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Objectives: An important pathogenic role for neutrophils in systemic lupus erythematosus (SLE) has been proposed. Neutrophils that lack brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1), one of the clock genes, are defective in aging and proinflammatory responses. We assessed the role of Bmal1 in clinical and immunologic manifestations of murine lupus and in human SLE neutrophils. Methods: Myeloid-conditional Bmal1 knockout mice (Bmal1Mye−/−) and wild type (WT) were treated with epicutaneous TLR7/8 agonist (imiquimod; IMQ) for 6 weeks to induce a lupus phenotype. Upon euthanasia, immune responses, autoantibodies and renal manifestations were evaluated. NET formation and gene expression of bone marrow (BM)-derived murine neutrophils were evaluated. BMAL1 expression was quantified in SLE neutrophils and compared with clinical disease. Results: IMQ-treated Bmal1Mye−/− and WT displayed comparable systemic inflammation. While renal function did not differ, serum anti-dsDNA levels and renal immune complex deposition were significantly increased in Bmal1Mye−/−. While no differences were observed in NET formation, expression levels of April in BM neutrophils were significantly higher in Bmal1Mye−/−. Bulk RNA-sequence data showed that BM neutrophils in IMQ-treated Bmal1Mye−/− were relatively immature when compared with IMQ-treated WT. BM showed an enhanced April protein expression in Bmal1Mye−/− mice. BMAL1 levels in human SLE peripheral blood neutrophils correlated positively with serum C3 and negatively with serum anti-dsDNA levels. Conclusion: Bmal1 is associated with lower disease activity in SLE. These results indicate that perturbation in the circadian rhythm of neutrophils can have pathogenic consequences in SLE. [ABSTRACT FROM AUTHOR]

Objective: Systemic lupus erythematosus (SLE) increases cardiovascular disease (CVD) risk, and this is not explained by traditional risk factors. Characterization of blood immunologic signatures that associate with subclinical CVD and predict its progression has been challenging and may help identify subgroups at risk. Methods: Patients with SLE (n = 77) and healthy controls (HCs) (n = 27) underwent assessments of arterial stiffness, vascular wall inflammation, and coronary atherosclerosis burden with cardio‐ankle vascular index (CAVI); fluorodeoxyglucose–positron emission tomography/computed tomography (CT) (target‐to‐background ratio [TBR]); and coronary CT angiography. Whole blood bulk RNA sequencing was performed in a subset of study participants (HC n = 10, SLE n = 20). In a partially overlapping subset (HC n = 24, SLE n = 64), serum inflammatory protein biomarkers were quantified with an Olink platform. Results: CAVI, TBR, and noncalcified coronary plaque burden (NCB) were increased in patients with SLE compared to HCs. When comparing patients with SLE with high CAVI scores to those with low CAVI scores or to HCs, there was a down‐regulation of genes in pathways involved in the cell cycle and differentially regulated pathways related to metabolism. Distinct serum proteins associated with increased CAVI (CCL23, colony‐stimulating factor 1, latency‐activating peptide transforming growth factor β1, interleukin 33 [IL‐33], CD8A, and IL‐12B), NCB (monocyte chemotactic protein 4 and FMS‐like tyrosine kinase 3 ligand [Flt3L]), and TBR (CD5, IL‐1α, AXIN1, cystatin D [CST5], and tumor necrosis factor receptor superfamily 9; P < 0.05). Conclusion: Blood gene expression patterns and serum proteins that associate with worse vascular phenotypes suggest dysregulated immune and metabolic pathways linked to premature CVD. Cytokines and chemokines identified in associations with arterial stiffness, inflammation, and NCB in SLE may allow for characterization of new CVD biomarkers in lupus. [ABSTRACT FROM AUTHOR]

The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host's hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission. Here, Pala et al. show that mosquito salivary apyrase regulates hemostasis in the mosquito blood meal, boosting malaria transmission. They demonstrate that immunizing against apyrase inhibits parasite transmission. [ABSTRACT FROM AUTHOR]

The article presents the discussion on editorial leadership at Arthritis & Rheumatology, highlighting the challenges and developments encountered, particularly during the COVID-19 pandemic. Topics include increase in manuscript submissions related to COVID-19, the importance of expert opinions in delivering quality care, and the introduction of new article types such as Expert Perspectives on Clinical Challenges and Notes From the Field.

