease(production(integrates(TollIlike(receptor(4((TLR4)(proteinI(and(Nlrp3(inflammasomeImediated(host(defense(against(enteropathogens.(J(Biol(Chem.(2012(Oct(5b287(41):34474I83.(

284.( Jabir(MS,(Ritchie(ND,(Li(D,(Bayes(HK,(Tourlomousis(P,(Puleston(D,(et(al.(CaspaseI1(cleavage(of(the(TLR(adaptor(TRIF(inhibits(autophagy(and(betaIinterferon(production(during(Pseudomonas(aeruginosa(infection.(Cell(host(&(microbe.(2014(Feb(12b15(2):214I27.(

285.( Bowen(WS,(Minns(LA,(Johnson(DA,(Mitchell(TC,(Hutton(MM,(Evans(JT.(Selective(TRIFIdependent(signaling(by(a(synthetic(tollIlike(receptor(4(agonist.(Sci(Signal.(2012(Feb(14b5(211):ra13.(

286.( Shalova(IN,(Kajiji(T,(Lim(JY,(GomezIPina(V,(FernandezIRuiz(I,(Arnalich(F,(et(al.(CD16(regulates(TRIFIdependent(TLR4(response(in(human(monocytes(and(their(subsets.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Apr(15b188(8):3584I93.(

287.( TeixeiraICoelho(M,(Guedes(J,(Ferreirinha(P,(Howes(A,(Pedrosa(J,(Rodrigues(F,(et(al.(Differential(postItranscriptional(regulation(of(ILI10(by(TLR2(and(TLR4Iactivated(macrophages.(Eur(J(Immunol.(2014(Marb44(3):856I66.(

288.( Rathinam(VA,(Appledorn(DM,(Hoag(KA,(Amalfitano(A,(Mansfield(LS.(Campylobacter(jejuniIinduced(activation(of(dendritic(cells(involves(cooperative(signaling(through(TollIlike(receptor(4((TLR4)IMyD88(and(TLR4ITRIF(axes.(Infection(and(immunity.(2009(Junb77(6):2499I507.(

289.( Gaddis(DE,(Michalek(SM,(Katz(J.(TLR4(signaling(via(MyD88(and(TRIF(differentially(shape(the(CD4+(T(cell(response(to(Porphyromonas(gingivalis(hemagglutinin(B.(Journal(of(immunology((Baltimore,(Md(:(1950).(2011(May(15b186(10):5772I83.(

290.( Nagarajan(UM,(Sikes(J,(Prantner(D,(Andrews(CW,(Jr.,(Frazer(L,(Goodwin(A,(et(al.(MyD88(deficiency(leads(to(decreased(NK(cell(gamma(interferon(production(and(T(cell(recruitment(during(Chlamydia(muridarum(genital(tract(infection,(but(a(predominant(Th1(response(and(enhanced(monocytic(inflammation(are(associated(with(infection(resolution.(Infection(and(immunity.(2011(Janb79(1):486I98.(

74(

291.( Yanagibashi(T,(Nagai(Y,(Watanabe(Y,(Ikutani(M,(Hirai(Y,(Takatsu(K.(Differential(requirements(of(MyD88(and(TRIF(pathways(in(TLR4Imediated(immune(responses(in(murine(B(cells.(Immunol(Lett.(2015(Janb163(1):22I31.(

292.( Kolanowski(ST,(Dieker(MC,(LissenbergIThunnissen(SN,(van(Schijndel(GM,(van(Ham(SM,(ten(Brinke(A.(TLR4Imediated(proIinflammatory(dendritic(cell(differentiation(in(humans(requires(the(combined(action(of(MyD88(and(TRIF.(Innate(Immun.(2014(Mayb20(4):423I30.(

293.( Janssen(E,(Ozcan(E,(Liadaki(K,(Jabara(HH,(Manis(J,(Ullas(S,(et(al.(TRIF(signaling(is(essential(for(TLR4Idriven(IgE(class(switching.(Journal(of(immunology((Baltimore,(Md(:(1950).(2014(Mar(15b192(6):2651I8.(

294.( Matsushita(K,(Yoshimoto(T.(B(cellIintrinsic(MyD88(signaling(is(essential(for(IgE(responses(in(lungs(exposed(to(pollen(allergens.(Journal(of(immunology((Baltimore,(Md(:(1950).(2014(Dec(15b193(12):5791I800.(

295.( Bolz(DD,(Sundsbak(RS,(Ma(Y,(Akira(S,(Kirschning(CJ,(Zachary(JF,(et(al.(MyD88(plays(a(unique(role(in(host(defense(but(not(arthritis(development(in(Lyme(disease.(Journal(of(immunology((Baltimore,(Md(:(1950).(2004(Aug(1b173(3):2003I10.(

296.( Liu(N,(Montgomery(RR,(Barthold(SW,(Bockenstedt(LK.(Myeloid(differentiation(antigen(88(deficiency(impairs(pathogen(clearance(but(does(not(alter(inflammation(in(Borrelia(burgdorferiIinfected(mice.(Infection(and(immunity.(2004(Junb72(6):3195I203.(

297.( Behera(AK,(Hildebrand(E,(Bronson(RT,(Perides(G,(Uematsu(S,(Akira(S,(et(al.(MyD88(deficiency(results(in(tissueIspecific(changes(in(cytokine(induction(and(inflammation(in(interleukinI18Iindependent(mice(infected(with(Borrelia(burgdorferi.(Infection(and(immunity.(2006(Marb74(3):1462I70.(

298.( Kim(TH,(Shin(SJ,(Park(YM,(Jung(ID,(Ryu(SW,(Kim(DJ,(et(al.(Critical(role(of(TRIF(and(MyD88(in(Mycobacterium(tuberculosis(Hsp70Imediated(activation(of(dendritic(cells.(Cytokine.(2015(Febb71(2):139I44.(

299.( ShaikIDasthagirisaheb(YB,(Huang(N,(Weinberg(EO,(Shen(SS,(Genco(CA,(Gibson(FC,(3rd.(Aging(and(contribution(of(MyD88(and(TRIF(to(expression(of(TLR(pathwayIassociated(genes(following(stimulation(with(Porphyromonas(gingivalis.(J(Periodontal(Res.(2015(Febb50(1):89I102.(

300.( Suet(Ting(Tan(R,(Lin(B,(Liu(Q,(TuckerIKellogg(L,(Ho(B,(Leung(BP,(et(al.(The(synergy(in(cytokine(production(through(MyD88ITRIF(pathways(is(coIordinated(with(ERK(phosphorylation(in(macrophages.(Immunol(Cell(Biol.(2013(Mayb91(5):377I87.(

75(

301.( Orr(MT,(Duthie(MS,(Windish(HP,(Lucas(EA,(Guderian(JA,(Hudson(TE,(et(al.(MyD88(and(TRIF(synergistic(interaction(is(required(for(TH1Icell(polarization(with(a(synthetic(TLR4(agonist(adjuvant.(Eur(J(Immunol.(2013(Sepb43(9):2398I408.(

302.( Trinchieri(G.(Type(I(interferon:(friend(or(foe?(J(Exp(Med.(2010(Sep(27b207(10):2053I63.(

303.( Nguyen(KB,(Cousens(LP,(Doughty(LA,(Pien(GC,(Durbin(JE,(Biron(CA.(Interferon(alpha/betaImediated(inhibition(and(promotion(of(interferon(gamma:(STAT1(resolves(a(paradox.(Nature(immunology.(2000(Julb1(1):70I6.(

304.( Braun(D,(Caramalho(I,(Demengeot(J.(IFNIalpha/beta(enhances(BCRIdependent(B(cell(responses.(Int(Immunol.(2002(Aprb14(4):411I9.(

305.( Monroe(KM,(McWhirter(SM,(Vance(RE.(Induction(of(type(I(interferons(by(bacteria.(Cell(Microbiol.(2010(Julb12(7):881I90.(

306.( Asano(M,(Hayashi(M,(Yoshida(E,(Kawade(Y,(Iwakura(Y.(Induction(of(interferonIalpha(by(interferonIbeta,(but(not(of(interferonIbeta(by(interferonIalpha,(in(the(mouse.(Virology.(1990(Mayb176(1):30I8.(

307.( Zuerner(RL.(Host(response(to(leptospira(infection.(Curr(Top(Microbiol(Immunol.(2015b387:223I50.(

308.( Brown(CR,(Reiner(SL.(Experimental(lyme(arthritis(in(the(absence(of(interleukinI4(or(gamma(interferon.(Infection(and(immunity.(1999(Julb67(7):3329I33.(

309.( Nguyen(KB,(Watford(WT,(Salomon(R,(Hofmann(SR,(Pien(GC,(Morinobu(A,(et(al.(Critical(role(for(STAT4(activation(by(type(1(interferons(in(the(interferonIgamma(response(to(viral(infection.(Science.(2002(Sep(20b297(5589):2063I6.(

310.( Rothfuchs(AG,(Trumstedt(C,(Mattei(F,(Schiavoni(G,(Hidmark(A,(Wigzell(H,(et(al.(STAT1(regulates(IFNIalpha(betaI(and(IFNIgammaIdependent(control(of(infection(with(Chlamydia(pneumoniae(by(nonhemopoietic(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2006(Jun(1b176(11):6982I90.(

311.( Vincent(MS,(Roessner(K,(Sellati(T,(Huston(CD,(Sigal(LH,(Behar(SM,(et(al.(Lyme(arthritis(synovial(gamma(delta(T(cells(respond(to(Borrelia(burgdorferi(lipoproteins(and(lipidated(hexapeptides.(Journal(of(immunology((Baltimore,(Md(:(1950).(1998(Nov(15b161(10):5762I71.(

312.( Tupin(E,(Benhnia(MR,(Kinjo(Y,(Patsey(R,(Lena(CJ,(Haller(MC,(et(al.(NKT(cells(prevent(chronic(joint(inflammation(after(infection(with(Borrelia(burgdorferi.(Proc(Natl(Acad(Sci(U(S(A.(2008(Dec(16b105(50):19863I8.(

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313.( Egli(A,(Santer(DM,(O'Shea(D,(Tyrrell(DL,(Houghton(M.(The(impact(of(the(interferonIlambda(family(on(the(innate(and(adaptive(immune(response(to(viral(infections.(Emerg(Microbes(Infect.(2014(Julb3(7):e51.(

314.( Odendall(C,(Dixit(E,(Stavru(F,(Bierne(H,(Franz(KM,(Durbin(AF,(et(al.(Diverse(intracellular(pathogens(activate(type(III(interferon(expression(from(peroxisomes.(Nature(immunology.(2014(Augb15(8):717I26.(

315.( Wen(H,(Miao(EA,(Ting(JP.(Mechanisms(of(NODIlike(receptorIassociated(inflammasome(activation.(Immunity.(2013(Sep(19b39(3):432I41.(

316.( Liu(D,(Rhebergen(AM,(Eisenbarth(SC.(Licensing(Adaptive(Immunity(by(NODILike(Receptors.(Front(Immunol.(2013b4:486.(

317.( PetnickiIOcwieja(T,(DeFrancesco(AS,(Chung(E,(Darcy(CT,(Bronson(RT,(Kobayashi(KS,(et(al.(Nod2(suppresses(Borrelia(burgdorferi(mediated(murine(Lyme(arthritis(and(carditis(through(the(induction(of(tolerance.(PloS(one.(2011b6(2):e17414.(

318.( Oosting(M,(Berende(A,(Sturm(P,(Ter(Hofstede(HJ,(de(Jong(DJ,(Kanneganti(TD,(et(al.(Recognition(of(Borrelia(burgdorferi(by(NOD2(is(central(for(the(induction(of(an(inflammatory(reaction.(The(Journal(of(infectious(diseases.(2010(Jun(15b201(12):1849I58.(

319.( Berke(IC,(Li(Y,(Modis(Y.(Structural(basis(of(innate(immune(recognition(of(viral(RNA.(Cell(Microbiol.(2013(Marb15(3):386I94.(

320.( Kawai(T,(Akira(S.(TollIlike(receptor(and(RIGIIIlike(receptor(signaling.(Ann(N(Y(Acad(Sci.(2008(Novb1143:1I20.(

321.( Loo(YM,(Gale(M,(Jr.(Immune(signaling(by(RIGIIIlike(receptors.(Immunity.(2011(May(27b34(5):680I92.(

322.( Reikine(S,(Nguyen(JB,(Modis(Y.(Pattern(Recognition(and(Signaling(Mechanisms(of(RIGII(and(MDA5.(Front(Immunol.(2014b5:342.(

323.( Cervantes(JL,(DunhamIEms(SM,(La(Vake(CJ,(Petzke(MM,(Sahay(B,(Sellati(TJ,(et(al.(Phagosomal(signaling(by(Borrelia(burgdorferi(in(human(monocytes(involves(TollIlike(receptor((TLR)(2(and(TLR8(cooperativity(and(TLR8Imediated(induction(of(IFNIbeta.(Proc(Natl(Acad(Sci(U(S(A.(2011(Mar(1b108(9):3683I8.(

324.( Zelensky(AN,(Gready(JE.(The(CItype(lectinIlike(domain(superfamily.(FEBS(J.(2005(Decb272(24):6179I217.(

77(

325.( Hovius(JW,(de(Jong(MA,(den(Dunnen(J,(Litjens(M,(Fikrig(E,(van(der(Poll(T,(et(al.(Salp15(binding(to(DCISIGN(inhibits(cytokine(expression(by(impairing(both(nucleosome(remodeling(and(mRNA(stabilization.(PLoS(pathogens.(2008(Feb(8b4(2):e31.(

326.( Salazar(JC,(Pope(CD,(Moore(MW,(Pope(J,(Kiely(TG,(Radolf(JD.(LipoproteinIdependent(and(Iindependent(immune(responses(to(spirochetal(infection.(Clin(Diagn(Lab(Immunol.(2005(Augb12(8):949I58.(

327.( Diament(D,(Brunialti(MK,(Romero(EC,(Kallas(EG,(Salomao(R.(Peripheral(blood(mononuclear(cell(activation(induced(by(Leptospira(interrogans(glycolipoprotein.(Infection(and(immunity.(2002(Aprb70(4):1677I83.(

328.( Berende(A,(Oosting(M,(Kullberg(BJ,(Netea(MG,(Joosten(LA.(Activation(of(innate(host(defense(mechanisms(by(Borrelia.(Eur(Cytokine(Netw.(2010(Marb21(1):7I18.(

329.( Oosting(M,(Buffen(K,(Cheng(SC,(Verschueren(IC,(Koentgen(F,(van(de(Veerdonk(FL,(et(al.(BorreliaIinduced(cytokine(production(is(mediated(by(spleen(tyrosine(kinase((Syk)(but(is(DectinI1(and(DectinI2(independent.(Cytokine.(2015(Aug(18.(

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78(

Chapter!2!

Title:!!The(adaptor(molecule(Trif(contributes(to(murine(host(defense(during(

Leptospiral(infection!

Priya(A.(Jayaraman*a,(Amy(A.(Devlina,(Jennifer(C.(Millera(and(Frank(Schollea(

!

aDepartment(of(Biological(Sciences,(North(Carolina(State(University,(Raleigh,(North(

Carolina,(United(States(of(America((

*Corresponding(Author(

EImail:(pajayara@ncsu.edu((

Present(address:(3510(Thomas(Hall,(112(Derieux(Place,(Raleigh(NC(27610(

(

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This!chapter!will!be!submitted!as!a!manuscript!to!Immunobiology!(©ElSevier)!

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79(

Abstract(

Leptospirosis(is(a(zoonotic(disease(and(is(caused(by(pathogenic(species(of(the(

Leptospira(genus,(including(Leptospira*interrogans*(L.*interrogans).(Humans,(

domestic(and(wild(animals(are(susceptible(to(acute(or(chronic(infection.(The(innate(

immune(response(is(a(critical(defense(mechanism(against(Leptospira(interrogans,(

and(has(been(investigated(in(mouse(models.(Murine(TollIlike(receptors((TLRs)(have(

been(shown(to(be(key(factors(in(sensing(and(responding(to(L.*interrogans*infection.(

Specifically,(TLR2,(TLR4(and(the(TLR(adaptor(molecule(MyD88(are(essential(for(

host(defense(against(L.*interrogansS*however,(the(role(of(the(TLR(adaptor(molecule(

TIRIdomainIcontaining(adaptorIinducing(interferon(β((TRIF)(in(the(response(to(L.*

interrogans(has(not(been(previously(determined.(In(the(present(study,(TRIF(was(

found(to(play(an(important(role(during(leptospiral(infection.((Following(challenge(with(

L.*interrogans,(Trif5/5(mice(exhibited(delayed(weight(gain(compared(to(wildItype(mice.((

Moreover,(TrifI/I(mice(exhibited(an(increase(in(L.*interrogans*burden(in(the(kidneys,(

lungs,(and(blood(at(early(time(points((less(than(7(days(post(infection).(((Multiple(

components(of(the(innate(immune(responses(were(dampened(in(response(to(

leptospiral(infection(including(transcription(and(production(of(cytokines,(and(the(

humoral(response,(which(suggested(that(TRIF(contributes(to(expression(and(

production(of(cytokines(important(for(the(host(defense(against(L.*interrogans.((

(

Keywords:!Leptospira*interrogans,(leptospirosis,(Innate(immunity,(TRIF(!

80(

Introduction!

