Immunoregulatory Defects In Inflammatory Bowel Disease

National Institute Of Allergy And Infectious Diseases

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Abstract

Project I - Blau Syndrome: In our initial studies to define the functional consequences of mutations of NOD2 in Blau Syndrome we assessed the function of NOD2 constructs expressing NOD2 with a Blau mutations in HEK293T cells. We found that NOD2 with these mutations exhibits a reduced ability to oligomerize, to interact with or phosphorylate and/or ubiquitinate RIPK2 and to activate NF-kappaB. Importantly, these studies were conducted under conditions that avoided effects of NOD2 over-expression that mat have characterized previous studies. We then conducted in vivo studies in which 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 or DSS-colitis. Whereas administration of intact NOD2 constructs 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 and, in fact, the mutated NOD2 allele exhibited dominant-negative effects on the normal NOD2 allele in heterozygous mice. Finally, we conducted studies to determined the molecular basis of these cross-regulation defects. Here we showed that MDP-stimulated cells from BS-NOD2 KI mice, as a result of the signaling abnormality described above, 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. Lack of IRF4-mediated cross-regulatory function in Blau KI cells was shown in vitro 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. Project II - Atg16L1 Polymorphism and Crohn's disease: 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). Inasmuch as the decreased autophagy associated with the presence of the polymorphism causes increased epithelial apoptosis perhaps due to accumulation of toxic intracellular substances, the main theory of how the polymorphism results in Crohn's disease is that it compromises epithelial survival. Results of the present study, however, support a very different conclusion, namely that the defective autophagy mediated by the polymorphism leads to enhanced NF-kB responses and pro-inflammatory cytokine production. In these studies we showed first that the effect of the Atg16L1 T300A 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 that intact autophagy in macrophages regulates NF-kB 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-kB activation and a concomitant increase in pro-inflammatory cytokine/chemokines. Next, we showed that defective autophagy due to the Atg16L1T300A polymorphism leads to increased ubiquitination of NF-kB 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-kB-mediated pro-inflammatory responses which itself can be a cause of the hyper-responsiveness to commensal organisms in the GI tract that underlies Crohn's 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.