Purpose of Review: In this review, we summarize the current evidence that suggests that neutrophils play a key role in facilitating damage to local bone structures. Recent Findings: Neutrophil infiltration is a hallmark of inflammatory bone diseases such as rheumatoid arthritis (RA) and periodontitis disease (PD). Both of these human diseases are marked by an imbalance in bone homeostasis, favoring the degradation of local bone which ultimately leads to erosions. Osteoclasts, a multinucleated resident bone cell, are responsible for facilitating the turnover of bone and the bone damage observed in these diseases. The involvement of neutrophils and neutrophil extracellular trap formation have recently been implicated in exacerbating osteoclast function through direct and indirect mechanisms. We highlight a recent finding that NET proteins such as histones and elastase can generate non-canonical, inflammatory osteoclasts, and this process is mediated by post-translational modifications such as citrullination and carbamylation, both of which act as autoantigens in RA. Summary: It appears that NETs, autoantibodies, modified proteins, cytokines, and osteoclasts all ultimately contribute to local and permanent bone damage in RA and PD. However, more studies are needed to fully understand the role of neutrophils in inflammatory bone diseases. [ABSTRACT FROM AUTHOR]

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans. [ABSTRACT FROM AUTHOR]

Systemic autoimmune diseases are characteristically associated with aberrant autoreactive innate and adaptive immune responses that lead to tissue damage and increased morbidity and mortality. Autoimmunity has been linked to alterations in the metabolic functions of immune cells (immunometabolism) and, more specifically, to mitochondrial dysfunction. Much has been written about immunometabolism in autoimmunity in general, so this Essay focuses on recent research into the role of mitochondrial dysfunction in the dysregulation of innate and adaptive immunity that is characteristic of systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Enhancing the understanding of mitochondrial dysregulation in autoimmunity will hopefully contribute to accelerating the development of immunomodulatory treatments for these challenging diseases. Systemic autoimmune diseases are linked to alterations in the metabolic functions of immune cells. This Essay addresses key aspects of the current knowledge on how mitochondrial dysfunction in particular affects human autoimmune diseases. [ABSTRACT FROM AUTHOR]

Summary: A body of evidence has re‐energized the interest on the role neutrophils in inflammatory and autoimmune conditions. For decades, neutrophils have been considered a homogenous population. Nevertheless, accumulating evidence suggests that neutrophils are more versatile and heterogeneous than initially considered. The notion of neutrophil heterogeneity has been supported by the identification of low‐density granulocytes (LDGs) in systemic lupus erythematosus (SLE) and other systemic autoimmune and autoinflammatory conditions. Transcriptomic, epigenetic, proteomic, and functional analyses support that LDGs are a distinct subset of proinflammatory neutrophils implicated in the pathogenesis of SLE and other autoimmune diseases. Importantly, it remains incompletely characterized whether LDGs detected in other inflammatory/autoimmune conditions display the same phenotype that those present in SLE. A shared feature of LDGs across diseases is their association with vascular damage, an important contributor to morbidity and mortality in chronic inflammatory conditions. Additionally, the lack of specific markers to identify LDGs in circulation or in tissue, makes it a challenge to elucidate their role in the pathogenesis of inflammatory and autoimmune conditions. In this review, we aim to examine the evidence on the biology and the putative pathogenic role of LDGs in systemic autoimmune diseases. [ABSTRACT FROM AUTHOR]

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)F1 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. [ABSTRACT FROM AUTHOR]

