STAT1 gain-of-function (GOF) mutations cause an inborn error of immunity with diverse phenotype ranging from chronic mucocutaneous candidiasis (CMC) to various non-infectious manifestations, the most precarious of which are autoimmunity and vascular complications. The pathogenesis centers around Th17 failure but is far from being understood. We hypothesized that neutrophils, whose functions have not been explored in the context of STAT1 GOF CMC yet, might be involved in the associated immunodysregulatory and vascular pathology. In a cohort of ten patients, we demonstrate that STAT1 GOF human ex-vivo peripheral blood neutrophils are immature and highly activated; have strong propensity for degranulation, NETosis, and platelet-neutrophil aggregation; and display marked inflammatory bias. STAT1 GOF neutrophils exhibit increased basal STAT1 phosphorylation and expression of IFN stimulated genes, but contrary to other immune cells, STAT1 GOF neutrophils do not display hyperphosphorylation of STAT1 molecule upon stimulation with IFNs. The patient treatment with JAKinib ruxolitinib does not ameliorate the observed neutrophil aberrations. To our knowledge, this is the first work describing features of peripheral neutrophils in STAT1 GOF CMC. The presented data suggest that neutrophils may contribute to the immune pathophysiology of the STAT1 GOF CMC.

• Keywords
• Neutrophils, STAT1 GOF, autoimmunity, candidiasis, platelets, ruxolitinib,
• MeSH
• Gain of Function Mutation * MeSH
• Autoimmunity MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Candidiasis, Chronic Mucocutaneous * drug therapy   genetics   MeSH
• Humans MeSH
• Neutrophils metabolism   MeSH
• STAT1 Transcription Factor * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• STAT1 protein, human MeSH Browser
• STAT1 Transcription Factor * MeSH

OBJECTIVE: Posttranslational modifications (PTMs) of proteins are crucial for regulating various biological processes. However, protein alteration via PTMs, and consequently, the creation of new epitopes, can induce abnormal autoimmune responses in predisposed individuals. Immunopathogenesis of several rheumatic diseases, including the most common childhood form, juvenile idiopathic arthritis (JIA), is associated with the generation of autoantibodies against such modified proteins. Dysregulated generation of neutrophil extracellular traps (NETs) can be a source of post-translationally altered proteins. Thus, we investigated the role of PTMs and the presence of NET-associated markers in JIA patients. METHODS: We recruited 30 pediatric patients with JIA (20 with active disease and 10 in remission) and 30 healthy donors. The serum concentrations of citrullinated histone H3 (citH3), peptidyl arginine deiminases (PADs), and NET-related products were detected using ELISA, and the number of citH3+ neutrophils was assessed using flow cytometry. RESULTS: The serum levels of citH3 and PADs were higher in active as well as in remission JIA patients than in healthy donors. Similarly, the number of citH3+ neutrophils was higher in the peripheral blood of patients with JIA, implying an enhanced process of NETosis. This was effectively reflected by elevated serum levels of NET-associated products, such as neutrophil elastase, LL37, and cell-free DNA-histone complexes. Additionally, 16.7% of active JIA patients were seropositive for carbamylated autoantibodies, the levels of which declined sharply after initiation of anti-TNFα therapy. CONCLUSION: Collectively, our data suggest that the accelerated process of NETosis and PTMs in JIA may result in the generation of anti-citrullinated/carbamylated autoantibodies against various epitopes later in life, which could be prevented by effectively regulating inflammation using immune therapy.

• Keywords
• NETosis, carbamylation, citrullination, histone, juvenile idiopathic arthritis, neutrophil, peptidyl arginine deiminases (PAD),
• Publication type
• Journal Article MeSH

Type 1 diabetes (T1D) is an autoimmune disorder with unambiguous involvement of both innate and adaptive immune mechanisms in the destruction of pancreatic beta cells. Recent evidence demonstrated that neutrophils infiltrate the pancreas prior to disease onset and therein extrude neutrophil extracellular traps (NETs), web-like structures of DNA and nuclear proteins with a strong pro-inflammatory biologic activity. Our previous work showed that T1D NETs activate dendritic cells, which consequently induce IFNγ-producing Th1 lymphocytes. The aim of this study was to assess direct ex vivo biomarkers of NETosis in the serum of recent onset and long-term pediatric T1D patients, their first-degree relatives and healthy controls. To this end we evaluated serum levels of myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3), protein arginine deiminase 4 (PAD4), LL37 and cell-free DNA-histone complexes in sex- and age-matched cohorts of T1D first-degree relatives, recent-onset T1D patients, and in patients 12 months after clinical manifestation of the disease. Our data shows that disease onset is accompanied by peripheral neutrophilia and significant elevation of MPO, NE, PR3, PAD4 and cell-free DNA-histone complexes. Most biomarkers subsequently decrease but do not always normalize in long-term patients. First-degree relatives displayed an intermediate phenotype, except for remarkably high levels of LL37. Together, this report provides evidence for the presence of ongoing NETosis in pediatric patients with T1D at time of clinical manifestation of the disease, which partly subsides in subsequent years.

• Keywords
• ELISA, NET, NETosis, PAD4, neutrophil extracellular trap, neutrophils, pediatric, type 1 diabetes,
• MeSH
• Biomarkers blood   MeSH
• Diabetes Mellitus, Type 1 blood   immunology   MeSH
• Child MeSH
• Adult MeSH
• Extracellular Traps * MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Neutrophils immunology   MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH