Transcript Mucosal Immunity Part 2

Mucosal Immunity Part 2
Sarah L. Gaffen
Spring, 2009
[email protected]
Mucosal Immunity & Infections
• Balancing act
– Tolerate commensals, food antigens
– Protect against infectious agents
• Gut is most frequent site of infection against
pathogenic bugs
• Emerging themes: Th17 cells in the mucosa
Mucosal pathogens exploit mucosal
surfaces
• Enteric pathogens often enter via M cells
– Salmonella – typhoid, food poisoning
– Shigellae – dysentery
– Yersiniae – plague
Oral Tolerance
• Oral tolerance is
– A general immunosuppressive state in the oral
mucosa to prevent reaction to harmless Ags
such as commensals or foods
– the generation of systemic immune
unresponsiveness by feeding of antigen
• Necessary to prevent excessive response to
normal flora and food antigens
AExperiment
modern
version of the
to demonstrate Oral Tolerance
same experiment:
o Mice are given naïve CD4+ T cells that
transgenically express TCR specific for OVA
peptide (OT-II mice)
o Mice are fed ovalbumin
o After tolerance induction, OVA-T cells are
anergized - they do not proliferate
to OVA/adjuvant in vitro.
o Smaller numbers of OVA-specific
T cells suggest clonal deletion
Limitations to Oral Tolerance
• Can be overcome with mucosal adjuvant (e.g.
Cholera toxin)
• Alter physical characteristics of antigen:
antigen in micro-spheres that target PP
• Feeding of attenuated enteric pathogen
expressing the antigen (Salmonella)
Commensals
• They outnumber us at least 10-100:1 (“you
are only 1-10% human”)
• Weight ~1 kg
• Normal commensal flora maintain health
– Help metabolize cellulose, etc.
– Fill niches to prevent pathogenic bacteria from
colonizing
– Required for immune development: germ-free
rodents have reduced lymphoid organs, low Ig,
reduced immune responses of all types
– Help maintain tolerance
Commensals are immunosuppressive
• Different microbiota in disease vs health
• Intestinal epithelial cells play role in regulating
response to commensals
– IECs deficient in SIGGIR (negative regulator of TLRs)
show increased cytokines, increased susceptibility to
intestinal inflammation
• Immune status of host also influences makeup
of commensal flora, thereby affecting immune
system function
– IgA-deficient mice have different microbial spp
– Tbet KO/scid mice have increased “colitogenic”
bacteria and UC symptoms
Specific commensals determine
inflammation vs. suppression
• Differentiation of lamina propria T cells into
Treg vs Th17 cells is determined by flora
– Bacterial DNA signals through TLR9 to suppress
Treg conversion, acts as a “natural adjuvant” (Hall,
Immunity 2008)
– Specific types of flora promote Th17 vs Treg
development in the intestine (Ivanov, Cell Host &
Microbe, 2008)
Commensals & Pathogens
influence NF-kB
Artis, Nat Rev Immunol 2008
Examples of mucosal diseases- role
for Th17 cells
• Gut: IBD – autoimmune diseases of the gut
• Vaginal mucosa:Th17 cells are protective against
Neisseria gonnorrheae
• Lung: Th17 cells are protective against various
pneumoniaes, TB, etc., promote airway
hyperreactivity/allergy
• Mouth: Th17 cells promote Sjogren’s
(autoimmune disease of salivary gland, tear
ducts), but protective in oropharyngeal
candidiasis (“thrush”)
The Spectrum of
Inflammatory Bowel Disease
Ulcerative colitis
Crohn’s disease
IBD: Evidence of Genetic
Influence
• Racial differences in incidence:
White > Black > Asian
• Ethnic influences:
Jewish > non-Jewish; Ashkanazi > Sephardic
• Twin studies: Monozygotic > dizygotic
• Association with specific HLA antigens
• Genes
– NOD2, IL-23R
Sartor RB. Inflammatory Bowel Dis. 1995;24:475.