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs. Synopsis: The FOXM1 transcription factor modulates NETs by regulating ROS during diabetic wound healing. TREM1 contributes to the clinical healing outcome and promotes FOXM1+ neutrophil recruitment to enhance diabetic wound healing. Diabetic foot ulcers (DFUs) are characterized by a suppressed inflammatory response with increased NET formation.Inhibition of FOXM1 in human neutrophils increases ROS levels leading to NET formation.Topical TREM1 application promotes recruitment of FOXM1+ neutrophils and enhances diabetic wound healing in mice.TREM1 expression and NET formation contributes to the clinical healing outcome of DFUs and may serve as potential biomarker. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) often features extensive cardiovascular (CV) comorbidity and patients with SLE are at significantly increased risk of CV event occurrence and CV-related mortality. While the specific mechanisms leading to this increased cardiovascular disease (CVD) risk remain to be fully characterized, this heightened risk cannot be fully explained by traditional CV risk factors and is likely driven by immunologic and inflammatory features of SLE. Widespread innate and adaptive immune dysregulation characterize SLE, and factors including excessive type I interferon burden, inappropriate formation and ineffective clearance of neutrophil extracellular traps, and autoantibody formation have been linked to clinical and metabolic features impacting CV risk in SLE and may represent pathogenic drivers of SLE–related CVD. Indeed, functional and phenotypic aberrations in almost every immune cell type are present in SLE and may impact CVD progression. As understanding of the contribution of SLE–specific factors to CVD in SLE improves, improved screening and monitoring of CV risk alongside development of therapeutic treatments aimed at prevention of CVD in SLE patients are required and remain the focus of several ongoing studies and lines of inquiry. [ABSTRACT FROM AUTHOR]

This article is a contribution to the special issue on: Inflammation in vascular diseases - Guest Editors: Mariana Kaplan & Peter Grayson In 1865, a 27-year-old man was hospitalized at the University of Freiburg, Germany, for a rapidly progressive, systemic illness. 8285055 11 Harper RL, Ferrante EA, Boehm M. Development of vascular disease models to explore disease causation and pathomechanisms of rare vascular diseases. I Harper i et al. detail various methods to create patient-specific vascular disease models including use of primary cells derived from various patient biospecimens coupled with in vitro and in vivo vascular models and provide examples of successful approaches to study various rare vascular diseases [[11]]. Using Takayasu's arteritis, a disease predominantly of the young, and giant cell arteritis, a disease exclusive to later adulthood, the authors compare age of the host as a critical intrinsic factor that influences the immunologic landscape of different inflammatory forms of vascular disease. [Extracted from the article]

Objective: Mechanisms leading to anti–citrullinated protein antibody (ACPA) generation in rheumatoid arthritis (RA) are hypothesized to originate in the lung. We undertook this study to understand associations between neutrophil extracellular trap (NET) formation in the lung and local ACPA generation in subjects at risk of developing RA. Methods: Induced sputum was collected from 49 subjects at risk of developing RA, 12 patients with RA, and 18 controls. Sputum neutrophils were tested for ex vivo NET formation, and sputum‐induced NET formation of control neutrophils was measured using immunofluorescence imaging. Sputum macrophages were tested for ex vivo endocytosis of apoptotic and opsonized cells. Levels of ACPA, NET remnants, and inflammatory proteins were quantified in sputum supernatant. Results: Spontaneous citrullinated histone H3 (Cit‐H3)–expressing NET formation was higher in sputum neutrophils from at‐risk subjects and RA patients compared to controls (median 12%, 22%, and 0%, respectively; P < 0.01). In at‐risk subjects, sputum IgA ACPA correlated with the percentage of neutrophils that underwent Cit‐H3+ NET formation (r = 0.49, P = 0.002) and levels of Cit‐H3+ NET remnants (r = 0.70, P < 0.001). Reduced endocytic capacity of sputum macrophages was found in at‐risk subjects and RA patients compared to controls. Using a mediation model, we found that sputum inflammatory proteins were associated with sputum IgA ACPA through a pathway mediated by Cit‐H3+ NET remnants. Sputum‐induced Cit‐H3+ NET formation also correlated with sputum levels of interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor in at‐risk subjects, suggesting a causal relationship. Conclusion: These data support a potential mechanism for mucosal ACPA generation in subjects at risk of developing RA, whereby inflammation leads to increased citrullinated protein–expressing NETs that promote local ACPA generation. [ABSTRACT FROM AUTHOR]

