SymbiontAssociated
Molecular
Patterns
SAMPs
Rafael / Cláudia / Thaís
We are (fortunately) not alone:
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.
Lee et al. 2010, Science
Inside us...
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
Ley et al. 2006, Cell
Symbiont microbiota
Phyla
Defhlefsen et al. 2007, Nature
These microbes have patterns...
CpG DNA
Flagelin
Lipopolysacharide
ssRNA
Peptideoglycan
Symbionts bacteria are bacteria, and they have patterns too!!!!
So, how do symbionts avoid triggering intestinal immunity in their mammalian hosts?
Ignorance
Tolerance or 3Ignorance?
2
1
...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
Germ-free mice: deficiency on IL-17+ cells
Atarashi et al. 2008 Nature Letters
Roun et al. 2009, Nat. Immunol
Exemple:
Inflammatory Bowel Disease
Roun 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
Nature, 2008
Bacteroides fragilis
Polysaccharide A, PSA
Proposed model
Roun et al. 2009, Nat. Immunol
12204–12209 | PNAS | July 6, 2010 | vol. 107 | no. 27
active role
Treg/Th17 axis
Objective: to evaluate the role of Bacteroides fragilis
in the induction of intestinal tolerance
Could B. fragilis colonization directly affects Treg
development?
lethally
C57BL/6 irradiated
or
Germ-free
Bone marrow
Foxp3-GFP
± B. fragilis or
B. fragilis ΔPSA
Analysis of percentage of
Treg cells and IL-10
production
MLN
Colon
Colon
Could B. fragilis colonization directly affects Treg
development?
lethally
C57BL/6 irradiated
or
Germ-free
Bone marrow
Foxp3-GFP
± B. fragilis or
B. fragilis ΔPSA
IL-10 production by Foxp3
Treg cells
MLN
lamina propria lymphocytes
Is PSA able to convert Foxp3+ T cells from Foxp3precursors?
CD4+Foxp3
T cells
Foxp3-GFP
-
± B. fragilis or
B. fragilis ΔPSA
Germ-free
Rag-/-
MLN
Expression of Foxp3
and IL-10 on CD4+ T
Does PSA promote inducible Foxp3+ Tregs with supressive
activity?
gavaged
Foxp3-GFP
purified PSA
or PBS
Analysis of CD4+ CD25+ Foxp3+ T
cell population from the MLN
How PSA affect the development of Foxp3+ Tregs ?
gavaged
Foxp3-GFP
purified PSA
or PBS
RNA extraction of CD4+ Foxp3+ and
CD4+ Foxp3- T cells from the MLN
*
*
By which mechanism does PSA promote Tregs ?
gavaged
TLR2-deficient
purified PSA
or PBS
Analysis of CD4+ Foxp3+ T
cell-development
Is there an effect of PSA on colitis development?
TNBS
BALB/c
Treated with
PSA or PBS
Analysis of CD4+ Foxp3+ T
cells from the MLN
Is there an effect of PSA on colitis development?
Treated with
PSA or PBS
WT or TLR2-/-
TNBS
Clinical score
Is there an effect of PSA on colitis development?
Treated with
PSA or PBS
TNBS
Cytokine production
WT or TLR2-/-
Percentage of Treg in TLR2-/-
Is PSA suitable as a treatment for estabilished colitis?
6d
PBS
50ug PSA
TNBS
TNBS
TNBS (pre-TNBS)
50ug PSA
(post-TNBS)
5d
Is PSA suitable as a treatment for estabilished colitis?
6d
PBS
50ug PSA
TNBS
TNBS
TNBS (pre-TNBS)
50ug PSA
(post-TNBS)
Is PSA suitable as a treatment for estabilished colitis?
6d
PBS
50ug PSA
TNBS
TNBS
TNBS (pre-TNBS)
50ug PSA
(post-TNBS)
High doses of TNBS
IL-10
TLR2-dependent
Inducible Foxp3+ Tregs
immunomodulation
Theraphy for IBD
Effector T cell
Thais Herrero
How do symbionts avoid triggering intestinal immunity
in their mammalian hosts?
