Microbial determination of mucosal homeostasis vs. chronic inflammation

Anna George

National Institute of ImmunologyNew Delhi

The common mucosal immune system

Gastrointestinal tract

Urinary/genital tract

Respiratory tract

Lactating mammary tissue

Immune cells generated at any one site can traffic to other sites

Primed T/B cells leave the localized lymphoid tissue, enter blood and re-circulate to seed the lamina propria and IEL compartments

Intestinal immunity

PPMLNsLPIELsIgA PCsMast cellsPMNsMFsMicrobesBarrier

Live Microbes: harmless, opportunists, frank pathogens

Dead microbes and microbial products (soluble, particulate)

Food (soluble, particulate, in various stages of digestion)

Enzymes, bile salts, mucus, etc

GF-mice show impaired: epithelial integrityGALT developmentinnate immunityadaptive immunity- esp. IgA

Q: Does our immune system respond to food antigens? Q: Does our immune system respond to our gut flora?

PPs are chronically activated

10 410 310 210 110 010 -10

10000

20000

30000OVA RxPBS Rx

OVA (µg/ml)

CP

M

CF

U/s

ple

en

*

*

10 4

10 5

10 6

10 7

10 8

OVA RxPBS Rx

NRR

Feed (or not) with soluble OVA

Immunize s.c with OVA/CFA

Assay T cell proliferation in DLNs

Feed (or not) with soluble OVA

Immunize i.p with Stm or rStm

Assess bacterial CFU in spleen

PHYLUM ACTINOBACTERIACLASS ACTINOBACTERIA: Bifidobacterium

PHYLUM BACTEROIDETES : FAM BPP (Genus Bacteroides)

PHYLUM FIRMICUTESCLASS BACILLI ORDER BACILLALES: Bacillus

ORDER LACTOBACILLALES: LactobacillusCLASS CLOSTRIDIA FAM CLOSTRIDIACEAE: Clostridium

FAM PEPTOSTREPTOCOCCACEAE: Peptostreptococcus

PHYLUM FUSOBACTERIM: F. prausnitzii

PHYLUM PROTEOBACTERIACLASS ALPHAPROTEOBACTERIACLASS BETAPROTEOBACTERIA: SutterellaCLASS GAMMAPROTEOBACTERIA

FAM ENTEROBACTERIACEAEFAM MORAXELLACEAE : Acinetobacter

CLASS DELTAPROTEOBACTERIACLASS EPSILON PROTEOBACTERIA: Helicobacter

PHYLUM VERRUCOMICROBIA: Akkermansia

CANDIDATUS ARTHROMITIS sp SFB

Phylum Bacteroidetes Phylum Firmicutes(20 genera) (250 genera)

Bacteroides Lactobacillus, Clostridium Mycoplasma,Bacillus

Organisms

Gram+ and Gram-Vegetative forms and spores (SFB)Aerobes, microaerophiles, facultative /strict anaerobesAutochthonous and allocthonous Changes with age (more diverse, Bifidobacterium↓, Bacteroides ↑)Formula-fed: more diverse and colonization quicker after weaning

Lactobacilli constitute only 0.01% of the total cultivable CFU and are not recoverable from 25% of individuals

Domain Bacteria

Flora provide competition for invading pathogens

� Niche occupancy (antibiotic Rx mice more susceptible to C. albicans infection)

�✪ GF mice: high susceptibility to L. monocytogenes: < 500 vs 1E9 (CNV)

Flora facilitate caloric extraction.

�✪ Colonization of adult GF mice with microbes from distal gut of CNV mice leads to weight gain in 10-14 days despite decreased food consumption.

�✪ Methanogenic archaea increase the efficiency of bacterial fermentation by removing one of its end products.

�✪ Flora can synthesize vitamins, especially of the B and K groups

�✪ Flora differences in obese and lean mice. B:F low in ob.

Flora generate SCFAs: main microbial metabolites of fiber

Accelerate cellular metabolismgenerate acetyl CoA

more ATP, more fatty acids, more respiration, more proliferation

Promote Ab responsesIncrease Tfh numbersRegulate gene expression to promote plasma cell generation

inhibit HDAC activity increased Blimp-1, Aid, Xbp-1, etcdecreased Mitf, CD27 etc

Increase Tregs and tolerogenic DCs

Promote neutrophil recruitment and function

Induce mucus secretion from goblet cells

Calibrate inflammation (inflammasome activation, anti-inflammatory

Prevent Y-H change

Kim et al 2016 and others

Flora stimulate the gut immune system

Do we mount immune responses to the microbes present in the gut?

Can we have a situation where the PPs are not chronically activated?

What happens when bacteria are introduced into the gut in such situations?

Colonization of adult GF mice- invasion, clearance, persistence, IgA

Secondary association of formerly GF miceWhat happens during neonatal life

Protection against cross-reactive pathogens (secreted IgA)

IgD

PNA

IgA

d0 d28d14 d107

Bacteria translocate, are cleared from systemic organs, persist-coated with IgA, evoke no chronic B cell response, but IgA memory cells persist.