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 (low‐density 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 LDG‐derived 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. [ABSTRACT FROM AUTHOR]

Objective: Antibodies against carbamylated proteins (anti-CarP) are associated with poor prognosis and the development of bone erosions in rheumatoid arthritis (RA). RA neutrophils externalize modified autoantigens through the formation of neutrophil extracellular traps (NETs). Increased levels of the cathelicidin LL37 have been documented in the synovium of RA patients, but the cellular source remains unclear. We sought to determine if post-translational modifications of LL37, specifically carbamylation, occur during NET formation, enhance this protein's autoantigenicity, and contribute to drive bone erosion in the synovial joint. Methods: ELISA and Western blot analyses were used to identify carbamylated LL37 (carLL37) in biological samples. Anti-carLL37 antibodies were measured in the serum of HLA-DRB1*04:01 transgenic mice and in human RA synovial fluid. Results: Elevated levels of carLL37 were found in plasma and synovial fluid from RA patients, compared to healthy controls. RA NETs release carLL37 and fibroblast-like synoviocytes (FLS) internalized NET-bound carLL37 and loaded it into their MHCII compartment. HLA-DRB1*04:01 transgenic mice immunized with FLS containing NETs developed autoantibodies against carLL37. Anti-carLL37 antibodies were present in RA sera and synovial fluid and they correlated with radiologic bone erosion scores of the hands and feet in RA patients. CarLL37-IgG immune complexes enhanced the ability of monocytes to differentiate into osteoclasts and potentiated osteoclast-mediated extracellular matrix resorption. Conclusions: NETs are a source of carLL37 leading to induction of anti-carbamylated autoantibody responses. Furthermore, carLL37-IgG immune complexes may be implicated in the bone damage characteristic of RA. These results support that dysregulated NET formation has pathogenic roles in RA. [ABSTRACT FROM AUTHOR]

The liver is the central organ for cholesterol synthesis and homeostasis. The effects of dietary cholesterol on hepatic injury, mainly of oxidized low-density lipoproteins (OxLDL), are not fully understood. Here, we show that the degree of cholesterol oxidation had different impacts on the global gene expression of human M2-like macrophages, with highly oxidized LDL causing the most dramatic changes. M2-like macrophages and Kupffer cells undergo M4-like polarization, decreasing the expression of important markers, such as IL10, MRC1, and CD163. These cells also displayed functional changes, with reduced phagocytic capacity, increased neutrophil recruitment, and more effective neutrophil extracellular traps (NETs) induction. Our findings provide a link between LDL oxidation and modification of peripheral and liver macrophage function. [ABSTRACT FROM AUTHOR]

An editorial is presented on improving clarity, resolution, and aesthetic value. Topics include received by readers and helping in balancing the research content with pragmatic advice from worldleading clinicians; and containing sham-controlled test of acupuncture for osteoarthritis and phase II testing of a new molecule for the treatment of systemic lupus erythematosus.

