Crohn's disease-like phenotype could be initiated by one gut microbe in genetically susceptible mice

1. The authors used doubly deficient Nod2−/−Cybb−/− [NADPH oxidase] (DKO) mice (genes thought to be altered in #Crohns). When fostered with Taconic dams, but not Jax dams, these DKO pups get spontaneous gut #inflammation after weaning (3 weeks post birth). pic.twitter.com/LdoBA8Dfao

— David Usharauli (@3DiMMUNE) April 20, 2019

3. using the #microbiota analysis tool (linear discriminant analysis
(LDA) effect size (#LEfSe) methodology) the author found that one particular microbiota species, M. schaedleri, showed 1349-fold enrichment in DKO mice but not in single KOs. pic.twitter.com/BzMUB63cGf

— David Usharauli (@3DiMMUNE) April 20, 2019

5. Time analysis showed that DKO pups fostered with Tac dams harbor M. schaedleri-specific IgA and IgG but only before weaning. These #antibody titers quickly decline post weaning that correlated with the onset of #CrohnsDisease-like phenotype. pic.twitter.com/elMRXdMdv3

— David Usharauli (@3DiMMUNE) April 20, 2019

In summary, this study showed that #spontaneous gut #inflammation could be recapitulated in mice doubly deficient for two specific genes, #NOD2 and #NADPH oxidase and exposed to specific gut #microbiota, Mucispirillum schaedleri.

— David Usharauli (@3DiMMUNE) April 20, 2019

So, here we have a complete model of spontaneous gut inflammation initiated by a particular gut microbe in a genetically susceptible host. Is it how #CrohnsDisease develops too in humans?

— David Usharauli (@3DiMMUNE) April 20, 2019

Flagellin-specific T cells induce colitis by recognizing antigen other than flagellin

This is very interesting study from Journal of Immunology (JI). Here, researcher led by Timothy Hand at the University of Pittsburgh Medical School, showed that CBir1 transgenic T cells, thought to cause gut inflammation by recognizing flagellin expressing microbiota, were in fact specific for non-flagellin antigens. 

CBir1 transgenic mouse have been used for past 10 years to model human Crohn's disease in mice. CBir1 tetramer+ T cells recognize epitope from flagellin, antigen thought to be a target in Crohn's disease. Interestingly, all of those experiments were done using CBir1 T cells on WT background. This could be an issue because it has been known for some time now that transgenic T cells on WT background could use alternative Vα chain to form TCR with new specificity.

To avoid such limitation, here the authors generated CBir1 mouse on RAG KO background that only expressed transgenic Vα:Vβ chains. Surprisingly, unlike CBir1:WT T cells, CBir1:RagKO T cells when transferred in immunodeficient hosts did not induce colitis, and it was not because CBir1:RagKO T cells differentiate into Foxp3+ T cells. It appears that CBir1:RagKO T cells just did not see flagellin epitope in adoptive host.

In vitro tests showed that both CBir1:WT T cells and CBir1:RagKO T cells could respond to DCs pulsed with flagellin epitope [it would have been better and more relevant here to use DCs pulsed with gut flora component].

Other tests showed that CBir1:WT T cells in gut tissue could express alternative Vα chain to form a completely new TCR specificity together with transgenic Vβ chain such as against Ags derived from Helicobacter (HH1713 tetramer). 

In summary, it appears that CBir1 T cells initiate colitis by recognizing non-flagellin antigen from the gut flora through non-CBir1 TCR and only following gut inflammation and gut leakage do flagellin-specific CBir1 transgenic T cells get activated and participate in overall colitis.

So, what is missing from this study? One, it would have been relevant to transfer in vitro activated CBir1:RagKO T cells or activate them in vivo directly to see if then they could initiate colitis. Second, the authors could have tried monocolonization of germ-free mice to see the source of non-flagellin microbiota. Third, there is inconsistency between Fig. 3B and Fig. 6A with regard of proliferation of  CBir1:WT T cells in response to Vanc-treated samples (in vitro it did not proliferate but in vivo it did).

posted by David Usharauli

CARD9 modulates gut inflammation via microbiota-specific tryptophan metabolism

In my previous blogpost I discussed new Science paper that showed relationship between autophagy pathway (ATG16L1) and microbiota-secreted product (PSA-OMV) in driving Crohn's disease (CD) risk. 

As it happens frequently in scientific publication, yesterday another prestigious journal Nature Medicine published another study from another research group that showed relationship between Card9 (C-type lectin sensor, also involved in NOD2 signaling) and microbiota-specific tryptophan metabolism in driving inflammatory bowel disease (Crohn’s disease and ulcerative colitis) risk.  

It is known that Card9^−/− mice are more susceptible to colitis. Here, the authors noticed that Card9^−/− mice are slow to recover after dextran sulfate sodium (DSS)-induced colitis (a self-limiting colitis model) and expressed fewer IL-22⁺ cells, a cytokine with well-known beneficial effects on intestinal homeostasis.

Examination of composition of the fecal bacterial microbiota using 16S rDNA sequencing revealed differences between WT and  Card9^−/− mice gut flora.

Interestingly, germ-free mice transplanted with gut flora from Card9^−/− mice were more susceptible to DSS-induced colitis. This indicated that unlike WT microbiota, Card9^−/− mice microbiota failed to provide "healing" signaling to host's gut epithelium.

