Liam J. O’Neil

Neutrophil dysregulation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). SLE is characterized by elevated levels of a pathogenic neutrophil subset known as low-density granulocytes (LDGs). The origin and phenotypic, functional, and pathogenic heterogeneity of LDGs remain to be systematically determined. Transcriptomics and epigenetic assessment of lupus LDGs, autologous normal-density neutrophils, and healthy control neutrophils was performed by bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. Functional readouts were compared among neutrophil subsets. SLE LDGs display significant transcriptional and epigenetic heterogeneity and comprise 2 subpopulations of intermediate-mature and immature neutrophils, with different degrees of chromatin accessibility and differences in transcription factor motif analysis. Differences in neutrophil extracellular trap (NET) formation, oxidized mitochondrial DNA release, chemotaxis, phagocytosis, degranulation, ability to harm the endothelium, and responses to type I interferon (IFN) stimulation are evident among LDG subsets. Compared with other immune cell subsets, LDGs display the highest expression of IFN-inducible genes. Distinct LDG subsets correlate with specific clinical features of lupus and with the presence and severity of coronary artery disease. Phenotypic, functional, and pathogenic neutrophil heterogeneity are prevalent in SLE and may promote immune dysregulation and prominent vascular damage characteristic of this disease.

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

Hidradenitis suppurativa (HS), also known as acne inversa, is an incapacitating skin disorder of unknown etiology manifested as abscess-like nodules and boils resulting in fistulas and tissue scarring as it progresses. Given that neutrophils are the predominant leukocyte infiltrate in HS lesions, the role of neutrophil extracellular traps (NETs) in the induction of local and systemic immune dysregulation in this disease was examined. Immunofluorescence microscopy was performed in HS lesions and detected the prominent presence of NETs. NET complexes correlated with disease severity, as measured by Hurley staging. Neutrophils from the peripheral blood of patients with HS peripheral also displayed enhanced spontaneous NET formation when compared to healthy control neutrophils. Sera from patients recognized antigens present in NETs and harbored increased antibodies reactive to citrullinated peptides. B cell dysregulation, as evidenced by elevated plasma cells and IgG, was observed in the circulation and skin from patients with HS. Peptidylarginine deiminases (PADs) 1 to 4, enzymes involved in citrullination, were differentially expressed in HS skin, when compared to controls, in association with enhanced tissue citrullination. NETs in HS skin coexisted with plasmacytoid dendritic cells, in association with a type I interferon (IFN) gene signature. Enhanced NET formation and immune responses to neutrophil and NET-related antigens may promote immune dysregulation and contribute to inflammation. This, along with evidence of up-regulation of the type I IFN pathway in HS skin, suggests that the innate immune system may play important pathogenic roles in this disease.

Abstract 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.