Objective: Neutrophil dysregulation and the type I interferon (IFN) axis have been proposed to contribute to premature cardiovascular disease, a leading cause of mortality in patients with systemic lupus erythematosus (SLE). In the present study, we evaluated the ability of anifrolumab, a type I IFN receptor–blocking antibody, to reduce neutrophil extracellular trap (NET) formation and modulate cardiometabolic disease markers in comparison to placebo. Methods: Study subjects comprised patients with moderate‐to‐severe SLE who were enrolled in phase IIb of the MUSE trial (A Phase II, Randomized Study to Evaluate the Efficacy and Safety of MEDI‐546 in Subjects with Systemic Lupus Erythematosus), with healthy individuals as controls. Blood samples were collected from SLE patients (n = 305) and healthy controls (n = 10–20) before the initiation of treatment (baseline) and from SLE patients after they had been treated with 300 mg of anifrolumab (n = 99) or placebo (n = 102). Baseline IFN gene signature test status was determined, and the IFN gene signature (21‐gene panel) was monitored over time. Serum proteins were measured by multiplex immunoassay or ultrasensitive Simoa assay. NET complexes, cholesterol efflux capacity (CEC), and glycoprotein acetylation (GlycA) and other lipid parameters were assessed in plasma. Results: Formation of NET complexes and levels of tumor necrosis factor (TNF) and interleukin‐10 (IL‐10) were correlated with extent of type I IFN pathway activity. NET complexes and IL‐10 levels were up‐regulated in SLE patients compared to healthy controls (P < 0.008). The cardiometabolic disease markers CEC and GlycA were also found to be dysregulated in patients with SLE (P < 0.001 versus healthy controls). Type I IFN receptor inhibition with anifrolumab significantly reduced NET complexes and GlycA and improved CEC compared to baseline (P < 0.05) whereas no improvements were seen with placebo. Levels of TNF and IL‐10 were reduced with anifrolumab compared to placebo (P < 0.05). Conclusion: These data support a key role for type I IFNs in modulating factors contributing to SLE vasculopathy and suggest that inhibition of this pathway could decrease cardiovascular risk in individuals with SLE. [ABSTRACT FROM AUTHOR]

Systemic rheumatic diseases are a heterogeneous group of disorders characterized by profound immune dysregulation. Recent discoveries have led to a significant resurgence of interest in neutrophils as shapers of immune dysregulation and as triggers of organ damage in rheumatic diseases. Neutrophils contribute to the initiation, promotion, and perpetuation of immune dysregulation through a variety of mechanisms including synthesis of proinflammatory cytokines, direct tissue damage through degranulation and synthesis of reactive oxygen species, and the formation of neutrophil extracellular traps (NETs). The identification of a subset of proinflammatory neutrophils, the low-density granulocytes (LDGs), which promote Th1 responses and cause endothelial dysfunction, has further strengthened the pathogenic role of neutrophils in various rheumatic diseases. The presence of autoantibodies targeting molecules commonly expressed in neutrophils suggests that neutrophils, particularly NETs, may be a source of autoantigens. An imbalance between NET formation and degradation, which leads to increased NET levels in the circulation and tissues, could enhance the exposure of the immune system to modified autoantigens, promote vascular disease, and increase tissue damage. This review will present an overview of recent advances in our understanding of how neutrophil dysregulation modulates the innate and adaptive immune responses in systemic rheumatic diseases and their putative contributions to pathogenicity. Understanding the potential pathogenic role of neutrophil dysregulation may provide better molecular candidates for therapeutic targeting, and ultimately promote improvements in the clinical outcomes in rheumatic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease in which type I interferons (IFN) play a key role. The IFN response can be triggered when oxidized DNA engages the cytosolic DNA sensing platform cGAS-STING, but the repair mechanisms that modulate this process and govern disease progression are unclear. To gain insight into this biology, we interrogated the role of oxyguanine glycosylase 1 (OGG1), which repairs oxidized guanine 8-Oxo-2′-deoxyguanosine (8-OH-dG), in the pristane-induced mouse model of SLE. Ogg1 −/− mice showed increased influx of Ly6Chi monocytes into the peritoneal cavity and enhanced IFN-driven gene expression in response to short-term exposure to pristane. Loss of Ogg1 was associated with increased auto-antibodies (anti-dsDNA and anti-RNP), higher total IgG, and expression of interferon stimulated genes (ISG) to longer exposure to pristane, accompanied by aggravated skin pathology such as hair loss, thicker epidermis, and increased deposition of IgG in skin lesions. Supporting a role for type I IFNs in this model, skin lesions of Ogg1 −/− mice had significantly higher expression of type I IFN genes (Isg15, Irf9 , and Ifnb). In keeping with loss of Ogg1 resulting in dysregulated IFN responses, enhanced basal and cGAMP-dependent Ifnb expression was observed in BMDMs from Ogg1 −/− mice. Use of the STING inhibitor, H151, reduced both basal and cGAMP-driven increases, indicating that OGG1 regulates Ifnb expression through the cGAS-STING pathway. Finally, in support for a role for OGG1 in the pathology of cutaneous disease, reduced OGG1 expression in monocytes associated with skin involvement in SLE patients and the expression of OGG1 was significantly lower in lesional skin compared with non-lesional skin in patients with Discoid Lupus. Taken together, these data support an important role for OGG1 in protecting against IFN production and SLE skin disease. [ABSTRACT FROM AUTHOR]