One mechanism for this "healing" could be the modulation of aryl hydrocarbon receptor (AhR) activation. Tryptophan can be metabolized either by the gut bacteria into indole derivatives (i.e IAA) or by host cells into kynurenine (Kyn) via indoleamine 2,3-dioxygenase 1 (IDO1). Indole derivatives are AHR ligands and promote IL-22. Indeed, WT microbiota, but not Card9^−/− microbiota, promoted AhR ligand production (Of note, exogenous IL-22 could normalize AhR ligand production and colitis susceptibility in Card9−/− mice).

In vitro assay with AhR reporter system confirmed that Card9^−/− microbiota were defective in activation of AhR signaling.  

Furthermore, supplementation with Lactobacilli strains that are able to produce AhR ligands could restore "healing" effect of Card9^−/− microbiota on gut epithelium.

Finally, the authors showed that fecal samples from IBD patients were indeed deficient in promoting AhR signaling via indole products (genotyping confirmed that fecal samples from IBD patients with Card9 risk allele was associated with reduced AHR activation in their in vitro reporter assay). Of note, the authors said that no such association was observed among other major IBD risk alleles, such as NOD2, ATG16L1 and LRRK2.

In summary, this new study suggests that alterations in IBD risk genes modifies microbiota composition that in turn could tip the balance in favor of "non-healing" microbiota resulting in reduced level of "healing"AhR ligands.

David Usharauli

Gut flora antigen packaged within outer membrane vesicles mediates natural tolerance to microbiota

Crohn’s disease (CD) and ulcerative colitis (UC) are gut inflammatory conditions of unknown etiology. Current hypothesis suggests that these conditions develop due to failure of body's immune system to tolerate antigen processed or generated within gut. 

New paper in Science has pointed to one of the mechanisms for such failure. This study showed that CD's risk gene, ATG16L1, is involved in response to microbiota and promotes gut tolerance via IL-10 producing Foxp3+ Tregs cells.

Initially the authors showed that DCs deficient for ATG16L1 failed to induce IL-10 producing Foxp3+ regulatory T cells in response to WT PSA-OMV (Polysaccharide A derived from gut commensal Bacteroides fragilis and packaged within outer membrane vesicles). Conversely, WT DCs failed to induce IL-10 producing regulatory T cells in response to OMVs harvested from isogenic B. fragilis mutant lacking PSA (ΔPSA-OMV).

Similarly, NOD2-deficient DCs pulsed with WT PSA-OMV failed to support IL-10 production from Foxp3+ Tregs during in vitro co-cultures (NOD2 has been shown to physically interact with ATG16L1).

In vivo experiments confirmed that unlike WT mice, mice deficient for DC-specific ATG16L1 or NOD2 were not protected by WT PSA-OMV from chemical-induced colitis (though it is not clear whether chemically-induced mouse colitis models, i.e. 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis or dextran sulfate sodium (DSS)-induced colitis, are representative of inflammations in Crohn's disease)

.

Finally, the authors showed that monocyte-derived dendritic cells (MoDC) from Crohn's patients harboring ATG16L1 T300A risk variant did not respond to WT PSA-OMV and failed to induce IL-10 production from Foxp3+ Tregs.

In summary, this study showed that failure to sense gut microbiota components by NOD2-ATG16L1 axis could predispose individuals to gut inflammation.

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

α-IL23, not α-IL17 antibody blockade, shows protection in IBD model

Previous studies indicated that cytokines IL-23 and IL-17 act in concert to induce and perpetuate autoimmune inflammation. This is certainly true for psoriatic skin inflammation.

However, two new back-to-back studies in journal Immunity provided evidence that when it comes to intestine inflammation, role of IL-23 and IL-17 goes in opposite direction. Here, blockade of IL-17 exacerbate while blockade of IL-23 inhibits intestine pathology and gut wall permeability.

Both studies came from research groups working in biotech/biopharma (Daniel Cua's group at Merck Research Laboratories and Jennifer Towne's group at Amgen [presently at Janssen]). Both studies reached similar conclusions. 

Here, the authors treated colitis-prone mice with blocking antibodies specific for IL-23 or IL-17 cytokine family. Surprisingly, both α-IL17A or α-IL17RA treatment worsened gut pathology, while α-IL23 (p40 or p19 subunits) antibody showed protection.

Unlike α-IL23, α-IL17RA antibody treatment was associated with increased gut wall permeability (serum sCD14 and LBP), implying IL-17 role in gut health.

Similar increased gut leakage (with FITC-dextran) was observed by Merck's team in chemical irritant DSS-induced GI inflammation model in IL-17KO mice (though Amgen's team did not observe it in DSS mouse model with α-IL17 treatment. It could be that α-IL17 antibody blockade did not fully inhibit IL-17 action as it could be expected in IL-17KO mice).

Additional experiments showed that γδ T cells were the main producers of gut IL-17 in DSS model and that γδ T cell KO mice showed the same increased GI tract permeability as IL-17KO mice.

Finally, Merck's team showed that (a) γδ T cell-derived IL-17 production in the gut were mostly IL-23 independent and (b) IL-23rKO mice were protected against worsening GI wall pathology.

In overall, these two studies suggest that local gut tissue associated IL-23 independent but γδ T cell-derived IL-17 production plays a protective role in gut permeability. Basically, this means that α-IL23 blockade, but not α-IL17 pathway inhibition, would most likely provide benefits to patients suffering from GI tract idiopathic inflammation.

David Usharauli