Research Article Low Dose BCG Infection as a Model for

Research ArticleLow Dose BCG Infection as a Model for MacrophageActivation Maintaining Cell Viability

Leslie Chaacutevez-Galaacuten12 Dominique Vesin1 Denis Martinvalet3 and Irene Garcia1

1Department of Pathology and Immunology Centre Medical Universitaire (CMU) Faculty of Medicine University of GenevaGeneva Switzerland2Laboratory of Integrative Immunology National Institute of Respiratory Diseases ldquoIsmael Cosio Villegasrdquo Mexico City Mexico3Department of Cell Physiology and Metabolism Centre Medical Universitaire (CMU) Faculty of Medicine University of GenevaGeneva Switzerland

Correspondence should be addressed to Irene Garcia irenegarcia-gabayunigech

Received 18 May 2016 Revised 26 August 2016 Accepted 15 September 2016

Academic Editor Ethan M Shevach

Copyright copy 2016 Leslie Chavez-Galan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Mycobacterium bovis BCG the current vaccine against tuberculosis is ingested by macrophages promoting the development ofeffector functions including cell death andmicrobicidalmechanisms Despite accumulating reports onM tuberculosis mechanismsof BCGmacrophage interaction remain relatively undefined In vivo few bacilli are sufficient to establish a mycobacterialinfection however in vitro studies systematically use high mycobacterium doses In this study we analyze macrophageBCGinteractions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activationwithout affecting viability We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels ofproinflammatory cytokines and transcription factors while MOI 01 was more efficient for early stimulation of IL-1120573 MCP-1 andKC Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrialstructures Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 01 infection In conclusionBCG infection at low dose is an efficient in vitromodel to studymacrophageBCG interactions that maintains macrophage viabilityand mitochondrial structures This represents a novel model that can be applied to BCG research fields including mycobacterialinfections cancer immunotherapy and prevention of autoimmunity and allergies

1 Introduction

Mycobacterium tuberculosis (M tuberculosis) the causativeagent of tuberculosis (TB) infection is a major global healthproblem including transmission of drug-resistant strains(MDR-TB) and the increased risk of TB among HIV-infectedpersons [1] The World Health Organization estimated thatin 2013 there were 9 million cases of TB and 15 million diedfrom the disease TB cases coinfected with HIV were around11 million with an estimated 480000 new cases of MDR-TB This report indicates the need to intensify the efforts inTB control and to give access to high-quality care for all TBpatients [2]

Mycobacterium bovis Bacillus Calmette-Guerin (BCG)the current vaccine used against TB is a live attenuated

mycobacteriumwhichwas isolated in 1908 and administratedfor the first time to newborn infants in 1921 [3] BCG genomecontains a deletion in the region of differentiation 1 (RD1) andits cytolytic activity is decreased compared toM tuberculosis[4 5] The BCG strains used in clinics were derived fromthe original BCG for example BCG Pasteur Danish JapanMoreau Tice and Connaught each vaccine has its ownimmunogenic ability in both animals and humans [6 7]BCG vaccine provides effective protection against childhoodTB but the level of protection against adult pulmonary TBcan be variable [8] To date there are significant advancesin development of new vaccines against TB however sofar BCG remains the only licensed vaccine to preventTB and over 2 billion people have been immunized withBCG

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 4048235 17 pageshttpdxdoiorg10115520164048235

2 Journal of Immunology Research

Currently BCG clinical application is not limited tothe field of mycobacterial infections Adjuvant instillationof BCG as standard immunotherapy used in non-muscle-invasive bladder cancer (NMIBC) results in a significantreduction in tumor and progression It has been describedthat high doses of BCG Connaught induce a high Th1response prime CD8+ T cells and efficiently prevent recur-rences of NMIBC [9] However the optimal dose and dura-tion of treatment in NMIBC are controversial and derivedside effects may lead to premature interruption of treatmentNew treatment schedules of BCGwith no negative impact onpatients are considered [10]

Macrophages are part ofmononuclear phagocytic systemand the alveolar macrophage is the first cell to encounterM tuberculosis after infection however the bacillus has theability to evade microbicidal activities For example Mtuberculosis inhibits phagolysosomal fusion consequentlymacrophage provides a niche for mycobacteria and althoughit is a hostile environment it is sufficient for bacillus growth[11 12]

Using human monocytes and macrophages and macro-phage cell lines it has been shown that avirulent strains ofmycobacteria induce higher levels of apoptosis than viru-lent strains which represent a mechanism of host defensewhich can be subverted by virulent M tuberculosis toescape from innate immunity [13ndash15] However data havealso reported that virulent and avirulent M tuberculosisstrains can both inhibit and promote apoptosis accordingto experimental conditions [16ndash19] Despite accumulatingdata on macrophage activation and death by M tuberculosisstrains BCG-mediated cellular activities in phagocytic cellsremain relatively unexplored Studies comparing BCG withM tuberculosis have frequently used high Multiplicity-of-Infection (MOI) In general studies use BCG infection atMOI 10 (10 bacillicell) pointing apoptosis as a cellularstrategy to eliminate mycobacteria [20 21]

BCG-induced activation ofmacrophages has been associ-ated with TNF production which is a critical cytokine in hostdefense mechanisms against mycobacterial infection [22]Both BCG-infected and uninfected macrophages produceTNF although in lower amount than macrophages infectedwith virulent strains in which TNF supports mycobacterialgrowth [23] BCG-induced TNF activates iNOS but TNF cancontrol intracellular BCG growth by iNOS-dependent andiNOS-independent pathways [24] Collectively these modelshave provided important insights into immune responseagainst mycobacteria but considering that a low dose ofbacteria (1 bacillus) is sufficient to establish a mycobacterialinfection in the host it is of interest to analyze macrophageresponses in terms of cell death activation and integrity tolow number of mycobacteria [25]

In the present work we compare RAW macrophageresponses upon infection with BCG Pasteur at MOI 1 andMOI 01 the last is considered as a very low dose of infectionThis study aims (1) to evaluate if low doses of BCG Pasteurare sufficient to activatemacrophages whilemaintaining theirviability and mitochondrial integrity and (2) to determineif under low MOIs the macrophage is able to retain theirfunctional activities over time after infection Our results

show that low BCG doses are an efficient in vitro infec-tiousmodel to study interactions betweenmacrophagesBCGmaintaining viablemacrophages andmitochondrial integrity

2 Material and Methods

21 Cell Culture The murine macrophage cell line RAW2647 (RAW macrophages) was purchased from Ameri-can Type Culture Collection (Rockville MD) The cellswere maintained in DMEM supplemented with 10 head-inactivated FBS penicillin streptomycin sodium pyruvateglutamine and HEPES (complete DMEM) at 37∘C in ahumidified atmosphere containing 5 CO

2

22 M bovis BCG M bovis BCG Pasteur strain 1172 P2(Pasteur Institute Paris France) was used and grown to thelog phase in 7H9 middlebrook medium supplemented witholeic albumin dextrose catalase (OADC) The bacteria werethen harvested washed and frozen at minus80∘C in PBS plus 10of glycerol Bacterial load was determined by plating serial10-fold dilutions on 7H10 middlebrook agar (supplementedwith OADC) and counting colonies after incubation forat least 3 weeks [26] A BCG-GFP M bovis BCG Pasteurstrain harboring phsp60-gfp expressing Green FluorescenceProtein (BCG-GFP)was grown in the presence of Kanamycinas previously reported [27]

23 BCG Infection RAW macrophages were cultured in24-well flat-bottomed cell culture plates (1 times 106mL) andinfected with BCG Pasteur at MOI 1 or MOI 01 along 25 18 24 30 48 and 72 hours (h) at 37∘C in a humidifiedatmosphere containing 5 CO

2

24 Evaluation of Cell Death by Flow Cytometry Endingthe culture cells were harvested by adding cold PBS toculture plates and maintaining for 10 minutes on ice todetach cells from plastic plates Cells were washed in PBA(phosphate buffered saline containing 01 Sodium Azideand 01 Albumin Bovine) and incubated with 3 120583L 7-AAD(eBioscience) solution for 20min at 4∘C in the dark washedin PBA suspended in binding buffer 1x (BD pharmingen)and incubated with 5 120583L Annexin-V FITC-conjugated orAPC-conjugated (when using BCG-GFP infection) (eBio-science) for 15min at room temperature in the dark Datawere collected using a FACs CyAn (Beckton Dickinson Inc)within one hour and then analyzed with FlowJo software(Tree Star Inc) 50000 events were acquired per sample

25 Flow Cytometry The percentage of RAW macrophagesexpressing transmembrane TNF (tmTNF) and intracellularTNF (iTNF)was assessed by flow cytometry Briefly for iTNFending the culture cells were harvested washed in PBAand fixed in 4 formaldehyde for 10min at 4∘C Cells wereincubated with saponin-containing buffer shaken gently for10min at 4∘C and then incubated with anti-TNF (MP6-XT22) conjugated PE Cy7 (eBioscience) for 30min at roomtemperature in the dark and washed with PBS-saponin FortmTNF label cells were harvested washed in PBA and fixedin 4 formaldehyde RAW macrophages were suspended in

