The prevailing view of the pathogenesis of the major inflammatory bowel diseases such as Crohns disease (CD) and ulcerative colitis (UC) is that they are caused by dysregulated and therefore enhanced responses to microbial organisms in the gut microbiome that ultimately result in gut inflammation. An important support for this idea is the fact that the various gene polymorphisms associated with these diseases affect the risk for disease development by their effects on mucosal immune homeostasis. In addition, several point mutations affecting genes contributing to the mucosal immune response have been identified that cause disease in a limited number of patients because of the relative rarity of the mutation. In the study completed during this period, we establish that a loss-of-function mutation in the CARD8 gene is one such mutation causing Crohn's disease. In these studies, we evaluated the contribution of the NLRP3 inflammasome to Crohns disease (CD) in a kindred containing three affected individuals, each having a missense mutation in CARD8 (also known as TUCAN or CARDINAL), a protein known to regulate (inhibit) this inflammasome. Whole exome sequencing and/or PCR studies identified the affected individuals as having a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1 in the affected individuals were increased compared with those in health controls, and their peripheral monocytes produced increased amounts of IL-1 when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 failed to down-regulate the NLRP3 inflammasome because it did not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerted a dominant-negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impeded their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevented NLRP3 deubiquitination and serine dephosphorylation, NLRP3 changes associated with activation. CD due to a CARD8 mutation was not effectively treated by antiTNF-, but did respond to IL-1 inhibitors. Thus, patients with antiTNF-resistant CD may respond to this treatment option. These studies have significance in that they show that abnormal NLRP3 inflammasome activity and exceessly Increased IL-1beta production is a possible cause of Crohn's disease and one that will respond to agents that block inflammasome activity. They thus resolve the question raised in studies of murine models as to whether gut inflammation is inflammasome-dependent. The answer to this question is that whereas normal inflammasome activity is not in itself a dominant cause of Crohn's disease, increase inflammasome activity due to a mutation can be a cause. In addition, these studies show that some patients with Crohn's disease whose disease does not response to anti-TNF-alpha or anti-IL-12 p40, may respond to inhibitors of IL-1beta.
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