Genetics, Pathophysiology, and Treatment of Dominant Autoinflammatory Diseases

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

Abstract

During the current reporting period we focused on two areas of investigation: 1) Disabling pansclerotic morphea Disabling pansclerotic morphea (DPM) is a severe systemic autoinflammatory disorder in the scleroderma continuum; it is characterized by poor wound healing with rapidly progressive deep fibrosis involving the mucous membranes, dermis, subcutaneous fat, fascia, muscles, and bone, leading to contractures, musculoskeletal atrophy, and articular ankylosis. Systemic manifestations include cytopenias and hypogammaglobulinemia, but scleroderma-associated autoantibodies are usually not present. DPM is refractory to therapy, including systemic glucocorticoids, immunosuppression, and autologous stem-cell transplantation. Disease pathogenesis has been attributed to abnormal collagen synthesis and deposition, vascular damage, and altered immunoregulation similar to that in other forms of scleroderma. Prior to this study, treatment integrated multiple, broad-spectrum pharmaceutical and ancillary therapies (such as methotrexate, mycophenolate mofetil, and ultraviolet A light therapy), which were directed at halting disease progression but had limited success and unacceptable side effects. DPM is associated with high morbidity and mortality due to squamous-cell carcinoma, restrictive pulmonary disease, sepsis, and gangrene, resulting in a post-diagnosis survival time of less than 10 years. Prior to this study, no genetic cause had been identified. In a collaboration involving our own research group, the University of California San Diego, the University of Pittsburgh, and the Undiagnosed Diseases Program, we evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Clinical features of these patients included mucosal lesions, inflammation and ulcerative lesions of the skin, joint swelling and contractures, histologic evidence of fibrosis, poor wound healing, muscular atrophy, recurrent infections, and squamous-cell carcinoma. Genomic sequencing independently identified three heterozygous variants in the gene that encodes the signal transducer and activator of transcription 4 (STAT4). In two of the families one of the parents exhibited a less severe phenotype, while in the third family the mutation was de novo. The three mutations were found at highly conserved residues of the SH2 domain of the STAT4 protein (p.H623Y, p.A635V, and p.A650D), and in silico modeling was consistent with a gain-of-function. We further evaluated the effects of the mutations in transfection studies. In a cell line stably transfected with a luciferase gene driven by the IL6 promoter (a target of STAT4), transfection with each of the three mutant variants of STAT4 led to greater luciferase activity than when cells were transfected with control STAT4. To analyze phosphorylated STAT4 levels, U3A cells (deficient in STAT1 and STAT4) were stably transfected with plasmids containing control or variant STAT4. In unstimulated cells with variant STAT4, pSTAT4 levels were higher than those in cells transfected with control STAT4. After interferon alfa stimulation, cells carrying control or variant STAT4 had similarly increased pSTAT4 levels at 30 minutes after exposure, but pSTAT4 levels remained elevated much longer in cells carrying variant STAT4. Gene expression studies and STAT4 fluorescence microscopy in transfected cells also supported the gain-of-function hypothesis. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. A manuscript describing these findings in greater detail was published in the New England Journal of Medicine during the current reporting period. 2) Pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) syndrome Although dominantly inherited mutations in PSTPIP1 were discovered in PAPA syndrome over 20 years ago, the molecular pathophysiology of this disorder has remained poorly understood, and targeted monotherapy has remained an elusive goal. During the current reporting period our group discovered that PAPA-associated mutant PSTPIP1 stimulates the noncanonical activation of the pyrin inflammasome, establishing an IFNgamma-dependent autocrine feedback loop and providing the conceptual basis for a novel therapeutic approach for PAPA syndrome. Although we were unable to establish a mouse model of PAPA syndrome, we were able to develop a human U937 myeloid cell line model. Utilizing this model, PAPA-associated human PSTPIP1 mutations activated the pyrin inflammasome, but not the NLRP3, NLRC4, or AIM2 inflammasomes, independently of the canonical pathway of pyrin dephosphorylation, but were blocked by the p.W232A PSTPIP1 mutation, which prevents the interaction of PSTPIP1 with pyrin. IFNgamma priming of monocytes from PAPA patients led to IL-18, but not IL-1beta, IL-6, or TNF release in a pyrin-dependent manner. IFNgamma was abundant in the inflamed dermal skin of PAPA patients, but not patients with idiopathic pyoderma gangrenosum. Ex vivo JAK inhibitor treatment attenuated IFNgamma-mediated pyrin induction and IL-18 release. In 3/3 PAPA patients, the addition of JAK inhibitor therapy to IL-1 inhibition was associated with clinical improvement. A manuscript describing these findings is in preparation.