Journal of Immunology Research 3

PBA and stained with anti-TNF 30min at 4∘C and washedwith PBA Fluorochrome-labeled isotype-matched controlantibody was used to evaluate background staining Afterincubation with antibodies cells were washed twice in PBAand data collected using a FACs CyAn and analyzed withFlowJo software 100000 events were acquired per sample

26 Cytokine and Chemokine Measurements At differenttime points after BCG infection cell-free supernatants werecollected and frozen at minus80∘C for cytokine or chemokineassessment Cytokine amounts were assessed by ELISA forTNFR1 TNFR2 TNF IL-6 IL-1120573 MIP-1120572 MCP-1 and KCin accordance with the manufacturerrsquos instructions

27Nitrite Assay Cell-free supernatantswere collected at dif-ferent time points after BCG infection and nitrite content wasmeasured using Griess method as previously described [28]The plate was incubated with Griess reagent (sulfonamide 1plus N-(1-naphthyl)-ethylenediamine dihydrochloride 01in phosphoric acid 25) at room temperature for 5min in thedark Absorbance at 570 nm was measured with a microplatereader NO concentrations were calculated using a standardcurve

28 Western Blotting Ending the culture cells were har-vested washed in PBS and lysed in RIPA buffer containingprotease inhibitor (Complete Mini Protease Inhibitor Cock-tail Tablet Roche) Cellular protein extracts were separatedby SDS-PAGE and transferred to 02120583mpore-size nitrocellu-lose membranes (Bio-Rad Laboratories Hercules CA USA)with 25mm Tris-base (pH 80) containing 150mm glycineand 20 (volumevolume) methanol as previously described[28] Membranes were incubated with antibodies to phos-pho-p4442 MAPK (ERK12) phospho-nuclear factorkappa-light-chain-enhancer of activated B cells (NF120581B p65)phospho-apoptosis signal-regulating kinase 1 (ASK1)(Ser967) (from Cell Signaling Technology) rabbit poly-clonal anti-iNOS (Calbiochem Merck) and anti-caspase-1(clone 5B10) (BioLegend) Protein bands were detected byincubating with horseradish peroxidase-labeled antibodiesand visualized with enhanced chemiluminescence reagent(Advantas) using an ImageQuant Las-4000mini (GEHealth-care Life Science) Band densities were analyzed bydensitometry using the online IMAGEJ 139c software(National Institutes of Health) (httprsbinfonihgovijindexhtml) as described by Luke Miller (httpwwwluke-millerorgjournal200708quantifying-western-blots-with-outhtml) Samples were normalized using tubulin as loadingcontrol

29 Macrophage Proliferation Assay RAWmacrophage pro-liferation was determined using the Click-iT EdU FlowCytometry assay kit (Invitrogen Life Technology Inc) inaccordance with the manufacturerrsquos instructions Brieflyfresh RAW macrophages were cultured in 24-well flat-bottomed cell culture plates (1 times 106mL) in DMEMF-12medium supplemented (volumevolume) with supernatantsrecovered from different time points and MOI cultures of

infected macrophages Three 120583M of EdU (5-ethylnyl-21015840-deoxyuridine) was added to cultures and cells were left for12 or 24 h in culture at 37∘C in a humidified atmosphere con-taining 5 CO

2 Cells were harvested fixed permeabilized

and stained with Alexa Fluor 488 dye for detention of DNAsynthesis As control of proliferation RAWmacrophages cul-tured in DMEMF-12 medium was used Data were collectedusing a FACs CyAn analyzed with FlowJo software 50000events were acquired per sample

210 Transfection of RAW Cells and Analysis by ConfocalMicroscopy RAW macrophages were transfected with plas-mid encoding mitochondrial targeted red fluorescein protein(mitoRFP) at final concentration of 3 120583g plasmid1times106 cellsto generate RAW-mitoRFP We used a Neon TransfectionSystem under conditions 1680 v (pulse voltage) 20ms (pulsewidth) and 1 (pulse number) andmaintained in DMEM cul-ture medium plus G418 antibiotic (500 120583gmL) (MediatechInc Manassas VA) Transfected macrophages were sortedbased on the expression of mitoRFP using a FACs-Aria IIwe obtained 97 of RAW cells which were RAW-mitoRFP+Sorted RAW-mitoRFP+ cells were then infected with BCG-GFP at MOI 01 and MOI 1 Cultures were done on ChamberSlide System (Thermo Scientific Inc Waltham MA) andmaintained 2 5 or 18 h at 37∘ in a humidified atmospherecontaining 5 CO

2 Cells were washed with PBS and fixed

in 4 formaldehyde during 15 minutes and mounted withmounting medium containing DAPI (Vector LaboratoriesInc Burlingame CA) The slides were examined by SP5confocal microscopy and the Leica Application Suite (LAS)software was used for analysis (Leica Microsystems Co)

211 Statistical Analysis Results are expressed as means plusmnSEM Data comparisons were performed using one-wayANOVA followed by a Dunnettrsquos post hoc test for multiplecomparisons Two-tailed unpaired Studentrsquos 119905-testwas used toevaluate differences between 2 independent groups (Graph-Pad Software Inc San Diego CA) A 119875 value lt005 wasconsidered to be statistically significant

3 Results

31 BCG Infection at MOI 1 but Not MOI 01 Induced CellDeath We have first assessed early and late apoptosis andnecrosis in RAW macrophages induced by BCG Pasteurinfection using MOI 1 (1 bacilluscell) versus MOI 01 (1bacillus10 cells) by flow cytometry Studies comparing BCGwith M tuberculosis have frequently used high MOI Ingeneral studies have used BCG infection at MOI 10 (10bacillicell) showing that apoptosis is a cell strategy toeliminate mycobacteria [20 21]

Cells were analyzed in a dot plot color where Annexin-V+ cells indicated early apoptotic cells Annexin-V+7-AAD+late apoptotic cells 7-AAD+ necrotic cells and negative cellsfor both markers were living cells as using a general gate(Figures 1(a) and 1(b)) At MOI 1 we observed an increaseof early apoptosis at 5 h after infection whereas late apoptosisincreased at 5 h and 18 h contrasting with MOI 01 infected


UninfectedA

nnex

in-V

7-AAD

Early apoptosis

Late apoptosis

Live cells

Necrotic cells

4 plusmn 05 3 plusmn 05


(a)

SSC

FSC

On general gate

(b)

Ann

exin

-VA

nnex

in-V

Ann

exin

-VA

nnex

in-V

7-AAD 7-AAD

7-AAD7-AAD

MO

I 1M

OI 0

1

MO

I 1M

OI 0

1

BCG (5h) BCG (18h)


86 plusmn 21 plusmn 005


87 plusmn 3

2 plusmn 02





(c)

Figure 1 Continued


Early

apop

totic

cells

()

0

2

4

6

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10

5 18

BCG (hour)

0

lowastlowast

MOI 1MOI 01

(d)

Late

apop

totic

cells

()

0

5

10

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5 18

BCG (hour)

0

lowast

MOI 1MOI 01

lowast

(e)

Figure 1 BCG infection atMOI 1 but notMOI 01 induces cell death (a) Uninfected RAWmacrophages were cultured or (b) and (c) infectedwith BCGPasteur atMOI 1 andMOI 01 during 2 5 and 18 hours and stainedwithAnnexin-V and 7-AAD to assess the percentage of cell deathby flow cytometry as shown in the representative plots (b) Representative FCS and SSC scatter plots of cells infected for 5 h with MOI 1 and(c) Annexin-V versus 7-AAD at 5 and 18 h Percentage of early (d) and late (e) apoptosis induced by BCG at 5 and 18 h Two-tailed unpairedStudentrsquos 119905-test was used to compare percentage of infected cells MOI 1 versus MOI 01 Bars indicate mean plusmn SEM from four independentexperiments lowastlowast119875 lt 001 lowast119875 lt 005 Light grey refers to uninfected cells

cells which did not exhibit apoptosis after infection comparedto uninfected cells (Figures 1(c) 1(d) and 1(e))

32 BCG Infection at MOI 1 and MOI 01 Activated theExpression of tmTNF sTNF and sTNF Receptors TNF is amajor proinflammatory cytokine induced by BCG infectionand can be observed intracellularly (iTNF) on the cell surfaceas transmembrane (tmTNF) and in a soluble TNF form(sTNF) [22 29ndash32] In vivo BCG infection has shown thattmTNF may interact with soluble TNF receptors (sTNFR1or sTNFR2) which may play a critical role in the infectionoutcome [32]

