Antigen-specific Foxp3+ Tregs control tolerance to gut pathobiont H. hepaticus

Helicobacter hepaticus, H. hepaticus, is an opportunist commensal, a pathobiont, that causes gut inflammation in IL-10 deficient but not in WT mice. A New study in journal Nature from Dan Littman's lab tried to understand basis for such dual nature of host response to H. hepaticus.

The authors have generated transgenic T cells specific for H. hepaticus-unique protein, HH_1713. Next, the authors co-transferred H. hepaticus-specific T cells (HH7-2tg cells) alongside with segmented filamentous bacteria, SFB-specific T cells (7B8tg cells) into WT mice exposed to H. hepaticus. Interestingly, HH7-2tg cells mostly differentiated into RORγt+ Foxp3+ Tregs while 7B8tg cells mostly differentiated into Th17 cells, as observed in earlier studies.

However, similar transfer of H. hepaticus-specific T cells into IL-10KO mice exposed to H. hepaticus mostly yielded inflammatory T cells (Th17 and Th1 cells).

It appears that transcription factor c-MAF control differentiation of H. hepaticus-specific T cells into RORγt+ Foxp3+ Tregs and its deficiency recapitulates H. hepaticus-specific T cells differentiation into inflammatory T helper cells observed in IL-10KO hosts when exposed to H. hepaticus.

In summary, this study indicates that in WT mice harbor H. hepaticus specific Foxp3+ T cells that keep tolerance to H. hepaticus. These induced Foxp3+ T cells are probably generated (or expand) by chain reaction orchestrated by existed antigen-specific natural Tregs since induced Tregs don't develop efficiently when transferred into RAG KO hosts lacking natural Tregs. IL-10 or c-MAF deficiency could alter overall microbiota composition (unrelated to H. hepaticus) and drive shift from Treg program into inflammatory pathway.

posted by David Usharauli

CD4 T cells silence innate over-activation to gut microbiota

A simple but very effective study was published in journal Nature from Ron Germain's lab. His group is known for publishing high quality in situ microscopy data combined with cellular analysis. New study continues this trend. 

In this study the authors analyzed pattern of phosphorylation of STAT3 transcription factor in small intestine derived from various immune deficient mouse strains. Compared to WT mice, pSTAT3 staining in RAG1 KO gut tissue (lacking adaptive immune system) was significantly up-regulated. 

Analysis of antibiotic-treated or germ-free mice indicated that pattern of pSTAT3 staining in RAG1-KO was correlated with the presence of gut microbiota.

Interestingly, longitudinal analysis showed that pSTAT3 staining inversely correlated with  maturation of adaptive immune system post weaning (between 4-20 weeks). 

Co-housing experiments showed that T cells, but not B cells, played a role in silencing innate pSTAT3 over-activation.

And out of T cells, it were CD4 T cells and class II antigen-presentation that played the role in pSTAT3 silencing.

Finally, both Tregs and SBF-specific Th17 cells (7B8 transgenic T cells) could mediate silencing of pSTAT3 over-activation. Both T cell type could down-regulate STAT3 phosphorylation in innate and epithelial cells but the mechanisms could be different. 

In summary, this study showed that persistent pSTAT3 over-activation observed in mice deficient for CD4 T cell function could explain some of chronic metabolic shifts observed in clinical settings. 

posted by David Usharauli

Worm infection tips the balance in favor of Crohn's disease soothing gut flora

This week Science magazine published yet another study that revealed a complex biological inter-species relationship called defensive symbiosis. Here, the authors showed that worm infection of mammalian host favors gut flora species that soothe intestinal inflammation in a T_H2-dependent manner.

Mice deficient in Nod2 develop small intestinal abnormalities that resemble human Crohn's disease (for example, goblet cell defect that compromises intestinal mucus layer). This new study showed that experimental infection of Nod2-deficient mice with the parasitic worm Trichuris muris (T. muris) could restore small intestinal goblet cell numbers and morphology.

Earlier reports showed that intestinal abnormalities in Nod2-deficient mice dependent on gut flora species, Bacteroides vulgatus. Now, the authors showed that T. muris worm infection could reduce Bacteroides vulgatus burden in Nod2-deficient mice in a manner dependent on STAT6 signaling and IL-13 (type II immunity). Similar results were seen with a second worm infection, Heligmosomoides polygyrus.

Finally, the authors showed that worm infection specifically expanded another gut flora species, Clostridiales, that could directly inhibit pro-inflammatory Bacteroides vulgatus.

In summary, this study showed that in Nod2-deficient mice gut species Clostridiales represent defensive symbionts with an antagonistic interaction with another commensal bacteria, Bacteroidales. Worm infection of Nod2-deficient mice tips the balance in favor anti-inflammatory Clostridiales species. This knowledge could be utilized in treatment of Crohn's disease (for example, therapeutic worm infection or application of its derivative that promote type II immunity). 

David Usharauli

Gut-associated IgA repertoire diversity is maintained independently of continuous gut flora exposure

Gut-associated immune system has a delicate task to maintain tolerance towards endogenous gut flora and food-associated antigens while at the same time to detect and mount protective immune response against invading pathogens.

Clonal size and repertoire diversity of gut-associated IgA pool are influenced by presence of flora antigens. However, little is known of the mechanisms that control gut IgA repertoire stability in response to antibiotic treatment or pathogen exposure.