Leptospira(interrogans*(L.*interrogans)(is(a(pathogenic(bacterium(that(causes(

the(zoonotic(disease(leptospirosis*(1I5).(Some(rodents,(such(as(rats(can(act(as(

reservoir(hosts(via(asymptomatic(carriage(of(bacteria((6,(7)(and(have(been(

established(as(vectors(for(transmission.((Humans,(as(well(as(both(domestic(and(wild(

animals,(are(deadIend(hosts,(and(are(susceptible(to(acute(or(chronic(infection((4).((

The(bacteria(are(transmitted(via(contaminated(urine(through(broken(skin,(eyes,(and(

mucous(membranes(and(can(cause(acute(infection((4,(8,(9).((Symptoms(include(

febrile(illness,(headaches,(and(myalgia((5,(9I11),(but(in(many(cases,(symptoms(of(

acute(infection(extend(to(pulmonary(hemorrhaging,(renal(failure,(liver(failure(and(

death((4,(9,(12).(Chronic(infection(results(from(colonization(of(the(kidneys(by(

leptospires,(and(failure(to(treat(infection(can(result(in(chronic(renal(fibrosis((13I15).((

Acute(and(chronic(phases(of(leptospirosis(have(both(been(extensively(studied(

in(several(animal(models((8,(16I18).(Guinea(pigs,(hamsters,(and(certain(strains(of(

inbred(mice(die(rapidly(following(injection(with(some(pathogenic(Leptospira*spp.((8,(

19I21).((Rats(and(other(inbred(mouse(strains(are(asymptomatic(carriers,(exhibiting(

persistent(colonization(of(the(kidneys(with(leptospires((6,(7,(17,(20).((Although(

rodents(are(a(natural(reservoir(for(leptospiral(infection,(select(strains(of(inbred(mice(

show(different(outcomes(of(infection(and(thus(have(been(used(to(elucidate(

mechanisms(of(leptospiral(pathogenesis(and(host(response((20I24).((Balb/c(mice(

are(asymptomatic(carriers,(but(C3H/HeJ(mice(die(rapidly(following(intraperitoneal(

81(

injection(with(Leptospira((20,(25,(26).((C57BL/6(mice(show(moderate(symptoms(

during(acute(infection(and(develop(chronic(fibrosis(after(infection(by(pathogenic(

Leptospira*spp..(Knockout(mice(with(the(C57BL/6(background(have(been(used(to(

assess(mechanisms(of(pathogenesis(during(acute(and(chronic(infection((14,(22,(23,(

27,(28).((Therefore,(inbred(mice(are(valuable(tools(for(studying(both(Leptospiral(

pathogenesis(and(the(associated(host(response(to(infection,(since(different(mouse(

strains(can(be(utilized(to(model(a(wide(spectrum(of(disease(severity.((

One(part(of(the(innate(immune(response(is(the(recognition(of(components(

known(as(pathogen(associated(molecular(patterns((PAMPs)(by(pattern(recognition(

receptors((PRR’s)((29,(30).((The(PAMPIPRR(interaction(initiates(a(signaling(cascade(

that(leads(to(the(production(of(cytokines(and(chemokines((30).((TollIlike(receptors(

(TLRs)(are(a(subset(of(PRRs(that(are(involved(in(the(innate(immune(response.((

Upon(ligand(binding,(TLRs(recruit(adaptor(molecules(MyD88(or(TIRIdomainI

containing(adaptorIinducing(interferon(β((TRIF)((30),(depending(on(the(specific(TLR(

involved.((MyD88(is(an(adaptor(molecule(involved(in(signaling(downstream(of(

multiple(TLRs(leading(to(Type(I(IFN(induction(by(TLR(7/8/9(or(proIinflammatory(

cytokine(induction(by(TLR2/4((29).((TRIF(is(also(an(adaptor(molecule(that(is(involved(

in(downstream(signaling(of(TLR3,(TLR2,(or(TLR4((29,(30).(TLR4(uses(both(MyD88(

and(TRIF,(with(signaling(via(MyD88(leading(to(expression(of(inflammatory(genes,(

and(signaling(via(TRIF(leading(to(the(induction(of(Type(I(IFN(and(interferon(

stimulated(genes((ISG),(respectively((30).(ProIinflammatory(cytokines,(like(TNFα(

82(

and(ILI6(often(appear(during(the(acute(phase(of(infection((31).((TNFα(levels(are(

elevated(in(patients(showing(symptoms(of(severe(leptospirosis(and(in(the(blood(of(

C3H/HeJ(mice(at(48(hours(post(infection((32I34).!(Also,(while(C57BL/6ITNFIreceptor(

deficient(mice(survived(infection(by(Leptospira*interrogans,*histopathological(

analysis(of(kidney(lesions(indicated(that(interstitial(nephritis(was(present((22).((ILI6(

has(been(detected(in(kidneys(at(elevated(levels(during(acute(infection((21,(35).(

Elevated(ILI6(levels(in(patient(sera(have(also(been(associated(with(increased(

disease(severity((36).(Lower(levels(of(IFNγ(and(ILI12(have(been(reported(during(

chronic(infection(in(mice((28).(

(Chassin(et(al.(showed(that(TLR4I/I(mice,(TLR2I/I/4I/I(mice,(and(MyD88I/I(mice(

die(after(injection(with(L.*interrogans*(26,(27).((In(addition(to(this,(TLR2/4(dependent(

induction(of(IFNγ(and(antiILeptospira(IgM(were(essential(for(bacterial(clearance((27).((

Several(studies(suggest(TRIF(is(a(key(player(in(the(immune(response(to(bacterial(

pathogens,(including(spirochetes((37I43)b(however,(the(role(of(TRIF(in(the(host(

defense(against(L.*interrogans(specifically(has(not(yet(been(elucidated.((In(this(

study,(the(pathogenesis(of(L.*interrogans*was(investigated(in(wildtype((WT)(and(Trif5/5(

mice(by(measurement(of(specific(markers(of(host(defense(including,(symptoms(after(

infection,(cytokine(production,(innate(and(adaptive(immune(responses(and(bacterial(

burden.((

(

!

83(

Materials!and!Methods(

!

Bacteria:!

Leptospira*interrogans(serovar(Copenhageni(strain(Fiocruz(L1I130(bacteria(were(

kindly(provided(by(Dr.(David(Haake(at(the(University(of(California,(Los(Angeles.((

EMJH(medium(for(bacterial(growth(was(prepared(as(previously(described((44),(and(

leptospires(were(grown(in(semiIsolid(and(liquid(EMJH(medium,(as(previously(

described((44).((The(spirochetes(were(passaged(no(more(than(4(times(in(liquid(

EMJH(before(use(in(the(in*vivo(mouse(experiments.((Leptospires(were(grown(to(

stationary(phase((5x108(per(mL),(and(counted(using(a(PetroffIHauser(counter(prior(

to(diluting(bacteria(to(4x108(per(mL(in(EMJH(for(intraIperitoneal(injections.(

(

!

Mice!and!in#vivo!experiments!

All(procedures(were(followed(according(to(the(IACUC(regulations(at(North(Carolina(

State(University((#(12I021B).((FourItoIfive(week(old(C57BL/6((WT)(mice(were(

purchased(from(Jackson(Laboratory((Bar(Harbor,(ME)(and(maintained(in(the(

Biological(Resources(Facility(at(North(Carolina(State(University.((WT(mice(were(

used(as(controls(for(all(mouse(experiments.(C57BL/6J5Ticam1Lps2/J((Trif5/5)(mice(

were(purchased(from(Jackson(Laboratory(and(colonies(were(continuously(

maintained(in(the(Biological(Resources(Facility(at(NCSU.((SixItoIeight(week(old(WT(

84(

and(Trif5/5(mice(were(injected(intraperitoneally((i.p.)(with(a(volume(of(0.5(mL(EMJH(

media(or(2x108!leptospires(in(EMJH(media.((Following(infection,(mice(were(weighed(

once(daily(and(euthanized(at(1,(3,(7,(or(14(days(post(infection((d.p.i.).((Mice(that(had(

lost(more(than(15%(of(their(body(weight(and(showed(signs(of(sickness(including(

hunched(posture,(ruffled(fur,(and(slow(movement,(were(euthanized.((Following(

euthanasia,(blood,(lungs,(and(kidneys(were(collected(and(flash(frozen(in(liquid(

nitrogen(prior(to(storage(at(I80°C(for(further(processing,(and(bladders(from(infected(

WT(and(Trif5/5(mice(at(1,(3,(7,(and(14(d.p.i.(were(cultured(in(tubes(containing(5mL(

EMJH(at(30°C,(and(examined(for(the(presence(of(leptospires(by(dark(field(

microscopy.(

(

Creatinine!and!Nitrate!in!serum!

Serum(Creatinine(levels(at(1(and(3(d.p.i.(were(measured(using(an(assay(kit(from(

Cayman(Chem(following(the(manufacturers(instructions.(Serum(nitrate(levels(were(

measured(using(the(Greiss(assay((14)(and(readings(were(measured(at(an(OD(of(540(

nm.((

(

RNA!and!qRTVPCR!

Kidneys(or(lungs(were(homogenized(each(in(1mL(Qiazol((Qiagen,(Germantown,(

MD)(and(RNA(was(isolated(following(the(manufacturer’s(instructions.((RNA(was(

isolated(from(blood(after(processing(with(Trizol(LS((Invitrogen,(Carlsbad,(CA)(

85(

according(to(the(manufacturer’s(instructions.((RNA(was(quantified(using(a(Nanodrop(

1000(spectrophotometer((NanoDrop,(Wilmington,(DE)(and(stored(at(I80°C.((RT(

Buffer((5X)(and(MIMLV(Reverse(Transcriptase((Affymetrix,(Santa(Clara,(CA),(

Random(Primers((Promega,(Madison,(WI),((and(dNTPs((Fermentas,(Maryland,(NY)(

were(used(in(a(50uL(reaction(containing(up(to(5ug(RNA(and(incubated(at(37°C(for(1(

hour(and(stored(at(I20°C.((SYBR(Green(for(qRTIPCR(was(purchased(from(BioRad(

(Hercules,(CA)(and(qRTIPCR(reactions(were(run(on(96(well(plates(using(the(MyiQ2(

TwoIcolor(RealItime(PCR(Detection(System(and(iQ5(software((BioIRad).((To(

quantify(bacterial(burden,(16s(mRNA(copies(of(the(rRNA(subunit(were(calculated(

using(a(standard(curve(and(normalized(to(the(housekeeping(gene(βIactin.*mRNA(

copies(of(transcripts(were(normalized(to(the(housekeeping(gene(βIactin*as(

previously(described(and(fold(induction(was(calculated(using(the(ΔΔCT(method(as(

reported(previously((40,(45).(The(following(forward(and(reverse(primer(sequences(

have(been(previously(described(and(were(used(in(this(study:(βIactin,(IL51β,*Stat1*

(46),(Oasl2,(Cxcl10,(Ifnγ,*IL56,(IL512p40*(47),(Ifit1(and(Gbp2*(40).((Additional(forward(

and(reverse(primer(sequences*are(listed(below:**

L.*interrogans(16s(rRNA((48)(F:(5’IGGCGGCGCGTCTTAAACATGI3’b(R:(5’I

TTCCCCCCATTGAGCAAGATTI3’)b((

Tnfα((F:(5′IATGAGCACAGAAAGCATGATCI3′(,(R:(5′I

TACAGGCTTGTCACTCGAATTI3′),((

86(

Irf3((F:5'I(ACGCACAGATGGCTGACTTTG(I3',(R:(5'I(

CTTCGGTAGGTTTTCCTGGGAG(I3')(.((

IL518((F:(5’ICAAAGAAAGCCGCCTCAAACC(I3’,(R:(5’I

CAAAGTTGTCTGATTCCAGGTCTCC(I3’)(

!

Soluble!Cytokine!Extracts!

One(whole(kidney,(or(one(whole(lung,(was(homogenized(in(3mL(of(protein(lysis(

buffer((0.5%(Triton(XI100,(150(mM(NaCl,(15(mM(Tris,(1(mM(CaCl2(and(1(mM(

MgCl2,(pH(7.4)(containing(protease(inhibitors((Thermo(Fisher,(Grand(Island,(NY)(as(

described(previously((20,(27,(49).((Homogenates(were(clarified(by(centrifugation(for(

10(minutes(at(10,000(RPM(and(supernatants(were(stored(at(I80°C.((The(

supernatants(were(assayed(for(cytokines(by(ELISA.(

(

Cytokine!ELISA!

Cytokine(concentration(in(serum(samples(or(soluble(tissue(extracts(was(detected(by(

sandwich(ELISAs(for(ILI6,(ILI1β,(IFNγ,(or(TNFα(as(previously(described((40,(50).((

Mouse(capture(cytokine(antibodies(to(ILI6,(IL12p70,(ILI1β,(and(IFNγ(were(

purchased(from(Biolegend((San(Diego,(CA)(and(TNFα(mouse(capture(antibody(was(

purchased(from(BD(Pharmingen((San(Jose,(CA).((Recombinant(cytokines(ILI6(

(eBioscience,(San(Diego(CA),(ILI1β((Gemini(Biosciences,(West(Sacramento,(CA),(

IFNγ,(IL12p70((BD(Pharmingen),(and(TNFα((eBioscience)(were(used(as(standards.((

87(

Serial(dilutions(of(standards(and(serum(samples(or(soluble(cytokine(extracts(were(

added(to(respective(wells(followed(by(overnight(incubation(at(4°C(and(subsequent(

washes(in(PBSITween20((PBSIT).((Biotinylated(detecting(antibodies(to(mouse(ILI6,(

IL12p70,(ILI1β,(and(TNFα(were(purchased(from(eBioscience,(and(biotinylated(

detecting(antiImouseIIFNγ(and(HRPIAvidin(were(purchased(from(BioLegend.((The(

substrate(was(citrate(buffer(containing(oIPhenylenediamine((MP(Biomedicals,(Santa(

Ana,(CA)(and(hydrogen(peroxide!and(1N(HCl(was(added(as(a(stop(solution(after(

plates(developed(for(5I10(minutes.((A(plate(reader((BioTek,(Winooski,(VT)(and(

accompanying(software((Gen5(v1.0)(were(used(to(read(the(plates(at(OD490(and(

calculations(were(made(according(to(the(standard(curve.((

(

Leptospiral!sonicate!!

Leptospires(were(grown(to(late(log(phase((1I1.4(x(108(per(mL)(in(250(mL(batch(

cultures.(Bacteria(were(centrifuged(at(4000(RPM(for(ten(minutes,(washed(twice(with(

PBS(and(resuspended(in(6(mL(PBSI5mM(MgCl2(supplemented(with(protease(

inhibitors((ThermoFisher).((Bacteria(were(sonicated(for(20(minutes(using(alternating(

30(second(burst/rest(cycles.((The(resulting(slurry(was(centrifuged(at(4000(RPM(to(

pellet(cellular(debris,(and(the(supernatant(was(transferred(to(a(new(polycarbonate(

tube.((The(supernatant(was(sonicated(again(and(centrifuged(as(before.((Protein(

content(of(the(Leptospiral(sonicate(was(quantified(using(a(BCA(kit((Thermo(Fisher)(

88(

with(BSA((stock(concentration(2mg/ml)(as(standard(and(aliquots(of(sonicate(were(

stored(at(I80°C(until(used(for(ELISAs.(

(

AntiVLeptospiral!Ig!ELISA!

AntiILeptospiral(IgIELISAs(were(performed(as(previously(described((27,(51),(with(the(

following(modifications.((COSTAR(HighIbinding(plates(were(coated(with(leptospiral(

sonicate(at(a(concentration(of(1ug/ml((100ng(of(sonicate(per(well)(in(1xPBS,(except(

for(one(column(which(was(coated(with(mouse(antiIIg(capture(antibody((Millipore,(

Billerica,(MA).((Plates(were(incubated(overnight(at(4°C(and(blocked(the(following(day(

with(1%(BSA(in(PBS,(followed(by(a(second(incubation(overnight(at(4°C.((Plates(were(

shaken(to(remove(the(blocking(solution(and(washed(3(times(with(PBSIT(prior(to(

addition(of(standards(or(samples.((Recombinant(IgM(standard(and(recombinant(IgG(

standard(were(purchased(from(Millipore.((Plates(were(incubated(at(37°C(for(1(hour(

followed(by(5(PBSIT(washes.((Biotinylated(antiImouse(IgA,(Biotinylated(antiImouse(

IgG1,(HRPIAvidin(were(purchased(from(BioLegend(and(HRPIconjugated(antiImouse(

IgG2c(was(purchased(from(Millipore.(Detection(was(performed(as(described(above.((

Leptospiral(sonicate(specific(IgM(or(IgG(levels(were(then(quantified(by(reading(the(

plates(on(a(reader((BioITek(Synergy(HT)(at(an(OD(of(490nm(and(calculating(the(

standard(curve.(AntiIleptospiral(IgA,(IgG1(and(IgG2c(measurements(were(read(at(an(

OD(of(490(as(previously(described((26,(27).(

(

89(

Statistics!

Statistical(analyses(were(completed(using(Graphpad(Prism(6.0((Valencia,(CA).((The(

ShapiroIWilk(test(was(used(to(determine(whether(data(points(in(each(group(were(

normally(distributed.((If(one(or(more(groups(did(not(pass(the(test(for(normal(

distributions,(the(MannIWhitney(test(for(pairIwise(non(parametric(comparisons(was(

performed(and(the(KruskalIWallis(test(was(performed(for(nonIparametric(

comparisons(across(multiple(groups.(

(

Results!

Trif!contributes!to!murine!host!defense!against!L.#interrogans!infection!

The(role(of(TRIF(in(host(defense(and(bacterial(clearance(of(L.*interrogans(

was(assessed(by(i.p.(injection(of(C57BL/6((WT)(mice(and(Trif5/5(mice(with(leptospires(

and(initial(observation(of(changes(in(weight(and(appearance,(including(hunched(

posture,(slow(movement,(and(ruffled(fur,(until(euthanasia(at(1,(3,(7,(or(14(days(post(

infection((d.p.i.).((WT(and(Trif5/5(infected(mice(had(lost(weight(at(1I2(d.p.i.((Figure!

1A).(Infected(WT(mice(began(to(gain(weight(before(6(d.p.i.,(and(their(weight(

gradually(increased(over(time.(Trif5/5(mice(did(not(begin(to(put(on(weight(until(after(6(

d.p.i.,(and(their(weight(increased(at(a(slower(rate(compared(to(the(WT(mice((Figure!

1A).((This(difference(in(weight(change(over(time(between(infected(WT(and(Trif5/5(

mice(was(significant((p(=(0.0366).(((

90(

It(is(possible(that(the(delay(in(weight(gain(of(the(infected(TrifI/I(mice(was(due(

to(differences(in(bacterial(burden,(hence(leptospiral(load(was(quantified(in(kidneys,(

lungs(and(blood(by(qRTIPCR.(Leptospira(16s(rRNA(copies(trended(higher(in(TrifI/I(

mice(than(WT(infected(mice(at(1(dpi(in(the(blood(and(the(kidneys,(but(not(the(lungs(

(Figure!1BVD).((At(3(d.p.i.(leptospiral(load(was(significantly(higher(in(the(kidneys,(

lungs,(and(blood(of(infected(TrifI/I(mice(compared(to(infected(WT(mice((Figure!1BV

D).(The(lower(levels(of(Leptospira(16s(rRNA(copies(in(the(kidneys(and(blood(WT(

mice(suggested(early(clearance(of(the(bacteria(compared(to(the(sustained(elevation(

of(Leptospira(16s(rRNA(copies(in(the(kidneys(and(blood(of(TrifI/I(mice(at(3(d.p.i..(No(

copies(of(Leptospira(16s(rRNA(were(detected(in(the(lungs(or(blood(at(7(or(14(days(

post(infection(in(both(strains(of(mice(and(no(significant(difference(in(copies(of(

Leptospira(16s(rRNA(was(detected(in(the(kidneys(of(either(infected(group(at(7(d.p.i.(

compared(to(3(d.p.i.(At(14(d.p.i.,(the(burden(in(the(kidneys(had(increased(in(both(of(

the(infected(groups(but(the(differences(in(burden(were(not(significant,(suggesting(an(

established(chronic(leptospiral(infection(by(renal(colonization(in(both(strains(of(mice.(

This(finding(also(correlates(with(results(in(C57BL/6(and(Balb/c(mice(from(a(study(

measuring(burden(by(monitoring(bioluminescent(leptospires,(which(showed(a(visible(

decrease(and(apparent(clearance(of(the(bacteria(between(6I8(days(before(reI

establishment(in(the(kidneys(and(subsequent(chronic(infection((52).(

(

91(

(In(order(to(verify(that(the(mice(were(actively(infected(with(Leptospira,(

bladders(were(cultured(up(to(14(days(post(infection(in(EMJH(and(examined(for(the(

presence(of(motile(Leptospira(by(dark(field(microscopy.((At(1(and(3(d.p.i.,(all(bladder(

cultures(from(both(infected(groups(contained(motile(leptospires((Supplemental!

Table!1).(At(7(d.p.i.,(no(leptospires(were(detected(in(the(bladders(of(Trif5/5(mice(and(

only(one(WT(bladder(was(positive(for(bacteria.(In(contrast,(Leptospires(were(

detected(in(bladder(cultures(from(both(infected(groups(at(14(d.p.i.,(indicating(a(failure(

to(clear(the(bacteria(and(establishment(of(persistent(infection(in(mice(

(Supplemental!Table!1).(Even(though(the(burden(was(elevated(in(the(kidneys(of(

Trif5/5(mice(at(1(and(3(d.p.i.(compared(to(WT(mice,(measurement(of(creatinine(in(

serum(and(observation(of(kidneys(did(not(indicate(any(significant(physiological(

differences(in(either(infected(group(at(1(or(3(d.p.i.((Supplemental!Table!1,!Data!not!

shown,!Supplemental!Figure!1).(Serum(Nitrite(levels(did(not(differ(between(WT(

and(infected(groups(at(7(or(14(d.p.i.,(further(suggesting(that(there(were(no(

physiological(differences(between(WT(and(Trif5/5(mice(at(7(or(14(d.p.i.(

(Supplemental!Figure!1).((These(results(suggest(that(chronic(leptospiral(infection(is(

ultimately(established(in(both(WT(and(Trif5/5(mice,(but(significant(differences(in(weight(

gain(and(spirochete(burden(exhibited(by(the(Trif5/5(mice(compared(to(WT(mice(at(1(

and(3(d.p.i.(indicate(that(TRIF(contributes(to(murine(host(defense(during(leptospiral(

infection.((

!

92(

ISG!and!IFNγ!induction!in!Trif3/3!infected!mice!and!WT!infected!mice!