To determine if low doses of BCGwere enough to activateTNF and sTNFRs we measured iTNF and tmTNF by flowcytometry and sTNF sTNFR1 and sTNFR2 levels by ELISAOur data showed that both MOI 1 and MOI 01 activatediTNF and responses were BCG dose dependent The maindifference betweenMOIs was that iTNF expression wasmoretransient using MOI 01 compared to MOI 1 (Figure 2(a))However the percentage of MOI 1 infected cells expressingtmTNF was higher than cells infected with MOI 01 at 5 hrsbut similar at 18 hrs after infection (Figure 2(b)) We thenassessed sTNF and observed induction in a dose dependentmanner and the levels were maintained longer with MOI1 than with MOI 01 (Figure 2(c)) Regarding sTNFRs weobserved that sTNFR1 was similarly regulated by the twoMOIs but in contrast sTNFR2 expression was BCG dosedependent (Figures 2(d) and 2(e)) Collectively our datashow that both MOI 1 and MOI 01 induce iTNF sTNF andsTNFR2 in a dose dependent manner but expression levels oftmTNF and sTNFR1 appear independent of MOIs

33 Different Activation Patterns of Cytokines and ChemokinesTriggered by BCG Infection at MOI 1 and MOI 01 Secretionof cytokines other than TNF as well as chemokines isnecessary for cellular activation and recruitment followingmycobacterial infection in vivo We have then comparedthe levels of secreted cytokines and chemokines after BCGinfection and observed that IL-6 is only activated with MOI1 but not with MOI 01 (Figure 3(a)) Unexpectedly IL-1120573was produced earlier with MOI 01 than with MOI 1 andpeaked at 18 h after infection and decreased at 24 h afterinfection In contrast MOI 1 induced IL-1120573 which reacheda maximum at 48 h but this high level was maintainedup to 72 h (Figure 3(b)) Western blot analyses showed anincreased level at 2 h after infection of procaspase-1 in cellsinfected with MOI 01 compared with those infected withMOI 1 However amounts of caspase-1 p20 subunit werenot significantly different using MOI 1 and MOI 01 (seeSupplementary Figure 1 in Supplementary Material availableonline at httpdxdoiorg10115520164048235) We haveassessed the concentration of three chemokines monocytechemotactic protein 1 (MCP-1) chemokine (C-X-C motif)ligand 1 or also called keratinocyte chemoattractant (KC) andmacrophage inflammatory protein-1 alpha (MIP-1120572) whichhave important chemoattractant activity for monocytes andneutrophils [33] BCG infection at MOI 01 induced highlevels of MCP-1 from 5 h and still increased at 18 h but at24 h MCP-1 was found downregulated Conversely MOI 1induced high level at 18 h that was maintained until 72 h afterinfection (Figure 3(c)) KCwas induced at 2 h byMOI 01 andin higher levels thanMOI 1 infection (Figure 3(d)) BothMOI1 andMOI 01 increasedMIP-1120572 level up to 5 h after infection


0 2 5 180

5

10

15

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25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

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5

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20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

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1000012000

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BCG (hour)

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F-120572

(pg

mL) lowastlowast

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(c)

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(pg

mL)

0 2 5 18 24 30 48 720

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BCG (hour)

(d)

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FR2

(pg

mL)

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1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 2 BCG infection at MOI 1 and MOI 01 activates TNF and soluble TNF receptors (a) and (b) RAW macrophages were infected atMOI 1 or MOI 01 during 2 5 and 18 h and stained for intracellular and transmembrane TNF with mAb against TNF (c) Concentrations ofsoluble form of TNF were assessed by ELISA (d) and (e) Soluble TNFR1 and TNFR2 were measured by ELISA in culture supernatant fromRAW macrophages infected with BCG Pasteur at MOI 1 and MOI 01 Two-tailed unpaired Studentrsquos 119905-test was used to evaluate statisticaldifferences Bars indicate mean plusmn SEM from four independent experiments in each case lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 lowast119875 lt 005


0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

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lowastlowast

lowastlowastlowastlowastlowastlowast

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BCG (hour)

IL-1120573

(pg

mL)

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lowastlowast lowastlowast

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-1(p

gm

L)

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lowastlowast

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(c)

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gm

L)

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lowast

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(d)

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BCG (hour)

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-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)

Figure 3 BCG infection at MOI 1 and MOI 01 is efficient to induce cellular activation but kinetics are different (a) Concentrations ofproinflammatory cytokines IL-6 (b) IL-1120573 and chemokines (c) MCP-1 (d) KC and (e) MIP-1120572 were measured by ELISA in culturesupernatant from infected RAW macrophages with BCG Pasteur at MOI 1 and MOI 01 during different time points Bars indicate meanplusmn SEM from four independent experiments ANOVA and Dunnettrsquos post hoc test compared to uninfected macrophages or MOI 1 versus MOI01 lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 and lowast119875 lt 005


(Figure 3(e))Our data show thatMOI 01 is efficient to induceearly IL-1120573 and caspase-1 activation and that cytokine andchemokine kinetics induced by MOI 1 versus MOI 01 isdifferent suggesting that intracellular pathways are regulatedby infection dosages

34 Differential Regulation of Phosphorylated ASK1 and NF120581Bby BCG Infection at MOI 1 and MOI 01 Following ourprevious results indicating that MOI 1 and MOI 01 mayactivate different intracellular pathways we examined tran-scription factors involved in cytokine expression includingnuclear factor kappa B (NF120581B) phosphorylation required forcytokine transcription [34] We assessed extracellular signal-regulated kinase (ERK12) and apoptosis signal-regulatingkinase (ASK1) two members of the MAPK family involvedin inflammatory process [35 36] Levels of phosphorylatedNF120581B (NF120581B-P) ASK1 (ASK1-P) ERK1 (ERK1-P) and ERK2(ERK2-P) were determined by western blot analyses at differ-ent time points after BCG infection We observed that MOI1 and MOI 01 similarly induced phosphorylation of NF120581B-P and ASK1-P and signaling was sustained with MOI 1 butnot with MOI 01 (Figures 4(a) and 4(b)) However ERK1-Pand ERK2-P pathways showed the same pattern of activationwhich was independent of BCG infection doses (Figures 4(c)and 4(d)) Together these data show that phosphorylatedNF120581B and ASK correlate with soluble cytokine expressionwhile ERK1-P and ERK2-P are activated similarly with bothlow and high BCG doses

35 Microenvironment Produced by BCG Infection at MOI1 Induces Effects on Macrophage Proliferation Our datashowing that cellular activation is MOI- and time-dependentcould suggest that the microenvironment generated by theinfection would affect macrophage proliferation To testthis hypothesis we recovered the supernatant from infectedcells at different time points which was added to freshRAWmacrophages and cell proliferation was evaluated after12 h and 24 h as previously described [37] Macrophagescultured in fresh medium were considered as control cellsfor proliferation Macrophage proliferation at 12 h was notaffected by supernatant of BCG-infected cells at MOI 1 butwas inhibited by supernatant of 18 h of BCG-infected cellsat MOI 01 (Figures 5(a) and 5(b)) In contrast macrophageproliferation at 24 hours was significantly increased with2 and 5 h supernatant of BCG-infected cells at MOI 1while again supernatant of 18 h of BCG-infected cells atMOI 01 inhibited the macrophage proliferation (Figures 5(c)and 5(d)) Thus these results show that microenvironmentgenerated with MOI 1 enhanced cell proliferation but notMOI 01

36 Low Dose BCG Infection Induced Microbicidal Effec-tor Functions Activation of inducible nitric oxide synthase(iNOS) to generate oxide nitric (NO) is an important effectormechanism to eliminate mycobacteria [38] We comparedmacrophage iNOS expression bywestern blot in cells infectedwith BCG at MOI 1 and MOI 01 Infection at MOI 1 inducedhigher levels of iNOS protein than MOI 01 at 18 h afterinfection (Figures 6(a) and 6(b))We alsomeasured the levels

of nitrite as an indirect manner to evaluate NO productionWe observed that although NO was similarly producedat early infection infection with BCG at MOI 1 yieldedsustained and higher NO levels at later time points Infectionwith BCG at MOI 01 triggered lower and transient NOproduction (Figure 6(c)) Our data showed that both MOIsactivate NO production however only MOI 1 maintainslonger and higher NO levels at late time points

