Symbiont- Associated Molecular Patterns SAMPs Rafael / Cláudia / Thaís.
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Transcript of Symbiont- Associated Molecular Patterns SAMPs Rafael / Cláudia / Thaís.
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Symbiont-Associated Molecular Patterns
SAMPs
Rafael / Cláudia / Thaís
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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
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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
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Phyla
Defhlefsen et al. 2007, Nature
Symbiont microbiota
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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
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Tolerance or Ignorance?
Ignorance
? 12
3
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...and tolerance?
Do we develop tolerance to commensal microbes?
or
Do commensal microbes induce that tolerance?
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EFFECTS OF MICROBIOTA ON THE HOST IMMUNE SYSTEM
GNOTOBIOLOGY (Greek for “know life”)
=
Selective colonization of germ-free (sterile) animals
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Roun et al. 2009, Nat. Immunol
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Atarashi et al. 2008 Nature Letters
Germ-free mice: deficiency on IL-17+ cells
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Roun et al. 2009, Nat. Immunol
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Exemple:
Inflammatory Bowel DiseaseRoun et al. 2009, Nat. Immunol
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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
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Bacteria and IBD
Roun et al. 2009, Nat. Immunol
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Pathways to Mucosal inflammation
Th17
Bouma et al. 2003, Nat Rev Immunol
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However, microbial molecules that coordinate
the Treg/Th17 axis remain to be described
Weaver et al. 2009, Nat Rev Immunol
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Bacteroides fragilis
Polysaccharide A, PSA
Nature, 2008
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?Proposed model
Roun et al. 2009, Nat. Immunol
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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
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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
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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
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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
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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?
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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 ?
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TLR2-deficient
purified PSA or PBSgavaged
Analysis of CD4+ Foxp3+ T cell-development
By which mechanism does PSA promote Tregs ?
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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?
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WT or TLR2-/-
TNBSTreated with PSA or PBS
Clinical score
Is there an effect of PSA on colitis development?
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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?
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PBS TNBS6d
TNBS 50ug PSA
(pre-TNBS)
(post-TNBS)
50ug PSA TNBS
5d
Is PSA suitable as a treatment for estabilished colitis?
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PBS TNBS6d
TNBS 50ug PSA
(pre-TNBS)
(post-TNBS)
50ug PSA TNBS
Is PSA suitable as a treatment for estabilished colitis?
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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
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Inducible Foxp3+ Tregs
IL-10
immunomodulation Theraphy for IBD
TLR2-dependent
Effector T cell
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Thais Herrero
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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.
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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.
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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.
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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.
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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.
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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
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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
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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.
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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.
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TLR2
CD4+
PSA
B. fragilis
Tregs
Tregs
TregsTregs
Tregs
Tregs
IL-10
Th17
Th17
Th17
Mucosal colonization
??
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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
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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.
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TLR2
CD4+
PSA
B. fragilis
Tregs
Tregs
TregsTregs
Tregs
Tregs
IL-10
Th17
Th17
Th17
Mucosal colonization
Conclusion
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Immunologic ignorance
Certain symbiotic bacteria adhere to the intestinal mucosal
Not explain why inflammation is averted by the microbiota
New insight...
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New insight...
SAMPs
(symbiont-associated molecular patterns)
PSA
To orchestrate immune responses to establish
host-commensal symbiosis.
Who has evolved?
PathogensSymbionts
?
Immunologic Tolerance