Because(signaling(downstream(of(TLR4(and(TRIF(leads(to(induction(of(Type(I(

IFN(via(IRFI3(activation,(we(hypothesized(that(elevated(leptospiral(burden(in(Trif5/5*

infected(mice*may(have(resulted(from(differences(in(IFNβ,(IFNγ,(and(ISG(induction.(

We(initially(attempted(to(detect(IFNβ(protein(by(ELISA(or(a(bioassay,(however,(both(

assays(proved(to(be(not(sensitive(enough((Data!not!shown).(Instead,(ISG(induction(

was(assayed(by(RTIPCR.(It(is(possible(that(elevated(burden(resulted(in(differences(

in(Type(I(and(II(IFN(mediated(ISG(induction,(including(the(expression(of(Ifit1,(Oasl2,(

and(Gbp2.(No(significant(differences(in(ISG(induction(were(detected(in(the(lungs(at(1(

d.p.i.((Figure!2C).(Expression(of(the(Type(I(ISG(Ifit1(was(elevated(in(the(blood(of(

Trif5/5(mice(at(1(d.p.i.(and(continued(to(trend(higher(in(the(blood(of(Trif5/5(mice(at(3(

d.p.i.((Figure!2B).(Oasl2(trended(higher(in(the(kidneys(of(Trif5/5(mice(at(1(d.p.i.(and(

although(relative(mRNA(levels(had(already(decreased(by(3(d.p.i.(remained(at(higher(

levels(in(the(kidneys,(lungs(and(the(blood(of(Trif5/5(mice(at(3(d.p.i.((Figure!2AVC).(

Transcript(induction(of(the(Type(I(and(II(ISG(Gbp2(trended(higher(in(the(kidneys(of(

Trif5/5(mice(at(1(d.p.i.,(but(was(elevated(in(the(blood(of(Trif5/5(mice(at(3(d.p.i.((Figure!

2A,!B).(These(results(suggested(that(the(changes(in(ISG(induction(in(the(kidneys(

and(the(blood,(specifically(Ifit1,(Oasl2,(and(Gbp2,(correlated(with(elevated(bacterial(

burden(in(Trif5/5(mice.((

We(sought(to(determine(whether(differences(in(Type(II(IFN(and(ISG(induction(

correlated(with(the(elevated(bacterial(burden(in(Trif5/5*mice.(mRNA(levels(of(Ifnγ(were(

93(

elevated(in(the(kidneys(of(Trif5/5(mice(at(1(d.p.i.,(and(the(Type(II(ISG(Cxcl10(was(

significantly(elevated(in(the(kidneys(and(the(blood(of(Trif5/5(mice(at(1(d.p.i.(compared(

to(WT((Figure!2DVF).(Ifnγ(also(trended(higher(in(the(blood(of(Trif5/5(mice(at(1(d.p.i(

and(3(d.p.i.((Figure!2DVF).((However,(at(3(d.p.i.,(Cxcl10*transcript(induction(was(

reduced(in(lungs(and(trended(lower(in(kidneys(of(Trif5/5*mice((Figure!2DVF).(IFNγ(

protein(levels(trended(higher(in(soluble(kidney(extracts(from(Trif5/5*mice((Figure!2G)(

correlating(with(the(mRNA(expression((Figure!2D).(These(results(suggest(the(

elevated(bacterial(burden(at(1(d.p.i.(and(3(d.p.i.(in(Trif5/5(infected(mice(correlates(with(

an(early(induction(of(Ifnγ(and(Cxcl10.((

(((It(is(possible(that(ISG(and(Type(II(IFN(induction(downstream(of(TRIF(might(

be(affected(by(differences(in(transcription(factor(expression(during(leptospiral(

infection(in(WT(and(Trif5/5(mice.((Generally,(infection(did(not(induce(transcript(levels(

of(IRF3(at(1d.p.i(and(relative(IRF3(mRNA(expression(was(decreased(at(3(d.p.i..(After(

infection,(relative(mRNA(expression(of(the(transcription(factor(Irf53(was(repressed(in(

the(blood(of(Trif5/5(mice(compared(to(WT(at(3(d.p.i.((Figure!2H)!but(no(significant(

differences(in(Irf53(mRNA(expression(were(seen(in(the(kidneys(or(lungs(of(either(WT(

or(Trif5/5*infected(mice((Figure!2H).((

ProVinflammatory!cytokine!responses!in!kidneys,!lungs,!and!blood!of!Trif#3/3!

mice!!

94(

Inflammatory(responses(independent(of(MyD88(have(been(previously(

observed(in(kidneys(of(mice((27),(and(an(increase(in(ILI1β(production(in(bone(

marrowIderived(macrophages(from(Trif5/5(mice(after(infection(with(Leptospira(was(

also(recently(documented((39).((It(is(possible(that(the(elevation(in(bacterial(burden,(

in(addition(to(signaling(through(MyD88(and(other(pathways,(might(result(in(a(higher(

proIinflammatory(response.(We(first(investigated(the(proIinflammatory(cytokine(

responses(in(the(kidneys(by(qRTIPCR((Figure!3).(TNFα(mRNA(expression(trended(

higher(in(the(kidneys(of(Trif5/5*infected(mice(compared(to(WT(mice,(but(fold(induction(

in(both(infected(groups(decreased(during(the(course(of(infection(and(no(significant(

differences(were(detected(between(either(infected(group(at(3,(7,(or(14(d.p.i.((Figure!

3A).(No(significant(differences(in(TNFα(protein(levels(were(detected(between(WT(

and(TrifI/I(mice((Figure!3B).(No(differences(in(ILI6(mRNA(expression(were(detected(

between(WT(and(TrifI/I(mice,(although(ILI6(protein(levels(trended(higher(in(TrifI/I(mice(

at(1(d.p.i.(by(ELISA((Figure!3C,!D).(Although(no(significant(differences(in(ILI1β(

protein(levels(were(detected(between(WT(and(TrifI/I(mice,(ILI1β(mRNA(levels(

trended(higher(in(the(kidneys(at(1(d.p.i.(and(were(significantly(reduced(at(3(d.p.i.(in(

TrifI/I(infected(mice((Figure!3E,!F).(We(extended(our(investigation(of(proI

inflammatory(cytokine(levels(to(ILI12,(which(is(involved(in(macrophage(activation(

and(Type(I(cytokine(responses.((IL12p40(mRNA(levels(were(elevated(at(1d.p.i.(in(the(

kidneys(of*TrifI/I(mice(compared(to(WT,(and(these(results(correlated(with(higher(

trending(ILI12p70(protein(levels(in(infected*TrifI/I(mice((Figure!3G,!H).(

95(

Because(the(overall(proIinflammatory(response(trended(higher(in(the(kidneys(

of(TrifI/I(mice(at(1(d.p.i.,(induction(of(mRNA(transcripts(via(qRTIPCR(was(assessed(

in(lungs(and(the(blood(of(mice(to(determine(whether(this(response(was(tissue(

specific((Figure!4,!Supplemental!Figure!2).((TNFα(levels(trended(higher(in(the(

blood(of(TrifI/I(mice(compared(to(WT(at(both(1(d.p.i.(and(3(d.p.i.((Figure!4A).(No(

differences(in(ILI6(mRNA(expression(were(detected(between(either(infected(group,(

although(ILI6(trended(lower(in(TrifI/I(mice(compared(to(WT(at(3(d.p.i.((Figure!4B).(

Serum(levels(of(ILI6(protein(were(also(elevated(in(both(Trif5/5*and(WT(infected(mice(

compared(to(uninfected(at(1(d.p.i.(and(continued(to(trend(higher(in(Trif5/5*infected(

mice(compared(to(Trif5/5*uninfected(mice(at(3(d.p.i.((Figure!4C,!D).(We(did(not(detect(

any(ILI1β(protein(in(serum(of(WT(or(Trif5/5(mice(at(1(or(3(d.p.i.((Data!not!shown)(

even(though(induction(of(ILI1β(mRNA(trended(higher(in(the(blood(at(1(d.p.i.((Figure!

4A).(No(significant(differences(in(proIinflammatory(cytokine(induction(between(either(

infected(group(were(detected(in(the(lungs(at(1(d.p.i.(or(3(d.p.i.((Supplemental!

Figure!2).(These(results(showed(that(elevated(leptospiral(burden(in(mice(lacking(

TRIF(might(be(associated(with(a(higher(proIinflammatory(response(in(the(kidneys(at(

1(d.p.i..((

(

AntiVLeptospiral!IgM!and!IgG!responses!in!WT!and!Trif3/3!mice!

The(critical(role(of(TLR2(and(TLR4(in(LeptospiraIspecific(antibody(production(to(

clear(bacterial(burden((27)(has(been(established.(Because(TRIF(is(an(adaptor(

96(

molecule(for(TLR2(and(TLR4((37,(41),(we(sought(to(determine(whether(leptospiral(

specific(IgM(and(IgG(responses(were(impaired(in(infected(Trif5/5(mice((Figure!5).(At(3(

d.p.i.,(although(antiILeptospira(IgM(antibodies(were(elevated(in(both(infected(groups(

of(mice(compared(to(uninfected,(the(antiILeptospira(IgM(levels(in(Trif5/5(infected(mice(

were(significantly(lower((p=0.0065)(compared(with(their(wildtype(counterparts(

(Figure!5A).((At(7(days(post(infection,(antiILeptospira(IgM(responses(were(

enhanced(in(Trif5/5(infected(mice(compared(to(WT(mice((p=(0.0424)((Figure!5B)(This(

response(decreased(in(both(infected(groups(at(14(d.p.i.,(but(antiILeptospira(IgM(in(

Trif5/5*mice(was(significantly(lower(compared(to(WT(mice((p=0.0146)((Figure!5C).(

These(results(suggested(that(Trif5/5*mice(showed(slower(kinetics(of*LeptospiraI

specific(IgM(production(and(ultimately(lower(levels(during(chronic(infection.((

In(order(to(determine(whether(the(kinetic(delay(in(antiILeptospira(IgM(

production(led(to(a(delay(in(class(switching(and(onset(of(the(adaptive(immune(

response,(we(assessed(serum(antiILeptospira(IgG(and(antiILeptospira(IgG(subclass(

levels(at(7(and(14(d.p.i.((Figure!5D,!E).((Both(infected(Trif5/5(and(WT(mice(had(

elevated(Leptospira*sonicateIspecific(IgG(levels,(but(there(was(no(statistically(

significant(difference(between(the(infected(groups(at(7(or(14(d.p.i.(Similar(results(

with(antiILeptospira(IgG2c(at(14(d.p.i.(and(antiILeptospira(IgG1(at(7(d.p.i.(were(

obtained(in(infected(Trif5/5(and(WT(mice((Figure!5F,!G).(((Interestingly,(by(14(d.p.i.(

antiILeptospira(IgG1(levels(remained(elevated(in(Trif5/5(infected(mice(but(not(WT(

infected(mice(compared(to(their(uninfected(counterparts((Figure!5H).(We(did(no(find(

97(

any(significant(differences(in(antiILeptospira(IgA(between(the(infected(Trif5/5(and(WT(

mice,(although(levels(trended(higher(compared(to(their(respective(uninfected(groups(

(Figure!5I).(Taken(together,(these(results(suggest(that(mice(lacking(TRIF(have(a(

delayed(antiILeptospira(IgM(response(during(the(course(of(infection,(but(this(delay(

may(not(affect(class(switching(during(leptospiral(infection.(((

(

(

Discussion!

Several(studies(have(identified(mechanisms(of(host(defense(and(key(players(

in(leptospiral(clearance.(Here,(we(have(shown(that(TRIF(contributes(to(murine(host(

defense(during(the(initial(response(to(leptospiral(infection,(and(our(results(are(

summarized(in(Figure(6.(Infected(WT(and(TrifI/I(mice(demonstrated(a(significant(loss(

of(weight(at(1(d.p.i.,(with(the(initial(weight(loss(being(more(pronounced(in(the(WT(

mice.(In(contrast,(infected(Trif5/5(mice(did(not(gain(back(their(weight(as(quickly(as(WT(

mice,(which(correlated(with(elevated(bacterial(burden(at(1(and(3(d.p.i.((Figure!6).!(

Interestingly,(this(elevation(in(leptospiral(burden(and(delay(in(clearance(

corresponded(with(elevated(tissueIspecific(proIinflammatory(mRNA(induction(in(the(

kidneys(of(Trif*5/5*mice(at(1(d.p.i.,(including(the(cytokines(IFNγ,(TNFα,(IL56(and(the(

chemokine(CXCL10.(Finally,(mice(lacking(TRIF(had(an(impaired(leptospiral(specific(

IgM(response(at(3(and(14(d.p.i.,(with(a(transient(elevation(at(7(d.p.i.,(but(this(did(not(

affect(class(switching(to(IgG2c(or(IgA.(((

98(

In(both(WT(and(Trif5/5(infected(mice,(L.*interrogans(transiently(appeared(in(the(

lungs(and(blood(at(1(d.p.i.(and(3(d.p.i.,(and(disappeared(by(7(d.p.i.,(which(is(

consistent(with(previous(work(showing(the(kidneys(as(the(primary(site(of(colonization(

when(spirochetes(are(no(longer(present(in(other(tissues(by(7(d.p.i.((14,(18,(22).(The(

reduction(of(bacterial(burden(in(kidneys(of(infected(mice(at(7(d.p.i.(and(reappearance(

at(14(d.p.i.(was(also(an(expected(outcome,(and(agrees(with(recent(work((52).((The(

initial(weight(loss(found(in(WT(and(Trif5/5*mice(at(1(d.p.i.(was(likely(caused(in(part(by(

the(elevated(bacterial(burden,(while(the(delay(in(increased(anti5Leptospira(IgM(

production(in(Trif5/5(mice(correlated(temporally(with(the(delayed(weight(gain(between(

3(and(7(d.p.i.((Figure!6).(The(WT(mice(had(initially(lost(more(weight(at(1d.p.i.,(but(

Trif5/5*mice(were(unable(to(put(on(weight(as(quickly(during(the(course(of(leptospiral(

infection.(Serum(creatinine(levels(are(an(established(marker(of(kidney(dysfunction,(

and(serum(nitrite(is(a(marker(of(macrophage(defense(and(inflammation,(but(because(

there(was(no(significant(elevation(of(serum(creatinine(or(serum(nitrite(in(Trif*5/5(

infected(mice(compared(to(WT(mice,(kidney(damage(does(not(appear(to(be(a(

primary(contributor(to(pathogenesis.(

The(innate(immune(response(includes(the(induction(and(expression(of(proI

inflammatory(cytokines,(and(the(proIinflammatory(response(has(been(extensively(

studied(in(response(to(leptospiral(infection((21I23,(39,(49).(During(the(course(of(

leptospiral(infection,(we(found(tissue(specific(differences(in(proIinflammatory(

cytokine(expression.(Specifically,(differences(in(ILI6,(ILI1β,(and(TNFα(were(detected(

99(

in(the(kidneys(during(early(stages(of(infection.(((ILI6(is(produced(early(during(

leptospiral(infection,(and(often(disappears(after(24(hours(post(infection((21).(The(

elevation(of(ILI6(at(3(d.p.i.(was(not(restricted(to(the(kidneys(as(shown(by(serum(ILI6(

ELISA((Figure!4).(TNFα(usually(appears(at(3I5(d.p.i.(in(C3H/HeJ,(C3H/Pas,(and(

Balb/C(mice((49).(We(found(similar(results(in(our(WT(mice,(but(TNFα(levels(were(

elevated(in(kidneys(of(TrifI/I(mice(at(1(d.p.i.,(and(no(significant(differences(in(TNFα(

induction(were(detected(in(the(lungs(or(the(blood(of(either(WT(or(Trif5/5(mice(at(1(or(3(

d.p.i..(Recent(studies(have(demonstrated(a(TLR(independent(mechanism(of(

inflammation(and(renal(fibrosis((14),(and(a(MyD88Iindependent(proIinflammatory(

response(in(Leptospira5infected(mice(has(also(been(shown((27).((Since(MyD88(is(an(

additional(adaptor(to(TLR4,(it(is(possible(that(enhanced(compensatory(signaling(

through(MyD88(occurs(in(the(absence(of(TRIF.(

ISGs(associated(with(Type(I(and(II(IFN(induction(trended(higher(in(the(kidneys(

of(Trif5/5(mice.(Interestingly,(elevated(IFNγ(levels(at(1(d.p.i.(were(specific(to(the(

kidneys(and(blood(of(infected(Trif5/5(mice,(as(was(the(early(elevation(of(CXCL10(at(1(

d.p.i..((We(had(found(that(IRF3(expression(levels(did(not(affect(Type(I(or(II(IFN(

induction,(but(this(does(not(mean(that(IRF3(activation(might(play(a(role(in(potential(

differences(at(1(d.p.i.(or(earlier(during(leptospiral(infection.(Additional(studies(to(

identify(cellular(responses(from(peripheral(blood(of(infected(mice(can(provide(insight(

into(the(mechanisms(underlying(the(negative(correlation(between(IRF3(expression,(

STAT1(expression,(and(activation(of(these(and(other(transcription(factors.((We(had(

100(

also(found(higher(trending(STAT1(expression(in(the(kidneys(and(the(blood(of(

infected(Trif5/5(mice(compared(to(WT(infected(mice.(Our(results(agree(with(previous(

studies(that(address(the(complex(relationship(between(IFNβ(and(IFNγ.(The(

regulation(of(IFNγ(by(IFNβ(during(viral(and(bacterial(infection(has(been(reported(

elsewhere((53I56),(including(a(distinct(mechanism(for(STAT1(mediated(inhibition(of(

IFNγ(by(IFNβ(during(viral(infection(which(has(been(previously(investigated((55).(

Additional(studies(also(showed(an(inhibition(of(IFNγ(by(IFNβ(in(lesions(from(human(

leprosy(patients,(as(well(as(an(inverse(correlation(between(Type(I(and(Type(II(IFN(

and(ISG((57).(We(did(not(detect(IFNβ(in(the(kidneys(or(the(blood(at(1(d.p.i..(It(is(

possible(that(IFNβ(might(have(appeared(earlier(in(blood(and(tissues(from(infected(

mice,(but(the(role(of(Type(I(IFN(remains(to(be(investigated.(IFNγ(is(not(upregulated(

in(peritoneal(macrophages(from(Balb/c(mice(during(infection,(but(IFNβ(is(

upregulated(as(early(as(one(hour(after(infection((58),(also(suggesting(potential(

negative(regulation(of(IFNγ.(However,(the(elevated(levels(of(IFNγ(and(CXCL10(in(

Trif5/5(infected(mice(compared(to(WT(infected(mice,(as(well(as(the(ISG(levels(in(both(

infected(groups,(seem(to(support(this(potential(mechanism(during(leptospiral(

infection.((

TNFα,(ILI6,(and(ILI1β(are(all(involved(in(multiple(innate(immune(responses(to(

infection((27,(59I61).(TNFα(is(a(proIinflammatory(cytokine(that(is(produced(by(

activated(macrophages,(CD4+T(cells,(and(NK(cells((22,(49,(62).(ILI6(is(produced(by(

macrophages(in(response(to(Leptospiral(LPS(mediated(TLR4ICD14(dependent(

101(

signaling((26,(63).((The(ILI1β(precursor(is(cleaved(by(neutrophilic(proteases(or(

caspaseI1(intracellularly((59,(60)(and(produced(by(monocytes(found(in(the(blood,(or(

macrophages(and(dendritic(cells(in(tissues((59,(60).(The(production(of(ILI1β(is(also(

associated(with(naïve(CD4+(T(cell(expansion((59,(60).(Elevation(of(TNFα(and(ILI1β(

has(been(previously(observed(in(murine(peritoneal(macrophages(in(response(to(

leptospiral(infection(and(the(induction(of(ILI1β(in(mouse(bone(marrow(derived(

macrophages(is(mediated(by(leptospiral(LPS((39,(58).(Other(proIinflammatory(

cytokines(like(IFNγ(and(CXCL10(are(produced(by(NK(cells(and(TH1(CD4+(T(cells(in(

response(to(leptospiral(infection(21,(64I69).(Elevated(CXCL10(and(Granzyme(B(

levels(have(been(detected(in(patients(with(leptospirosis(and(both(chemokines(are(

indicators(of(activated(NK(cells(and(T(cells,(respectively((67).(Human(PBMCs(

stimulated(with(leptospires(produce(TH1(cytokines,(including(TNFα(and(IFNγ((62).(In(

addition(to(this,(IFNβ(plays(a(critical(role(in(the(immune(response(to(bacteria,(

including(the(spirochete(Borrelia(burgdorferi((37,(57,(70,(71).(Macrophages(are(able(

to(phagocytose(leptospires,(but(neutrophils(are(not(able(to(kill(pathogenic(leptospires(