37 BCG Infection with Low Bacterial Burden Does Not AlterMitochondrial Distribution and Nuclear Integrity Host celldeath mediated by infectious agents involves modulation ofmitochondria Using M tuberculosis infection model it hasbeen reported that high dose (MOI ge 10) induced cell deathwhere mitochondria and nucleus were the first organellesshowing damage [39 40] We asked if BCG infection withlow dose affects mitochondrial integrity To explore thisquestion RAWmacrophages were transfected with mitoRFP(RAW-mitoRFP) infected with BCG-GFP and examined byconfocal microscopy to evaluate nuclear structure and local-ization of mitochondria Results showed that mitochondrialdistribution and nuclear integrity after BCG-GFP infectionwere maintained in a similar form than in uninfected cells(Figures 7(a) and 7(b)) We observed that the number ofRAW-mitoRFP cells decreased after the infection To betterexamine this result RAW-mitoRFP+ cells were sorted andenriched to 97 and then infected with BCG-GFP (Figures8(a) and 8(b)) A reduction of 25ndash40 of mitoRFP+ cells wasstill observed (Figure 7(c)) In addition we observed that cellintegrity and size assessed by flow cytometry were similar ininfected and uninfected cells but only a discretemodificationof the cell structure was observed at 18 h MOI 1 postinfection(Figure 7(d)) In order to evaluate if the decrease of mitoRFPexpression was due to mitochondrial loss we measured thepresence of the constitutive mitochondrial molecule TOM40bywestern blot analyses TOM40 expression only increased at5 h after infection with BCG at bothMOIs indicating that lossof mitoRFP expression was not due to loss of mitochondrialmass during BCG infection (Figures 8(c) and 8(d)) Togetherthese results show that low doses of BCG do not affectthe nuclear integrity mitochondrial quantity or distributioneven if MOI 1 induces a discrete increase of cell death

4 Discussion

The present study analyzes the effect of very low dose BCGinfection on macrophages in terms of cell viability cell acti-vation and mitochondrial integrity Our results demonstratethat although BCG infection at MOI 1 triggers some celldeath there was a clear activation of the viable macrophageIn contrast BCG infection at MOI 01 was sufficient forpromoting macrophage activation with a distinct cytokinepattern in the absence of cell death and mitochondrialdamage

Monocytes andmacrophages are among the most impor-tant cells of the innate immunity involved in host protectionagainst mycobacterial infections Recent studies have shownthat BCG infection induces epigenetic reprogramming inmonocytes and macrophages that defines the molecular


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MOI 1MOI 01

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elat

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2h 5h 18h

(e)

Figure 4 BCG infection at MOI 1 and MOI 01 activates NF120581B and MAPK pathways (a) Phosphorylated forms of NF120581B (b) ASK (c)ERK1 and (d) ERK2 were evaluated by western blot analyses Band densities were normalized against tubulin by densitometry analyses(e) Representative western blot from three independent experiments Results are shown in relative units of concentration using IMAGEJsoftware Two-tailed unpaired Studentrsquos 119905-test was used to evaluate statistical differences Bars indicate mean plusmn SEM lowastlowast119875 lt 001 lowast119875 lt 005Light grey bar indicates uninfected cells (0)


MOI 1 MOI 01

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ed ce

lls(0h)(2h)(5h)(18h)

Click-iT EdU Alexa Fluor 488 Click-iT EdU Alexa Fluor 488

(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100

+ supernatant from infected cellsmed

ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)

Figure 5 Microenvironment produced by BCG infection at MOI 1 affects macrophage proliferation (a) and (b) Representative histogramsmeasuringmean fluorescence intensity (MFI) of Alexa Fluor 488 after 12 h and 24 h (c) and (d) Supernatant from infected cells was recoveredat different time points after infection and added to fresh RAWmacrophages and proliferationwas evaluated at 12 h and 24 h in the presence ofEdU RAWmacrophages were stained with Alexa Fluor 488 and proliferation was evaluated by flow cytometry Two-tailed unpaired Studentrsquos119905-test was used to evaluate differences versus macrophages proliferation in new medium Bars indicate mean plusmn SEM from three independentexperiments lowastlowast119875 lt 001 lowast119875 lt 005 The light grey bar refers to uninfected cells or 0 h

mechanisms involved in trained immunity conferring anonspecific protection against a secondary infection [41]BCG-induced epigenetic modifications in innate cells suchas NOD receptor activation and histone methylation pro-vide long term functional state of circulating monocyteswhich may explain the nonspecific beneficial effects alreadydescribed many years ago for children vaccinated with BCG[42] In this context the present work illustrates several of theBCG-induced functional changes on macrophages showingthe importance of the selected dosage of BCG which couldbe relevant in this process

Studies on human alveolar macrophages have shownthat infection with nonpathogenic or pathogenic strainsof M tuberculosis (MOI 5ndash10) as well as with M bovisBCG induced apoptosis playing an important role in host-pathogen interaction and contributing to host defense mech-anisms against mycobacterial infection [43 44] Mycobacte-ria-induced apoptosis was shown to affect both the infectedand uninfected macrophages and be mediated by cell contactand independent of TNF TGF-120573 and TLRs [45] Apoptosisof macrophages during mycobacterial infection has beenattributed to different factors including mycobacterial vir-ulence bacillary load time points of observation and theamount of activated cytokines

Our work shows that the amount of BCG correlates withmacrophage apoptosis Using a BCG-GFP strain cell deathwas observed on infected and uninfected macrophages (datanot shown) as reported under infection with M tuberculosis[45] The balance of apoptosisnecrosis is an importantmechanism to control intracellular mycobacterial growthand cell activation [46] Nonpathogenic strains such as BCG

were reported to induce apoptosis whereas pathogens strainsuch as H37Rv promoted necrosis in cultured macrophages[44 47 48] However it has been reported that the bal-ance of apoptosis and necrosis in M tuberculosis infectedmacrophages depends on bacterial virulence and bacterialload [19]

Macrophage activation involves the induction ofcytokines chemokines and bactericidal mechanisms TNFis one of the main cytokines activated during BCG infectionin macrophages which has been associated with apoptosisinduced by M tuberculosis infection [43] Mechanisms ofevasion developed by virulentM tuberculosis versus avirulentstrains involve both TNF production and TNF inactivationby released soluble TNFR2 from activated macrophages[23 49] However human monocytes and macrophagestreated with clinically used TNF inhibitors showed that Mtuberculosis-induced cell death was independent of TNF andnot modulated by TNF inhibition [50] In mice macrophageapoptosis has been related to TNF production in the lungof BCG-infected mice using low dose of attenuated versusvirulent M bovis which induced higher TNF and highermacrophage apoptosis at early time points but was reversedat late time points of infection indicating a dynamic responsein vivo [47] Our data on macrophages show that earlyapoptosis was independent of intracellular and soluble TNFbut correlated with transmembrane TNF expression Resultsalso show that whereas soluble TNFR1 was independentof BCG dose soluble TNFR2 was released in a BCG dosedependent manner

In the attempt to evaluate the main differences inactivation between MOI 1 and MOI 01 proinflammatory


5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01

lowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

(c)

Figure 6 BCG infection at MOI 1 and MOI 01 induces iNOS and NO production but only MOI 1 maintains this function for long timeRAW macrophages were infected with BCG Pasteur at MOI 1 and MOI 01 (a) Cells were recovered at 2 5 and 18 h after infection andiNOS expression was evaluated by western blot Band densities were normalized against tubulin by densitometry analysis Results are shownin relative units of concentration using IMAGEJ software (b) Representative western blot for iNOS and tubulin as a loading control (c)Nitrite was evaluated in culture supernatant from each condition using Griess reagent Two-tailed unpaired Studentrsquos 119905-test was used toevaluate differences versus uninfected cells Bars indicate mean plusmn SEM from three (a) or five (c) independent experiments lowastlowastlowastlowast119875 lt 00001lowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 Grey bar refers to uninfected cells indicated by 0


Uni

nfec

ted

MOI 1 MOI 01

NucleusMitochondriaBCG-GFP

BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)

Figure 7 BCG infection at MOI 01 and MOI 1 maintains undisturbed mitochondrial and nuclear integrity (a) Transfected RAW-mitoRFPmacrophages and (b) transfected and sorted (enriched to 97 or RAW-mitoRFP+) were infected atMOI 1 andMOI 01 BCG-GFP Slides wereprepared with DAPI and mounting medium and analyzed by confocal SP5 microscopy using Leica Application Suite software Mitochondria(red) BCG (green) and nucleus (blue) (left panel) Scale bar 10 120583m (c) RAW-mitoRFP cells were sorted enriched to 97 (RAW-mitoRFP+)and infected with BCG-GFP and quantified for RAW-mitoRFP+ cells before and after BCG-GFP infection at MOI 1 and MOI 01 (d) Flowcytometry analyses of cell integrity and size using side scatter (SSC)forward scatter (FSC) Representative image from three independentexperiments

cytokines molecules other than TNF have been analyzedOur data showed that IL-6 was highly produced by BCGinfection at MOI 1 while no expression was observed withinfection at MOI 01 Our data revealed that IL-1120573 andcaspase-1 were also differentially modulated by different

infectious dosages BCG infection at MOI 01 was more effi-cient to induce the early production of IL-1120573 while infectionat MOI 1 induced a delay but high level of IL-1120573 In this con-text the microenvironment generated by BCG infection atthe two MOIs affected differently macrophage proliferation


Before sorting RAW-RFPMOI 1 MOI 01

NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)

