A major project that involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration with Dan Kastner and Ivona Aksentijevich, we have measured serum cytokines levels in patients with a novel autoinflammatory disease caused by haploinsufficiency of A20 (encoded by TNFAIP3). The syndrome resembles Behcets disease (BD) and our data showed that these patients have increased expression of NFkappaB-mediated proinflammatory cytokines. The manuscript reporting these findings was published in Nature Genetics. In collaboration with Ivona Aksentijevich and Qing Zhou we have reported another novel inflammatory disease caused by the malfunction of otulin, a single gene on chromosome 5. As a result of the mutations, the otulin protein is unable to remove the ubiquitin from various molecules, resulting in an increased production of inflammatory cytokines, ultimately leading to inflammation. The manuscript describing this new syndrome has been published in the Proceedings of the National Academy of Sciences. Furthermore, in collaboration with the Kastner group and Elaine Remmers we have been studying a new Behcets disease-associated loci that we identified using the Immunochip to densely interrogate immune/inflammatory disease loci. We found a noncoding SNP in IL1A associated with disease at genome-wide significance. We found that the disease-associated SNP is also a reported eQTL; the disease risk allele is significantly associated with decreased expression of IL1A in lymphoblastoid cell lines and skin, and the lead disease-associated SNP is also the lead IL1A eQTL SNP. The TIS has been responsible for confirming decreased lipopolysaccharide-stimulated IL1A mRNA expression in healthy control monocytes from disease risk allele homozygotes compared with individuals with 0 or 1 risk allele. Association of the lead disease SNP with IL1B expression was not statistically significant in the published eQTL study. IL-1 is highly expressed in the epidermis and plays an important role in skin barrier functions against pathogens. A manuscript reporting this study is currently being reviewed. With the Goldbach-Mansky group, we studied patients with Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature (CANDLE), a Proteasome-Associated Autoinflammatory Syndrome (PRAAS), who were shown to have mutations in the inducible proteasome-subunit, PSMB8 and PSMB4. The TIS was responsible for measuring patient serum cytokine levels, which revealed an increase in type I IFNs as well as inflammatory cytokines such as IL-6 and TNF. A manuscript was published in the Journal of Clinical Investigation. In collaboration with the Colbert group we have investigated the cytokine profile and the immunologic phenotype of a patient with a de-novo gain-of-function mutation in MyD88, which results in early-onset severe arthritis. We continue to collaborate with NHGRI investigators assessing the cytokine-secreting capabilities of patients with Erdheim-Chester Disease (ECD), a rare, non-familial multisystem disorder characterized by proliferation and infiltration of non-Langerhans histocytes into multiple organs. We compared the cytokine secreted by ECD patients to normal controls after stimulation of PBMCs. The TIS has also been investigating novel approaches for the treatment of autoimmune diseases. In collaboration with the O'Shea group, the Kaplan group, and Pfizer (via a CRADA), we evaluated the efficacy of tofacitinib on a murine model of systemic lupus erythematous (SLE) using the MRL/lpr mouse. Tofacitinib treatment results in a significant amelioration of SLE phenotype, besides reducing autoantibody levels, nephritis, skin inflammation and inflammatory cytokines, tofacitinib mitigates the dysregulation of neutrophil function and induces significant improvements in endothelium-dependent vasorelaxation, which is relevant to vascular health. A manuscript reporting these results was published in Arthritis and Rheumatism. We renewed the CRADA and received second-generation JAK-selective inhibitors, which we are currently evaluating for their activity on T cells. We generated induced pluripotent stem cells (iPSC) from a patient with HLA B-27+ Ankylosing Spondylitis and demonstrated their pluripotency. We then differentiated iPSCs into MSC and osteoblasts, and found that MSC-derived osteoblasts from patients showed greater mineralization capacity compared to normal controls. We have continued the gene-editing project using CRISPR-Cas9 techniques in iPSCs. We have undertaken an effort to generate isogenic human iPS cell lines from AxSpA patients with specific HLA-B*27:04 gene knock-out to assess its role in disease. We used CRISPR/Cas9 gene editing technology to target HLA-B*27:04 through non-homologous end joining by editing the coding sequence at exon 3. We transfected patient-derived iPSCs (HT19) with a CRISPR/Cas9 expression vector containing guide RNA consisting of a 20-nt sequence. The transfected cells were sorted based on GFP expression, and the SURVEYOR nuclease assay was used as a functional test to evaluate Cas9 cutting efficiency. Specifically, we used flow cytometry as a screening method to examine cell surface HLA-B27 expression by developing a method to enrich and screen out knockout or mutant HLA-B27 iPSC colonies by using GFP sorting and single colony selection. RNAseq was then used to identify in-del mutations, frameshifts, missense mutations, or premature stop codons. Two types of mutations were identified by RNAseq on the targeting site of exon 3 of the B27 allele that were predicted to disrupt B27 protein expression. Western Blot results were consistent with the genomic data indicating that B27 protein expression is decreased significantly in the knock-out cell lines. Further studies are currently ongoing to confirm the allele-specific loss of HLA-B expression and to investigate osteoblast development and its function from isogenic B27 knock-out iPS cell lines. The newly generated isogenic HLA-B27 knock-out IPS cell lines are also in the process of being characterized.
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