Immunoregulatory Defects In Inflammatory Bowel Disease

National Institute Of Allergy And Infectious Diseases

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Abstract

To define the immunologic abnormalities that arise from mutations of NBD (donain) of NOD2 in Blau Syndrome we first 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 and to interact with or phosphorylate and ubiquitinate the immediate down-stream signaling component of NOD2, RIPK2; this, in turn, was associated with a reduced ability to activate NF-kappaB. We next conducted in vivo studies in which we determined the capacity of intact NOD2 or NOD2 with a Blau mutation (BS-NOD2) (R314W) to cross (down)-regulate TLR responses necessary for the development of colonic inflammation in TNBS-colitis or in DSS-colitis. Whereas mice over-expressing intact NOD2 constructs by several strategems exhibited greatly reduced TNBS-colitis (as shown in extensive previous studies) over-expression of BS-NOD2 didn't exhibit such reduced TNBS-colitis. 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; in fact, this was also observed in heterozygous mice, indicating that the mutated NOD2 allele exhibited dominant-negative effects on the normal NOD2 allele. Finally, in studies to determined the molecular basis of these cross-regulation defects 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 suggest 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 induce inflammation at sterile tissue sites. In our current studies we are taking advantage of recent work showing that stimulation of monocytes with CpG to induce IFN-alpha is inhibited by co-stimulation with MDP due to MDP-mediated induction of deubiquitinating enzyme A (DUBA), a factor that normally impedes ubiquitination of TRAF3 and thus down-regulates translocation of IRF3 and IRF7 necessary for IFN-alpha transcription; on this basis MDP administration inhibits exacerbation of DSS-colitis by CPG administration. In addition we took advantage of prior work showing that DUBA is generated during induction of RORgt in T cells and down-regulates the level of this transcription factor during IL-17 production; thus, DUBA expression puts a brake on T cell IL-17 synthesis. In studies applying this information to the study of Blau syndrome we showed first that whereas CpG stimulation of IFNalpha production by control peripheral blood monocytes was reduced by co-stimulation with MDP such production by Blau patient monocytes was not so reduced. In addition, we showed that TCR-stimulation of T cells from Blau patients or spleen/mesenteric lymphoid cells from Blau KI mice produced increased amounts of IL-17 compared to control human or murine cells respectively and this correlated in mouse cells with an reduced DUBA expression during peak RORgt transcriptional activity. Thus, Blau CARD15 mutations lead to altered NOD2-mediated DUBA induction and this, in turn leads to increased pro-inflammatory production of IL-17 and IFN-alpha. This mechanism of NOD2 dysfunction conforms with the concept that NOD2 has important immunoregulatory properties under normal circumstances. In accompanying studies of Yao syndrome we have established a relation with Dr. Qingping Yao at Stonybrook University, the discoverer of Yao syndrome, to investigate the immunologic defects present in the large cohort of patients with Yao syndrome under Dr. Yao's care. Dr. Yao has sent blood samples to us to conduct a broad survey of Yao syndrome patient responses to innate (TLR) stimulation. This has shown that whereas responses to innate stimulation is generally normal, IFN-alpha responses to CpG are reduced (in most patients). This suggests that inflammation in Yao syndrome is caused by reduced IFN-alpha-mediated tolerogenic responses to CpG induced by commensal organisms, particularly B. fragilis. On this basis, we are now assessing control and patient IFN-alpha, IL-27 and IL-10 responses following in vitro exposure to live B. fragilis.