After sorting RAW-RFPMOI 1 MOI 01

NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin

MOI Uninfected Uninfected Uninfected1 01 1 01 1 01

2h 5h 18h

(d)

Figure 8 Amount of TOM40 is maintained at both MOI 1 and MOI 01 (a) RAW-mitoRFP macrophages were infected with BCG-GFP atMOI 1 andMOI 01 for 5 and 18 h or (b) sorted for mitoRFP and then infected (c) Western blots for TOM40 band densities were normalizedagainst tubulin (d) Representativewestern blot from three independent experiments Two-tailed unpaired Studentrsquos 119905-test was used to evaluatestatistical differences Bars indicate mean plusmn SEM from three experiments lowast119875 lt 005


Previous data reported that BCG (MOI 3 and MOI 10) failedto stimulate release of IL-1120573 from human macrophages [51]The different results could be also attributed to differentcellular origins It has been reported that BCG infectionof human macrophages induced very low levels of IL-1120573however human monocytes infected with M tuberculosis orBCG delivered comparable levels indicating that the IL-1120573response is influenced by the host cell type [52] Similar toIL-1120573 we observed that MOI 01 was a better stimulus toinduceMCP-1 andKC expression at early time after infectionHowever at late time points BCG infection at MOI 1 inducedhigher protein amounts than infection atMOI 01 From thesedata we conclude that MOI 01 is a good early inducer oftwo important chemokinesMCP-1 andKCwhich are relevantin maintaining the integrity of granuloma in asymptomaticindividuals and also mediate host defense via activationof transcription factors MAPK and adhesion molecules[53 54] We have then analyzed transcription factors andfound that NF120581B and ASK1 were similarly activated at earlytime by both BCG MOIs and at 18 h and using MOI 01cytokines production and NF120581B were downregulated Nodifferenceswere found in ERK12 phosphorylation patterns atthe two infection dosages In addition both MOIs were ableto activate bactericidal mechanisms required for bacterialelimination and as expected MOI 1 induced higher iNOSprotein and NO production

Previous report has shown thatmycobacterial infection inmacrophages caused mitochondrial perturbation Compari-son of the effects of virulent versus avirulent M tuberculosisstrains showed that virulent strain increased mitochondrialactivity whereas avirulent strains resulted in mitochondriaexhaustion suggesting that virulent strains could maintain aniche for sustained survival [40] Our data show that bothBCG infections transiently increased the amount of themito-chondrial protein TOM40 suggesting a transient increase inmitochondrialmassThis could be a consequence of the stressinduced by the infection however more experiments will benecessary to test this hypothesis

In this study we also used parameters such as cellulargranularity and size and chromatin condensation that classi-cally has been described as indicators of cellular integrity [5556] Even if macrophages under condition MOI 1 showed adiscrete loss of SSC compared to uninfectedmacrophages thecondensed chromatin and mitochondria were not affectedWe concluded that low BCG doses are enough to activatemacrophages and to maintain cellular viability and mito-chondrial integrity

Although our study has limitations due to the fact thatit has been developed in a cell line it can be consideredthat data provide new insights into macrophage activation byBCG infection with very low number of bacilli that can beused as an in vitro infection system and applied to studies onmacrophage early activationmechanismmaintaining cell via-bility More extensive studies are needed to confirm that verylowBCGdosemay induce changes in innate cells and providebeneficial effects In support to our data the use of one-halfor one-third of BCG for standard instillation has recentlybeen proposed A study in NMIBC-patients treated withlow dose BCG had lower toxicity and higher quality of life

comparedwithNMIBC-patients instilled with standard-dose[57 58]Thus low dose BCG can be applied to different BCGresearch fields of interest including mycobacterial infectionscancer immunotherapy and prevention of autoimmunity andallergies [59]

Competing Interests

The authors declare no competing interests regarding thepublication of the present manuscript

Authorsrsquo Contributions

L Chavez-Galan designed and performed the experimentsD Vesin performed experiments D Martinvalet helpedwith designed mitochondria studies L Chavez-Galan andI Garcia wrote the manuscript D Martinvalet gave criticalreviews of the manuscript

Acknowledgments

This work was supported by FNS Grant 310033-166662 to IGarcia L Chavez-Galan received a fellowship (207760) fromthe Consejo Nacional de Ciencia y Tecnologıa (CONACyT)Mexico The authors are grateful to CMU Core Facilitiesbioimaging and flow cytometry (Faculty of Medicine Uni-versity of Geneva) for help with the analysis of confocalmicroscopy multiparametric cytometry and cellular sorterused in this study The authors thank Guillaume Blaser andHusnu Uysal for excellent assistance

References

[1] H Getahun C Gunneberg R Granich and P Nunn ldquoHIVinfection-associated tuberculosis the epidemiology and theresponserdquo Clinical Infectious Diseases vol 50 no 3 pp S201ndashS207 2010

[2] A Zumla AGeorge V Sharma RHNHerbert AOxley andM Oliver ldquoThe WHO 2014 Global tuberculosis report-furtherto gordquoThe Lancet Global Health vol 3 no 1 pp e10ndashe12 2015

[3] M J King andW H Park ldquoEffect of Calmettersquos BCG vaccine onexperimental animalsrdquo American Journal of Public Health andthe Nations Health vol 19 no 2 pp 179ndash192 1929

[4] G G Mahairas P J Sabo M J Hickey D C Singh and CK Stover ldquoMolecular analysis of genetic differences betweenMycobacterium bovis BCG and virulent M bovisrdquo Journal ofBacteriology vol 178 no 5 pp 1274ndash1282 1996

[5] T Hsu S M Hingley-Wilson B Chen et al ldquoThe primarymechanism of attenuation of bacillus Calmette-Guerin is aloss of secreted lytic function required for invasion of lunginterstitial tissuerdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 21 pp12420ndash12425 2003

[6] M R R Lagranderie A-M Balazuc E Deriaud C D Leclercand M Gheorghiu ldquoComparison of immune responses ofmice immunized with five different mycobacterium bovis BCGvaccine strainsrdquo Infection and Immunity vol 64 no 1 pp 1ndash91996


[7] B Wu C Huang L Garcia et al ldquoUnique gene expressionprofiles in infants vaccinated with different strains ofMycobac-terium bovis bacille Calmette-Guerinrdquo Infection and Immunityvol 75 no 7 pp 3658ndash3664 2007

[8] C Gernez Rieux andMGervois ldquoProtection conferred by BCGduring the 20 years following vaccinationrdquo Bulletin of theWorldHealth Organization vol 48 no 2 pp 139ndash154 1973

[9] C A Rentsch F D Birkhauser C Biot et al ldquoBacillus calmette-guerin strain differences have an impact on clinical outcome inbladder cancer immunotherapyrdquo European Urology vol 66 no4 pp 677ndash688 2014

[10] M Decobert H LaRue F Harel F Meyer Y Fradet and LLacombe ldquoMaintenance bacillus Calmette-Guerin in high-risknonmuscle-invasive bladder cancer how much is enoughrdquoCancer vol 113 no 4 pp 710ndash716 2008

[11] O Olakanmi L S Schlesinger A Ahmed and B E Briti-gan ldquoThe nature of extracellular iron influences iron acqui-sition by mycobacterium tuberculosis residing within humanmacrophagesrdquo Infection and Immunity vol 72 no 4 pp 2022ndash2028 2004

[12] A S Davis I Vergne S S Master G B Kyei J Chua andV Deretic ldquoMechanism of inducible nitric oxide synthaseexclusion from mycobacterial phagosomesrdquo PLoS Pathogensvol 3 no 12 article e186 2007

[13] I Durrbaum-Landmann J Gercken H-D Flad and M ErnstldquoEffect of in vitro infection of human monocytes with lownumbers of Mycobacterium tuberculosis bacteria on monocyteapoptosisrdquo Infection and Immunity vol 64 no 12 pp 5384ndash5389 1996

[14] J Keane H G Remold and H Kornfeld ldquoVirulent Mycobac-terium tuberculosis strains evade apoptosis of infected alveolarmacrophagesrdquo Journal of Immunology vol 164 no 4 pp 2016ndash2020 2000

[15] C Loeuillet F Martinon C Perez M Munoz M Thomeand P R Meylan ldquoMycobacterium tuberculosis subverts innateimmunity to evade specific effectorsrdquo Journal of Immunologyvol 177 no 9 pp 6245ndash6255 2006

[16] K Klingler K-M Tchou-Wong O Brandli et al ldquoEffectsof mycobacteria on regulation of apoptosis in mononuclearphagocytesrdquo Infection and Immunity vol 65 no 12 pp 5272ndash5278 1997

[17] M B Santucci M Amicosante R Cicconi et al ldquoMycobac-terium tuberculosis-induced apoptosis in monocytesmacro-phages early membrane modifications and intracellularmycobacterial viabilityrdquo Journal of Infectious Diseases vol 181no 4 pp 1506ndash1509 2000

