Significance: Despite clear differences in female and male immunity that may contribute to variations in response to infections and predisposition to cancer and autoimmunity, the exact mechanisms that drive this stark contrast remain insufficiently characterized. Neutrophils play essential roles in homeostasis and disease, but little is known about how sex differences modulate their phenotype and function. Using transcriptomic and functional approaches, we report that healthy young adult females have an activated/mature neutrophil profile characterized by enhanced type I IFN pathway activity, enhanced proinflammatory responses, and distinct bioenergetics. We further show that these differences are cell specific and likely driven by sex hormones. Modulation of these pathways in neutrophils may provide more individualized, sex-specific therapeutic options in a variety of disease states.

Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease.

Innate and adaptive immune systems are dysregulated in hidradenitis suppurativa.

Rheumatoid arthritis (RA) is characterized by synovial joint inflammation, cartilage damage, and dysregulation of the adaptive immune system. While neutrophil extracellular traps (NETs) have been proposed to play a role in the generation of modified autoantigens and in the activation of synovial fibroblasts, it remains unknown whether NETs are directly involved in cartilage damage. Here, we report a new mechanism by which NET-derived elastase disrupts cartilage matrix and induces release of membrane-bound peptidylarginine deiminase-2 by fibroblast-like synoviocytes (FLSs). Cartilage fragments are subsequently citrullinated, internalized by FLSs, and then presented to antigen-specific CD4 ⁺ T cells. Furthermore, immune complexes containing citrullinated cartilage components can activate macrophages to release proinflammatory cytokines. HLA-DRB1*04:01 transgenic mice immunized with NETs develop autoantibodies against citrullinated cartilage proteins and display enhanced cartilage damage. Inhibition of NET-derived elastase rescues NET-mediated cartilage damage. These results show that NETs and neutrophil elastase externalized in these structures play fundamental pathogenic roles in promoting cartilage damage and synovial inflammation. Strategies targeting neutrophil elastase and NETs could have a therapeutic role in RA and in other inflammatory diseases associated with inflammatory joint damage.

Reduction of adenosine deaminase 2 (ADA2) activity due to autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1) results in a systemic vasculitis known as deficiency of ADA2 (DADA2). Neutrophils and a subset of neutrophils known as low-density granulocytes (LDGs) have been implicated in the pathogenesis of vasculitis, at least in part, through the formation of neutrophil extracellular traps (NETs). The study objective was to determine whether neutrophils and NETs play a pathogenic role in DADA2. In vivo evidence demonstrated NETs and macrophages in affected gastrointestinal tissue from patients with DADA2. An abundance of circulating LDGs prone to spontaneous NET formation was observed during active disease in DADA2 and were significantly reduced after remission induction by anti–tumor necrosis factor (TNF) therapy. Increased circulating LDGs were identified in unaffected family members with monoallelic ADA2 mutations. Adenosine triggered NET formation, particularly in neutrophils from female patients, by engaging A1 and A3 adenosine receptors (ARs) and through reactive oxygen species– and peptidylarginine deiminase–dependent pathways. Adenosine-induced NET formation was inhibited by recombinant ADA2, A1/A3 AR antagonists, or by an A2A agonist. M1 macrophages incubated with NETs derived from patients with DADA2 released significantly greater amounts of TNF-α. Treatment with an A2AAR agonist decreased nuclear translocation of NF-κB and subsequent production of inflammatory cytokines in DADA2 monocyte-derived macrophages. These results suggest that neutrophils may play a pathogenic role in DADA2. Modulation of adenosine-mediated NET formation may contribute a novel and directed therapeutic approach in the treatment of DADA2 and potentially other inflammatory diseases.

Objective Neutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology. Methods NETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed. Results We found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers. Conclusions Our results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.