(72)((73).(The(early(elevation(of(these(cytokines(suggests(the(possibility(of(an(

increase(in(recruitment(of(NK(cells,(T(cells(and(macrophages(during(leptospiral(

infection(in(Trif5/5(mice.(It(is(possible(that(mice(lacking(TRIF(have(an(elevated(NK(and(

TH1(response(due(to(an(early(elevation(of(TNFα,(IFNγ(and(CXCL10,(but(the(

relationship(of(IFNβ(and(IFNγ(and(associated(cellular(responses(during(leptospiral(

102(

infection(in(mice(is(unknown.((Our(results(suggest(that(IFNβ(may(be(protective(

during(the(immune(response(to(Leptospira(in(mice(by(an(unknown(mechanism.((

Surprisingly,(we(found(that(infected(mice(lacking(TRIF(had(an(altered(antiI

Leptospiral(IgM(response.(The(critical(role(of(B(cells(during(infection(with(spirochetes(

has(been(previously(studied(in(various(knockout(strains(lacking(the(ability(to(produce(

B(cells(or(have(mature(B(cells((27,(74).((In(addition,(mice(lacking(TLR2(and(TLR4(

had(an(impaired(adaptive(response,(with(a(reduced(level(of(CD4+(and(CD8+(T(cells,(

and(CD19+(B(cells((27),(and(serum(antiILeptospira*IgM(and(IgG(levels(were(not(

reduced(in(TLR2I/I(mice,(but(were(reduced(in(TLR4I/I(mice(and(TLR2/4(I/I(mice((26,(

27).(MyD88(and(TRIF(are(adaptors(to(both(TLR2(and(TLR4,(and(it(is(possible(that(

MyD88(and(TRIF(are(acting(synergistically(through(different(mechanisms(in(murine(

host(defense(against(L.*interrogans.((Braun(et(al.(had(previously(determined(a(role(

for(Type(I(IFN(during(B(cell(development,(proliferation(and(survival((75).(TrifI/I(mice(

exhibited(elevated(levels(of(IFNγ(and(CXCL10,(and(a(delayed,(but(transient(

production(of(antiILeptospira(IgM.(In(addition(to(this,*TrifI/I(mice(had(elevated(antiI

Leptospira(IgG1(and(IgG2c(levels(at(14(d.p.i.,(compared(to(WT(mice(which(only(had(

elevated(IgG2c(at(14(d.p.i..(It(is(possible(that(a(Type(I(IFN(response(occurring(before(

1(d.p.i.(may(have(contributed(to(BIcells(producing(antiILeptospiral(IgM(during(

infection.(Further(studies(are(required(to(elucidate(the(protective(role(of(IFNβ(during(

Leptospiral(infection(by(supporting(B(cell(development(and(a(sustained(leptospiral(

specific(humoral(and(cellular(response.(To(our(knowledge,(this(study(is(the(first(to(

103(

suggest(that(Type(I(and(II(ISG(responses(may(be(protective(during(leptospiral(

infection.(This(is(also(the(first(study(to(show(an(association(between(TRIF(dependent(

signaling(and(protective(humoral(responses,(specifically(antiILeptospira(IgM(

responses,(during(infection(by(Leptospira*interrogans.(((

(

(

(

(

(

(

(

(

(

(

(

104(

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25.( Nally(JE,(Fishbein(MC,(Blanco(DR,(Lovett(MA.(Lethal(infection(of(C3H/HeJ(and(C3H/SCID(mice(with(an(isolate(of(Leptospira(interrogans(serovar(copenhageni.(Infection(and(immunity.(2005(Octb73(10):7014I7.(

26.( Viriyakosol(S,(Matthias(MA,(Swancutt(MA,(Kirkland(TN,(Vinetz(JM.(TollIlike(receptor(4(protects(against(lethal(Leptospira(interrogans(serovar(icterohaemorrhagiae(infection(and(contributes(to(in(vivo(control(of(leptospiral(burden.(Infection(and(immunity.(2006(Febb74(2):887I95.(

27.( Chassin(C,(Picardeau(M,(Goujon(JM,(Bourhy(P,(Quellard(N,(Darche(S,(et(al.(TLR4I(and(TLR2Imediated(B(cell(responses(control(the(clearance(of(the(bacterial(pathogen,(Leptospira(interrogans.(Journal(of(immunology((Baltimore,(Md(:(1950).(2009(Aug(15b183(4):2669I77.(

28.( Ferrer(MF,(Scharrig(E,(Alberdi(L,(Cedola(M,(Pretre(G,(Drut(R,(et(al.(DecayIaccelerating(factor(1(deficiency(exacerbates(leptospiralIinduced(murine(chronic(nephritis(and(renal(fibrosis.(PloS(one.(2014b9(7):e102860.(

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31.( VernelIPauillac(F,(Goarant(C.(Differential(cytokine(gene(expression(according(to(outcome(in(a(hamster(model(of(leptospirosis.(PLoS(neglected(tropical(diseases.(2010b4(1):e582.(

107(

32.( Goris(MG,(Wagenaar(JF,(Hartskeerl(RA,(van(Gorp(EC,(Schuller(S,(Monahan(AM,(et(al.(Potent(innate(immune(response(to(pathogenic(leptospira(in(human(whole(blood.(PloS(one.(2011b6(3):e18279.(

33.( Kyriakidis(I,(Samara(P,(Papa(A.(Serum(TNFIalpha,(sTNFR1,(ILI6,(ILI8(and(ILI10(levels(in(Weil's(syndrome.(Cytokine.(2011(Mayb54(2):117I20.(

34.( Wang(H,(Wu(Y,(Ojcius(DM,(Yang(XF,(Zhang(C,(Ding(S,(et(al.(Leptospiral(hemolysins(induce(proinflammatory(cytokines(through(TollIlike(receptor(2Iand(4Imediated(JNK(and(NFIkappaB(signaling(pathways.(PloS(one.(2012b7(8):e42266.(

35.( Marinho(M,(Monteiro(C,(Peiro(J,(Machado(GF,(OliveiraIJunior(I.(TNFIα(and(ILI6(immunohistochemistry(in(rat(renal(tissue(experimentaly(infected(with(Leptospira(interrogans(serovar(Canicola.(Journal(of(Venomous(Animals(and(Toxins(including(Tropical(Diseases.(2008b14(3):533I40.(

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37.( Aubry(C,(Corr(SC,(Wienerroither(S,(Goulard(C,(Jones(R,(Jamieson(AM,(et(al.(Both(TLR2(and(TRIF(contribute(to(interferonIbeta(production(during(Listeria(infection.(PloS(one.(2012b7(3):e33299.(

38.( Elsner(RA,(Hastey(CJ,(Baumgarth(N.(CD4+(T(cells(promote(antibody(production(but(not(sustained(affinity(maturation(during(Borrelia(burgdorferi(infection.(Infection(and(immunity.(2015(Janb83(1):48I56.(

39.( LacroixILamande(S,(d'Andon(MF,(Michel(E,(Ratet(G,(Philpott(DJ,(Girardin(SE,(et(al.(Downregulation(of(the(Na/KIATPase(pump(by(leptospiral(glycolipoprotein(activates(the(NLRP3(inflammasome.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Mar(15b188(6):2805I14.(

40.( Miller(JC,(MaylorIHagen(H,(Ma(Y,(Weis(JH,(Weis(JJ.(The(Lyme(disease(spirochete(Borrelia(burgdorferi(utilizes(multiple(ligands,(including(RNA,(for(interferon(regulatory(factor(3Idependent(induction(of(type(I(interferonIresponsive(genes.(Infection(and(immunity.(2010(Julb78(7):3144I53.(

41.( PetnickiIOcwieja(T,(Chung(E,(Acosta(DI,(Ramos(LT,(Shin(OS,(Ghosh(S,(et(al.(TRIF(mediates(TollIlike(receptor(2Idependent(inflammatory(responses(to(Borrelia(burgdorferi.(Infection(and(immunity.(2013(Febb81(2):402I10.(

108(

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43.( Zuerner(RL.(Host(response(to(leptospira(infection.(Curr(Top(Microbiol(Immunol.(2015b387:223I50.(

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52.( Ratet(G,(Veyrier(FJ,(Fanton(d'Andon(M,(Kammerscheit(X,(Nicola(MA,(Picardeau(M,(et(al.(Live(imaging(of(bioluminescent(leptospira(interrogans(in(mice(reveals(renal(colonization(as(a(stealth(escape(from(the(blood(defenses(and(antibiotics.(PLoS(neglected(tropical(diseases.(2014(Decb8(12):e3359.(

109(

53.( Brown(CR,(Blaho(VA,(Fritsche(KL,(Loiacono(CM.(Stat1(deficiency(exacerbates(carditis(but(not(arthritis(during(experimental(lyme(borreliosis.(J(Interferon(Cytokine(Res.(2006(Junb26(6):390I9.(

54.( Miyagi(T,(Gil(MP,(Wang(X,(Louten(J,(Chu(WM,(Biron(CA.(High(basal(STAT4(balanced(by(STAT1(induction(to(control(type(1(interferon(effects(in(natural(killer(cells.(J(Exp(Med.(2007(Oct(1b204(10):2383I96.(

55.( Nguyen(KB,(Cousens(LP,(Doughty(LA,(Pien(GC,(Durbin(JE,(Biron(CA.(Interferon(alpha/betaImediated(inhibition(and(promotion(of(interferon(gamma:(STAT1(resolves(a(paradox.(Nature(immunology.(2000(Julb1(1):70I6.(

56.( Nguyen(KB,(Watford(WT,(Salomon(R,(Hofmann(SR,(Pien(GC,(Morinobu(A,(et(al.(Critical(role(for(STAT4(activation(by(type(1(interferons(in(the(interferonIgamma(response(to(viral(infection.(Science.(2002(Sep(20b297(5589):2063I6.(

57.( Teles(RM,(Graeber(TG,(Krutzik(SR,(Montoya(D,(Schenk(M,(Lee(DJ,(et(al.(Type(I(interferon(suppresses(type(II(interferonItriggered(human(antiImycobacterial(responses.(Science.(2013(Mar(22b339(6126):1448I53.(

58.( Xue(F,(Zhao(X,(Yang(Y,(Zhao(J,(Cao(Y,(Hong(C,(et(al.(Responses(of(murine(and(human(macrophages(to(leptospiral(infection:(a(study(using(comparative(array(analysis.(PLoS(neglected(tropical(diseases.(2013b7(10):e2477.(

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61.( Barry(KC,(Fontana(MF,(Portman(JL,(Dugan(AS,(Vance(RE.(ILI1alpha(signaling(initiates(the(inflammatory(response(to(virulent(Legionella(pneumophila(in(vivo.(Journal(of(immunology((Baltimore,(Md(:(1950).(2013(Jun(15b190(12):6329I39.(

62.( Klimpel(GR,(Matthias(MA,(Vinetz(JM.(Leptospira(interrogans(activation(of(human(peripheral(blood(mononuclear(cells:(preferential(expansion(of(TCR(gamma(delta+(T(cells(vs(TCR(alpha(beta+(T(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2003(Aug(1b171(3):1447I55.(

63.( Werts(C,(Tapping(RI,(Mathison(JC,(Chuang(TH,(Kravchenko(V,(Saint(Girons(I,(et(al.(Leptospiral(lipopolysaccharide(activates(cells(through(a(TLR2Idependent(mechanism.(Nature(immunology.(2001(Aprb2(4):346I52.(

110(

64.( Naiman(BM,(Alt(D,(Bolin(CA,(Zuerner(R,(Baldwin(CL.(Protective(killed(Leptospira(borgpetersenii(vaccine(induces(potent(Th1(immunity(comprising(responses(by(CD4(and(gammadelta(T(lymphocytes.(Infection(and(immunity.(2001(Decb69(12):7550I8.(

65.( de(Fost(M,(Hartskeerl(RA,(Groenendijk(MR,(van(der(Poll(T.(Interleukin(12(in(part(regulates(gamma(interferon(release(in(human(whole(blood(stimulated(with(Leptospira(interrogans.(Clin(Diagn(Lab(Immunol.(2003(Marb10(2):332I5.(

66.( Chierakul(W,(de(Fost(M,(Suputtamongkol(Y,(Limpaiboon(R,(Dondorp(A,(White(NJ,(et(al.(Differential(expression(of(interferonIgamma(and(interferonIgammaIinducing(cytokines(in(Thai(patients(with(scrub(typhus(or(leptospirosis.(Clin(Immunol.(2004(Novb113(2):140I4.(

67.( De(Fost(M,(Chierakul(W,(Limpaiboon(R,(Dondorp(A,(White(NJ,(van(Der(Poll(T.(Release(of(granzymes(and(chemokines(in(Thai(patients(with(leptospirosis.(Clin(Microbiol(Infect.(2007(Aprb13(4):433I6.(

68.( Jongyota(W,(Wigraipat(C,(Nontapa(S,(Taweechaisupapong(S,(WaraIAswapati(NC,(Wongratanacheewin(S,(et(al.(Differential(response(of(cytokines(induced(by(Leptospira(interrogans,(serogroup(Pomona,(serovar(Pomona,(in(mouse(and(human(cell(lines.(Asian(Pac(J(Allergy(Immunol.(2008(Decb26(4):229I36.(

69.( Werts(C.(Leptospirosis:(a(Toll(road(from(B(lymphocytes.(Chang(Gung(Med(J.(2010(NovIDecb33(6):591I601.(

70.( Miller(JC,(Ma(Y,(Bian(J,(Sheehan(KC,(Zachary(JF,(Weis(JH,(et(al.(A(critical(role(for(type(I(IFN(in(arthritis(development(following(Borrelia(burgdorferi(infection(of(mice.(Journal(of(immunology((Baltimore,(Md(:(1950).(2008(Dec(15b181(12):8492I503.(

71.( KrupnaIGaylord(MA,(Liveris(D,(Love(AC,(Wormser(GP,(Schwartz(I,(Petzke(MM.(Induction(of(type(I(and(type(III(interferons(by(Borrelia(burgdorferi(correlates(with(pathogenesis(and(requires(linear(plasmid(36.(PloS(one.(2014b9(6):e100174.(

72.( Evangelista(KV,(Coburn(J.(Leptospira(as(an(emerging(pathogen:(a(review(of(its(biology,(pathogenesis(and(host(immune(responses.(Future(Microbiol.(2010(Sepb5(9):1413I25.(

73.( Wang(B,(Sullivan(J,(Sullivan(GW,(Mandell(GL.(Interaction(of(leptospires(with(human(polymorphonuclear(neutrophils.(Infection(and(immunity.(1984(Mayb44(2):459I64.(

111(

74.( Hastey(CJ,(Ochoa(J,(Olsen(KJ,(Barthold(SW,(Baumgarth(N.(MyD88I(and(TRIFIindependent(induction(of(type(I(interferon(drives(naive(B(cell(accumulation(but(not(loss(of(lymph(node(architecture(in(Lyme(disease.(Infection(and(immunity.(2014(Aprb82(4):1548I58.(

75.( Braun(D,(Caramalho(I,(Demengeot(J.(IFNIalpha/beta(enhances(BCRIdependent(B(cell(responses.(Int(Immunol.(2002(Aprb14(4):411I9.(

(

(

(

Ethical!Statement:!There(are(no(conflicts(of(interest(in(the(present(study.!

Acknowledgements(

We(thank(Dr.(David(Haake(for(providing(bacterial(cultures(for(this(project,(Dr.(Ben(

Adler(for(insight(and(help(in(developing(the(AntiILeptospira(IgM(ELISA,(and(Dr.(

Bryan(Troxell(for(critical(review(of(the(manuscript.((

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112(

!

Figure!1.(Leptospiral!infection!and!changes!in!weight!of!infected!mice.(WT(and(Trif5/5(mice(were(i.p.(injected(with(2x(108(bacteria/mouse(and(weighed(daily((A).(Changes(in(weight(over(time(are(described(in(methods(and(averaged(across(multiple(experiments.(Kinetics(of(leptospiral(load(in(kidneys((B),(lungs((C),(and(blood((D)(as(measured(by(copies(of(L.*interrogans(16s(rRNA(per(1000(copies(of(betaIactin(using(qRTIPCR.(*p<0.05,(***p<0.005.(ND(=(Not(detected.(

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14-15

-10

-5

0

5

10

15

Days post infection (d.p.i.)

% W

eigh

t Cha

nge

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

*

1 d.p

.i.

3 d.p

.i.

7 d.p

.i.

14 d

.p.i.

0.0

5.0×105

1.0×106

1.5×106

2.0×106

2.5×106

Lungs

Cop

ies

16s

rRN

A/ 1

000 β

- Act

in

WT, EMJHWT, L.i.

Trif-/- , EMJHTrif-/- , L.i.

ND ND

****

1 d.p

.i.

3 d.p

.i.

7 d.p

.i.

14 d

.p.i.

0.0

5.0×105

1.0×106

1.5×106

2.0×106

2.5×106

KidneysC

opie

s 16

s rR

NA

/ 100

0 β

- Act

in

WT, EMJHWT, L.i.

Trif-/- , EMJHTrif-/- , L.i.

***

1 d.p

.i.

3 d.p

.i.

7 d.p

.i.

14 d

.p.i.

0.0

5.0×106

1.0×107

1.5×107

2.0×107

Blood

Cop

ies

16s

rRN

A/ 1

000 β

- Act

in

WT, EMJHWT, L.i.

Trif-/- , EMJHTrif-/- , L.i.

ND ND

***

A

C

B

D

113(

!

Figure!2.!Induction!of!Type!I!and!II!IFN!and!ISG!in!WT!and!Trif3/3!infected!mice.!(ISG((Ifit1,*Oasl2,*Gbp2)(Transcripts(from(kidneys((A),(blood((B)(and(lungs((C)(were(measured(using(qRTIPCR(in(addition(to(levels(of(Ifnγ(and(Cxcl10(transcripts(from(kidneys((D),(blood((E)(and(lungs((F)(and(normalized(to(copies(per(1000(copies(of(beta(actin,(prior(to(foldIchange(calculations(as(described(in(methods.(IFNγ(in(soluble(extracts(from(kidneys(of(mice(at(1(d.p.i.((G)(were(determined(by(cytokine(sandwich(ELISAs.(Levels(of(Irf3(in(the(kidneys,(blood(and(lungs,(were(measured(using(qRTIPCR(as(described(above.((Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01,(***p<0.005.

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.0

5

10

15

20

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

IfitOasl2Gbp2

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.0

5

1050

100

150

Blood

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

Ifit1Oasl2Gbp2

***

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.05

101520

50

100

150

Lungs

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

Ifit1Oasl2Gbp2

*

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.0

10

20

30

40

50

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

IfnγCxcl10

*****

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.0

5

10

15

Blood

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

IfnγCxcl10

**

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.0

5

10

50100150

Lungs

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

IfnγCxcl10

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

2000

4000

6000

8000

10000

IFNγ

Kidneys, 1 d.p.i.

pg/m

L

WT, 1 d.p.i.

Trif-/- , 1

d.p.i.

WT, 3 d.p.i.

Trif-/- , 3

d.p.i.012345

15

20

25

30

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

Irf3

KidneysBloodLungs

*

A D

B E

C F

G

H

114(

!

Figure!3.!ProVinflammatory!cytokine!induction!in!kidneys!of!WT!and!Trif3/3!infected!mice.!!ProIinflammatory(transcripts(from(kidneys((A,C,(E,(G)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(copies(of(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Levels(of((B)(TNFα,((D)(ILI6((F)(ILI1β,((and((H)(ILI12p70(in(soluble(extracts(from(kidneys(of(mice(at(1(d.p.i.(were(determined(by(cytokine(sandwich(ELISAs.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(

1 d.p.i.