[18] R Placido G Mancino A Amendola et al ldquoApoptosis ofhumanmonocytesmacrophages inMycobacterium tuberculosisinfectionrdquo Journal of Pathology vol 181 no 1 pp 31ndash38 1997

[19] R E Butler P Brodin J Jang et al ldquoThe balance of apoptoticand necrotic cell death in Mycobacterium tuberculosis infectedmacrophages is not dependent on bacterial virulencerdquo PLoSONE vol 7 no 10 Article ID e47573 2012

[20] X Wu G Deng X Hao et al ldquoA caspase-dependent pathwayis involved in Wnt120573-catenin signaling promoted apoptosis inbacillus Calmette-Guerin infected RAW2647 macrophagesrdquoInternational Journal of Molecular Sciences vol 15 no 3 pp5045ndash5062 2014

[21] T Palaga S Ratanabunyong T Pattarakankul et al ldquoNotchsignaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophagesrdquo Cellular and Molecular Immunology vol10 no 5 pp 444ndash452 2013

[22] I Garcia M L Olleros V F J Quesniaux et al ldquoRoles ofsoluble and membrane TNF and related ligands in mycobac-terial infections effects of selective and non-selective TNFinhibitors during infectionrdquoAdvances in ExperimentalMedicineand Biology vol 691 pp 187ndash201 2011

[23] M Engele E Stoszligel K Castiglione et al ldquoInduction of TNF inhuman alveolar macrophages as a potential evasionmechanismof virulentMycobacterium tuberculosisrdquo Journal of Immunologyvol 168 no 3 pp 1328ndash1337 2002

[24] L-G Bekker S Freeman P J Murray B Ryffel and G KaplanldquoTNF-120572 controls intracellular mycobacterial growth by bothinducible nitric oxide synthase-dependent and inducible nitricoxide synthase-independent pathwaysrdquo Journal of Immunologyvol 166 no 11 pp 6728ndash6734 2001

[25] G S Dean S G Rhodes M Coad et al ldquoMinimum infectivedose ofMycobacterium bovis in cattlerdquo Infection and Immunityvol 73 no 10 pp 6467ndash6471 2005

[26] M L Olleros R Guler N Corazza et al ldquoTransmembraneTNF induces an efficient cell-mediated immunity and resistanceto Mycobacterium bovis bacillus Calmette-Guerin infection inthe absence of secreted TNF and lymphotoxin-120572rdquo Journal ofImmunology vol 168 no 7 pp 3394ndash3401 2002

[27] S Dhandayuthapani L E Via C A Thomas P M HorowitzD Deretic and V Deretic ldquoGreen fluorescent protein as amarker for gene expression and cell biology of mycobacterialinteractions withmacrophagesrdquoMolecularMicrobiology vol 17no 5 pp 901ndash912 1995

[28] R Guler M L Olleros D Vesin et al ldquoInhibition of induciblenitric oxide synthase protects against liver injury induced bymycobacterial infection and endotoxinsrdquo Journal of Hepatologyvol 41 no 5 pp 773ndash781 2004

[29] R Z Murray J G Kay D G Sangermani and J L StowldquoCell biology A role for the phagosome in cytokine secretionrdquoScience vol 310 no 5753 pp 1492ndash1495 2005

[30] DCastano L FGarcıa andMRojas ldquoIncreased frequency andcell death of CD16+ monocytes withMycobacterium tuberculo-sis infectionrdquo Tuberculosis vol 91 no 5 pp 348ndash360 2011

[31] L Chavez-Galan M L Olleros D Vesin and I Garcia ldquoMuchmore than M1 and M2 macrophages there are also CD169+andTCR+macrophagesrdquo Frontiers in Immunology vol 6 article263 2015

[32] M L Olleros D Vesin R Bisig et al ldquoMembrane-boundTNF induces protective immune responses to M bovis BCGinfection regulation of memtnf and TNF receptors comparingtwomemTNFmoleculesrdquoPLoSONE vol 7 no 5 article e314692012

[33] M D Turner B Nedjai T Hurst and D J PenningtonldquoCytokines and chemokines at the crossroads of cell signallingand inflammatory diseaserdquo Biochimica et Biophysica ActamdashMolecular Cell Research vol 1843 no 11 pp 2563ndash2582 2014

[34] M R Gwinn and V Vallyathan ldquoRespiratory burst role in sig-nal transduction in alveolarmacrophagesrdquo Journal of Toxicologyand Environmental Health Part B Critical Reviews vol 9 no 1pp 27ndash39 2006

[35] M Torres andH J Forman ldquoActivation of several MAP kinasesupon stimulation of rat alveolar macrophages role of theNADPH oxidaserdquo Archives of Biochemistry and Biophysics vol366 no 2 pp 231ndash239 1999

[36] Y Gotoh and J A Cooper ldquoReactive oxygen species- anddimerization-induced activation of apoptosis signal-regulatingkinase 1 in tumor necrosis factor-120572 signal transductionrdquo Journalof Biological Chemistry vol 273 no 28 pp 17477ndash17482 1998


[37] M Jung M-K Shin Y-K Jung and H S Yoo ldquoModulationof macrophage activities in proliferation lysosome and phago-some by the nonspecific immunostimulator micardquo PLoS ONEvol 10 no 2 Article ID e0117838 2015

[38] I Garcia R Guler D Vesin et al ldquoLethal Mycobacterium bovisBacillus Calmette Guerin infection in nitric oxide synthase 2-deficient mice cell-mediated immunity requires nitric oxidesynthase 2rdquo Laboratory Investigation vol 80 no 9 pp 1385ndash1397 2000

[39] M Chen H Gan and H G Remold ldquoA mechanism of viru-lence Virulent Mycobacterium tuberculosis strain H37Rv butnot attenuated H37Ra causes significant mitochondrial innermembrane disruption in macrophages leading to necrosisrdquoJournal of Immunology vol 176 no 6 pp 3707ndash3716 2006

[40] S Jamwal M KMidha H N Verma A Basu K V S Rao andV Manivel ldquoCharacterizing virulence-specific perturbationsin the mitochondrial function of macrophages infected withMycobacterium tuberculosisrdquo Scientific Reports vol 3 article1328 2013

[41] M G Netea J Quintin and J W M Van Der Meer ldquoTrainedimmunity a memory for innate host defenserdquo Cell Host andMicrobe vol 9 no 5 pp 355ndash361 2011

[42] J Kleinnijenhuis J Quintin F Preijers et al ldquoBacille Calmette-Guerin induces NOD2-dependent nonspecific protection fromreinfection via epigenetic reprogramming of monocytesrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 109 no 43 pp 17537ndash17542 2012

[43] J Keane M K Balcewicz-Sablinska H G Remold et alldquoInfection by Mycobacterium tuberculosis promotes humanalveolarmacrophage apoptosisrdquo Infection and Immunity vol 65no 1 pp 298ndash304 1997

[44] C J Riendeau and H Kornfeld ldquoTHP-1 cell apoptosis inresponse to Mycobacterial infectionrdquo Infection and Immunityvol 71 no 1 pp 254ndash259 2003

[45] D M Kelly A M C ten Bokum S M OrsquoLeary M POrsquoSullivan and J Keane ldquoBystander macrophage apoptosisafter Mycobacterium tuberculosis H37Ra infectionrdquo Infectionand Immunity vol 76 no 1 pp 351ndash360 2008

[46] A Molloy P Laochumroonvorapong and G Kaplan ldquoApop-tosis but not necrosis of infected monocytes is coupled withkilling of intracellular bacillus Calmette-Guerinrdquo Journal ofExperimental Medicine vol 180 no 4 pp 1499ndash1509 1994

[47] M F Rodrigues MM Barsante C C S Alves et al ldquoApoptosisof macrophages during pulmonaryMycobacterium bovis infec-tion correlation with intracellular bacillary load and cytokinelevelsrdquo Immunology vol 128 no 1 pp e691ndashe699 2009

[48] L Chavez-Galan L A Ramon-Luing L Torre-Bouscoulet RPerez-Padilla and I Sada-Ovalle ldquoPre-exposure of Mycobac-terium tuberculosis-infected macrophages to crystalline silicaimpairs control of bacterial growth by deregulating the balancebetween apoptosis and necrosisrdquo PLoS ONE vol 8 no 11Article ID e80971 2013

[49] M K Balcewicz-Sablinska J Keane H Kornfeld and HG Remold ldquoPathogenic Mycobacterium tuberculosis evadesapoptosis of host macrophages by release of TNF-R2 resultingin inactivation of TNF-120572rdquo Journal of Immunology vol 161 no5 pp 2636ndash2641 1998

[50] N Reiling D Schneider and S Ehlers ldquoMycobacterium tuber-culosis-induced cell death of primary human monocytes andmacrophages is not significantly modulated by tumor necrosisfactor-targeted biologicalsrdquo Journal of InvestigativeDermatologySymposium Proceedings vol 12 no 1 pp 26ndash33 2007

