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.
Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in 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.
Increased risk of premature cardiovascular disease (CVD) is well recognized in systemic lupus erythematosus (SLE). Aberrant type I-Interferon (IFN)-neutrophil interactions contribute to this enhanced CVD risk. In lupus animal models, the Janus kinase (JAK) inhibitor tofacitinib improves clinical features, immune dysregulation and vascular dysfunction. We conducted a randomized, double-blind, placebo-controlled clinical trial of tofacitinib in SLE subjects (ClinicalTrials.gov NCT02535689). In this study, 30 subjects are randomized to tofacitinib (5 mg twice daily) or placebo in 2:1 block. The primary outcome of this study is safety and tolerability of tofacitinib. The secondary outcomes include clinical response and mechanistic studies. The tofacitinib is found to be safe in SLE meeting study’s primary endpoint. We also show that tofacitinib improves cardiometabolic and immunologic parameters associated with the premature atherosclerosis in SLE. Tofacitinib improves high-density lipoprotein cholesterol levels (p = 0.0006, CI 95%: 4.12, 13.32) and particle number (p = 0.0008, CI 95%: 1.58, 5.33); lecithin: cholesterol acyltransferase concentration (p = 0.024, CI 95%: 1.1, −26.5), cholesterol efflux capacity (p = 0.08, CI 95%: −0.01, 0.24), improvements in arterial stiffness and endothelium-dependent vasorelaxation and decrease in type I IFN gene signature, low-density granulocytes and circulating NETs. Some of these improvements are more robust in subjects with STAT4 risk allele.
Objective A role for mitochondrial dysfunction has been proposed in the immune dysregulation and organ damage characteristic of systemic lupus erythematosus (SLE). Idebenone is a coenzyme Q10 synthetic quinone analog and an antioxidant that has been used in humans to treat diverse diseases in which mitochondrial function is impaired. This study was undertaken to assess whether idebenone ameliorates lupus in murine models. Methods Idebenone was administered orally to MRL/lpr mice at 2 different doses (1 gm/kg or 1.5 gm/kg idebenone‐containing diet) for 8 weeks. At peak disease activity, clinical, immunologic, and metabolic parameters were analyzed and compared to those in untreated mice (n = 10 per treatment group). Results were confirmed in the lupus‐prone NZM2328 mouse model. Results In MRL/lpr mice, idebenone‐treated mice showed a significant reduction in mortality incidence (P < 0.01 versus untreated mice), and the treatment attenuated several disease features, including glomerular inflammation and fibrosis (each P < 0.05 versus untreated mice), and improved renal function in association with decreased renal expression of interleukin‐17A (IL‐17A) and mature IL‐18. Levels of splenic proinflammatory cytokines and inflammasome‐related genes were significantly decreased (at least P < 0.05 and some with higher significance) in mice treated with idebenone, while no obvious drug toxicity was observed. Idebenone inhibited neutrophil extracellular trap formation in neutrophils from lupus‐prone mice (P < 0.05) and human patients with SLE. Idebenone also improved mitochondrial metabolism (30% increase in basal respiration and ATP production), reduced the extent of heart lipid peroxidation (by one‐half that of untreated mice), and significantly improved endothelium‐dependent vasorelaxation (P < 0.001). NZM2328 mice exposed to idebenone also displayed improvements in renal and systemic inflammation, reducing the kidney pathology score (P < 0.05), IgG/C3 deposition (P < 0.05), and the gene expression of interferon, proinflammatory, and inflammasome‐related genes (at least P < 0.05 and some with higher significance). Conclusion Idebenone ameliorates murine lupus disease activity and the severity of organ damage, supporting the hypothesis that agents that modulate mitochondrial biologic processes may have a therapeutic role in human SLE.