3 d.p.i.

7d.p.i.

14 d.p.i.

0

50

100

150

TNFα

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTrif-/-

1 d.p.i.

3 d.p.i.

7d.p.i.

14 d.p.i.

0

100

200

300

IL-6

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTrif-/-

1 d.p.i.

3 d.p.i.

7d.p.i.

14 d.p.i.

0

5

10

15

IL-1β

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTrif-/-**

1 d.p.i.

3 d.p.i.

7d.p.i.

14 d.p.i.

0

10

20

30

40

IL-12p40

Kidneys

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTrif-/-

*

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

50

100

150

200

TNFα

Kidneys, 1 d.p.i.

pg/m

L

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

200

400

600

800

IL-6

Kidneys, 1 d.p.i.

pg/m

L

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

1000

2000

3000

IL-1β

Kidneys, 1 d.p.i.

pg/m

L

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

10

20

30

40

IL-12p70

Kidneys, 1 d.p.i.

pg/m

L

A

E

G

C

B

F

H

D

115(

(

!!

Figure!4.!ProVinflammatory!cytokine!induction!in!blood!of!WT!and!Trif3/3!infected!mice.!ProIinflammatory(transcripts(from(blood(at(1(d.p.i.((A)(and(3(d.p.i.((B)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(copies(of(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Levels(of(ILI6(in(sera(of(mice(at(1(d.p.i.((C)(and(3(d.p.i.((D)(were(determined(by(cytokine(sandwich(ELISA.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(!

TNFα IL-6IL-1β

IL-12p40

0

5

10

15

20

25

Blood

1 d.p.i.

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTRIF-/-

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

50

100

150

IL-6

1 d.p.i.

pg/m

L

** **

TNFα IL-6IL-1β

IL-12p40

0

20

40

60

80

Blood

3 d.p.i.

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTrif-/-

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i.0

50

100

150

200

IL-6

3 d.p.i.

pg/m

L

A

C

B

D

116(

!

Figure!5.!Leptospiral!specific!IgM!and!IgG!responses!between!WT!and!Trif3/3!Mice.!Levels(of(leptospiralIspecific(IgM((at(3,(7,(and(14(d.p.i.((A,(B,(C),(leptospiralIspecific(IgG(at(7(and(14(d.p.i.((D,(E)(were(quantified(by(ELISA(as(described(in(Methods.(OD(490(measurements(of(leptospiralIspecific((IgG2c((F)(,(IgG1((G,(H)(and(IgA((I)(were(determined(by(ELISA(as(described(in(Methods.(Data(are(depicted(as(Mean(+/I(SEM(averaged(across(multiple(independent(experiments.(*p>0.05,(**p<0.01,(***p<0.005.!

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0

2000

4000

6000 **

**

***

3 d.p.i.

Ant

i-Lep

tosp

ira

IgM

ng/

mL

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0

10000

20000

30000 ***

*

***

7 d.p.i.

Ant

i-Lep

tosp

ira

IgM

ng/

mL

A GD

B HE

C IF

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0

5000

10000

15000 *****

***

*

14 d.p.i.

Ant

i-Lep

tosp

ira

IgM

ng/

mL

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0

2000

4000

6000 ******

7 d.p.i.

Ant

i-Lep

tosp

ira

IgG

ng/

mL

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0

10000

20000

30000

40000

50000 ******

14 d.p.i.

Ant

i-Lep

tosp

ira

IgG

ng/

mL

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

-0.1

0.0

0.1

0.2

0.3

0.4

Anti-Leptospira IgA

7 d.p.i.

OD

(490

)

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0.0

0.5

1.0

7 d.p.i.

OD

(490

)

Anti-Leptospira IgG1

** **

Anti-Leptospira IgG1

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

0.0

0.2

0.4

0.6

0.8

1.0*

14 d.p.i.

OD

(490

)

Anti-Leptospira IgG2c

WT EMJH

WT L.i.

Trif-/- EMJH

Trif-/- L.i.

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

******

14 d.p.i.

OD

(490

)

117(

!

Figure!6.!Diagram!of!Leptospiral!pathogenesis!in!WT!and!TrifV/V!mice.!!(A)(Bacterial(burden(in(the(kidneys((16s)(,(and(Weight(change(during(the(course(of(leptospiral(infection(in(WT(and(TrifI/I(mice((B)(Immune(response(for(WT(and(TrifI/I((mice((by(AntiILeptospira(IgM((ALIIgM)(TNF(in(the(kidneys(as(a(marker(for(renal(inflammation(in(WT(and(TrifI/I(mice.(

!

!

!

!

!

!

!

0 d.p

.i.

1 d.p

.i.

3 d.p

.i.

7 d.p

.i.

14 d

.p.i.

0

50

100

150

-10

-5

0

5

10

WT-16s-KidneyTrif-/- 16s-Kidney

WT-weightTrif-/- weight

Days post infection (d.p.i.)

16s

Cop

ies

per

1000

β-a

ctin

Weight C

hange (%)

0 d.p

.i.

1 d.p

.i.

3 d.p

.i.

7 d.p

.i.

14 d

.p.i.

0

5000

10000

15000

0

10

20

30

WT TNFα-KidneyTrif-/- TNFα-Kidney

WT AL-IgMTrif-/- AL-IgM

Days post infection (d.p.i.)

Ant

i-Lep

tosp

ira

IgM

ng/

mL

Relative m

RN

A expression

(Fold Change)

A B

118(

Supporting!information!

Supplemental!Table!1.!Functional!observation!of!leptospiral!infection!in!bladders!.!(Bladders(were(removed(from(mice(postIeuthanasia(and(incubated(in(5mL(EMJH(as(described(in(methods(for(10(days.(10uL(of(cultured(medium(was(observed(on(a(slide(via(Dark(Field(microscopy(for(motile(Leptospires.(*One(mouse(died(at(4(d.p.i.(

Experimental!Replicate!

Days!Post!Infection!(d.p.i.)!

#!positive!cultured!bladders!/!Total!#!bladders!WT! Trif3/3#

1( 1( 5/5( 6/6(2( 1( 5/5( 5/5(2( 3( 8/8( 7/7(3( 3( 5/5( 6/6(3( 7( 1/5( 0/6(1( 14( 5/5( 5/5(2( 14( 10/10( 8/9*((Supplemental!Figure!1.!Creatinine!levels!in!serum!of!WT!and!Trif3/3!infected!mice.!Creatinine(was(measured(in(serum(of(mice(at(3(d.p.i.((A)(using(a(kit(from(Cayman(Chem(following(the(manufacturers(instructions.(Serum(Nitrite(levels(were(measured(in(mice(at(7(d.p.i.((B)(and(14(d.p.i.((C)(as(described(in(methods.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.((((

(

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i. -0.005

0.000

0.005

0.010

Serum Creatinine

3 d.p.i.

OD

(500

)

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i. -0.2

0.0

0.2

0.4

0.6

0.8

Serum Nitrite

7 d.p.i.

OD

(540

)

*

WT EMJH

WT L.i.

Trif-/-

EMJH

Trif-/-

L.i. -0.5

0.0

0.5

1.0

1.5

Serum Nitrite

14 d.p.i.

OD

(540

)

A B C

119(

!!Supplemental!Figure!2.!ProVinflammatory!cytokine!induction!in!lungs!of!WT!and!Trif3/3!infected!mice.(ProIinflammatory(transcripts(from(lungs(at(1(d.p.i.((A),(3(d.p.i.((B),(and(7(d.p.i.((C)(were(measured(using(qRTIPCR(and(normalized(to(copies(per(1000(beta(actin.(FoldIchange(was(calculated(as(described(in(methods.(Results(are(depicted(as(Mean(+/I(SEM(and(are(described(in(methods(and(averaged(across(multiple(experiments.(*p>0.05,(**p<0.01.(((

!

!

WT, T

NFα

Trif-/-

, TNFα

WT, IL

-6

Trif-/-

, IL-6

WT, IL

-1β

Trif-/-

, IL-1β

WT, IL

12p40

Trif-/-

, IL12

p400

100

200

300

400

Lungs

1 d.p.i.

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

*

WT, T

NFα

Trif-/-

, TNFα

WT, IL

-6

Trif-/-

, IL-6

WT, IL

-1β

Trif-/-

, IL-1β

WT, IL

12p40

Trif-/-

, IL12

p400

5

10

15

20

25

Lungs

3 d.p.i.

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

*

**

WT, T

NFα

Trif-/-

, TNFα

WT, IL

-6

Trif-/-

, IL-6

WT, IL

-1β

Trif-/-

, IL-1β

WT, IL

12p40

Trif-/-

, IL12

p400

10

20

30

40

Lungs

7 d.p.i.

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

A

B

C

120(

!

Chapter!3!

Murine!TLR7!dependent!ISG!and!cytokine!responses!to!B.#burgdorferi!and!B.#

burgdorferi!RNA!

Abstract!

Lyme(Disease(is(prevalent(throughout(the(United(States(and(is(caused(by(the(

vectorIborne(spirochete(Borrelia*burgdorferi.(B.*burgdorferi(is(transmitted(by(Ixodes*

spp.*ticks(to(humans(and(domestic(and(wild(animals.(Mice(are(reservoir(hosts,(and(

have(been(used(to(elucidate(mechanisms(of(the(immune(response(to(infection(

including(innate(immune(responses.(TLR(signaling(is(an(important(component(of(the(

innate(immune(response(during(pathogenesis(of(B.*burgdorferi*and(has(been(

studied(in(mice.(Multiple(TLRs(recognize(lipoproteins,(flagellin,(and(nucleic(acids(

from(B.*burgdorferi,(which(results(in(Type(I(IFN(induction.(A(role(for(human(TLR8(

and(TLR7(in(B.*burgdorferi*RNA(mediated(immune(responses(has(been(established,(

but(the(role(of(murine(TLR7(in(the(innate(immune(response(and(host(defense(is(not(

known.(We(hypothesized(that(TLR7(might(play(a(role(in(murine(response(to(infection(

by(B.*burgdorferi.(Mice(lacking(TLR7(did(not(clear(bacterial(infection,(but(displayed(a(

dampened(humoral(response(to(infection(by(B.*burgdorferi.(Additional(studies(in(

bone(marrow(derived(macrophages(showed(B.*burgdorferi(RNA(mediated(ISG(and(

cytokine(induction(in(a(TLR7IMyD88(dependent(manner,(but(the(immune(response(

might(not(be(restricted(to(this(mechanism.(((

121(

!

Introduction:!

The(gramInegative(spirochete(Borrelia*burgdorferi(is(transmitted(between(

rodent(reservoir(and(human(hosts(and(Ixodes*scapularis(ticks,(and(causes(Lyme(

Disease((LD)((1I4).(LD(is(prevalent(in(the(Northeast(and(Midwest(and(regions(of(the(

United(States,(although(cases(have(been(reported(nationwide((5I11).!Symptoms(are(

variable,(including(a(bull’sIeye(rash(at(the(site(of(a(tick(bite((erythema(migrans),(and(

extend(to(Bell’s(palsy,(uveitis(and(arthritis.(LD(is(treatable(with(antibiotics,(but(no(

human(vaccines(are(available,(and(failure(to(treat(with(antibiotics(can(lead(to(chronic(

Lyme(arthritis,(and(carditis((4,(12).(The(number(of(cases(may(actually(be(significantly(

higher(that(previously(reported(because(of(the(variability(in(symptoms,(and(reports(of(

asymptomatic(patients(with(LD((5I11).((The(genome(of(B.*burgdorferi*has(one(linear(

chromosome(and(varying(number(of(circular(and(linear(plasmids(by(strain.(

Interestingly,(genes(that(code(for(proteins(associated(with(sophisticated(secretion(

systems(or(exotoxins(have(not(been(identified(in(the(genome(of(B.*burgdorferi((13I

15).(Because(of(this,(the(resulting(inflammation(and(tissue(damage(following(

infection(may(occur(because(of(an(elevated(immune(response(in(mammalian(hosts(

(16I18).(!

In(the(innate(immune(response,(pathogen(associated(molecular(patterns(

(PAMPs),(like(lipoproteins,(lipopolysaccharide((LPS),(flagellin(or(nucleic(acids,(are(

recognized(by(pathogen(recognition(receptors((PRRs)((19I21),(which(initiate(a(

122(

signaling(cascade(resulting(in(the(production(of(proIinflammatory(cytokines(or(

interferon((IFN)(depending(on(the(PRRIspecific(pathway((19I22).(TollIlike(receptors(

(TLRs)(are(a(subset(of(PRRs(that(are(bound(to(the(plasma(membrane(or(in(the(

endosome((19,(21).(Plasma(membrane(bound(TLRs(1/2/6,(TLR4,(and(TLR5(

recognize(lipoproteins,(LPS,(and(flagellin,(respectively((19,(21).(Endosomal(TLR3,(

TLR7/8,(and(TLR9(recognize(double(stranded(RNA,(single(stranded(RNA,(and(DNA,(

respectively((19,(21).(Myeloid(differentiation(factor(88((MyD88)(and(TIRIdomainI

containing(adapterIinducing(interferonIβ((Trif)(are(adaptor(proteins(to(TLRs,(

including(the(nucleic(acid(sensing(TLRs,(and(signaling(downstream(of(either(adaptor(

can(result(in(Type(I(IFN(and(ISG(induction((19,(23).((

PAMPIPRR(recognition(during(infection(by(B.*burgdorferi(has(been(

extensively(studied((22,(24I35).((((Outer(surface(proteins(like(OspA(and(OspC(are(

recognized(by(TLR2((36I39).(TLR5(which(recognizes(flagellin,(and(TLR9,(which(

recognizes(DNA,(have(both(also(been(implicated(in(the(inflammatory(response(to(B.*

burgdorferi*(29I32,(35,(36,(40,(41).(Mice(lacking(TLR2(and(mice(lacking(MyD88(have(

enhanced(bacterial(burden(in(the(joints,(and(an(inability(to(mount(an(effective(

immune(response.(Arthritis(severity(during(LD(infection(is(governed(by(a(Type(I(

Interferon((IFN)(response,(IFNIreceptor(deficient(mice(and(C3H/HeJ(mice(display(

more(severe(symptoms(compared(to(C57BL/6(mice((22,(27,(28,(42I45).(The(roles(of(

other(TLRs(in(murine(host(defense(during(B.*burgdorferi*pathogenesis(have(not(yet(

been(determined.((

123(

In(addition(to(mouse(models(of(host(defense(to(B.*burgdorferi,(mechanisms(of(

the(innate(immune(response(have(been(studied(in*vitro((30,(31,(46I50).((In(murine(

boneImarrow(derived(macrophages((BMDMs)(and(human(peripheral(blood(

mononuclear(cells((PBMCs),(recognition(of(multiple(ligands,(including(RNA(from(B.*

burgdorferi(results(in(induction(of(Type(I(IFN(and(Interferon(Stimulatory(Genes((ISG)(

(30,(32,(33,(35).(Human(TLR8(and(TLR7,(which(recognize(RNA,(have(been(

implicated(in(recognition(of(RNA(from(B.*burgdorferi*and(the(subsequent(induction(of(

Type(I(IFN(and(NFIκB(mediated(cytokines((30,(32,(33,(35).((The(role(of(murine(TLR7(

in(B.*burgdorferi*and(B.*burgdorferi*RNA(mediated(Type(I(ISG(induction(is(not(yet(

known.(We(sought(to(evaluate(the(role(of(murine(TLR7(in(host(defense(to(B.*

burgdorferi,(and(in(B.*burgdorferi*RNAImediated(Type(I(ISG(expression.(We(found(

that(TLR7(may(have(a(contributing(role(in(host(defense(during(B.*burgdorferi(

pathogenesis,(and(RNA(from(B.*burgdorferi*induces(ISG(expression(in(a(TLR7I

MyD88(dependent(manner.((

(

Materials!and!Methods(

(Bacteria!!

Low(passage(isolates((<(4(passages(per(experiment)(of(B.*burgdorferi(B31IMII16(

were(grown(at(33°C(to(midIlogarithmic(phase((3I4(x107(/mL)(for(mouse(and(BMDM(

experiments,(or(late(logarithmic(phase((6x107(–(1x108)(for(BMDM(experiments.(

Plasmid(content(was(monitored(by(PCR(to(ensure(that(plasmid(loss(did(not(occur(

124(

during(in*vitro(cultivation.(BarbourIStoennerIKelley(Media((BSKIII)(was(prepared(as(

previously(described(and(supplemented(with(6%(rabbit(serum((Gemini(Biosciences,(

West(Sacramento,(CA)((31).((

(

Mice!(

(((((((((( C57BL/6((WT),(and(C57BL/6ITg(Vil1IMyd88)1Lvh/J((Myd885/5)(mice(were(purchased(

from(The(Jackson(Laboratory((Bar(Harbor,(ME).(Tlr75/5(mice(were(provided(by(Frank(

Scholle((North(Carolina(State(University,(Raleigh,(NC).(Mice(were(housed(in(the(

North(Carolina(State(University(Biological(Resources(Facility((Raleigh,(NC)(and(all(

animal(procedures(were(approved(by(the(North(Carolina(State(University(Institutional(

Animal(Care(and(Use(Committee((Protocol(numbers:(10I099IB,(and(13I066IB).(((

((((((((( Groups(of(ageImatched(5(to(7(week(old(B6(and(Tlr75/5(mice((n=5I7/group)(

were(intradermally((i.d.)(injected(with(20uL(containing(either(5x104(midIlog(phase(

B31IMII16,(or(BSKIII(medium.(Mice(were(monitored(for(signs(of(infection,(including(

hunched(posture,(slow(movement,(and(ruffled(fur.(Mice(were(euthanized(at(one(or(

four(weeks(postIinfection,(and(rear(ankle(joints(were(collected(in(Eppendorf(tubes(

and(flash(frozen(in(a(dry(ice/ethanol(for(RNA(isolation(and(subsequent(quantitative(

RTIPCR(analysis.(Bladders(were(removed(and(cultivated(in(BSKII(containing(50(

mg/ml(rifampicin(and(100(mg/ml(phosmomycin(for(verification(of(bacterial(infection(

via(dark(field(microscopy.(Blood(was(collected(from(mice(postIeuthanasia(and(

processed(for(serum(by(centrifugation(at(5000(RPM(for(10(minutes(at(room(

125(

temperature(prior(to(storage(at(I20°C.(Serum(samples(were(then(analyzed(for(antiI

Borrelia(IgG(and(antiIBorrelia(IgM(ELISAs.(

!(

Reagents!

R848,(poly(IC,(and(poly(A:U(were(purchased(from(Invivogen((San(Diego,(CA).(

RNAse(A(was(obtained(from(Roche(Applied(Science((Indianapolis,(IN)(and(DNase(I(

was(purchased(from(New(England(BioLabs((Ipswich,(MA).(Mouse(cytokine(capture(

antibodies(to(ILI6,(ILI1β,(and(IFNγ(were(purchased(from(Biolegend((San(Diego,(CA)(

and(TNFα(mouse(capture(antibody(was(purchased(from(BD(Pharmingen(San(Jose,(

CA).((Recombinant(ILI6(and(TNFα(were(purchased(from(eBioscience((San(Diego(

CA).(Recombinant(ILI1β(was(purchased(from(Gemini(Biosciences((West(

Sacramento,(CA)(and(recombinant(IFNγ(and(ILI10(were(purchased(from(BD(

Pharmingen.((Biotinylated(detecting(antibodies(to(mouse(ILI6,(ILI1β,(and(TNFα(were(

purchased(from(eBioscience,(and(biotinylated(detecting(antiImouseIIFNγ(and(HRPI

Avidin(were(purchased(from(BioLegend.((oIPhenylenediamine(was(purchased(from(

MP(Biomedicals((Santa(Ana,(CA).(Recombinant(mouse(IgG,(recombinant(mouse(

IgM,(rabbit(antiImouse(IgM/G/A(capture(antibody,(and(HRPIrabbit(antiImouse(IgM(

were(purchased(from(Millipore((Billerica,(MA).(HRPIrabbit(antiImouse(IgM(was(

purchased(from(BioLegend.(

Total(RNA(from(B.*burgdorferi*(Bb(RNA)(was(extracted(from(late(logarithmic(

phase(B31IMII16(batch(cultures((250I300(mL)(using(Qiazol((Qiagen,(Germantown,(

126(

MD)(following(the(manufacturer’s(instructions(and(quantified(using(the(Nanodrop(

1000.(Following(isolation(of(total(RNA,(the(manufacturer’s(specifications(were(

followed(to(remove(DNA(from(Bb(RNA(via(treatment(with(DNase(I((NEB).(Quality(of(

RNA(was(assessed(via(1%(agarose(gel(electrophoresis,(both(before(and(following(

DNase(I(digestion,(to(ensure(RNA(degradation(did(not(occur(during(isolation(by(

confirmation(of(the(23s(and(16s(rRNA(bands(on(the(gel.((((

!