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis. [ABSTRACT FROM AUTHOR]

The effects of cell size, shape and deformability on cellular function have long been a topic of interest. Recently, mechanical phenotyping technologies capable of analysing large numbers of cells in real time have become available. This has important implications for biology and medicine, especially haemato‐oncology and immunology, as immune cell mechanical phenotyping, immunologic function, and malignant cell transformation are closely linked and potentially exploitable to develop new diagnostics and therapeutics. In this review, we introduce the technologies used to analyse cellular mechanical properties and review emerging findings following the advent of high throughput deformability cytometry. We largely focus on cells from the myeloid lineage, which are derived from the bone marrow and include macrophages, granulocytes and erythrocytes. We highlight advances in mechanical phenotyping of cells in suspension that are revealing novel signatures of human blood diseases and providing new insights into pathogenesis of these diseases. The contributions of mechanical phenotyping of cells in suspension to our understanding of drug mechanisms, identification of novel therapeutics and monitoring of treatment efficacy particularly in instances of haematologic diseases are reviewed, and we suggest emerging topics of study to explore as high throughput deformability cytometers become prevalent in laboratories across the globe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) patients exhibit accelerated development of atherosclerosis and increased incidents of cardiovascular disease (CVD) that cannot be explained by traditional risk factors alone. Accumulating evidence suggests that reduced levels of high‐density lipoproteins (HDLs), along with altered HDL composition and function, may contribute to the accelerated atherosclerosis in SLE patients. Normally, HDLs play various atheroprotective roles through facilitating cholesterol efflux, inhibiting vascular inflammation, and scavenging oxidative species. However, systemic inflammation, oxidative stress, and autoimmunity in SLE patients induce changes in HDL size distribution and proteomic and lipidomic signatures. These compositional changes in HDLs result in the formation of proinflammatory, dysfunctional HDL. These lupus‐altered HDLs have impaired antiatherogenic function with reduced cholesterol efflux capacities, impaired antioxidation abilities, and diminished antiinflammatory properties. In fact, dysfunctional HDL may promote atherogenesis by inducing inflammation. Thus, dysfunctional HDLs could be an important biomarker of accelerated atherosclerosis in lupus. Additionally, HDL‐targeted therapies, especially infusion of reconstituted HDLs, may serve as a potential therapeutic intervention for SLE patients with CVD. [ABSTRACT FROM AUTHOR]

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 IFNinducible 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. [ABSTRACT FROM AUTHOR]

Implicating the immune system in hidradenitis suppurativa: The cause of the painful skin condition hidradenitis suppurativa (acne inversa) is unknown. To better understand pathogenesis of this debilitating disease, Byrd et al. examined blood and lesional skin of patients and healthy controls. They observed prominent neutrophil extracellular traps (NETs), plasma cells, autoantibodies, and a type I IFN signature in patient samples. Inflammation and netting neutrophils may promote B cell activation and autoantibody secretion, and all of these mechanisms may contribute to tissue damage. These results suggest that multiple arms of the immune system are dysregulated in hidradenitis suppurativa and could be potentially targeted in new therapies. 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 FROM AUTHOR]

