Symbiont-Associated Molecular Patterns

SAMPs

Rafael / Cláudia / Thaís

Bacteria populated Earth 2 billion years before the first signs of eukaryotic life.

They occupy almost every terrestrial and aquatic niche on our planet.

Mitochondria and chloroplasts of eukaryotic cells are descended from bacteria.

Animals represent a stable, nutrient-rich ecosystem for microbes to thrive; hence, host health is paramount to the microbiota.

In turn, the host benefits from a diverse commensal microbiota that helps to digest complex carbohydrates and provide essential nutrients to mammals.

We are (fortunately) not alone:

Lee et al. 2010, Science

Our intestinal tract is a nutrient-rich environment packed with up to 100 trillion (1014) microbes.

The vast majority reside in our colon where densities approach 1011–1012 cells/ml, the highest recorded for any microbial habitat

Today, there are 6.5 billion humans living on Earth.

Together, we represent a gut reservoir of 1023–1024 microbial cells.

This number is just five orders of magnitude less than the world’s oceans, which contain an estimated 1029 cells

Inside us...

Ley et al. 2006, Cell

Phyla

Defhlefsen et al. 2007, Nature

Symbiont microbiota

These microbes have patterns...

Symbionts bacteria are bacteria, and they have patterns too!!!!

So, how do symbionts avoid triggering intestinal immunity in their mammalian hosts?

Peptideoglycan

Lipopolysacharide ssRNA

CpG DNAFlagelin

Tolerance or Ignorance?

Ignorance

? 12

3

...and tolerance?

Do we develop tolerance to commensal microbes?

or

Do commensal microbes induce that tolerance?

EFFECTS OF MICROBIOTA ON THE HOST IMMUNE SYSTEM

GNOTOBIOLOGY (Greek for “know life”)

=

Selective colonization of germ-free (sterile) animals

Roun et al. 2009, Nat. Immunol

Atarashi et al. 2008 Nature Letters

Germ-free mice: deficiency on IL-17+ cells

Roun et al. 2009, Nat. Immunol

Exemple:

Inflammatory Bowel DiseaseRoun et al. 2009, Nat. Immunol

Model: IBD (Inflammatory Bowel Disease)

Crohn’s disease and ulcerative colitis together refereed to as IBD, lead to long term and sometimes irreversible impairment gastrointestinal structure and function.

Prevalence range of 10-200 case per 100 000 individuals on North America and Europe.

Innapropriate and exagerated mucosal immune response to normal constituents of mucosal microbiota.

Bouma et al. 2003, Nat Rev Immunol

Bacteria and IBD

Roun et al. 2009, Nat. Immunol

Pathways to Mucosal inflammation

Th17

Bouma et al. 2003, Nat Rev Immunol

However, microbial molecules that coordinate

the Treg/Th17 axis remain to be described

Weaver et al. 2009, Nat Rev Immunol

Bacteroides fragilis

Polysaccharide A, PSA

Nature, 2008

?Proposed model

Roun et al. 2009, Nat. Immunol

12204–12209 | PNAS | July 6, 2010 | vol. 107 | no. 27

Objective: to evaluate the role of Bacteroides fragilis in the induction of intestinal tolerance

active role Treg/Th17 axis

C57BL/6or

Germ-free

± B. fragilis or B. fragilis ΔPSA

Analysis of percentage of Treg cells and IL-10

production

MLN

Colon

Could B. fragilis colonization directly affects Treg development?

lethally irradiated

Bone marrow Foxp3-GFP

Colon

lamina propria lymphocytes

MLN

C57BL/6or

Germ-free

± B. fragilis or B. fragilis ΔPSA

IL-10 production by Foxp3 Treg cells

Could B. fragilis colonization directly affects Treg development?

lethally irradiated

Bone marrow Foxp3-GFP

Foxp3-GFP

CD4+Foxp3-

T cellsGerm-free

Rag-/-

Expression of Foxp3 and IL-10 on CD4+ T

MLN

Is PSA able to convert Foxp3+ T cells from Foxp3- precursors?

± B. fragilis or B. fragilis ΔPSA

Foxp3-GFP

purified PSA or PBS

Analysis of CD4+ CD25+ Foxp3+ T cell population from the MLNgavaged

Does PSA promote inducible Foxp3+ Tregs with supressive activity?

Foxp3-GFP

purified PSA or PBSgavaged

RNA extraction of CD4+ Foxp3+ and

CD4+ Foxp3- T cells from the MLN

* *

How PSA affect the development of Foxp3+ Tregs ?

TLR2-deficient

purified PSA or PBSgavaged

Analysis of CD4+ Foxp3+ T cell-development

By which mechanism does PSA promote Tregs ?

BALB/c

TNBSTreated with PSA or PBS

Analysis of CD4+ Foxp3+ T cells from the MLN

Is there an effect of PSA on colitis development?

WT or TLR2-/-

TNBSTreated with PSA or PBS

Clinical score

Is there an effect of PSA on colitis development?

WT or TLR2-/-

Cytokine production

Percentage of Treg in TLR2-/-

Treated with PSA or PBS

TNBS

Is there an effect of PSA on colitis development?

PBS TNBS6d

TNBS 50ug PSA

(pre-TNBS)

(post-TNBS)

50ug PSA TNBS

5d

Is PSA suitable as a treatment for estabilished colitis?

