Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism.

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.

Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus.

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.

A High-Throughput Real-Time Imaging Technique To Quantify NETosis and Distinguish Mechanisms of Cell Death in Human Neutrophils.

Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes. Neutrophils undergoing NETosis induced by various physiological stimuli showed distinct changes, with a loss of multilobulated nuclei, as well as nuclear decondensation followed by membrane compromise, and were accurately counted by applying filters based on fluorescence intensity and nuclear size. Findings were confirmed and validated with the established method of immunofluorescence microscopy. The platform was also validated to rapidly assess and quantify the dose-dependent effect of inhibitors of NETosis. In addition, this method was able to distinguish among neutrophils undergoing NETosis, apoptosis, or necrosis based on distinct changes in nuclear morphology and membrane integrity. The IncuCyte ZOOM platform is a novel real-time assay that quantifies NETosis in a rapid, automated, and reproducible way, significantly optimizing the study of neutrophils. This platform is a powerful tool to assess neutrophil physiology and NETosis, as well as to swiftly develop and test novel neutrophil targets.