Neutrophils in STAT1 Gain-Of-Function Have a Pro-inflammatory Signature Which Is Not Rescued by JAK Inhibition

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TL;DR

This study shows that neutrophils from STAT1 gain-of-function (GOF) patients are highly activated, with enhanced degranulation, NETosis, and pro-inflammatory cytokine production, despite normal STAT1 phosphorylation. These neutrophils exhibit a pronounced interferon signature and increased platelet-neutrophil aggregates, suggesting they contribute to autoimmune and vascular complications in STAT1 GOF. Importantly, treatment with the JAK inhibitor ruxolitinib did not ameliorate these neutrophil alterations, indicating a STAT1-independent mechanism.

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**Background:** STAT1 gain-of-function (GOF) mutations cause chronic mucocutaneous candidiasis (CMC) and are associated with autoimmunity, vascular abnormalities, and increased susceptibility to infections. While Th17 impairment explains fungal susceptibility, the mechanisms behind autoimmune and vascular complications are poorly understood. Neutrophils are implicated in various autoimmune diseases, but their role in STAT1 GOF had not been investigated. This study aimed to characterize the phenotype, function, and transcriptome of neutrophils from STAT1 GOF patients and assess the effect of JAK inhibition with ruxolitinib.

**Methods:** The study included 10 STAT1 GOF patients (3 male, 7 female; median age not specified, range 8–52 years) from 7 families, with mutations in N-terminal (p.E29A, p.Y68C), coiled-coil (p.A267V, p.T288N), and DNA-binding (p.N357D, p.M390T) domains. Three patients were treated with ruxolitinib (RUXO). Neutrophils were isolated from peripheral blood. STAT1 and STAT3 phosphorylation was assessed by phosphoflow and western blot after IFNα or IFNγ stimulation. Transcriptomic analysis was performed using NanoString Myeloid Innate Immunity panel (770 genes). Neutrophil phenotype was analyzed by flow cytometry for activation markers (CD16, CD62L, HLA-DR), subsets (mature CD10+, immature CD10−, suppressive CD62L^lo^CD16^hi^, aged CXCR4+CD62L−, G-MDSC), and platelet-neutrophil aggregates (PNAs). Functional assays included phagocytosis (zymosan, E. coli), ROS production (DHR123), NETosis (Sytox Green), and cytokine production (Luminex) after LPS or zymosan stimulation. Serum levels of degranulation products (MPO, NE, MMP8, lactoferrin, lipocalin, PR3), IFN-related chemokines (CXCL9, CXCL10), adhesion molecules (sICAM-1, sVCAM-1, sCD62E, sCD62L), and NETosis markers (DNA-histone complexes, PAD4, CXCL4) were measured by ELISA or Luminex.

**Key Results:** - **STAT1 phosphorylation:** Unlike monocytes, neutrophils from STAT1 GOF patients did not show increased pSTAT1 upon IFNα or IFNγ stimulation compared to HD. However, unstimulated pSTAT1 was higher in patient neutrophils (Fig. 1F, G). IFNα-induced pSTAT3 was increased in patient neutrophils (Fig. 1C). - **Transcriptome:** 57 genes were differentially expressed between STAT1 GOF and HD neutrophils, involved in TLR and cytokine signaling (IL-2, IL-3, IL-7, IFNs) and JAK-STAT pathway (Fig. 2A–C). RUXO-treated neutrophils had 82 DEGs vs. HD, with no amelioration of the pro-inflammatory signature. - **Interferon response:** Increased expression of IFN-stimulated genes (8/10 tested, e.g., IFIT, ISG15, MX1, IRF1) was detected in patient neutrophils (Fig. 2F). Serum IFNα, CXCL9, and CXCL10 were elevated (Fig. 2G). - **Degranulation:** Serum levels of MPO, NE, MMP8, lipocalin, and PR3 were significantly increased in STAT1 GOF patients (Fig. 3A). Isolated neutrophils released more MMP8 and lipocalin spontaneously and upon LPS/zymosan (Fig. 3C, D). - **Phenotype:** Patient neutrophils showed activation markers: increased CD16 and HLA-DR, decreased CD62L (Fig. 3E). Serum sCD62L was elevated (Fig. 3F). S100A8/A9, CXCL1, and IL-8 were increased (Fig. 3G). CXCR2 expression was increased (Fig. 3I). - **Subsets:** Immature (CD10−) and suppressive (CD62L^lo^CD16^hi^) neutrophils were expanded; mature, aged, and G-MDSC subsets were decreased (Fig. 4A–C). - **Function:** Phagocytosis and ROS production were unaltered (Fig. 5A, B). Cytokine production (IL-1β, IL-6, TNFα) was significantly higher after LPS/zymosan (Fig. 5C). - **NETosis:** Serum DNA-histone complexes and PAD4 were elevated (Fig. 5D, F). PMA-induced NET release was increased (Fig. 5E). CXCL4 was also increased (Fig. 5F). - **Platelet-neutrophil aggregates:** PNAs were elevated in STAT1 GOF patients (Fig. 6A), with increased CD62P expression (Fig. 6B). Serum sCD62P and sCD40L were increased (Fig. 6C). Adhesion molecules sICAM-1, sVCAM-1, sCD62E, and sCD62L were elevated (Fig. 6D). - **Effect of ruxolitinib:** RUXO treatment did not reverse most neutrophil alterations; in some assays, it even amplified differences from HD.

**Clinical Implications:** This study provides the first evidence that neutrophils in STAT1 GOF are pro-inflammatory, with enhanced degranulation, NETosis, and cytokine production, likely contributing to autoimmune and vascular complications. The lack of effect of ruxolitinib on neutrophil alterations suggests that these changes are not primarily driven by STAT1 hyperphosphorylation and may explain the incomplete response to JAK inhibition in some patients. Targeting neutrophil-specific pathways could be a therapeutic strategy for STAT1 GOF-associated autoimmunity and vascular disease.