Immunoregulatory Defects In Inflammatory Bowel Disease

National Institute Of Allergy And Infectious Diseases

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Abstract

Project I - Blau Syndrome: To define the functional consequences of mutations of NOD2 centered in the nucleotide binding domain of NOD2, such as those found in Blau syndrome, we conducted in vitro studies of HEK293T cells bearing plasmids expressing NOD2 constructs with a Blau mutation. We found that NOD2 with this mutation exhibits a reduced ability to oligomerize, to interact with or activate RIPK2 and to activate NF-kappaB. Importantly, these studies were conducted under conditions that avoided effects of NOD2 over-expression that had characterized previous studies. In complementary in vivo studies we determined the capacity of over-expressed intact NOD2 or NOD2 with a Blau mutation (BS-NOD2) (R314W) to cross (down)-regulate TLR responses underlying TNBS-colitis. Whereas administration of intact NOD2 protected mice from TNBS-colitis, over-expression of BS-NOD2 failed to protect. These findings were corroborated by studies of mice bearing a knock-In mutation of NOD2 similar to that in patients with BS in which we showed that such mice were not as protected from DSS-colitis by NOD2-ligand (muramyl dipeptide, MDP) administration as was comparably treated littermate control mice. Finally, we conducted studies to determine the molecular basis of the failure of NOD2 bearing a Blau mutation to mediate cross-regulation. We showed that MDP-stimulated cells from BS-NOD2 KI mice fail to up-regulate expression of IRF4, a factor that has been shown to mediate NOD2 cross-regulation by de-ubiquitination of NF-kappaB signaling components. Indeed, lack of IRF4-mediated cross-regulatory function in KI cells was shown by the fact that enhanced TLR responses exhibited by these cells are suppressed by lentivirus transduction of IRF4. In addition, whereas WT mice expressed IRF4 in inflamed gut and joint tissue following MDP administration, Blau KI mice failed to do so under similar conditions. Overall, these studies indicate that NOD2 bearing a BS mutation lacks the ability to cross-regulate TLR responses via its inability to activate IRF4. The mutation thus renders BS patients susceptible to excessive TLR responses that have the potential to support inflammation at sterile tissue sites. Thus, the Blau mutation affecting NOD2 causes inflammatory disease by affecting NOD2 immunoregulatory function. Project II - Atg16L1 Polymorphism and Crohn's disease: The studies of the functional effects of the Blau mutation were conducted in part with mice bearing a R314W NOD2 mutation. Such mice exhibit reduced levels of NOD2 probablybecause this mutation causes increased NOD2 degradation. To overcome this potential problem we constructed a second Blau KI mouse strain bearing a R307W mutation. Interestingly, this mouse exhibits T cells with increased resting and stimulated cytokine secretory activity again reflecting lack of NOD2 regulatory function. The Atg16L1 gene encodes a protein essential to the function of autophagy, an evolutionarily conserved phagosome-like process that facilitates the disposal of discarded intra-cellular proteins and participates in some aspects of host defense. Over a decade ago it was shown that a single nucleotide polymorphism (SNP) in the Atg16L1 gene (Atg16L1T300A) confers increased risk for the development of Crohns disease (CD). In the present study, we showed that the effect of the Atg16L1T300A polymorphism on TLR- or NLR-mediated signaling in macrophages (and perhaps in dendritic cells as well), is likely to be a major contributor to Crohns disease inflammation. The key findings reported here supporting this view were first that intact autophagy in macrophages regulates NF-B activation, and that defective autophagy in macrophages caused by the polymorphism or indeed caused by other molecular abnormalities resulting in loss of autophagy is accompanied by increased TLR- or NLR-induced NF-B activation and a concomitant increase in pro-inflammatory cytokine/chemokines. Second, we showed that defective autophagy due to the Atg16L1T300A polymorphism leads to increased ubiquitination of NF-B activating factors (TRAF6 and RIP2) as a result of cellular accumulation of p62 (SQSTM1), a sequesterosome receptor molecule that has the capacity to bind to and ubiquitinate TRAF6 and RIP2. Finally, we showed that deletion of p62 in autophagy-defective cells results in substantial normalization of the enhanced NF-B activation otherwise displayed by these cells and thus prove that the autophagy effect on NF-B activation is mainly related to p62 accumulation. The conclusion that emerged from these findings is that defective autophagy in TLR- or NLR-stimulated macrophages causes increased NF-B-mediated pro-inflammatory responses which itself can be a cause of the hyper-responsiveness to commensal organisms in the GI tract that underlies Crohns disease. Of very considerable interest was the fact that the same increase in NF-kB activation was displayed by macrophages obtained from normal individuals bearing the Crohn's disease-associated Atg16L1 polymorphism. This suggests that this polymorphism is a genetically determined host-defense mechanism that confers upon the bearing individual an enhanced response to pathologic agents at the expense os a greater risk for excess mucosal responses and Crohn's disease.