No..No..no... I´m
symbiontic!!!
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?
Conventional
Stained with anti-CD4 and antiIL-17A
Germ-free
Flow cytometry
B. Fragilis
B. fragilisΔPSA
LPLs
B. fragilis mono-associated animals did not induce Th17 cell development in the colon.
Does Bacteroides fragilis has molecular mechanisms to supress Th17 responses?
PSA or
PBS
Germ-free
Collected the RNA
B. Fragilis
B. fragilisΔPSA
Levels of IL-17A and
RORγt transcript
Stained with anti-CD4 and antiIL-17A
qRT-PCR
Flow cytometry
LPLs
B. fragilisΔPSA
Thus, B. fragilis actively restrains Th17 cell responses during colonization.
Does Tregs prevent immune response during B. fragilis colonization ?
BM from Foxp3-DTR
+
B. fragilis
Treatment with PBS (DT) or
diphtheria toxin (+DT)
Restimulated with PMAionomycin + brefeldin A
Stained for CD4, IL17A and Foxp3
LPLs
Germ-free Rag-/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?
4 days
+
WT or Tlr2-/-
Supernatants
ELISA
Splenic CD4+ T cells
TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA.
Does Treg suppression function is mediated by TLR2 signaling?
Anti-CD3 and TGF-β + PSA or
TLR ligands
CFSE pulsed CD4+Fop3-
+
Foxp3+EGFP or
CD4+Foxp3+Tregs
Proliferation
Flow
cytometry
CD4+Foxp3+Tregs
Tlr2-/- X Foxp3+EGFP
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.
Is the mechanism responsible to suppress Th17 cell responses?
CD4+ Tcells from WT or Tlr2-/+
B. Fragilis or B. FragilisΔPSA
Stained with anti-CD4
and anti-IL-17A
2 months
Germ-free Rag-/-
Flow cytometry
LPLs
These data demonstrate that B. fragilis actively suppress Th17 responses through
engagement of TLR2 specifically on T cells.
Can B. fragilis able to associate with the intestinal epithelium?
Germ-free mice
Stained with chicken antibodies
against B. fragilis and DAPI
Colon
B. fragilis mono-associated mice
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?
Colon
RNA from colon
homegenates
qRT-PCR
GF, B. frag, ΔPSA and
ΔPSA+PSA
Yes, PSA is important for maintaining host-bacterial symbiosis at the epithelial surface of the gut.
?
B. fragilis
PSA
Th17
Th17
Mucosal colonization
Th17
TLR2
CD4+
IL-10
Tregs
Tregs
Tregs
Tregs
Tregs
Tregs
To test this model.....
CD4+ Tcells from WT or Tlr2-/Foxp3+ - DTR
+ + B. Fragilis
PBS or DT (i.p)
B. Fragilis or B. FragilisΔPSA
2 months
Germ-free Rag-/Germ-free Rag-/-
Colon
2 months
RNA from colon
Colon
homegenates (B. fragilisqRT-PCR
or
RNA from colon
B. fragilis ΔPSA)
homegenates (B. fragilis or
B. fragilis
ΔPSA)
Rag-/- Foxp3-DTR
monoassociated with B. fragilis
qRT-PCR
Finally,
To determine the role of IL-17 resposnses in mucosal association.....
Isotype control or anti-IL-17
Days 0, 5, 10, 15 and 20)
24 days
B. fragilisΔPSA
Colon homegenates
CFU
qRT-PCR
These data indicate that IL-17 suppression by PSA is required by B. fragilis during association
with its host.
Conclusion
B. fragilis
Th17
PSA
Th17
Mucosal colonization
Th17
TLR2
CD4+
IL-10
Tregs
Tregs
Tregs
Tregs
Tregs
Tregs
New insight...
Immunologic
ignorance
Certain symbiotic bacteria adhere to
the intestinal mucosal
Not explain why inflammation is
averted by the microbiota
New insight...
SAMPs
(symbiont-associated
molecular patterns)
has
?Who
evolved?
PSA
Immunologic
Tolerance
To orchestrate immune
responses to establish
host-commensal symbiosis.
Symbionts
Pathogens