In the past year the Mucosal Immunity Section has been engaged in a number of research studies involving both inflammatory bowel disease and common variable immunodeficiency. In the area of IBD, we have conducted studies of IBD associated with LRRK2 risk polymorphisms, as described in detail in a previous annual report. In these studies we have established that the polymorphism is associated with increased levels of LRRK2 and this elevation is accompanied by increased gut inflammation in an induced colitis model driven by Dectin-1 induced NF-kappaB responses. Several observations derived from these studies have direct clinical significance. The first is that cells derived from patients who do not bear the LRRK2 risk polymorphism exhibit reduced pro-inflammatory cytokine responses in vitro when the cells are exposed to inhibitors of the kinase activity of LRRK2. The second is that induced colitis of normal mice or mice with elevated levels of LRRK2 is inhibited by administration of various LRRK2 inhibitors. These observations suggest that treatment of patients with inhibitors of LRRK2 can have a therapeutic effect in all patients with IBD regardless of their LRRK2 status. In this period, we have commenced a study of the safety and and immunologic effects of the administration of vorinostat, an HDAC inhibitor. The Clinical Protocol guiding the execution of this study (Protocol # 17-I-0101) has now obtained NIAID IRB and FDA approval. The study will ultimately enroll 20 patients who have failed other forms of Crohn's disease therapy; these patients will undergo a wide array of studies to evaluate the immunologic effects of HDAC inhibitor therapy including effects on regulatory T cells. So far one patient has been enrolled in the study and has completed its treatment phase (12 weeks of oral Vorinostat 100 mg BID); he will shortly return to complete the followup phase of the study. The patient has reported improvement in abdominal pain and cramping symptoms as well as some improvement in diarrhea; however, he still experiences intermittent watery stools. Importantly, his CDAI idex has decreased from > 300 to 180, a greater than 100 point drop. This represents a clinical response short of remission. No change in Treg cell number was observed in studies of cells present in peripheral blood. In the area of CVID we have continued to focus on gastrointestinal manifestations of this heterogeneous disease, i.e., patients with a CVID-entropathy characterized by diarrhea, malabsorption and villous atrophy. In prior studies of CVID we established that the CVID enteropathy is driven by cells producing IFN-gamma. In addition, we collaborated with Drs. Andriy Morgan and Natalia Stulzhenko in the Laboratory of Cellular and Molecular Immunology in studies of CVID that included microarray analyses of biopsy tissue of CVID patients with enteropathy showing that CVID enteropathy is characterized by up-regulation of genes related to various interferons. Based on this prior work, we have applied for and have obtained a U01 NIH cooperative research grant (PAR-13-029) with Drs. Morgan and Shulzhenko (now at Oregon State University) to expand on these findings. A major outcome of this collaborative study has emerged from analysis of patient immunoglobulin levels in gut specimens. We have found that those patients with enteropathy have little or no expression of IgA in intestinal biopsies whereas those patients without enteropathy have only moderately reduced levels of IgA. Moreover, this deficiency does not extend to mucosal IgG levels. This finding indicates that enteropathy occurs in the subset of CVID patients with severe mucosal B cell immunodeficiency affecting the mucosal immunoglobulin, IgA. A second major outcome from these studies is the finding the duodenal specimens from patients with enteropathy contain Acinetobacter baumanii, an organism that, acting as an pathobiont in immunodeficient patients, induces an excessive IFN-gamma synthesis and resultant enteropathy. In the clinical studies we evaluated whether CVID patients with enteropathy would respond to a monoclonal antibody which inhibits the IL-12/ IFN-gamma pathway (Ustekinumab) . CVID enteropathy patients received an induction dose of 270 mg ustekinumab given subcutaneously at Week 0 followed by a maintenance dose of 90 mg ustekinumab every 8 weeks through the Week 40 study point. Patients were then re-assessed at Week 48 for evaluation of efficacy of treatment. A total of 5 patients have been enrolled and completed the study. All patients demonstrated significant improvement in stool pattern with a change from watery consistency to that of soft-formed consistency. All patients concomitantly have observed a decrease in the number of stools per day with a decrease on average from >6-8 stools per day to < 2 stools per day. Most significantly, patients have had observable weight gain (range of 1.7 kg to 9 kg above baseline weight). Histology examination of duodenal area revealed improvement in previous villous blunting changes. All patients had an improvement in serum albumin and total protein levels. In 4/5 patients this was a sustained response. The fifth patient had a significant improvement in the above parameters until week 40 at which time significant diarrhea re-occurred thought to be due to concomitant infection with Enteropathic E Coli (EPEC) intestinal infection. Patient improved after therapy to be address the EPEC infection. All patients have been maintained on Ustekinumab after completion of study protocol. On this basis of these data, it is fair to say that usteinumab administration is an effective treatment ofCVID enteropathy. In this period we also collaborated with Dr. Tomohiro Watanabe of Kindai University in Osaka, Japan in the performance of studiers focusing on the role of NOD2 and NOD1 and the downstream signaling components of these innate immunity recognition factors in the mechanism of inflammation occurring in IBD. Previous studies have shown that inhibition of receptor-interacting serine/threonine kinase (RICK), (also known as RIP2) results in amelioration of experimental colitis. This role has largely been attributed to nucleotide-binding oligomerization domain 2 (NOD2) signaling since the latter is considered a major inducer of RICK activation. In this study, we explored the molecular mechanisms accounting for RICK-mediated inhibition of inflammatory bowel disease (IBD). In an initial series of studies focused on trinitrobenzene sulfonic acid (TNBS)-colitis and dextran sodium sulfate (DSS)-colitis we showed that down-regulation of intestinal RICK expression in NOD2-intact mice by intra-rectal administration of a plasmid expressing RICK-specific siRNA was accompanied by down-regulation of pro-inflammatory cytokine responses in the colon and protection of the mice from experimental colitis. Somewhat surprisingly, intra-rectal administration of RICK-siRNA also inhibited TNBS-colitis and DSS-colitis in NOD2-deficient and in NOD1/NOD2-double deficient mice. In complementary studies of humans with IBD we found that expression of RICK, cellular inhibitor of apoptosis protein 2 (cIAP2) and down-stream signaling partners were markedly increased in inflamed tissue of IBD compared to controls without marked elevations of NOD1 or NOD2 expression. In addition, the increase in RICK expression correlated with disease activity and pro-inflammatory cytokine responses. These studies thus suggest that NOD1 or NOD2-independenent activation of RICK plays a major role in both murine experimental colitis and human IBD.
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