Bone!marrow!derived!Macrophages(

((((((((( Bone(marrow(was(harvested(from(the(femurs(and(tibias(of(8I12(week(old(

mice(and(BMDMs(were(differentiated(by(growth(in(RPMI(medium((HyClone,(Logan,(

UT)(containing(30%(L929(culture(supernatant(and(20%(horse(serum((Gemini(

BioProducts)(at(37°C,(5%(CO2.(BMDMs(were(harvested(and(dispensed(at(7.2(x(

105/ml(in(serumIfree(RPMI(medium(containing(1%(Nutridoma((Roche)(into(12Iwell(

plates.(All(cell(culture(reagents,(including(the(L929(culture(supernatants,(utilized(in(

this(study(were(assayed(by(either(Hoechst(staining((Invitrogen,(Carlsbad,(CA)(and(

confirmed(to(be(negative(for(contaminating(Mycoplasma(species.(Following(an(

overnight(incubation,(medium(was(removed,(and(RPMI,(B.*burgdorferi*(MOI(of(10),(

Bb(RNA,(R848,(pIC(or(pAU(were(added(in(triplicate.(Plates(were(centrifuged(at(300(x(

g(for(10(min(to(facilitate(B.*burgdorferi*contact(with(BMDMs(and(incubated(at(37°C,(

5%(CO2(for(4,(6(or(24(hours.(Following(stimulation,(cellular(supernatants(were(

collected(for(measurement(of(secreted(cytokine(levels(by(sandwich(ELISA.(

127(

Cytokines(were(measured(in(harvested(supernatants(by(sandwich(ELISAs(as(

previously(described((51,(52).((

!

RNA!isolation!and!qVRTVPCR(

((((((((( At(1(week(postIinfection,(both(rear(ankle(joints(from(B6(or(TLR7I/I(mice(were(

excised,(and(separately(homogenized(each(in(2(ml(of(Qiazol(reagent((Qiagen)(for(

RNA(isolation,(following(the(manufacturer’s(protocol.((RNA(was(isolated(from(

BMDMs(cultured(in(12(well(plates(using(1(ml(of(Qiazol(reagent.((Up(to(5µg(of(RNA(

isolated(from(joints(or(BMDMs(was(reverse(transcribed(to(cDNA(using(random(

primers((Promega,(Madison,(WI)(and(MIMLV(reverse(transcriptase((Affymetrix,(

Santa(Clara,(CA).(Quantitative(PCR((qRTIPCR)(was(conducted(to(amplify(mRNA(

copies(using(the(iQ(SYBR(Green(Supermix(and(the(MyiQ2(TwoIcolor(RealItime(PCR(

Detection(System((BioIRad,(Hercules,(CA).(For(each(transcript(of(interest(fold(

change(was(calculated(using(the(2IΔΔCT(method.(The(following(primer(sets(were(

used(for(qPCR(and(are(described(elsewhere:(βIactin,(Stat1,(Iigp,(Igtp((31)(Oasl2,(

Cxcl9,(Cxcl10,*IFNg*(22),(Ifit1(and(Gbp2((45).(

!!

Tissue!DNA!extraction!and!qPCR!analysis(

!((((((((( ((DNA(was(extracted(from(the(rear(ankle(joints(of(B6(and(TLR7I/I(mice(

euthanized(at(4(weeks(postIinfection.(The(B.*burgdorferi*recA(gene(was(amplified(by(

qIPCR(using(the(iQ(SYBR(Green(Supermix(and(the(MyiQ2(TwoIcolor(RealItime(

128(

PCR(Detection(System((BioIRad).(The(number(of(B.*burgdorferi*genome(equivalents(

by(RecA(copies(was(calculated(from(the(starting(template(sample(and(normalized(to(

1000(copies(of(the(mouse(housekeeping(gene(Nidogen,(as(previously(described((71,(

72).((

!

Bioinformatics!and!Statistical!analysis(

!Sequences(of(Mouse(PRR’s((by(Uniprot(ID)(and(whole(genome(sequences(of(B31(

and(N40(were(analyzed(for(RNAIProtein(Interactions(using(the(RPIISeq(program(

(53,(54).((A(positive(prediction(probability(for(RNAIprotein(binding(was(considered(

greater(than(0.5(across(both(algorithms.((All(statistical(analyses(were(conducted(

using(GraphPad(Prism(Version(6(for(Windows((GraphPad(Software,(San(Diego,(

CA).(The(ShapiroIWilk(test(was(used(to(determine(whether(data(points(in(each(

group(met(criteria(for(a(normal(distribution.(The(MannIWhitney(Test(for(pairIwise(

nonIparametric(comparisons(and(the(Kruskal(Wallis(test(for(nonIparametric(

comparisons(across(multiple(groups(were(used(for(statistical(analysis.(For(all(tests(a(

p(value(of(less(than(0.05(was(considered(significant.((

(

Results:!

TLR7#contributes#to#the#immune#response#during#B.!burgdorferi#pathogenesis#

We(initially(sought(to(determine(whether(TLR7(was(required(for(bacterial(

clearance(or(host(defense.((Groups(of(4I6(mice(were(injected(intradermally((i.d.)(with(

129(

20μL(of(5x104(Bb(or(20μL(BSK(II(as(described(in(methods.(All(groups(of(WT(and(

Tlr75/5(mice(appeared(healthy,(with(no(changes(in(movement,(ruffled(fur,(or(other(

signs(of(disease(up(to(1(or(4(weeks(post(infection.(At(4(weeks(post(infection,(neither(

WT(nor(Tlr75/5showed(any(differences(in(RecA(induction(in(the(rear(ankle(joints(

(Figure!1A).!!Live(B.*burgdorferi*were(detected(in(all(infected(bladders(of(WT(and(

TLR7I/I(mice(at(1(week(and(4(weeks(post(infection((Data(not(shown),(which(

suggested(that(TLR7(was(not(required(for(bacterial(clearance(in(mice(during(

infection.(Anti5Borrelia(IgG(and(AntiIBorrelia(IgM(ELISAs(were(used(to(determine(the(

humoral(responses(to(B.*burgdorferi*infection(in(WT(and(Tlr75/5*mice((Figure!1B,!1C).(

There(were(no(differences(in(AntiI(Borrelia5IgG(levels(at(4(weeks(post(infection(

between(either(infected(group((Figure!1B).(Infected(Tlr75/*5mice(had(lower(AntiI

Borrelia(IgM(levels(compared(to(WT(at(1(week(post(infection((Figure!1C),(

suggesting(that(TLR7(contributes(to(the(immune(response(during(pathogenesis(of(

B.burgdorferi*in(mice.(ISG(levels(were(determined(by(qRTIPCR(in(WT(and(Tlr75/5*

mice((Figure!2)(at(1(week(post(infection(by(B.burgdorferi(before(the(peak(of(arthritis(

severity.(No(significant(differences(were(detected,(although(levels(of(Stat1(and(IFNγ(

trended(higher,(while(levels(of(Type(1(ISG(including(Gbp2,(Oasl2,(Cxcl9,(Iigp,(and(

Igtp(trended(lower(in(Tlr75/5mice(compared(to(WT.(Taken(together,(these(results(

suggest(that(TLR7(may(contribute(to(ISG(induction(during(infection(by(B.burgdorferi.**

#

#

130(

TLR7#is#required#for!B.#burgdorferi#RNA#mediated#ISG#induction#

Because(Tlr75/5mice(had(an(altered(AntiIBorrelia(IgM(response(and(a(lower(

trending(ISG(response,(we(sought(to(determine(whether(TLR7(was(required(for(B.*

burgdorferi*mediated(ISG(induction(using(BMDMs(as(a(previously(established(in*

vitro(model(system((31).((TLR7(is(located(in(the(endosome(and(recognizes(RNA(

from(bacteria(and(viruses((19,(55)(and(B.*burgdorferi*isolated(RNA((Bb(RNA)(was(

shown(to(induce(Type(I(IFN(and(ISG((31,(48,(56),(so(it(is(possible(that(BMDMs(

lacking(TLR7(might(not(induce(Type(I(IFN(and(ISG(in(response(to(RNA(from(B.*

burgdorferi.(BMDMs(were(stimulated(with(Media,(late(logarithmic(phase(B.*

burgdorferi*(Bb),(Bb(RNA,(or(the(TLR7(ligand(R848(for(6(hours(or(24(hours((Figure!

3).(Significantly(lower(levels(of(Cxcl10,(Gbp2,(and(Ifit1(were(observed(in(Tlr75/5

BMDMs(treated(with(Bb(RNA(or(R848(compared(to(WT(BMDMs((Figure!3).(Taken(

together,(these(results(suggest(that(TLR7(contributes(to(Bb(mediated(ISG(induction(

in(BMDMs.(

The(contribution(of(TLR7(to(Bb(and(Bb(RNA(mediated(NFIκB(dependent(

cytokine(production(was(determined(by(cytokine(Sandwich(ELISAs(for(IL6,(Il10(and(

IFNγ(and(4(and(24(hours((Figure!4).(WT(BMDMs(were(producing(ILI6(and(ILI10(as(

early(as(4(hours,(but(these(cytokines(were(not(detected(in(Tlr75/5*supernatants(

(Figure!4AVB).(ILI6(and(ILI10(cytokine(levels(trended(lower(in(Bb(stimulated(Tlr75/5

BMDM(supernatants(compared(to(WT.(IFNγ(was(enhanced(in(Tlr75/5*BMDMs(

stimulated(with(Bb(RNA.(Collectively(these(data(suggest(that(murine(TLR7(

131(

contributes(to(both(Bb(and(Bb(RNA(mediated(Type(I(IFN(induction,(and(NFIkB(

dependent(cytokine(production.((

#

Bb#RNA#mediated#innate#immune#response#requires#signaling#predominantly#

through#TLR7#and#MyD88#

MyD88(is(an(adaptor(molecule(for(downstream(signaling(of(TLR7(resulting(in(

Type(I(IFN(and(cytokine(production((23).(The(role(of(MyD88(during(Bb(pathogenesis(

has(also(been(explored(in(the(context(of(murine(host(defense,(and(elucidation(of(Bb(

RNA(mediated(induction(of(Type(I(IFN(in(human(PBMCs((32I35,(57).(It(is(not(known(

whether(Bb(RNA(mediated(induction(of(Type(I(IFN(or(cytokines(in(mouse(BMDMs(is(

MyD88(dependent.(Type(I(IFN(and(ISG(levels(were(assessed(in(Bb,(Bb(RNA,(R848(

or(Media(treated(BMDMs(via(qRTIPCR((Figure!5).(Cxcl10(mRNA(expression(levels(

were(reduced(in(Bb(RNA(and(R848(treated(MyD885/5(BMDMs(at(6(hours(compared(to(

Bb(treated(BMDMs.(The(levels(of(Gbp2(trended(lower(in(Myd885/5(BMDMs(stimulated(

with(Bb(and(Bb(RNA(at(6(hours(post(stimulation.(Similar(results(were(obtained(with(

lateIlogarithmic(phase(isolated(Bb((Data!not!shown).(These(results(suggested(that(

Bb(RNA(induced(Type(I(IFN(and(ISG(in(mouse(BMDMs(predominantly(through(

TLR7IMyD88,(but(induction(of(ISGs(was(not(exclusive(to(this(pathway.(ILI6(levels(

were(measured(in(cell(culture(supernatants(at(24(hours(by(ELISA(in(order(to(

determine(whether(MyD88(was(required(for(Bb(or(Bb(RNA(mediated(NFIκB(

dependent(cytokine(production.(The(difference(in(ILI6(production(from(Bb(treated(

132(

BMDM(supernatants(of(WT(and(MyD885/5*mice(at(24(hours(was(greater(than(the(

differences(detected(in(Bb(RNA(treated(BMDM(supernatants((Figure!6).(Taken(

together,(these(results(indicated(that(B.*burgdorferi(mediated(induction(of(ISG(and(

NFIκB(cytokines(like(ILI6(occurred(predominantly(through(MyD88,(but(Bb(RNA(

mediated(cytokine(and(ISG(induction(occurred(through(TLR7(and(MyD88.((

(

Prediction#of#Bb#RNA3PRR#interactions#

Because(IFNβ(and(ISG(induction,(and(NFIκB(dependent(cytokine(production(trended(

lower(in(Tlr75/5*BMDMs(we(considered(the(possibility(that(TLR7(might(not(be(the(only(

PRR(that(contributes(to(Bb(mediated(ISG(and(cytokine(induction(by(recognizing(

RNA(sequences(from(Bb.(Predicted(interactions(between(the(Borrelia(RNA(

chromosomal(sequences(for(B31(and(N40,(and(known(PRRs(were(determined(using(

a(sequence(based(prediction(algorithm(as(described(in(methods((Table!1).(Mouse(

TLR7(was(one(of(the(PRRs(with(a(higher(positive(prediction(probability(compared(to(

other(PRRs(for(both(B31(and(N40((Table!1).(We(also(noticed(that(TLR7(was(not(the(

only(PRR(to(have(a(high(positive(prediction(probability.((The(Retinoic(acid(Inducible(

gene((RIGII),(TLR8,(the(rRNA(recognizing(PRR(TLR13,(and(RNA(editing(enzyme(

ADAR(also(had(high(prediction(probabilities.((

!

!

!

133(

Discussion!

We(have(previously(shown(that(Bb(RNA(induces(a(Type(I(IFN(response(

during(B.*burgdorferi*pathogenesis((Miller(et(al(2010).(In(this(study,(we(have(shown(

that(Bb(RNA(is(recognized(by(murine(TLR7,(and(induces(Type(I(IFN,(ISG(and(NFIκB(

cytokine(production(in(a(TLR7IMyD88(dependent(manner.(Our(results(also(suggest(

this(mechanism(as(a(possible(result(for(the(impaired(immune(response(in(Tlr75/5*mice(

by(AntiIIgM(ELISA,(but(also(show(that(this(may(not(be(the(only(pathway(involved(in(

the(Type(I(IFN(response.((

Previous(studies(implicated(TLR2(and(MyD88(in(host(defense(against(B.(

burdorferi(pathogenesis.(Because(the(majority(of(genes(in(B.*burgdorferi*code(for(

lipoproteins,(and(because(B.*burgdorferi*lacks(LPS,(it(is(generally(thought(that(the(

immune(response(may(occur(via(a(TLR2IMyD88(dependent(manner.(Therefore(it(is(

not(surprising(that(we(did(not(find(any(differences(in(bacterial(burden(in(joints(or(AntiI

Borrelia(IgG(levels(in(sera.(We(did(find(an(impaired(AntiIBorrelia(IgM(response(in(

Tlr75/5*mice(compared(to(WT(mice(at(1(week.(Tlr75/5*B(cells(are(not(able(to(enhance(

antigenIspecific(IgM(production(when(reconstituted(in(SCID(mice,(and(TLR7(and(

BCR(coIactivation(contribute(to(effector(B(cell(functions,(including(Ig(production(((58I

60).(Additional(studies(are(required(to(elucidate(the(function(of(TLR7(in(B(cell(

development(and(antigen(specific(IgM(production(during(infection(by(B.*burgdorferi(

in(mice.(Because(signaling(downstream(of(TLR7(can(lead(to(Type(I(IFN(production(

and(IFN(enhances(B(cell(proliferation(and(survival((61),(it(is(possible(that(lower(IFN(

134(

and(ISG(induction(led(to(the(altered(B(cell(response(in(Tlr75/5*mice(at(1(week.(The(

relationship(between(B(cells,(TLR7(and(Type(I(IFN(responses(has(been(explored(

(58I60,(62,(63),(but(has(not(been(previously(addressed(in(the(context(of(murine(host(

defense(against(spirochetes.(The(lower(trending(ISG(levels(in(joints(of(Tlr75/5*mice(

also(support(the(resulting(impaired(antiIBorrelia(IgM(responses.(((

Because(the(ISG(levels(trended(lower(in(TLR7I/I(joints(at(1(week(p.i.,(we(had(

used(BMDMs(as(a(model(system(to(elucidate(the(mechanism(of(Type(I(IFN(induction(

during(B.*burgdorferi*pathogenesis.(Previous(studies(have(assessed(the(roles(of(

human(TLR8(and(TLR7((32I34,(64).(Human(TLR8(in(PBMCs(recognizes(B.*

burgdorferi*(RNA,(which(is(found(in(the(endosome(during(phagocytosis(of(the(

bacteria((32I34,(64).(Recent(studies(showed(that(human(TLR7(is(required(for(B.*

burgdorferi(RNA(mediated(Type(I(and(III(IFN,(ISG,(IRF7(expression,(and(NFIκB(

cytokine(production((35,(57).(We(found(differences(in(B.*burgdorferi*RNA(mediated(

induction(of(IFN(and(ISG(in(BMDMs(at(4(and(24(hours,(and(a(delay(in(NFIκB(

cytokine(production(for(both(B.*burgdorferi*and(B.*burgdorferi*RNA(stimulated(TLR7I/I(

BMDMs(at(4(and(24(hours(during(infection.((

It(is(also(expected(that(the(induction(of(Type(I(IFN(and(cytokines(was(MyD88(

dependent.(Previous(studies(in(PBMCs(and(Mouse(BMDMs(have(shown(a(MyD88I

dependent(induction(of(Type(I(IFN(and(production(of(NFIκB(cytokines(during(

infection(by(B.*burgdorferi.(We(had(expected(to(see(a(decrease(in(B.*burgdorferi*(

RNA(mediated(ISG(induction,((but(because(CXCL10(is(also(induced(by(IFNy(

135(

expression,(it(was(not(surprising(that(B.*burgdorferi*(RNA(mediated(CXCL10(

induction(was(increased(in(MyD88I/I(BMDMs.(All(NFIκB(cytokine(levels(were(lower(in(

MyD88I/I(BMDMs(compared(to(WT,(which(agrees(with(previous(studies(

demonstrating(the(critical(role(of(MyD88(during(B.*burgdorferi*pathogenesis.((

We(had(used(a(webIbased(prediction(program(to(determine(whether(the(

impaired(response(might(be(due(to(recognition(of(Bb(RNA(by(TLR7.(Sequence(

based(prediction(programs(have(been(developed(for(interaction(between(RNA(and(

Proteins((53,(54),(but(additional(biochemical(studies(would(be(required(to(verify(

these(predictions.(In(addition(to(a(high(prediction(probability(for(TLR7IBb(RNA(

interactions,(other(PRR(sequences(also(had(a(positive(prediction(probability,(

including(Adenosine(deaminase(reacting(on(RNA((ADAR)(and(TLR13.(ADAR(is(

expressed(as(an(ISG(involved(in(RNA(editing(by(changing(Adenines(to(Inosines,(

which(subsequently(show(up(as(AIG(mismatches(in(sequence(data((65I67).(ADAR(

expression(is(also(increased(earlier(in(C3H/HeJ(mice(compared(to(C57BL/6(mice,(

which(correlates(with(phenotypic(differences(in(disease((44,(45).(Recent(work(has(

also(suggested(that(TLR7(may(be(able(to(recognize(inosines(in(response(to(infection(

(66).(Human(PBMCs(treated(with(ssRNA(had(an(elevated(cytokine(and(IFN(

response(to(infection(and(the(immune(response(is(elevated(when(inosine(levels(in(

viral(RNA(are(high((66).(The(specific(RNA(sequence(s)(that(are(recognized(by(TLR7(

have(also(not(been(addressed,(but(additional(characterization(of(RNA(is(in(progress(

(Devlin(A,(unpublished(data).(TLR13(recognizes(the(rRNA(23s(subunit!from(bacteria(

136(

(68I70),(which(results(in(the(induction(of(NFIκB(dependent(cytokines.(RNA(editing(is(

generally(thought(of(as(an(antiviral(response,(but(this(has(not(yet(been(addressed(in(

the(context(of(bacterial(infection.(Additional(studies(are(required(to(determine(

whether(this(is(the(case(in(the(context(of(spirochete(infection.(

Several(studies(have(addressed(the(mechanism(of(Bb(RNA(recognition(by(

human(TLR7(and(TLR8,(but(our(study(is(the(first(to(explore(it(in(the(context(of(murine(

host(defense(and(suggest(that(other(PRRs(including(TLR13(and(ADAR(may(play(an(

additional(role(in(Bb(RNA(recognition(in(mice.(Moving(forward,(we(propose(that(the(

mechanism(of(ISG(induction(and(B(cell(responses(during(spirochete(infection(should(

be(further(explored.((

(

(

(

(

(

(

(

137(

Figures!and!Figure!Legends:!