NOD2
• First identified Crohn’s disease
susceptibility gene.
• Chromosome 16q12.
• One frameshift mutation (Europe and
USA) and 2 point mutations (Europe)
identified.
• Allele frequency 8.2% in CD, 4.0% in
controls, 3.2% in UC.
Ougura et al; Nature 2001; 411: 603
Hugot et al; Nature 2001; 411: 599
IL23R
NOD2
ATG16L1
5p13
IRGM
IBD5
NKX2-3
5q33
MHC
3p21
10q21
PTPN2
SBNO2
Innate Mucosal defenses- Pattern
Recognition Receptors
• TLRs – Toll-like receptors
– Bind LPS, flagellin, bacterial (CpG) DNA, dsRNA,
etc.
– “extracellular” pattern recognition
• NLRs – NOD-like receptors
– Bind muramyl di- and tri-peptides (from LPS)
– “intracellular” pattern recognition
• C-type lectin receptors
– Dectins
– Clec’s
Kawai and Akira (2006) Cell Death and Differentiation
NODs complement function of TLRs
• NODs (nucleotide-binding oligomerization domain)
recognize microbial components found in cytosol
• NODS are intracellular pattern recognition receptors
• NODs bind peptidoglycan in bacterial cell walls
• NODs lead to activation of Caspase-1, cleavage of inactive
forms of IL-1 (and other related cytokines)
• NODs can also downregulate immune responses
• Mutations in NOD genes associated with Crohn’s disease,
inflammatory bowel disease, asthma
NLRs
“Loss of Function” Mutations in NOD2
• How could this lead to IBD?
– Bacterial products from commensal flora deliver
anti-inflammatory signals through NOD2
– Intracellular defect in LPS signaling (NOD2) may
lead to increased extracellular signaling (TLRs)
– Decreased apoptosis of bacterially activated cells
– Defective killing of bacteria leading to persistent
immune response
– Maeda et al, Science 2005
Local Environmental
Factors in IBD
• Germ-free-derived colitis-prone animals do not
develop IBD unless commensal enteric bacteria are
re-introduced
• Restriction of bacterial colonization reduces the
incidence of IBD
• Antibiotic administration reduces the incidence of
IBD
• Enteric bacterial antigens activate T cells and
induce colitis
• A selected subset of bacterial proteins triggers
immune reactions
• No specific bacterial or viral infection has been
identified
Cytokines in IBD
• Spontaneous IBD models
– IL-10 -/– IL-10R -/– TGFb -/– IL-2 -/– Transgenic TNF
– IL-12p40-KO mice are resistant
• Long interpreted to mean that Th1 cells
mediate pathology in IBD, CD
- anti-IL-12p40 used in humans to treat CD!
Chronic Inflammation:
Imbalance Between Mediators
IFN-g
IL-12/23
IL-17
IL-1b
TNF-a
IL-10
TGF-b
IL-1ra
IL-4/IL-13
IL-10
• Immunosuppressive cytokine
– Inhibits DC maturation
– Inhibits TNF production by macrophages
– Inhibits IL-12 production
– Limits “collateral damage” in infection
• IL-10-KO mice have severe, spontaneous IBD
• Produced by all Th subsets (not just Th2)
• IL-23 inhibits IL-10 production in Th17 cells
• O’Garra and Vieira, Nat Rev Immunol, June 2007
Cytokines in IBD
• Spontaneous IBD models
– IL-10 -/– IL-10R -/– TGFb -/– IL-2 -/– Transgenic TNF
– IL-12p40-KO mice are resistant
• Long interpreted to mean that Th1 cells
mediate pathology in IBD, CD
- anti-IL-12p40 used in humans to treat CD!