This new paper in Nature Immunology tried to do exactly this. The authors serially sampled and sequenced individual mouse gut IgA pool longitudinally, after (a) antibiotic, (b) pathogen or (c) defined flora colonization, to determine changes in IgA repertoire diversity.

Initially, the authors showed that IgA repertoire diversity in germ-free mice exposed to various combination of flora (mono or poly-colonization) correlated with richness of colonized flora.

However, when GF mouse gut IgA pool was analyzed, before and after flora exposure, the authors noticed no major IgA repertoire change, implying flora-independent IgA repertoire maintenance (through it is not quite clear here how the authors differentiate IgA repertoire "diversity" versus repertoire "similarity").

Interestingly, the authors made similar observation in gut IgA pool from healthy volunteers exposed to antibiotics.

In addition, the article contains several other experiments, but I was not able to decipher their meaning or relevance to the concept, so I did not discuss them here. In general, paper is poorly written and lacks natural flow between experimental hypothesis and experimental results.

In summary, my interpretation of this paper is following: (a) gut IgA repertoire diversity is non-overlapping between individuals, including genetically identical mice, (b) gut IgA repertoire stability is minimally influenced by exposure to antibiotics or new flora (however if it is true and gut immune system maintains its integrity upon antibiotic exposure, then how can we account for frequent GI issues following antibiotic treatments?)

David Usharauli

Specific food additives modify gut microflora and cause metabolic syndrome

Here is another paper that highlights unsuspected health risks of commonly used food emulsifiers, carboxymethylcellulose (CMC) and polysorbate-80 (P80).

Ordinarily, mucus separates gut epithelial cells from the gut microflora. New study in Nature provided evidence suggesting that this separation of gut epithelial cells from the gut microflora is disrupted by CMC and P80, two commonly used food emulsifiers (but not by sodium sulfite, another commonly used food additive).

The authors showed that presence of either CMC or P80 in either drinking water or dry food pellets could promote development of colitis in susceptible mouse strain (IL-10 KO).

In addition, the authors  showed that presence of either CMC or P80 in water or dry pellets could promote development of metabolic syndrome in wild-type as well as in susceptible mouse strain (TLR5 KO).

Interestingly, metabolic syndrome did not develop in germ-free mice treated with CMC or P80.

More importantly, effects of CMC or P80 on animal health were transferable to germ-free mice by fecal transplantation from treated mice, implying direct role of modified gut microflora (note, CMC or P80 did not directly modify mucus thickness in germ-free mice).

In summary, these results support the growing evidence that chemically-processed food carries long-term health risks (development of type II diabetes, metabolic syndrome, inflammatory bowel disease).

David Usharauli

Sweet rejection: sugar-coated malaria parasite

Life is a competition, even for a parasite like a malaria. To infect the host the parasite needs to overcome not just host's defense system but to out-compete the local resident micro-flora as well. 

This new paper in Cell is a thorough research about the initial events of of malaria infection and the role of sugar molecules, called glycans, in the host defense against malaria.

It is well-known that hosts and their parasites may share molecular signature. Blood types, ABO system is one such example. Burnet's clonal selection theory predicts that any such similarity between host-pathogen prevents the host to mount an efficient immune response against shared antigens. As a consequence, based on pathogen burden and evolutionary pressure, the hosts started to loose the capacity to express such shared molecules.

It appears that at some point in their evolution human ancestors lost the capacity to make one type of sugar, alpha-gal, expressed by malaria. This change conferred an improved capacity to defend against malaria. 

This papers shows how exactly such modification provided protection. It turns out to be dependent of natural immunization conferred by gut resident microbes expressing the same exact sugars.

Analysis of serum samples across different age group from children in Mali (malaria endemic region), the authors noticed a gradual increase in anti-alpha-gal IgM level over time. Interestingly, 6-months parasite free condition correlated with higher level of anti-alpha-gal IgM in the serum.

To study this observation in laboratory setting, the authors used mice deficient in the capacity to make alpha-gal (alpha-gal KO). This "human-like" mice can produce anti-alpha-gal antibodies upon colonization with alpha-gal-positive E.coli O86:B7, but not alpha-gal-negative E.coli K12 strain. 

The authors showed that colonization of alpha-gal KO mice with alpha-gal-positive E.coli O86:B7 provided a protection against malaria transmission.

This protection after gut flora colonization was conferred by soluble germ-line, non-mutated IgM.

Immunization of alpha-gal KO mice with  (a) alpha-gal conjugated to BSA (protein carrier) or (b) rabbit RBC (naturally expressing high levels of alpha-gal) conferred protection against malaria transmission.

Deep analyses of mechanism of protection after immunization revealed it was dependent on T cell help and on both IgM and IgG (of note, immunization with rabbit RBC conferred protection even in IgM-deficient mice, unlike protection conferred after colonization with alpha-gal expressing E.coli O86:B7).

The protection could be conferred by passive transfer with IgM, IgG3 and IgG2b, but not IgG1 and IgG2a.

In summary, this study provides evidence how gut flora affects host's defense against parasites by a way of natural immunization. Of course, it is remains to be determined whether immunization against alpha-gal will protect humans as well as it does for "human-like" mice.

David Usharauli