!

Figure!1.!infection!of!B.#burgdorferi#in!WT!and!Tlr73/3!mice.!Mice(were(i.d.(injected(with(BSKIII(or(B.*burgdorferi*(Bb)(for(1(or(4(weeks(as(described(in(methods.((A)(Quantification(of(RecA(in(joints(as(a(marker(of(bacterial(burden(and(detection(of(host(response(in(serum(by(AntiIBorrelia(IgG(ELISA((B)(or(AntiIBorrelia(IgM(ELISA((C).(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(multiple(experiments.(*p<0.05,(***p<0.005(

(

Figure!2.!ISG!Induction!in!WT!and!Tlr73/3!mice!at!1!week!post!infection.!ISG(transcript(expression(was(measured(in(the(joints(of(WT((filled(bars)(and(Tlr75/5(gray(bars)(mice(by(qRTIPCR(as(described(in(methods.(Dashed(line(at(2(indicates(a(threshold(for(fold(induction(over(uninfected.(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(2(independent(experiments.((

(

WT, B

SK II

WT, B

b B31

Tlr7-/- , B

SK II

Tlr7-/- , B

b B31

0

25

50

75200225250

4 Weeks post infection

Cop

ies

Rec

A/1

000

mN

idog

en

WT, B

SK II

WT, B

b B31

Tlr7-/- , B

SK II

Tlr7-/- , B

b B31

0

1×104

2×104

3×104

4×104

5×104

4 Weeks post infection

Ant

i-Bor

relia

IgG

pg/

mL

WT, B

SK II

WT, B

b B31

Tlr7-/- , B

SK II

Tlr7-/- , B

b B31

0

1×107

2×107

3×107

1 week post infection

Ant

i-Bor

relia

IgM

pg/

mL

*** ****

A B C

Ifit1Gbp2Stat1 Ifn

γOasl2

Cxcl10Cxcl9 Igt

pIigp

0

5

10

15

20

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WTTlr7-/-

138(

(

(Figure!3.!ISG!induction!in!BMDMs!of!WT!and!Tlr73/3!mice.!(BMDMs(were(stimulated(with(RPMI(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(2ug,(or(Imiquimod((R848)(at(1ug(as(described(in(methods(for(6(hours.(ISG(mRNA(expression(was(measured(using(qRTIPCR(and(fold(change(was(calculated(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(are(averaged(across(2(independent(experiments.((

B. burg

dorferi,

MOI = 10

Bb RNA 1u

g/mL

R848 1

ug/mL

0

25

50

75

100

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

Cxcl10

WT Tlr7-/-

**** **

B. burg

dorferi,

MOI = 10

Bb RNA 1u

g/mL

R848 1

ug/mL

0

5

10

15

20

Gbp2

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WT Tlr7-/-

** ** **

B. burg

dorferi,

MOI = 10

Bb RNA 1u

g/mL

R848 1

ug/mL

0

5

10

15

Ifit1

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

WT Tlr7-/-

** ** **

A

B

C

139(

(Figure!4.!Cytokine!production!in!culture!supernatants!from!BMDMs!of!WT!and!TLR73/3!mice.!BMDMs(were(stimulated(with(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(1ug,(or(Imiquimod((R848)(at(1ug(for(4(or(24(hours(as(described(in(methods.(Cytokine(production(was(measured(by(sandwich(ELISA(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.((

Media

Bb MOI =

10

RNA 1ug/m

L

R848 1

ug/mL

0

1000

2000

3000

4000

IFNγ

4 hours

pg/m

L

WTTlr7-/-

Media

Bb MOI =

10

RNA 1ug/m

L

R848 1

ug/mL

0

500

1000

1500

2000

2500

IFNγ

24 hours

pg/m

L

WTTlr7-/-

Media

Bb MOI =

10

RNA 1ug/m

L

R848 1

ug/mL

0

5000

10000

15000

IL-6

24 hours

pg/m

L

WTTlr7-/-

Media

Bb MOI =

10

RNA 1ug/m

L

R848 1

ug/mL

0

20

40

60

IL-10

4 hours

pg/m

L

WTTlr7-/-

Media

Bb MOI =

10

RNA 1ug/m

L

R848 1

ug/mL

0

500

1000

1500

2000

2500

IL-10

24 hours

pg/m

L

WTTlr7-/-

A B

C D

E

140(

(((

((Figure!5.!ISG!induction!in!BMDMs!of!WT!and!Myd883/3!mice.!(BMDMs(were(stimulated(with(media((Mock),(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(2ug,(or(Imiquimod((R848)(at(1ug(as(described(in(methods(for(6(hours.(ISG(induction(was(measured(using(qRTIPCR(and(fold(change(was(calculated(as(previously(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.(

((

WT, Cxcl10

MyD88

-/-, Cxcl10

WT, Gbp2

MyD88

-/-, Gbp2

0

20

40

60

6 hours

Rel

ativ

e m

RN

A e

xpre

ssio

n (F

old

Cha

nge)

B. burgdorferi, MOI = 10Bb RNA 2ugR848 1ug

***

141(

((Figure!6.!ILV6!production!in!WT!and!Myd883/3!BMDMs.!BMDMs(were(stimulated(with(media,(B.*burgdorferi*(Bb)(at(an(MOI(=(10,(RNA(from(B.*burgdorferi*(Bb(RNA)(at(1ug,(or(the(TLR7(agonist(Imiquimod((R848)(at(1ug(for(24(hours(as(described(in(methods.(Cytokine(production(was(measured(by(sandwich(ELISA(as(described(in(methods.(Results(are(shown(as(Mean(±(SEM(and(from(one(representative(of(two(independent(experiments.(

(((((((((((((

142(

Table!1.!RNA!from!B.#burgdorferi#is!predicted!to!bind!TLR7.!!Sequences(of(Mouse(PRR’s((by(Uniprot(ID)(and(whole(genome(sequences(of(B31(and(N40(were(analyzed(for(RNAIProtein(Interactions(using(the(RPIISeq(program((53,(54).((RF,(SVM)>0.5(indicates(a(positive(predicted(binding(probability(between(Bb(RNA(and(the(PRR(in(the(left(column.(!

Host(Identifier(PRR(( Predicted(Binding(classifier((RF,(SVM)(B31((

Predicted(Binding(classifier((RF,(SVM)(N40(

>sp|P58681|TLR7_MOUSE!( 0.7( 0.85( 0.65( 0.992(

>sp|Q99MB1|TLR3_MOUSE!( 0.5( 0.92( 0.6( 0.996(

>sp|P58682|TLR8_MOUSE!( 0.5( 0.828( 0.65( 0.991(

>sp|Q6R5N8|TLR13_MOUSE!( 0.7( 0.806( 0.7( 0.992(

>sp|Q8VCW4|UN93B_MOUSE!( 0.55( 0.955( 0.8( 0.997(

>sp|Q6Q899|DDX58_MOUSE!( 0.75( 0.939( 0.65( 0.995(

>sp|Q8R5F7|IFIH1_MOUSE!( 0.65( 0.835( 0.75( 0.99(

>sp|P30204|MSRE_MOUSE!( 0.55( 0.938( 0.65( 0.994(

>sp|Q5ND28|SREC_MOUSE!( 0.6( 0.674( 0.75( 0.88(

>sp|Q8BV57|SRCRL_MOUSE!( 0.55( 0.93( 0.65( 0.995(

>sp|P59222|SREC2_MOUSE!( 0.5( 0.745( 0.65( 0.96(

>sp|A1L0T3|SRB4D_MOUSE!( 0.7( 0.72( 0.75( 0.968(

>sp|Q61009|SCRB1_MOUSE!( 0.75( 0.833( 0.7( 0.993(

>sp|Q8K299|SCAR5_MOUSE!( 0.55( 0.608( 0.65( 0.946(

>sp|Q2VLH6|C163A_MOUSE!( 0.6( 0.583( 0.55( 0.955(

>sp|Q8C850|SCAR3_MOUSE!( 0.35( 0.881( 0.65( 0.995(

>sp|Q3UP24|NLRC4_MOUSE!( 0.75( 0.888( 0.55( 0.995(

>sp|Q3TL44|NLRX1_MOUSE!( 0.6( 0.978( 0.75( 0.999(

((!!!

143(

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144(

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13.( Casjens(SR,(Mongodin(EF,(Qiu(WG,(Luft(BJ,(Schutzer(SE,(Gilcrease(EB,(et(al.(Genome(stability(of(Lyme(disease(spirochetes:(comparative(genomics(of(Borrelia(burgdorferi(plasmids.(PloS(one.(2012b7(3):e33280.(

14.( Di(L,(Pagan(PE,(Packer(D,(Martin(CL,(Akther(S,(Ramrattan(G,(et(al.(BorreliaBase:(a(phylogenyIcentered(browser(of(Borrelia(genomes.(BMC(Bioinformatics.(2014b15:233.(

15.( Fraser(CM,(Casjens(S,(Huang(WM,(Sutton(GG,(Clayton(R,(Lathigra(R,(et(al.(Genomic(sequence(of(a(Lyme(disease(spirochaete,(Borrelia(burgdorferi.(Nature.(1997(Dec(11b390(6660):580I6.(

16.( Marques(AR.(Lyme(disease:(a(review.(Curr(Allergy(Asthma(Rep.(2010(Janb10(1):13I20.(

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18.( Weis(JJ.(HostIpathogen(interactions(and(the(pathogenesis(of(murine(Lyme(disease.(Curr(Opin(Rheumatol.(2002(Julb14(4):399I403.(

19.( Kumar(H,(Kawai(T,(Akira(S.(TollIlike(receptors(and(innate(immunity.(Biochem(Biophys(Res(Commun.(2009(Oct(30b388(4):621I5.(

20.( Koening(CL,(Miller(JC,(Nelson(JM,(Ward(DM,(Kushner(JP,(Bockenstedt(LK,(et(al.(TollIlike(receptors(mediate(induction(of(hepcidin(in(mice(infected(with(Borrelia(burgdorferi.(Blood.(2009(Aug(27b114(9):1913I8.(

21.( Monroe(KM,(McWhirter(SM,(Vance(RE.(Induction(of(type(I(interferons(by(bacteria.(Cell(Microbiol.(2010(Julb12(7):881I90.(

22.( Miller(JC,(Ma(Y,(Bian(J,(Sheehan(KC,(Zachary(JF,(Weis(JH,(et(al.(A(critical(role(for(type(I(IFN(in(arthritis(development(following(Borrelia(burgdorferi(infection(of(mice.(Journal(of(immunology((Baltimore,(Md(:(1950).(2008(Dec(15b181(12):8492I503.(

23.( Khoo(JJ,(Forster(S,(Mansell(A.(TollIlike(receptors(as(interferonIregulated(genes(and(their(role(in(disease.(J(Interferon(Cytokine(Res.(2011(Janb31(1):13I25.(

145(

24.( Hirschfeld(M,(Kirschning(CJ,(Schwandner(R,(Wesche(H,(Weis(JH,(Wooten(RM,(et(al.(Cutting(edge:(inflammatory(signaling(by(Borrelia(burgdorferi(lipoproteins(is(mediated(by(tollIlike(receptor(2.(Journal(of(immunology((Baltimore,(Md(:(1950).(1999(Sep(1b163(5):2382I6.(

25.( Lien(E,(Sellati(TJ,(Yoshimura(A,(Flo(TH,(Rawadi(G,(Finberg(RW,(et(al.(TollIlike(receptor(2(functions(as(a(pattern(recognition(receptor(for(diverse(bacterial(products.(J(Biol(Chem.(1999(Nov(19b274(47):33419I25.(

26.( Wooten(RM,(Weis(JJ.(HostIpathogen(interactions(promoting(inflammatory(Lyme(arthritis:(use(of(mouse(models(for(dissection(of(disease(processes.(Curr(Opin(Microbiol.(2001(Junb4(3):274I9.(

27.( Wooten(RM,(Ma(Y,(Yoder(RA,(Brown(JP,(Weis(JH,(Zachary(JF,(et(al.(TollIlike(receptor(2(plays(a(pivotal(role(in(host(defense(and(inflammatory(response(to(Borrelia(burgdorferi.(Vector(Borne(Zoonotic(Dis.(2002(Winterb2(4):275I8.(

28.( Bolz(DD,(Sundsbak(RS,(Ma(Y,(Akira(S,(Kirschning(CJ,(Zachary(JF,(et(al.(MyD88(plays(a(unique(role(in(host(defense(but(not(arthritis(development(in(Lyme(disease.(Journal(of(immunology((Baltimore,(Md(:(1950).(2004(Aug(1b173(3):2003I10.(

29.( Shin(OS,(Isberg(RR,(Akira(S,(Uematsu(S,(Behera(AK,(Hu(LT.(Distinct(roles(for(MyD88(and(TollIlike(receptors(2,(5,(and(9(in(phagocytosis(of(Borrelia(burgdorferi(and(cytokine(induction.(Infection(and(immunity.(2008(Junb76(6):2341I51.(

30.( Petzke(MM,(Brooks(A,(Krupna(MA,(Mordue(D,(Schwartz(I.(Recognition(of(Borrelia(burgdorferi,(the(Lyme(disease(spirochete,(by(TLR7(and(TLR9(induces(a(type(I(IFN(response(by(human(immune(cells.(Journal(of(immunology((Baltimore,(Md(:(1950).(2009(Oct(15b183(8):5279I92.(

31.( Miller(JC,(MaylorIHagen(H,(Ma(Y,(Weis(JH,(Weis(JJ.(The(Lyme(disease(spirochete(Borrelia(burgdorferi(utilizes(multiple(ligands,(including(RNA,(for(interferon(regulatory(factor(3Idependent(induction(of(type(I(interferonIresponsive(genes.(Infection(and(immunity.(2010(Julb78(7):3144I53.(

32.( Cervantes(JL,(DunhamIEms(SM,(La(Vake(CJ,(Petzke(MM,(Sahay(B,(Sellati(TJ,(et(al.(Phagosomal(signaling(by(Borrelia(burgdorferi(in(human(monocytes(involves(TollIlike(receptor((TLR)(2(and(TLR8(cooperativity(and(TLR8Imediated(induction(of(IFNIbeta.(Proc(Natl(Acad(Sci(U(S(A.(2011(Mar(1b108(9):3683I8.(

33.( Cervantes(JL,(La(Vake(CJ,(Weinerman(B,(Luu(S,(O'Connell(C,(Verardi(PH,(et(al.(Human(TLR8(is(activated(upon(recognition(of(Borrelia(burgdorferi(RNA(in(the(phagosome(of(human(monocytes.(J(Leukoc(Biol.(2013(Decb94(6):1231I41.(

146(

34.( Cervantes(JL,(Hawley(KL,(Benjamin(SJ,(Weinerman(B,(Luu(SM,(Salazar(JC.(Phagosomal(TLR(signaling(upon(Borrelia(burgdorferi(infection.(Frontiers(in(cellular(and(infection(microbiology.(2014b4:55.(

35.( Love(AC,(Schwartz(I,(Petzke(MM.(Borrelia(burgdorferi(RNA(induces(type(I(and(III(interferons(via(TollIlike(receptor(7(and(contributes(to(production(of(NFIkappaBIdependent(cytokines.(Infection(and(immunity.(2014(Junb82(6):2405I16.(

36.( Fikrig(E,(Barthold(SW,(Marcantonio(N,(Deponte(K,(Kantor(FS,(Flavell(RA.(Roles(of(OspA,(OspB,(and(flagellin(in(protective(immunity(to(Lyme(borreliosis(in(laboratory(mice.(Infection(and(immunity.(1992(Febb60(2):657I61.(

37.( Radolf(JD,(Goldberg(MS,(Bourell(K,(Baker(SI,(Jones(JD,(Norgard(MV.(Characterization(of(outer(membranes(isolated(from(Borrelia(burgdorferi,(the(Lyme(disease(spirochete.(Infection(and(immunity.(1995(Junb63(6):2154I63.(

38.( Sellati(TJ,(Bouis(DA,(Kitchens(RL,(Darveau(RP,(Pugin(J,(Ulevitch(RJ,(et(al.(Treponema(pallidum(and(Borrelia(burgdorferi(lipoproteins(and(synthetic(lipopeptides(activate(monocytic(cells(via(a(CD14Idependent(pathway(distinct(from(that(used(by(lipopolysaccharide.(Journal(of(immunology((Baltimore,(Md(:(1950).(1998(Jun(1b160(11):5455I64.(

39.( Wooten(RM,(Morrison(TB,(Weis(JH,(Wright(SD,(Thieringer(R,(Weis(JJ.(The(role(of(CD14(in(signaling(mediated(by(outer(membrane(lipoproteins(of(Borrelia(burgdorferi.(Journal(of(immunology((Baltimore,(Md(:(1950).(1998(Jun(1b160(11):5485I92.(

40.( Benach(JL,(Coleman(JL,(GarciaIMonco(JC,(Deponte(PC.(Biological(activity(of(Borrelia(burgdorferi(antigens.(Ann(N(Y(Acad(Sci.(1988b539:115I25.(

41.( Dennis(VA,(Dixit(S,(O'Brien(SM,(Alvarez(X,(Pahar(B,(Philipp(MT.(Live(Borrelia(burgdorferi(spirochetes(elicit(inflammatory(mediators(from(human(monocytes(via(the(TollIlike(receptor(signaling(pathway.(Infection(and(immunity.(2009(Marb77(3):1238I45.(

42.( Liu(N,(Montgomery(RR,(Barthold(SW,(Bockenstedt(LK.(Myeloid(differentiation(antigen(88(deficiency(impairs(pathogen(clearance(but(does(not(alter(inflammation(in(Borrelia(burgdorferiIinfected(mice.(Infection(and(immunity.(2004(Junb72(6):3195I203.(

43.( Wooten(RM,(Ma(Y,(Yoder(RA,(Brown(JP,(Weis(JH,(Zachary(JF,(et(al.(TollIlike(receptor(2(is(required(for(innate,(but(not(acquired,(host(defense(to(Borrelia(burgdorferi.(Journal(of(immunology((Baltimore,(Md(:(1950).(2002(Jan(1b168(1):348I55.(

147(

44.( Miller(JC,(Ma(Y,(Crandall(H,(Wang(X,(Weis(JJ.(Gene(expression(profiling(provides(insights(into(the(pathways(involved(in(inflammatory(arthritis(development:(murine(model(of(Lyme(disease.(Exp(Mol(Pathol.(2008(Augb85(1):20I7.(

45.( Crandall(H,(Dunn(DM,(Ma(Y,(Wooten(RM,(Zachary(JF,(Weis(JH,(et(al.(Gene(expression(profiling(reveals(unique(pathways(associated(with(differential(severity(of(lyme(arthritis.(Journal(of(immunology((Baltimore,(Md(:(1950).(2006(Dec(1b177(11):7930I42.(