Q: what happens when a second microbe is introduced?

% specific Total

SFB 1.3% 2460

Morganella morganii 4.8% 924

Listeria monocytogenes 14.6% 2200

Ochrobactrum anthropi 0 560

Helicobacter muridarum 0.8 491

Specific vs aspecific IgA

Does the immune status of the mother affect the immune response of the neonate?

response to environmental antigens

response to specific oral infection

Set up two sets of matings as follows:

Assay serum Ig over time

IgM

IgG2b

IgA

IgG1

Serum Ig in pups born to scid () or +/+ () dams

Set up matings and foster nursing as follows

Infect all pups with reovirus 10 days after birth

Look at the ability of pups to generate antiviral IgA

How does the immune status of the mother influence the response of the pups?

Flora may cause pathogenic or opportunistic invasion.

Systemic infection: S. typhimurium

Inflammatory bowel disease

Crohn’s disease: Th1 response. IL-12, IFN, TNF (+IL17)

Ulcerative colitis : Ab-mediated hypersensitivity (+IL17)

Associations with microbes: Crohn’s: Eubacterium, Peptostreptococcus, Coprococcus high

UC: Streptococcus fecium (GrpD) concentration high in lumen

Enteric commensals known to initiate IBD in model animals

B. vulgatis HLA-B27 transgenic rats

E. fecalis in IL10-/- mice

H. muridarum in germ-free SCID mice

Spontaneous C3H/HeJ-Bir

KO mice IL2, IL10, TNFα TGFβ, SMAD3, Tcell, cathepsinD

Tnsgenics HLA-B27(rat), some TCR Tgs (Cytc vs MBP)

Leaky gut N-cadherin DN chimeras, mdr1a KOepithelium Chemically carageenan, DSS (MyD88 KO mice sensitive) induced colitis

Transfer Naive CD4 cells into SCID or RAG-/- mice

Tregs Disruption of regulatory T cell function

Mouse models for IBD

Transfer of CD25-depleted CD4 cells into athymic mice: autoimmunity

gastritis, colitis, thyroditis, insulitis, glomerulonephritis, sialadenitis, oophoritis, polyarthritis

Role of intestinal microbiota

naive CD4 cells -> CNV scids Colitis +

-> GF scids Colitis -

-> GF scids +H. muridarum Colitis +

-> GF scids +SFB /Mm /Lm /Oa Colitis -

Germ-free HLA-B27 transgenic rats colonized with:

Colitis Gastritis

Cocktail of 6 microbes including B. vulgatis ++++ ++++

Cocktail minus B. vulgatis + +

B. vulgatis (monoassociation) ++++ -

E.coli (monoassociation) + -

B. vulgatis (with caecum excluded from - - fecal stream)

disease

Barrier integrity and cell death in intestinal homeostasis

Low (A) and high (B) magnification images of inflamed colonic mucosal resection and biopsy specimens depicting ulceration (black arrow) and neutrophils migrating across the epithelium that results in formation of a crypt abscess (yellow arrow).

Neutrophil infiltration of intestinal mucosa is associated with tissue injury, disrupted barrier, and disease symptoms.

Spontaneous C3H/HeJ-Bir

KO mice IL2, IL10, TNFTGF, SMAD3, Tcell cathepsinD

Transgenics HLA-B27(rat), some TCR Tgs (Cytc vs MBP)

Leaky gut N-cadherin DN chimeras, mdr1a KOepithelium Chemically carageenan, DSS (MyD88 KO mice sensitive) induced colitis

Transfer Naive CD4 cells into SCID or RAG-/- mice

Tregs Disruption of regulatory T cell function

From Sumagin et al 2014, 2016

IRT5 (combination of 5 probiotic strains: Kwon et al 2010

B and T cell hyporesponsiveness

No induced apoptosis

Increased generation Tregs in MLNs via induction of CD11c+ rDCs(increased IL-10, COX-2, IDO, TGF-b)

Suppresses progression of experimental IBD.

Special considerations for mucosal immunity

Immunization has to be mucosal

Antigen intake is hard to predict or evaluate

No adjuvants available

Cannot boost for IgA responses

Chronic activation by gut microbes, food etc. Hence the precise immune status of individuals is unknown

Maternal status

Bifidobacterium longum AH1206: Maldonado-Gomez et al, 2016:

SAM strain: includeSAMP (accelerated senescence-prone, short-lived) and

SAMR (accelerated senescence-resistant, longer-lived

SAMP mice: develop CD like symptoms (chronic ileitis) with age

Administration of VSL3 (probiotic mixture of 8 different strains)

prevention protocol: in 3-week old miceTreatment protocol: in 30-week old mice

Effective in: prevention protocol (given for 6 weeks), not Rx protocol

VSL3 supports ‘physiological’ inflammation

Stimulates TNFa production and NFkB activation in epithelial cellsImproves barrier function

VSL3 in SAMP mice: Pagnini et al 2010

From Rooks and Garrett, 2016

SCFAs

From Brestoff and Artis, 2013