[51] A Dorhoi G Nouailles S Jorg et al ldquoActivation of the NLRP3inflammasome by Mycobacterium tuberculosis is uncoupledfrom susceptibility to active tuberculosisrdquo European Journal ofImmunology vol 42 no 2 pp 374ndash384 2012

[52] A Novikov M Cardone R Thompson et al ldquoMycobacteriumtuberculosis triggers host type I IFN signaling to regulate IL-1120573 production in humanmacrophagesrdquo Journal of Immunologyvol 187 no 5 pp 2540ndash2547 2011

[53] R Hussain A Ansari N Talat Z Hasan and G DawoodldquoCCL2MCP-I genotype-phenotype relationship in latenttuberculosis infectionrdquo PLoS ONE vol 6 no 10 Article IDe25803 2011

[54] S Cai S Batra S A Lira J K Kolls and S Jeyaseelan ldquoCXCL1regulates pulmonary host defense to Klebsiella infection viaCXCL2 CXCL5 NF-120581B and MAPKsrdquo Journal of Immunologyvol 185 no 10 pp 6214ndash6225 2010

[55] N I Dmitrieva and M B Burg ldquoAnalysis of DNA breaksDNA damage response and apoptosis produced by high NaClrdquoAmerican Journal of PhysiologymdashRenal Physiology vol 295 no6 pp F1678ndashF1688 2008

[56] P Brousseau J Pellerin Y Morin et al ldquoFlow cytometry as atool to monitor the disturbance of phagocytosis in the clamMya arenaria hemocytes following in vitro exposure to heavymetalsrdquo Toxicology vol 142 no 2 pp 145ndash156 1999

[57] C Pfister W Kerkeni J Rigaud et al ldquoEfficacy and toleranceof one-third full dose bacillus Calmette-Guerin maintenancetherapy every 3 months or 6 months two-year results of URO-BCG-4 multicenter studyrdquo International Journal of Urology vol22 no 1 pp 53ndash60 2015

[58] A Yokomizo Y Kanimoto T Okamura et al ldquoRandomizedcontrolled study of the efficacy safety and quality of lifewith low dose bacillus Calmette-Guerin instillation therapy fornonmuscle invasive bladder cancerrdquo Journal of Urology vol 195no 1 pp 41ndash46 2016

[59] D L Faustman The Value of BCG and TNF in AutoimmunityElsevier Academic Press 1st edition 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Currently BCG clinical application is not limited tothe field of mycobacterial infections Adjuvant instillationof BCG as standard immunotherapy used in non-muscle-invasive bladder cancer (NMIBC) results in a significantreduction in tumor and progression It has been describedthat high doses of BCG Connaught induce a high Th1response prime CD8+ T cells and efficiently prevent recur-rences of NMIBC [9] However the optimal dose and dura-tion of treatment in NMIBC are controversial and derivedside effects may lead to premature interruption of treatmentNew treatment schedules of BCGwith no negative impact onpatients are considered [10]

Macrophages are part ofmononuclear phagocytic systemand the alveolar macrophage is the first cell to encounterM tuberculosis after infection however the bacillus has theability to evade microbicidal activities For example Mtuberculosis inhibits phagolysosomal fusion consequentlymacrophage provides a niche for mycobacteria and althoughit is a hostile environment it is sufficient for bacillus growth[11 12]

Using human monocytes and macrophages and macro-phage cell lines it has been shown that avirulent strains ofmycobacteria induce higher levels of apoptosis than viru-lent strains which represent a mechanism of host defensewhich can be subverted by virulent M tuberculosis toescape from innate immunity [13ndash15] However data havealso reported that virulent and avirulent M tuberculosisstrains can both inhibit and promote apoptosis accordingto experimental conditions [16ndash19] Despite accumulatingdata on macrophage activation and death by M tuberculosisstrains BCG-mediated cellular activities in phagocytic cellsremain relatively unexplored Studies comparing BCG withM tuberculosis have frequently used high Multiplicity-of-Infection (MOI) In general studies use BCG infection atMOI 10 (10 bacillicell) pointing apoptosis as a cellularstrategy to eliminate mycobacteria [20 21]

BCG-induced activation ofmacrophages has been associ-ated with TNF production which is a critical cytokine in hostdefense mechanisms against mycobacterial infection [22]Both BCG-infected and uninfected macrophages produceTNF although in lower amount than macrophages infectedwith virulent strains in which TNF supports mycobacterialgrowth [23] BCG-induced TNF activates iNOS but TNF cancontrol intracellular BCG growth by iNOS-dependent andiNOS-independent pathways [24] Collectively these modelshave provided important insights into immune responseagainst mycobacteria but considering that a low dose ofbacteria (1 bacillus) is sufficient to establish a mycobacterialinfection in the host it is of interest to analyze macrophageresponses in terms of cell death activation and integrity tolow number of mycobacteria [25]

In the present work we compare RAW macrophageresponses upon infection with BCG Pasteur at MOI 1 andMOI 01 the last is considered as a very low dose of infectionThis study aims (1) to evaluate if low doses of BCG Pasteurare sufficient to activatemacrophages whilemaintaining theirviability and mitochondrial integrity and (2) to determineif under low MOIs the macrophage is able to retain theirfunctional activities over time after infection Our results

show that low BCG doses are an efficient in vitro infec-tiousmodel to study interactions betweenmacrophagesBCGmaintaining viablemacrophages andmitochondrial integrity

2 Material and Methods

21 Cell Culture The murine macrophage cell line RAW2647 (RAW macrophages) was purchased from Ameri-can Type Culture Collection (Rockville MD) The cellswere maintained in DMEM supplemented with 10 head-inactivated FBS penicillin streptomycin sodium pyruvateglutamine and HEPES (complete DMEM) at 37∘C in ahumidified atmosphere containing 5 CO

2

22 M bovis BCG M bovis BCG Pasteur strain 1172 P2(Pasteur Institute Paris France) was used and grown to thelog phase in 7H9 middlebrook medium supplemented witholeic albumin dextrose catalase (OADC) The bacteria werethen harvested washed and frozen at minus80∘C in PBS plus 10of glycerol Bacterial load was determined by plating serial10-fold dilutions on 7H10 middlebrook agar (supplementedwith OADC) and counting colonies after incubation forat least 3 weeks [26] A BCG-GFP M bovis BCG Pasteurstrain harboring phsp60-gfp expressing Green FluorescenceProtein (BCG-GFP)was grown in the presence of Kanamycinas previously reported [27]

23 BCG Infection RAW macrophages were cultured in24-well flat-bottomed cell culture plates (1 times 106mL) andinfected with BCG Pasteur at MOI 1 or MOI 01 along 25 18 24 30 48 and 72 hours (h) at 37∘C in a humidifiedatmosphere containing 5 CO

2

24 Evaluation of Cell Death by Flow Cytometry Endingthe culture cells were harvested by adding cold PBS toculture plates and maintaining for 10 minutes on ice todetach cells from plastic plates Cells were washed in PBA(phosphate buffered saline containing 01 Sodium Azideand 01 Albumin Bovine) and incubated with 3 120583L 7-AAD(eBioscience) solution for 20min at 4∘C in the dark washedin PBA suspended in binding buffer 1x (BD pharmingen)and incubated with 5 120583L Annexin-V FITC-conjugated orAPC-conjugated (when using BCG-GFP infection) (eBio-science) for 15min at room temperature in the dark Datawere collected using a FACs CyAn (Beckton Dickinson Inc)within one hour and then analyzed with FlowJo software(Tree Star Inc) 50000 events were acquired per sample

25 Flow Cytometry The percentage of RAW macrophagesexpressing transmembrane TNF (tmTNF) and intracellularTNF (iTNF)was assessed by flow cytometry Briefly for iTNFending the culture cells were harvested washed in PBAand fixed in 4 formaldehyde for 10min at 4∘C Cells wereincubated with saponin-containing buffer shaken gently for10min at 4∘C and then incubated with anti-TNF (MP6-XT22) conjugated PE Cy7 (eBioscience) for 30min at roomtemperature in the dark and washed with PBS-saponin FortmTNF label cells were harvested washed in PBA and fixedin 4 formaldehyde RAW macrophages were suspended in














3 Results




UninfectedA

nnex

in-V

7-AAD

Early apoptosis

Late apoptosis

Live cells

Necrotic cells



(a)

SSC

FSC

On general gate

(b)

Ann

exin

-VA

nnex

in-V

Ann

exin

-VA

nnex

in-V

7-AAD 7-AAD

7-AAD7-AAD

MO

I 1M

OI 0

1

MO

I 1M

OI 0

1

BCG (5h) BCG (18h)




87 plusmn 3

2 plusmn 02





(c)

Figure 1 Continued


Early

apop

totic

cells

()