46.( Sahay(B,(Patsey(RL,(Eggers(CH,(Salazar(JC,(Radolf(JD,(Sellati(TJ.(CD14(signaling(restrains(chronic(inflammation(through(induction(of(p38IMAPK/SOCSIdependent(tolerance.(PLoS(pathogens.(2009(Decb5(12):e1000687.(

47.( Salazar(JC,(Pope(CD,(Sellati(TJ,(Feder(HM,(Jr.,(Kiely(TG,(Dardick(KR,(et(al.(Coevolution(of(markers(of(innate(and(adaptive(immunity(in(skin(and(peripheral(blood(of(patients(with(erythema(migrans.(Journal(of(immunology((Baltimore,(Md(:(1950).(2003(Sep(1b171(5):2660I70.(

48.( Lochhead(RB,(Sonderegger(FL,(Ma(Y,(Brewster(JE,(Cornwall(D,(MaylorIHagen(H,(et(al.(Endothelial(cells(and(fibroblasts(amplify(the(arthritogenic(type(I(IFN(response(in(murine(Lyme(disease(and(are(major(sources(of(chemokines(in(Borrelia(burgdorferiIinfected(joint(tissue.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Sep(1b189(5):2488I501.(

49.( PetnickiIOcwieja(T,(Kern(A.(Mechanisms(of(Borrelia(burgdorferi(internalization(and(intracellular(innate(immune(signaling.(Frontiers(in(cellular(and(infection(microbiology.(2014b4:175.(

50.( PetnickiIOcwieja(T,(Chung(E,(Acosta(DI,(Ramos(LT,(Shin(OS,(Ghosh(S,(et(al.(TRIF(mediates(TollIlike(receptor(2Idependent(inflammatory(responses(to(Borrelia(burgdorferi.(Infection(and(immunity.(2013(Febb81(2):402I10.(

51.( Brown(CR,(Reiner(SL.(Experimental(lyme(arthritis(in(the(absence(of(interleukinI4(or(gamma(interferon.(Infection(and(immunity.(1999(Julb67(7):3329I33.(

52.( Brown(JP,(Zachary(JF,(Teuscher(C,(Weis(JJ,(Wooten(RM.(Dual(role(of(interleukinI10(in(murine(Lyme(disease:(regulation(of(arthritis(severity(and(host(defense.(Infection(and(immunity.(1999(Octb67(10):5142I50.(

53.( Muppirala(UK,(Honavar(VG,(Dobbs(D.(Predicting(RNAIprotein(interactions(using(only(sequence(information.(BMC(Bioinformatics.(2011b12:489.(

148(

54.( Suresh(V,(Liu(L,(Adjeroh(D,(Zhou(X.(RPIIPred:(predicting(ncRNAIprotein(interaction(using(sequence(and(structural(information.(Nucleic(Acids(Res.(2015(Feb(18b43(3):1370I9.(

55.( Shin(OS,(Miller(LS,(Modlin(RL,(Akira(S,(Uematsu(S,(Hu(LT.(Downstream(signals(for(MyD88Imediated(phagocytosis(of(Borrelia(burgdorferi(can(be(initiated(by(TRIF(and(are(dependent(on(PI3K.(Journal(of(immunology((Baltimore,(Md(:(1950).(2009(Jul(1b183(1):491I8.(

56.( Lochhead(RB,(Ma(Y,(Zachary(JF,(Baltimore(D,(Zhao(JL,(Weis(JH,(et(al.(MicroRNAI146a(provides(feedback(regulation(of(lyme(arthritis(but(not(carditis(during(infection(with(Borrelia(burgdorferi.(PLoS(pathogens.(2014(Junb10(6):e1004212.(

57.( KrupnaIGaylord(MA,(Liveris(D,(Love(AC,(Wormser(GP,(Schwartz(I,(Petzke(MM.(Induction(of(type(I(and(type(III(interferons(by(Borrelia(burgdorferi(correlates(with(pathogenesis(and(requires(linear(plasmid(36.(PloS(one.(2014b9(6):e100174.(

58.( Bikker(A,(Kruize(AA,(van(der(WurffIJacobs(KM,(Peters(RP,(Kleinjan(M,(Redegeld(F,(et(al.(InterleukinI7(and(TollIlike(receptor(7(induce(synergistic(B(cell(and(T(cell(activation.(PloS(one.(2014b9(4):e94756.(

59.( Hanten(JA,(Vasilakos(JP,(Riter(CL,(Neys(L,(Lipson(KE,(Alkan(SS,(et(al.(Comparison(of(human(B(cell(activation(by(TLR7(and(TLR9(agonists.(BMC(Immunol.(2008b9:39.(

60.( Hashimoto(Y,(Abu(Lila(AS,(Shimizu(T,(Ishida(T,(Kiwada(H.(B(cellIintrinsic(tollIlike(receptor(7(is(responsible(for(the(enhanced(antiIPEG(IgM(production(following(injection(of(siRNAIcontaining(PEGylated(lipoplex(in(mice.(J(Control(Release.(2014(Jun(28b184:1I8.(

61.( Braun(D,(Caramalho(I,(Demengeot(J.(IFNIalpha/beta(enhances(BCRIdependent(B(cell(responses.(Int(Immunol.(2002(Aprb14(4):411I9.(

62.( Maglione(PJ,(Simchoni(N,(Black(S,(Radigan(L,(Overbey(JR,(Bagiella(E,(et(al.(IRAKI4(and(MyD88(deficiencies(impair(IgM(responses(against(TIindependent(bacterial(antigens.(Blood.(2014(Dec(4b124(24):3561I71.(

63.( Poovassery(JS,(Bishop(GA.(Type(I(IFN(receptor(and(the(B(cell(antigen(receptor(regulate(TLR7(responses(via(distinct(molecular(mechanisms.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Aug(15b189(4):1757I64.(

64.( Hawley(KL,(Olson(CM,(Jr.,(IglesiasIPedraz(JM,(Navasa(N,(Cervantes(JL,(Caimano(MJ,(et(al.(CD14(cooperates(with(complement(receptor(3(to(mediate(

149(

MyD88Iindependent(phagocytosis(of(Borrelia(burgdorferi.(Proc(Natl(Acad(Sci(U(S(A.(2012(Jan(24b109(4):1228I32.(

65.( George(CX,(John(L,(Samuel(CE.(An(RNA(editor,(adenosine(deaminase(acting(on(doubleIstranded(RNA((ADAR1).(J(Interferon(Cytokine(Res.(2014(Junb34(6):437I46.(

66.( Sarvestani(ST,(Tate(MD,(Moffat(JM,(Jacobi(AM,(Behlke(MA,(Miller(AR,(et(al.(InosineImediated(modulation(of(RNA(sensing(by(TollIlike(receptor(7((TLR7)(and(TLR8.(J(Virol.(2014(Janb88(2):799I810.(

67.( Sedger(LM.(microRNA(control(of(interferons(and(interferon(induced(antiIviral(activity.(Mol(Immunol.(2013(Decb56(4):781I93.(

68.( Hidmark(A,(von(Saint(Paul(A,(Dalpke(AH.(Cutting(edge:(TLR13(is(a(receptor(for(bacterial(RNA.(Journal(of(immunology((Baltimore,(Md(:(1950).(2012(Sep(15b189(6):2717I21.(

69.( Li(XD,(Chen(ZJ.(Sequence(specific(detection(of(bacterial(23S(ribosomal(RNA(by(TLR13.(Elife.(2012b1:e00102.(

70.( Oldenburg(M,(Kruger(A,(Ferstl(R,(Kaufmann(A,(Nees(G,(Sigmund(A,(et(al.(TLR13(recognizes(bacterial(23S(rRNA(devoid(of(erythromycin(resistanceIforming(modification.(Science.(2012(Aug(31b337(6098):1111I5.(

71.( Morrison(TB,(Ma(Y,(Weis(JH,(Weis(JJ.(Rapid(and(sensitive(quantification(of(Borrelia(burgdorferiIinfected(mouse(tissues(by(continuous(fluorescent(monitoring(of(PCR.(J(Clin(Microbiol.(1999(Aprb37(4):987I92.(

72.( Morrison(TB,(Weis(JJ,(Wittwer(CT.(Quantification(of(lowIcopy(transcripts(by(continuous(SYBR(Green(I(monitoring(during(amplification.(Biotechniques.(1998(Junb24(6):954I8,(60,(62.(

(

(

((

(

(

150(

Chapter!4!

Discussion!The(overall(objective(of(the(research(presented(in(this(dissertation(was(to(

identify(how(TLR(signaling(contributed(to(immune(responses(during(infection(by(L.*

interrogans*and(B.*burgdorferi.(Previous(work(has(implicated(TLR2,(TLR4,(and(the(

adaptor(MyD88(in(an(effective(immune(response(to(leptospiral(infection.(Here,(we(

have(shown(that(TRIF(contributes(to(the(immune(response(to(L.*interrogans(in(mice(

in(the(kidneys((Chapter(2).(Additional(studies(have(implicated(numerous(TLRs(in(

immune(responses(during(borrelial(infection.(The(results(from(Chapter(3(suggest(

that(TLR7(is(another(PRR(that(plays(a(role(in(murine(host(defense.((

Our(results(support(previous(studies(that(demonstrate(the(importance(of(TLR(

signaling,(and(identify(a(novel(contributing(role(for(TRIF(during(the(early(responses(

to(leptospiral(pathogenesis.((Our(data(extend(this(work(by(suggesting(distinct(

mechanisms(of(infection(that(generate(a(synergistic(role(for(TLR(adaptors(during(

leptospiral(infection.(The(combined(role(of(both(MyD88(and(TRIF(during(leptospiral(

infection(remains(to(be(determined.(((L.*interrogans(infection(has(been(studied(in(

several(tissues,(including(the(livers,(spleens(and(lymph(nodes,(but(the(primary(site(of(

colonization(and(establishment(of(persistent(infection(is(the(kidney.(Livers(and(

spleens(were(among(the(tissues(not(examined(in(our(model(of(infection,(but(

differences(in(serum(antiILeptospira(IgM(between(WT(and(Trif5/5(mice(indicate(that(

additional(experiments(in(the(spleens(and(lymph(nodes(would(provide(greater(

understanding(of(TRIF(mediated(B(cell(activation(during(leptospiral(infection.(Gross(

151(

observations(of(other(tissues(in(Trif5/5(infected(mice(revealed(discolored(spleens(at(1(

d.p.i.(and(3(d.p.i.(and(spotted(livers(at(1(d.p.i.,(and(no(differences(in(appearance(of(

other(tissues(were(observed(at(7(d.p.i.(or(14(d.p.i.(((Jayaraman,(unpublished(data).((

We(had(also(identified(higher(trending(proIinflammatory(cytokine(levels(in(kidneys(

using(qRTIPCR,(but(by(7(d.p.i.,(these(responses(are(not(increased(in(Trif5/5(mice(

compared(to(WT(in(multiple(tissues.(Collectively,(these(results(point(to(TRIF(as(an(

early(modulator(of(the(immune(response(to(leptospires.(

We(were(not(able(to(detect(IFNβ(in(sera(or(kidney(lysates(of(our(mice,(but(it(is(

possible(that(IFNβ(may(also(appear(earlier(during(infection.(((IFNβ(levels(are(

upregulated(in(Murine(Peritoneal(Macrophages(from(Balb/c(mice(earlier(with(L.*

interrogans(but(upregulation(is(detected(later(in(human(blood(monocytes((1).(

C57BL/6(mice(lacking(IFNγ(do(not(die(after(injection(with(up(to(2x(108(leptospires(

and(colonization(in(the(renal(tubules(had(been(detected(by(staining(of(tissues(at(28(

d.p.i.((2).((Responses(were(identified(by(histopathological(analysis(of(kidney(sections(

to(identify(interstitial(nephritis,(but(there(were(no(differences(detected(between(IFNγI/I(

mice(and(C57BL/6(mice((2).(Our(results(showed(an(elevated(Type(II(IFN(response(

as(well(as(increased(STAT1(expression(in(the(kidneys(and(blood(of(Trif5/5(infected(

mice(at(1(d.p.i..(This(may(suggest(a(potential(mechanism(in(Trif5/5(mice(during(

infection,(where(upregulation(of(STAT1(results(in(IFNγ(production(instead(of(IFNβ(

production,(and(subsequent(recruitment(of(IFNγ(producing(cells(like(NK(and(CD4(T(

cells(during(acute(leptospiral(infection.(((Additional(studies(with(Stat15/5(mice(and(

152(

IFNAR5/5(mice(are(required(to(determine(if(this(is(the(case,(where(similar(experiments(

with(IFNAR5/5(mice(would(determine(whether(Type(I(IFN(affects(immune(responses(

during(acute(Leptospiral(infection,(including(differences(in(Type(II(IFN(and(proI

inflammatory(cytokine(responses,(as(well(as(activation(of(humoral(responses(and(

adaptive(immunity.((Mice(lacking(STAT1(have(a(higher(proIinflammatory(response(in(

hearts(following(infection(by(B.*burgdorferi*compared(to(resistant(models,(and(

inflammation(is(mediated(by(IFNγ(production((3).((Since(the(Type(I(and(II(IFN(

responses(have(not(been(collectively(explored(in(the(context(of(Leptospiral(infection,(

additional(experiments(with(Stat15/5(mice(would(determine(how(interferons(regulate(

inflammatory(responses(during(Leptospiral(infection.(

Our(TRIF(mice(showed(increased(proIinflammatory(responses(in(the(Type(I(

cytokine(levels,(where(mRNA(results(correlated(with(protein(levels.(Increased(levels(

of(proIinflammatory(transcripts(like(IL18(and(NLRP3(have(also(been(identified(in(the(

kidneys(at(3(d.p.i.,(whereas(preliminary(results(show(a(downregulation(of(

inflammatory(cytokines(in(the(livers(at(7(d.p.i.((Jayaraman,(unpublished(data).(These(

results(are(consistent(with(our(model(and(with(previous(studies(addressing(ILI1(

family(proteins(and(NLRP3(signaling(during(Leptospiral(infection(of(bone(marrow(

derived(macrophages((4).(We(have(additionally(showed(that(the(proIinflammatory(

responses(are(kidney(specific,(even(after(detecting(early(ILI6(responses(are(found(in(

both(kidneys(and(sera(of(infected(groups.(Similar(results(have(recently(been(

153(

published(in(a(sublethal(model(of(leptospiral(infection(implicating(the(importance(of(

understanding(mechanisms(that(underly(acute(Leptospiral(infection((5).(((

The(proposed(studies(with(mice(lacking(STAT1,(Type(I(or(II(IFN,(in(addition(to(

a(newly(proposed(animal(model(and(whole(tissue(responses(would(provide(insight(

into(the(mechanisms(of(host(defense(against(leptospiral(infection.(It(is(important(to(

note(that(the(primary(site(of(colonization(is(the(renal(tubules,(and(so(understanding(

mechanisms(of(pathogenesis(in(that(specific(cell(type(would(enable(a(greater(

understanding(of(what(underlies(innate(responses(and(acute(infection.(Moving(

forward,(novel(techniques(like(live(bioluminescent(imaging(have(allowed(scientists(to(

visualize(the(initial(mechanism(of(bacterial(dissemination(and(renal(colonization(that(

results(in(persistent(infection((6).(Additional(studies(addressing(differential(gene(and(

protein(expression(in(cell(types(that(are(more(relevant(to(colonization,(like(renal(

proximal(tubules(in(mice,(might(provide(more(insight(into(mechanisms(of(acute(

infection(and(leptospiral(persistence(in(mice.(((

!

Borrelia#burgdorferi!

Previous(studies(had(shown(that(TLR(signaling(is(an(essential(component(of(

the(innate(immune(response(against(B.*burgdorferi((7).(In(addition(to(this,(total(RNA(

from(B.*burgdorferi(was(identified(as(an(additional(ligand(that(induced(TLR(mediated(

immune(responses((8).(We(have(extended(our(findings(from(the(previously(

established(Human(TLR7(and(TLR8(to(murine(TLR7IMyD88,(providing(insight(into(

154(

Bb(RNA(mediated(Type(I(IFN(and(ISG(induction(in(a(TLR7IMyD88(dependent(

manner(in(mice,(which(supports(previous(findings(in(human(cell(types((9,(10).(

Additional(studies(are(ongoing(to(characterize(the(RNA(ligands(that(bind(murine(

TLR7((Devlin(et(al(unpublished(data).(The(Bb(RNA(response(to(the(MyD88(has(

previously(been(studied(in(human(cell(types.(We(had(also(found(dampened(

responses(to(Bb(RNA(in(MyD88I/I(macrophages,(which(supports(previous(studies(in(

human(cell(types(and(recent(work(in(mice((9,(11,(12).((Because(RNA(from(B.(

burgdorferi(also(induces(a(Type(I(IFN(response,(which(is(a(hallmark(of(the(innate(

immune(response,(characterizing(the(RNA(may(identify(additional(targets(for(

treatment(of(infection.(It(is(also(possible(that(other(mechanisms(targeting(Bb(RNA(

might(induce(a(Type(I(IFN(response.(The(roles(of(cytosolic(PRRs(including(RIGII(

and(MDA5(in(RNA(recognition(have(not(yet(been(elucidated.(Collectively(this(body(of(

work(demonstrates(the(importance(of(PRR(signaling,(specifically(TLR(signaling,(in(

host(defense(and(innate(immunity(during(Leptospiral(or(Borrelial(infection.((

!

!

!

!

!

!

155(

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6.( Ratet(G,(Veyrier(FJ,(Fanton(d'Andon(M,(Kammerscheit(X,(Nicola(MA,(Picardeau(M,(et(al.(Live(imaging(of(bioluminescent(leptospira(interrogans(in(mice(reveals(renal(colonization(as(a(stealth(escape(from(the(blood(defenses(and(antibiotics.(PLoS(neglected(tropical(diseases.(2014(Decb8(12):e3359.(

7.( PetnickiIOcwieja(T,(Kern(A.(Mechanisms(of(Borrelia(burgdorferi(internalization(and(intracellular(innate(immune(signaling.(Frontiers(in(cellular(and(infection(microbiology.(2014b4:175.(

8.( Miller(JC,(MaylorIHagen(H,(Ma(Y,(Weis(JH,(Weis(JJ.(The(Lyme(disease(spirochete(Borrelia(burgdorferi(utilizes(multiple(ligands,(including(RNA,(for(interferon(regulatory(factor(3Idependent(induction(of(type(I(interferonIresponsive(genes.(Infection(and(immunity.(2010(Julb78(7):3144I53.(

9.( Cervantes(JL,(La(Vake(CJ,(Weinerman(B,(Luu(S,(O'Connell(C,(Verardi(PH,(et(al.(Human(TLR8(is(activated(upon(recognition(of(Borrelia(burgdorferi(RNA(in(the(phagosome(of(human(monocytes.(J(Leukoc(Biol.(2013(Decb94(6):1231I41.(

156(

10.( Love(AC,(Schwartz(I,(Petzke(MM.(Borrelia(burgdorferi(RNA(induces(type(I(and(III(interferons(via(TollIlike(receptor(7(and(contributes(to(production(of(NFIkappaBIdependent(cytokines.(Infection(and(immunity.(2014(Junb82(6):2405I16.(

11.( PetnickiIOcwieja(T,(Chung(E,(Acosta(DI,(Ramos(LT,(Shin(OS,(Ghosh(S,(et(al.(TRIF(mediates(TollIlike(receptor(2Idependent(inflammatory(responses(to(Borrelia(burgdorferi.(Infection(and(immunity.(2013(Febb81(2):402I10.(

12.( Hastey(CJ,(Ochoa(J,(Olsen(KJ,(Barthold(SW,(Baumgarth(N.(MyD88I(and(TRIFIindependent(induction(of(type(I(interferon(drives(naive(B(cell(accumulation(but(not(loss(of(lymph(node(architecture(in(Lyme(disease.(Infection(and(immunity.(2014(Aprb82(4):1548I58.(

(

(