Objective. Neutrophil extracellular traps (NETs) are extracellular lattices composed of nucleic material bound to neutrophil granule proteins. NETs may play pathogenic roles in the development and severity of autoimmune diseases such as systemic lupus erythematosus (SLE), at least in part, through induction of type I interferon (IFN) responses via externalization of oxidized immunostimulatory DNA. A distinct subset of SLE proinflammatory neutrophils (lowdensity granulocytes [LDGs]) displays enhanced ability to form proinflammatory NETs that damage the vasculature. We undertook this study to assess whether NET-bound RNA can contribute to inflammatory responses in endothelial cells (ECs) and the pathways that mediate this effect.Methods. Expression of newly synthesized and total RNA was quantified in NETs from healthy controls and lupus patients. The ability of ECs to take up NET-bound RNA and downstream induction of type I IFN responses were quantified. RNAs present in NETs were sequenced and specific small RNAs were tested for induction of endothelial type I IFN pathways.Results. NETs extruded RNA that was internalized by ECs, and this was enhanced when NET-bound nucleic acids were oxidized, particularly in lupus LDG-derived NETs. Internalization of NET-bound RNA by ECs was dependent on endosomal Toll-like receptors (TLRs) and the actin cytoskeleton and induced type I IFN-stimulated genes (ISGs). This ISG induction was dependent on NET-associated microRNA let-7b, a small RNA expressed at higher levels in LDGderived NETs, which acted as a TLR-7 agonist.Conclusion. These findings highlight underappreciated roles for small RNAs externalized in NETs in the induction of proinflammatory responses in vascular cells, with implications for lupus vasculopathy.
Objective Gasdermin D (GSDMD) is the key executioner of an inflammatory cell death mechanism known as pyroptosis. Recent reports have also implicated GSDMD in other mechanisms of cell death, including apoptosis, necroptosis, and NETosis. Given the role of dysregulated cell death in autoimmune syndromes such as systemic lupus erythematosus (SLE), this study was undertaken in a murine lupus model to investigate whether GSDMD plays a pathogenic role in systemic autoimmunity by promoting inflammatory cell death, leading to increased generation of nuclear autoantigens and autoantibodies. Methods An imiquimod‐induced model of SLE was tested in GSDMD−/− mice (n = 30), with wild‐type (WT) mice as controls (n = 34), on a C57BL/6 background. At the time of euthanasia, the mice were examined for serum autoantibodies, immune complex deposition, organ inflammation, immune dysregulation, and type I interferon responses. A model of pristane‐induced lung injury in GSDMD−/− mice (n = 7), with WT mice as controls (n = 10), was used to confirm the pulmonary phenotype. Regulation of various mechanisms of cell death by GSDMD was investigated in the mice. Results Unexpectedly, GSDMD−/− mice developed enhanced mortality, more severe renal and pulmonary inflammation, and exacerbated autoantibody production in response to imiquimod. Pulmonary involvement was also more severe in the absence of GSDMD in mice with pristane‐induced lung injury. Compared to WT mice, lack of GSDMD was associated with increased levels of circulating nuclear autoantigens (P < 0.01), anti–double‐stranded DNA autoantibodies (P < 0.01), tissue immune complex deposition (P < 0.05), expansion of myeloid cell subsets (P < 0.05), and enhanced B cell activation and plasma cell differentiation (P = 0.001). Moreover, in the absence of GSDMD, enhanced autoantigen generation was associated with increased local induction of cell death in vivo. Conclusion GSDMD negatively regulates autoantigen generation and immune dysregulation in response to tissue injury and may play previously unappreciated protective roles in systemic autoimmunity.
Objective. Itaconic acid, a Krebs cycle-derived immunometabolite, is synthesized by myeloid cells in response to danger signals to control inflammasome activation, type I interferon (IFN) responses, and oxidative stress. As these pathways are dysregulated in systemic lupus erythematosus (SLE), we investigated the role of an itaconic acid derivative in the treatment of established murine lupus.Methods. Female (NZW × NZB)F 1 lupus-prone mice were administered 4-octyl itaconate (4-OI) or vehicle starting after clinical onset of disease (30 weeks of age) for 4 weeks (n = 10 mice /group). At 34 weeks of age (peak disease activity), animals were euthanized, organs and serum were collected, and clinical, metabolic, and immunologic parameters were evaluated.Results. Proteinuria, kidney immune complex deposition, renal scores of severity and inflammation, and anti-RNP autoantibodies were significantly reduced in the 4-OI treatment group compared to the vehicle group. Splenomegaly decreased in the 4-OI group compared to vehicle, with decreases in activation markers in innate and adaptive immune cells, increases in CD8+ T cell numbers, and inhibition of JAK1 activation. Gene expression analysis in splenocytes showed significant decreases in type I IFN and proinflammatory cytokine genes and increased Treg cell-associated markers in the 4-OI group compared to the vehicle group. In human control and lupus myeloid cells, 4-OI in vitro treatment decreased proinflammatory responses and B cell responses.Conclusions. These results support targeting immunometabolism as a potentially viable approach in autoimmune disease treatment, with 4-OI displaying beneficial roles attenuating immune dysregulation and organ damage in lupus.
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