Objectives: The presence of proinflammatory low-density granulocytes (LDG) has been demonstrated in autoimmune and infectious diseases. Recently, regulatory neutrophilic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) were identified in systemic lupus erythematosus (SLE). Because LDG and PMN-MDSC share a similar phenotype with contrasting functional effects, we explored these cells in a cohort of patients with SLE.Methods: LDG and normal-density granulocytes (NDG) were isolated from fresh blood of healthy donors (HD) and patients with SLE. Associations between LDG and clinical manifestations were analysed. Multicolor flow cytometry and confocal imaging were performed to immunophenotype the cells. The ability of LDG and NDG to suppress T cell function and induce cytokine production was quantified.Results: LDG prevalence was elevated in SLE versus HD, associated with the interferon (IFN) 21-gene signature and disease activity. Also, the LDG-to-lymphocyte ratio associated better with SLE disease activity index than neutrophil-to-lymphocyte ratio. SLE LDG exhibited significantly heightened surface expression of various activation markers and also of lectin-like oxidised low-density lipoprotein receptor-1, previously described to be associated with PMN-MDSC. Supernatants from SLE LDG did not restrict HD CD4+ T cell proliferation in an arginase-dependent manner, suggesting LDG are not immunosuppressive. SLE LDG supernatants induced proinflammatory cytokine production (IFN gamma, tumour necrosis factor alpha and lymphotoxin alpha) from CD4+ T cells.Conclusions: Based on our results, SLE LDG display an activated phenotype, exert proinflammatory effects on T cells and do not exhibit MDSC function. These results support the concept that LDG represent a distinct proinflammatory subset in SLE with pathogenic potential, at least in part, through their ability to activate type 1 helper responses. [ABSTRACT FROM AUTHOR]

Objective: Autoreactive IgE antibodies have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesize that omalizumab, a monoclonal antibody binding IgE, may improve SLE activity by reducing type I interferon (IFN) production by hampering plasmacytoid dendritic cells and basophil activation. This study was undertaken to assess the safety, tolerability, and clinical efficacy of omalizumab in mild to moderate SLE. Methods: Sixteen subjects with SLE and a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI‐2K) score of ≥4 and elevated autoreactive IgE antibody levels were randomized to receive omalizumab or placebo (2:1) for 16 weeks, followed by 16 weeks of open‐label treatment and a 4‐week washout period. The SLEDAI‐2K score, British Isles Lupus Assessment Group index (BILAG 2004) score, and physician's global assessment of disease activity were recorded at each visit. The type I IFN–induced gene signature was determined using quantitative polymerase chain reaction. Results: Omalizumab was well tolerated with no allergic reactions, and mostly mild adverse events comparable to those experienced with placebo treatment. SLEDAI‐2K scores improved in the omalizumab group compared to the placebo group at week 16 (P = 0.038), as well as during the open‐label phase in subjects initially receiving placebo (P = 0.02). No worsening in BILAG scores or the physician's global assessment was detected. There was a trend toward a reduction in IFN gene signature in subjects treated with omalizumab (P = 0.11), especially in subjects with a high baseline IFN signature (P = 0.052). Conclusion: Our findings indicate that omalizumab is well tolerated in SLE and is associated with improvement in disease activity. Larger randomized clinical trials will be needed to assess the efficacy of omalizumab in patients with SLE. [ABSTRACT FROM AUTHOR]

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape‐changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho‐rheological (MORE) changes capable of altering cell function. We employ real‐time deformability cytometry (RT‐DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT‐DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT‐DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un‐primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+/H+ antiport‐dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease. [ABSTRACT FROM AUTHOR]

Objective: Recognition of citrullinated antigens such as vimentin, fibrinogen, and α‐enolase is associated with rheumatoid arthritis (RA). Emerging data suggest that the matrix protein aggrecan is also recognized as a citrullinated antigen. This study was undertaken to directly visualize Cit‐aggrecan–specific T cells and characterize them in patients with RA. Methods: Citrullinated aggrecan peptides with likely DRB1*04:01 binding motifs were predicted using a previously published scanning algorithm. Peptides with detectable binding were assessed for immunogenicity by HLA tetramer staining, followed by single cell cloning. Selectivity for citrullinated peptide was assessed by tetramer staining and proliferation assays. Ex vivo tetramer staining was then performed to assess frequencies of aggrecan‐specific T cells in peripheral blood. Finally, disease association was assessed by comparing T cell frequencies in RA patients and controls and correlating aggrecan‐specific T cells with levels of aggrecan‐specific antibodies. Results: We identified 6 immunogenic peptides, 2 of which were the predominant T cell targets in peripheral blood. These 2 epitopes were citrullinated at HLA binding residues and shared homologous sequences. RA patients had significantly higher frequencies of Cit‐aggrecan–specific T cells than healthy subjects. Furthermore, T cell frequencies were significantly correlated with antibodies against citrullinated aggrecan. Conclusion: Our findings indicate that T cells that recognize citrullinated aggrecan are present in patients with RA and correlate with antibodies that target this same antigen. Consequently, aggrecan‐specific T cells and antibodies are potentially relevant markers that could be used to monitor patients with RA or at‐risk subjects. [ABSTRACT FROM AUTHOR]