IL-17-producing T cells
Th1- IFNg (Cell-mediated
immunity)
IL-12
p40/p35
Thp
IL-2,
TGFb
IL-4
Th2- IL-4, IL-5, IL-13 (Humoral
immunity, allergy)
TGFb
+ IL-6, IL-1, IL-23 (p40/p19)
Th17- IL-17, IL-17F, IL-21, IL-22, IL26 (Inflammation, autoimmunity)
IL-23R
Treg- IL-10, TGFb (Immune Suppression)
IL-12 family of cytokines
IL-23 rather than IL-12 per se in IBD
• IL-10KO
– Strong disease
• IL-10KO x IL-12p35KO
– Still get disease
• IL-10KO x IL-23p19KO
– Protected from disease
J. Clin. Invest. David Yen, et al. 116:1310 doi:10.1172/JCI21404
IL23R
NOD2
ATG16L1
5p13
IRGM
IBD5
NKX2-3
5q33
MHC
3p21
10q21
PTPN2
SBNO2
Probiotics and IBD
• Lactobacillus prevents colitis in mouse models of
IBD; can inhibit NF-kB and hence inflammation
• E. coli as effective as mesalazine in remission
maintenance in UC.
• Enteric helminths are anti-inflammatory;
generate strong Th2 response (inhibits Th17?)
(“eat worms!”)
• Epidemiologic data incidence of IBD increases with
improved sanitation = “hygiene hypothesis” (more
Th1 responses, inhibits Th17?)
IL-17-producing T cells
Th1- IFNg (Cell-mediated
immunity)
IL-12
p40/p35
Thp
IL-2,
TGFb
IL-4
Th2- IL-4, IL-5, IL-13 (Humoral
immunity, allergy)
TGFb
+ IL-6, IL-1, IL-23 (p40/p19)
Th17- IL-17, IL-17F, IL-21, IL-22, IL26 (Inflammation, autoimmunity)
IL-23R
Treg- IL-10, TGFb (Immune Suppression)
Examples of mucosal diseases- role
for Th17 cells
• Gut: IBD – autoimmune diseases of the gut
• Vaginal mucosa:Th17 cells are protective against
Neisseria gonnorrheae
• Lung: Th17 cells are protective against various
pneumoniaes, TB, etc., promote airway
hyperreactivity/allergy
• Mouth: Th17 cells promote Sjogren’s
(autoimmune disease of salivary gland, tear
ducts), but protective in oropharyngeal
candidiasis (“thrush”)
Immunity in the oral mucosa:
Lick your wounds
Candida albicans
•Common fungal commensal of the human
oral cavity
•Pathogenic when host immune response
compromised
•HIV+
•Cancer-radiation and chemotherapy
•HIES (hyper-IgE syndrome)
•Infants and elderly
•Progression from commensal state to
pathogen is poorly understood
(Berman, 2002)
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CD4+ T cell are vital for protection against OPC
Mucocutaneous
Systemic
Immune Components:
CD4+ T cells, neutrophils,
monocytes, macrophages,
dendritic cells, gd T cells,
mucosal epithelial cells,
antimicrobial peptides,
cytokines
Vaginal
CD4+ T cells
Oropharyngeal candidiasis
“OPC”
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Which CD4+ Th cell subset is involved in
immunity to OPC?
Th1
CD4+
IFN-g
Cell-mediated
Immunity
Intracellular
bacteria,
protozoa, viruses
IL-12
Thp
Th2
IL-4
CD4+
IL-23
CD4+
Th17
CD4+
IL-4,5,13
Humoral
Immunity
Helminthes
IL-17
IL-17F
IL-21
IL-22
Acute Inflammation
Autoimmunity
Extracellular
microbes
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Th1 vs. Th17
p40
p40
p35
IL-12
Th1
IL-12p35KO mice
p19
vs.
