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

Absence of NOD2 signaling protects against chemical[STZ]-induced diabetes

Experimental models developed in "clean" laboratory mice rarely translates to human diseases. For example, NOD mice spontaneously develop Type 1 diabetes, as susceptible humans do, however pancreatic tissue pathology does not match. 

There is another model of T1D in mice called treptozotocin (STZ)-induced diabetes. STZ is toxic to insulin producing β cells in pancreas and drive T1D in a T cell-dependent manner. In new study published in Journal of Experimental Medicine the authors showed that treptozotocin induced T1D depended on gut flora derived muramyl dipeptide → NOD2 signaling.

The authors found that NOD2^-/- mice, but not NOD1^-/- or Rip2^-/- mice, were resistant to STZ effect.

Interestingly, similar protective effect against T1D were seen in WT mice treated with broad-spectrum antibiotics and STZ. However, addition of NOD2 ligand muramyl dipeptide reversed protection in [antibiotic + STZ] treated mice.

This study showed that NOD2 ligand, muramyl dipeptide derived from gut microbiota, contributes to T1D in STZ-treated mice.

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)

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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

Adjuvant activity of cholera toxin relies on endogenous microbiota-dependent Nod2 activation

Cholera toxin (CT), derived from Vibrio cholerae, is still widely used as a potent mucosal adjuvant in experimental [rodent] immunology. CT binds GM1 ganglioside receptor and activates cyclic AMP (cAMP) production. This mechanism is thought to stimulate T_H2-associated cytokines and robust IgG₁ production. However, toxicity of CT prevents its implementation in the clinic (it is not uncommon that scientists are studying active molecules that have no clinical use, but all "in hope" that "the mechanisms underlying its [CT's] potent adjuvant activity may lead to the development of nontoxic and effective adjuvants for mucosal vaccination."

This time, new study in Nature Medicine showed that CT's adjuvant activity relied on Nod2 signaling initiated by endogenous microbial flora.

Initially, the authors reported that nasal or oral immunization of germ-free (GF) mice [that harbor no endogenous microbiota] with protein antigen + CT yielded reduced levels of antigen-specific IgG₁ and T cell effector differentiation. Similar results were obtained with antibiotic-treated mice.

Effect of endogenous microbiota on CT's adjuvant activity could be replicated in Ripk2 (the adaptor required for Nod1 and Nod2 signaling) or Nod2 deficient mice, but not in MyD88-KO or Nod1-KO mice.

It is known that Nod2 recognizes peptidoglycan molecules that contain muramyl dipeptide (MDP). Indeed, GF mice immunized with a combination of protein Ag + CT + MDP produced high level of antigen-specific IgG₁, suggesting the role of MDP in promoting CT's adjuvanticity.

Finally, the authors found that several members of MDP-rich endogenous microbiota (i.e. Staphylococcus sciuri) promoted CT's adjuvant activity in GF mice in a Nod2-dependent manner.

In summary, this study indicates that adjuvanticity of cholera toxin relies on Nod2 signaling triggered by MDP-rich endogenous microbiota.

David Usharauli