Gene Regulatory Events in Establishing Mature T Cell Tolerance

National Institute Of Allergy And Infectious Diseases

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Abstract

Inborn errors of immunity (IEI) can affect global cellular regulatory systems. We looked at two projects where IEI can cause devastating disease. For the first project, we discovered a new cause of infantile inflammatory bowel disease (I-IBD), which is defined by the onset of Crohns disease, ulcerative colitis disease, or IBD-unclassified disease in children under the age of 2 years old. I-IBD often presents with severe colitis that is refractory to treatments, immunodysregulation, and is etiologically linked to Mendelian mutations. IL-37 plays an anti-inflammatory role in the innate immune response. It is an IL-1 family cytokine that suppresses inflammation, unlike the other proinflammatory members in this gene family. The IL-37 receptor consists of IL-1R8 and IL-18R1, which are highly expressed in the GI tract. Interestingly, IL-37 signaling can occur via two mechanisms: intracellularly through nuclear translocation with SMAD3 and extracellularly after secretion and binding to its receptor. This study was the first to form a monogenic link between IL-37 and I-IBD and investigate the physiological function of IL-37 in humans. Our patient is a 2-year-old boy who presented at four months of age with recurrent bloody diarrhea eight to nine times per day. Given the familial consanguinity and diagnosis of I-IBD, we performed whole-exome sequencing analysis. While we found no rare variants in known very early onset-IBD disease-causing genes, there was a IL-37 missense variant. Interestingly, the patient's peripheral blood mononuclear cells (PBMCs) showed higher levels of IL-37 protein compared to healthy controls. Further assays demonstrated the patient's IL-37 protein was less stable than wild-type IL-37, could not be secreted, and was unable to suppress pro-inflammatory signals. This research sheds light on the role of IL-37 in homeostatic control of colonic inflammation in humans and begs the question of whether IL-37 can be used as a novel cytokine therapeutic for IBD and potentially other inflammatory conditions. The second project looked at mutations within the IL-17 signaling pathway. IL-17 mediated responses are necessary for protective mucosal immunity against fungal pathogens. Chronic mucocutaneous candidiasis (CMC) is a noninvasive and persistent infection of the skin and oral surfaces that can occur when IL-17 signaling is deficient. TRAF3IP2 is an adapter protein that interacts with IL-17R following IL-17 stimulation. We reported 2 patients who were both diagnosed with CMC at less than one year of age. Sequencing results revealed that both patients harbored rare, homozygous nonsense mutations in TRAF3IP2. We predicted that premature termination in the second exon of TRAF3IP2 transcripts would lead to loss of TRAF3IP2 at the protein level. Indeed, immunoblot of lysates derived from patient T cells revealed a complete loss of protein, and reduced mRNA levels, suggesting nonsense-mediated decay. We therefore investigated the effects of protein loss on IL-17 signaling. Patient cells showed a good cytokine response to IL-2 stimulation alone but no synergistically enhanced response following combined IL-2/IL-25 stimulation. Consistent with the cell stimulation, IL-5 and IL-13 were present at a lower level in the patient serum with relatively normal levels of other Th cell cytokines. We also observed a higher proportion of IL-17A producing Th17 cells in our patients compared to controls. However, we detected a lower proportion of IFN-gamma producing Th1 cells in our patients, which may reflect a Th1/Th17 counterbalance in vivo. To further clarify the proportion of Th cells in our TRAF3IP2 patients, we measured regulatory T cells and IL-13 producing Th2 cells. We found that the patients presented a higher proportion of regulatory T cells and a lower proportion of Th2 cells. Failure to initiate signaling downstream of IL-17R engagement likely underlies this defective IL-25 response, which could contribute to the patients' CMC. IL-25 signals though the IL-17 receptor, which requires TRAF3IP2 to transmit a signal. This defect will be present for all IL-17R mediated signaling. Defective IL-17 signaling in epithelial cells likely is an important contributor of host susceptibility to fungal infections.