Otubain 2 promotes muramyl dipeptide-mediated anti-colitogenic effects due to de-ubiquitination of receptor interaction protein 2

Abstract

Immune responses in the gut need to be tightly controlled in order to maintain mucosal immune tolerance and proper interactions with intestinal microbiota. Disruptions of these immune-microbiota circuits presumably underlie different immune-mediated disorders including inflammatory bowel disease (IBD). Distinct genetic traits that alter the expression and/or function of molecules and consequently the immune signalling networks they are embedded in can disrupt immune-microbiota interactions and microbe recognition and thus, promote mucosal inflammation. However, the molecular mechanisms and cellular circuits underlying the pathogenesis of IBD are only incompletely understood. Du and colleagues investigated the influence of posttranslational modifications on the complex signalling network in the gut in an experimental colitis model and in patients with ulcerative colitis.¹

The nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor which contributes to intestinal homeostasis through the regulation of epithelial cell functions and innate and adaptive immune responses. Hematopoietic cells of both myeloid and lymphoid origin as well as intestinal epithelial cells and Paneth cells express NOD2.² NOD2 senses intracellular muramyl dipeptide (MDP), a peptidoglycan component conserved in Gram-positive and Gram-negative bacteria. Following engagement by MDP, NOD2 undergoes oligomerization and subsequently attracts and activates receptor interaction protein 2 (RIPK2) through homotypic interactions, followed by transforming growth factor-beta-activated kinase 1 recruitment and activation which engages nuclear factor kappa-beta (NFκB) and mitogen-activated protein kinase pathways for pro-inflammatory cytokine production.³

Allelic variations of the gene encoding NOD2 have been associated with IBD. The three most common risk variants of the more than 2400 NOD2 variant genes reported to date are typically present in a heterozygous state and account for more than 80% of the NOD2 variations.⁴ These allelic risk variants are predicted to encode loss-of-function mutations that impair NFκB activation in response to MDP and to promote the onset and progression of IBD by altering the interaction with and the composition of intestinal microbiota.^{5, 6} Along with this assumption, MDP administration protects from experimental colitis and this protective effect of MDP is lost when NOD2 signalling is defective,^{7, 8} Thus, proper NOD2 signalling is pivotal for the maintenance of intestinal immune tolerance and the restriction of inflammatory insults.

Next to genetic NOD2 mutations, the versatile and complex signalling network NOD2 is embedded in as well as various posttranslational modifications regulate NOD2 function and thus, influence the outcome of disease. These posttranslational modifications include ubiquitination and de-ubiquitination processes, reversible and counter-regulable enzymatic cascades comprising a panoply of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), kinases and de-ubiquitinating enzymes (DUBs).⁹ Recent data have stressed the important role of DUBs in IBD pathogenesis.¹⁰

Ovarian tumour domain-containing ubiquitin aldehyde-binding protein Otubain 2 (OTUB2) is a DUB of the ovarian tumour protease (OTU) subfamily. It can deubiquitinase several molecules in different signalling networks including the Hippo pathway, the NFκB pathway and the hedgehog pathway.¹¹ Although presumably being involved in malignant diseases, its role in health and disease is not clear. Particularly, its influence on inflammatory processes has not yet been addressed.

Du and colleagues investigated the role of OTUB2 in RIPK2 signalling following NOD2 engagement by MDP.¹ They observed that OTUB2 removes polyubiquitin chains from RIPK2 to inhibit its degradation via the 26S proteasome, thereby stabilizing RIPK2 protein for efficient signal transduction (Figure 1). Intraperitoneal application of MDP attenuated dextran sodium sulfate- (DSS-) mediated colitis, epithelial damage, leukocyte infiltration and goblet cell loss in Otub2^+/+ littermates, but failed to do so in Otub2^−/− mice. Using bone marrow chimaeras, the authors attributed this protective effect of MDP signalling in the presence of Otub2 to the hematopoietic compartment. Indeed, OTUB2 was highly expressed in colon infiltrating macrophages of IBD patients and in DSS-treated mice, although overall Otub2 expression was reduced in colon samples following DSS application as compared to naive mice. Most importantly, immunoprecipitation and immunofluorescence studies in bone marrow-derived macrophages of Otub2^−/− and wildtype mice suggested colocalization and direct physical interactions of OTUB2 and RIPK2. In summary, these findings identify OTUB2 as the important gatekeeper of intestinal inflammation and a key mediator of NOD2 signals following MDP recognition.

The authors elegantly describe a novel mechanism of posttranslational RIPK2 stabilization following NOD2 engagement in the context of inflammatory disease. Most importantly, their study suggests that defective NOD2 signalling can occur even in patients who do not carry genetic NOD2 mutations themselves. In addition, certain genetic variants including Otub2 variants that affect NOD2 signalling might contribute to IBD susceptibility.

The study predominantly focused on hematopoietic, but not non-hematopoietic cells in a chemically induced colitis model and on NOD2 signalling in bone marrow-derived macrophages. It remained unclear whether OTUB2-mediated NOD2 signalling is preserved in macrophages purified from intestinal tissues of DSS-treated mice. It also warrants further investigation to explain how MDP reduces inflammatory immune responses in the DSS colitis model despite enhancing the release of inflammatory cytokines in macrophages in vitro. Thus, the exact mechanism(s) underlying OTUB-2-mediated protection from mucosal inflammation warrants further investigation. One aspect could be that certain pathobiontic bacteria might be preferentially eliminated following macrophage activation dampening secondarily inflammatory processes in the gut. To address this question in detail, infection-driven colitis models might be helpful. Further studies need to assess also the role of OTUB2 in other cell populations and how the functional and structural composition of microbiota is affected following MDP signalling in individual cell populations.

Jochen Mattner wrote the manuscript and prepared the figure.

The author declares no conflict of interest.

Not applicable.