PBS TNBS6d

TNBS 50ug PSA

(pre-TNBS)

(post-TNBS)

50ug PSA TNBS

Is PSA suitable as a treatment for estabilished colitis?

PBS TNBS6d

TNBS 50ug PSA

(pre-TNBS)

(post-TNBS)

50ug PSA TNBS

Is PSA suitable as a treatment for estabilished colitis?

High doses of TNBS

Inducible Foxp3+ Tregs

IL-10

immunomodulation Theraphy for IBD

TLR2-dependent

Effector T cell

Thais Herrero

No..No..no... I´m symbiontic!!!

How do symbionts avoid triggering intestinal How do symbionts avoid triggering intestinal immunity in their mammalian hosts?immunity in their mammalian hosts?

Objective: To demonstrate the mechanisms by which our immune system differentiates between the

microbiota and pathogenic microbes.

Does Bacteroides fragilis has molecular mechanisms to supress Th17 response?

LPLs

Conventional

Germ-free

B. Fragilis

B. fragilisΔPSA

Stained with anti-CD4 and anti-IL-17A Flow cytometry

B. fragilis mono-associated animals did not induce Th17 cell development in the colon.

Does Bacteroides fragilis has molecular mechanisms to supress Th17 responses?

qRT-PCRGerm-free

B. Fragilis

B. fragilisΔPSA

Collected the RNA Levels of IL-17A and RORγt transcript

B. fragilisΔPSA

PSA or PBS

LPLs

Stained with anti-CD4 and anti-IL-17A

Flow cytometry

Thus, B. fragilis actively restrains Th17 cell responses during colonization.

Does Tregs prevent immune response during B. fragilis colonization ?

Germ-free Rag-/-

BM from Foxp3-DTR+

B. fragilisTreatment with PBS (-

DT) or diphtheria toxin (+DT)

LPLs

Restimulated with PMA-ionomycin + brefeldin A Stained for CD4, IL-

17A and Foxp3

Flow cytometry

These results suggests that Foxp3+ Tregs are required for supression of Th17 cells during B. fragilis colonization.

What is the mechanism whereby B. fragilis suppresses Th17 cells responses?

WT or Tlr2-/-

+

Splenic CD4+ T cells

4 daysSupernatants ELISA

TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA.

These studies show that unlike other TLR2 ligands, PSA enhances Tregs function and gene expression in the absence of APCs through TLR2 signaling directly on CD4+Foxp3+ Treg cells.

Does Treg suppression function is mediated by TLR2 signaling?

Foxp3+EGFP or

Tlr2-/- X Foxp3+EGFP

CD4+Foxp3+Tregs

Anti-CD3 and TGF-β + PSA or TLR ligands CFSE pulsed CD4+Fop3-

CD4+Foxp3+Tregs

+Flow

cytometryProliferation

Is the mechanism responsible to suppress Th17 cell responses?

Germ-free Rag-/-

CD4+ Tcells from WT or Tlr2-/-+

B. Fragilis or B. FragilisΔPSA

LPLs

Stained with anti-CD4 and anti-IL-17A Flow cytometry

These data demonstrate that B. fragilis actively suppress Th17 responses through engagement of TLR2 specifically on T cells.

2 months

Can B. fragilis able to associate with the intestinal epithelium?

Colon

Germ-free mice

B. fragilis mono-associated mice

Stained with chicken antibodies against B. fragilis and DAPI

Confocal microscopy

B. fragilis can associates with the intestinal epithelium and these data indentify a previously unappreciated mucosal niche for B. fragilis.

Is PSA important to association of B. fragilis with the intestinal epithelium?

GF, B. frag, ΔPSA and ΔPSA+PSA

Colon RNA from colon

homegenatesqRT-PCR

Yes, PSA is important for maintaining host-bacterial symbiosis at the epithelial surface of the gut.

TLR2

CD4+

PSA

B. fragilis

Tregs

Tregs

TregsTregs

Tregs

Tregs

IL-10

Th17

Th17

Th17

Mucosal colonization

??

To test this model.....

Germ-free Rag-/-

CD4+ Tcells from WT or Tlr2-/-+

B. Fragilis or B. FragilisΔPSA

Colon RNA from colon

homegenates (B. fragilis or B. fragilis ΔPSA)

qRT-PCR2 months

Germ-free Rag-/-

Foxp3+ - DTR + B. Fragilis

2 monthsRag-/- Foxp3-DTR mono-associated with B. fragilis

PBS or DT (i.p)

Colon RNA from colon homegenates (B. fragilis or

B. fragilis ΔPSA)qRT-PCR

Finally,

To determine the role of IL-17 resposnses in mucosal association.....

B. fragilisΔPSA

Isotype control or anti-IL-17Days 0, 5, 10, 15 and 20)

24 days Colon homegenatesCFU

qRT-PCR

These data indicate that IL-17 suppression by PSA is required by B. fragilis during association with its host.

TLR2

CD4+

PSA

B. fragilis

Tregs

Tregs

TregsTregs

Tregs

Tregs

IL-10

Th17

Th17

Th17

Mucosal colonization

Conclusion

Immunologic ignorance

Certain symbiotic bacteria adhere to the intestinal mucosal

Not explain why inflammation is averted by the microbiota

New insight...

New insight...

SAMPs

(symbiont-associated molecular patterns)

PSA

To orchestrate immune responses to establish

host-commensal symbiosis.

Who has evolved?

PathogensSymbionts

?

Immunologic Tolerance