0

2

4

6

8

10

5 18

BCG (hour)

0

lowastlowast

MOI 1MOI 01

(d)

Late

apop

totic

cells

()

0

5

10

15

5 18

BCG (hour)

0

lowast

MOI 1MOI 01

lowast

(e)







0 2 5 180

5

10

15

20

25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

0

5

10

15

20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

0 2 5 18 24 30 48 720

1000

2000

3000

400060008000

1000012000

MOI 1MOI 01

BCG (hour)

sTN

F-120572

(pg

mL) lowastlowast

lowastlowast


(c)

sTN

FR1

(pg

mL)

0 2 5 18 24 30 48 720

200

400

600

800

1000

MOI 1MOI 01

BCG (hour)

(d)

sTN

FR2

(pg

mL)

0 2 5 18 24 30 48 720

500

1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)



0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results




UninfectedA

nnex

in-V

7-AAD

Early apoptosis

Late apoptosis

Live cells

Necrotic cells



(a)

SSC

FSC

On general gate

(b)

Ann

exin

-VA

nnex

in-V

Ann

exin

-VA

nnex

in-V

7-AAD 7-AAD

7-AAD7-AAD

MO

I 1M

OI 0

1

MO

I 1M

OI 0

1

BCG (5h) BCG (18h)




87 plusmn 3

2 plusmn 02





(c)

Figure 1 Continued


Early

apop

totic

cells

()

0

2

4

6

8

10

5 18

BCG (hour)

0

lowastlowast

MOI 1MOI 01

(d)

Late

apop

totic

cells

()

0

5

10

15

5 18

BCG (hour)

0

lowast

MOI 1MOI 01

lowast

(e)







0 2 5 180

5

10

15

20

25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

0

5

10

15

20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

0 2 5 18 24 30 48 720

1000

2000

3000

400060008000

1000012000

MOI 1MOI 01

BCG (hour)

sTN

F-120572

(pg

mL) lowastlowast

lowastlowast


(c)

sTN

FR1

(pg

mL)

0 2 5 18 24 30 48 720

200

400

600

800

1000

MOI 1MOI 01

BCG (hour)

(d)

sTN

FR2

(pg

mL)

0 2 5 18 24 30 48 720

500

1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)



0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















UninfectedA

nnex

in-V

7-AAD

Early apoptosis

Late apoptosis

Live cells

Necrotic cells



(a)

SSC

FSC

On general gate

(b)

Ann

exin

-VA

nnex

in-V

Ann

exin

-VA

nnex

in-V

7-AAD 7-AAD

7-AAD7-AAD

MO

I 1M

OI 0

1

MO

I 1M

OI 0

1

BCG (5h) BCG (18h)




87 plusmn 3

2 plusmn 02





(c)

Figure 1 Continued


Early

apop

totic

cells

()

0

2

4

6

8

10

5 18

BCG (hour)

0

lowastlowast

MOI 1MOI 01

(d)

Late

apop

totic

cells

()

0

5

10

15

5 18

BCG (hour)

0

lowast

MOI 1MOI 01

lowast

(e)







0 2 5 180

5

10

15

20

25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

0

5

10

15

20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

0 2 5 18 24 30 48 720

1000

2000

3000

400060008000

1000012000

MOI 1MOI 01

BCG (hour)

sTN

F-120572

(pg

mL) lowastlowast

lowastlowast


(c)

sTN

FR1

(pg

mL)

0 2 5 18 24 30 48 720

200

400

600

800

1000

MOI 1MOI 01

BCG (hour)

(d)

sTN

FR2

(pg

mL)

0 2 5 18 24 30 48 720

500

1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)



0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Early

apop

totic

cells

()

0

2

4

6

8

10

5 18

BCG (hour)

0

lowastlowast

MOI 1MOI 01

(d)

Late

apop

totic

cells

()

0

5

10

15

5 18

BCG (hour)

0

lowast

MOI 1MOI 01

lowast

(e)







0 2 5 180

5

10

15

20

25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

0

5

10

15

20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

0 2 5 18 24 30 48 720

1000

2000

3000

400060008000

1000012000

MOI 1MOI 01

BCG (hour)

sTN

F-120572

(pg

mL) lowastlowast

lowastlowast


(c)

sTN

FR1

(pg

mL)

0 2 5 18 24 30 48 720

200

400

600

800

1000

MOI 1MOI 01

BCG (hour)

(d)

sTN

FR2

(pg

mL)

0 2 5 18 24 30 48 720

500

1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)



0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 2 5 180

5

10

15

20

25

MOI 1MOI 01

BCG (hour)

iTN

F+ce

lls (

)lowastlowast

lowastlowast

(a)

0 2 5 18

0

5

10

15

20

MOI 1MOI 01

BCG (hour)

mTN

F+ce

lls (

) lowastlowast

(b)

0 2 5 18 24 30 48 720

1000

2000

3000

400060008000

1000012000

MOI 1MOI 01

BCG (hour)

sTN

F-120572

(pg

mL) lowastlowast

lowastlowast


(c)

sTN

FR1

(pg

mL)

0 2 5 18 24 30 48 720

200

400

600

800

1000

MOI 1MOI 01

BCG (hour)

(d)

sTN

FR2

(pg

mL)

0 2 5 18 24 30 48 720

500

1000

15002000

4000

6000

MOI 1MOI 01

BCG (hour)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(e)



0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-6

(pg

mL)

4000

3000

2000

1000

150

100

50

0

lowastlowast


lowastlowastlowast

lowastlowastlowast

(a)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

IL-1120573

(pg

mL)

350

300

250

200

150

100

50

0


lowastlowast

lowast

lowast

(b)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MCP

-1(p

gm

L)

20000

15000

10000

5000

2000

1500

1000

500

0

lowastlowast

lowastlowast


(c)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

KC (p

gm

L)

50

40

30

20

10

0

lowast

lowast

(d)

0 2 5 18 24 30 48 72

MOI 1MOI 01

BCG (hour)

MIP

-1120572

(pg

mL)

250000

200000

150000

100000

50000

30000

15000

0

(e)









4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion




00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

5 180BCG (hour)

2

MOI 1MOI 01

NF120581

B-P

(rel

ativ

e uni

ts)lowastlowast

(a)A

SK1-

P (r

elat

ive u

nits)

00

02

04

06

08 lowast

5 180BCG (hour)

2

MOI 1MOI 01

(b)

ERK1

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(c)

ERK2

-P (r

elat

ive u

nits)

00

05

10

15

20

5 180BCG (hour)

2

MOI 1MOI 01

(d)

011 011 011 MOI

Tubulin

0NF120581B-P

ASK1-P

ERK1-PERK2-P

2h 5h 18h

(e)



MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MOI 1 MOI 01

100 101 102 103 104100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


supe

rnat

ant f

rom

infe

cted

cells(0h)

(2h)(5h)(18h)


(a)

MOI 1 MOI 01

100 101 102 103 104 100 101 102 103 104


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


+ su

pern

atan

t fro

min

fect

ed ce

lls(0h)(2h)(5h)(18h)


(b)

Figure 5 Continued


EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EdU

Ale

xa F

luor

488

(MFI

)

0

10

20

30

40

50708090

100


ium

New

0h 2h 5h 18h

lowastlowast

MOI 1MOI 01

12h after culture

(c)

0

20

40

60

80

100

lowastlowast

lowastlowast

EdU

Ale

xa F

luor

488

(MFI

)


ium

New

0h 2h 5h 18h

MOI 1MOI 01

24h after culture

(d)









5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 18BCG (hour)

20

MOI 1MOI 01

06

04

02

00iN

OS

(rel

ativ

e uni

ts)

lowastlowast

(a)

iNOS

Tubulin

011 011 011 MOI02h 5h 18h

(b)

Nitr

ite(120583

M)

40

30

20

101086420

0 2 5 18 24 30 48 72

BCG (hour)

MOI 1MOI 01




(c)



Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(a)

Uni

nfec

ted

MOI 1 MOI 01


BCG(2

h)

(b)

0

20

40

60

80

100

5 180

BCG (hour)

2

RAW

-RFP

+ce

lls (

)

MOI 1Uninfected

MOI 01

(c)

MOI 1 MOI 01

FSC FSC

FSCFSC

FSC FSC

SSC

SSC

SSC

SSC

SSC

SSC

BCG(5

h)BC

G(2

h)BC

G(18

h)

BCG(5

h)BC

G(2

h)BC

G(18

h)

(d)






NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)(a)


NucleusMitochondria

BCG-GFP

BCG(5

h)BC

G(18

h)

(b)

5 18BCG (hour)

2

MOI 1Uninfected

MOI 01

20

15

10

05

00

TOM40

(rel

ativ

e uni

ts)

lowast lowast

(c)

TOM40

Tubulin


2h 5h 18h

(d)








Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Competing Interests




Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Low Dose BCG Infection as a Model for