IL-23
Th17
IL-23p19KO mice
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Immunity to OPC
• Long considered to be Th1-dependent
– IL-12p40KO (Th1-deficient) mice are susceptible
– IL-4KO (Th2-deficient) mice are resistant
• BUT…
– IFNgKO mice (Th1-deficient) are resistant
– IL-12p40 is part of IL-23 (therefore, Th1 and Th17deficient)
• AND …
– IL-17RAKO mice are susceptible to disseminated
candidiasis
– IL-17 and IL-23 drive pathogenesis in gastric
candidiasis
• SO… What is the role of Th17 cells in OPC?
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Experimental Mouse Model of OPC
Monitor weight, appearance, activity level, etc.
Sac mice
collect blood,
LN, spleen,
tongue
Infection
Cortisone
Day: -1
Cortisone
0
1
Tongue-YPD
Agar plates
Cortisone
2
3
4
Mean:
5
3
Colony Count
6
6.4X105
7
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Th17-deficient mice are more susceptible to
OPC than Th1-deficient mice
Conclusion:
Th17 cells, not Th1 cells, are
essential for mucosal host
defense against oral candidiasis
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Th17-deficient mice are more susceptible to
OPC than Th1-deficient mice
IL-12KO + Candida
IL-23KO + Candida
WT-No Cort, Sham
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Th17 cells control PMNs, anti-microbial
defenses
(Kolls et al, Nature Reviews Immunology Nov 2008)
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Host defense against oral C. albicans is mediated mainly
by ab-T cells, rather than gd-T cells
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Affymetrix GeneChip Mouse Genome 430 2.0
Array analysis of tongue tissue
Monitor weight, appearance, activity level
Sac mice
collect blood,
LN, spleen,
tongue, etc.
Infection
Cortisone
Day: -1
Cortisone
0
1
Tongue-YPD
Agar plates
Cortisone
2
3
4
5
Colony Count
6
7
Microarray Microarray
Comparisons:
WT Day 0 vs. WT Day 1
IL-17RAKO Day 0 vs. IL-17RAKO Day 1
WT Day 0 vs. IL-17RAKO Day 0
WT Day 1 vs. IL-17RAKO Day 1
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IL-17 induces expression of
antimicrobial proteins
b-defensin 3 (BD3, DEFB3)
• small inducible cationic protein expressed by salivary gland and
epithelial cells
• Interacts with CCR6
• Exerts potent candidacidal activity in vitro
• Induced by IL-17 and IL-22 in the gut and lungs
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Anti-microbial peptides
• Small, cationic peptides (6-60 a.a.) typically
found in mucosal surfaces and skin, can
directly kill microbes
• Found in frogs, flies, humans, plants, etc.
• Regulated by TLRs, NODs, inflammation
• Often work by disrupting target membranes,
mechanisms not full known
KO
IL-17RA mice show decreased
expression of mBD3
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(Experiment performed in collaboration with Jianing Sun)
IL-23 and IL-17, not IL-12, regulate the antimicrobial
activity of saliva
+ carbachol
+
Add Candida (104 cells)
Incubate 1 hour, 37°C
Plate 500 cells on YPD agar
Count colonies 48 hours later
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(Experiment performed in collaboration with Namrata Nayyar)
Hyper-IgE syndrome
HIES or ‘Job’s syndrome’
•Rare multi-system disorder (incidence <1/106) with immunologic and nonimmunologic features:
•Extremely elevated serum IgE
•Staphylococcal abscesses
•Mucocutaneous and oral candidiasis
•Bone and connective tissue abnormalities
•Characteristic facial features (prominent forehead, fleshy nasal
tip)
•Pathological fractures
•Retention of primary teeth
•Lesions of hard palate and dorsal tongue
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Defective response to IL-6, IL-10, IL-21
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Th cell subset defining transcription factors
Th1
STAT1,4
T-bet
Th2
Thp
STAT6
GATA3
Th17
RORgt
STAT3
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Th17 cells/IL-17 receptor signaling and not Th1 cells are
essential for host defense against OPC
Adapted from O’quinn et al. Advances in Immunonolgy Vol 99, 2008
Oral Cavity
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