An editorial is presented on the medical publishing world have observed an infodemic occurring alongside the COVID-19 pandemic. Topics include the Arthritis & Rheumatology (A&R) has tasked with conducting, reviewing, and translating science to the rheumatic disease community has agree with this diagnosis, and the most clearly by the Randomized Evaluation of COVID-19 Therapy trial which found that dexamethasone reduced mortality in patients requiring respiratory support.

Objectives: Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterised by flares of sterile arthritis with neutrophil infiltrate and the overproduction of interleukin (IL)-1β. The purpose of this study was to elucidate the potential role of neutrophil subsets and neutrophil extracellular traps (NET) in the pathogenesis of PAPA.Methods: Neutrophils and low-density granulocytes (LDG) were quantified by flow cytometry. Circulating NETs were measured by ELISA and PAPA serum was tested for the ability to degrade NETs. The capacity of NETs from PAPA neutrophils to activate macrophages was assessed. Skin biopsies were analysed for NETs and neutrophil gene signatures.Results: Circulating LDGs are elevated in PAPA subjects. PAPA neutrophils and LDGs display enhanced NET formation compared with control neutrophils. PAPA sera exhibit impaired NET degradation and this is corrected with exogenous DNase1. Recombinant human IL-1β induces NET formation in PAPA neutrophils but not healthy control neutrophils. NET formation in healthy control neutrophils is induced by PAPA serum and this effect is inhibited by the IL-1 receptor antagonist, anakinra. NETs from PAPA neutrophils and LDGs stimulate IL-6 release in healthy control macrophages. NETs are detected in skin biopsies of patients with PAPA syndrome in association with increased tissue IL-1β, IL-8 and IL-17. Furthermore, LDG gene signatures are detected in PAPA skin.Conclusions: PAPA syndrome is characterised by an imbalance of NET formation and degradation that may enhance the half-life of these structures in vivo, promoting inflammation. Anakinra ameliorates NET formation in PAPA and this finding supports a role for IL-1 signalling in exacerbated neutrophil responses in this disease. The study also highlights other inflammatory pathways potentially pathogenic in PAPA, including IL-17 and IL-6, and these results may help guide new therapeutic approaches in this severe and often treatment-refractory condition. [ABSTRACT FROM AUTHOR]

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that neutrophil extracellular traps (NETs) may play a role in promoting atherosclerotic plaque lesions in humans and in murine models. The exact pathways involved in NET-driven atherogenesis remain to be systematically characterized. To assess the extent to which myeloid-specific peptidylarginine deiminase 4 (PAD4) and PAD4-dependent NET formation contribute to atherosclerosis, mice with myeloid-specific deletion of PAD4 were generated and backcrossed to Apoe−/− mice. The kinetics of atherosclerosis development were determined. NETs, but not macrophage extracellular traps, were present in atherosclerotic lesions as early as 3 weeks after initiating high-fat chow. The presence of NETs was associated with the development of atherosclerosis and with inflammatory responses in the aorta. Specific deletion of PAD4 in the myeloid lineage significantly reduced atherosclerosis burden in association with diminished NET formation and reduced inflammatory responses in the aorta. NETs stimulated macrophages to synthesize inflammatory mediators, including IL-1β, CCL2, CXCL1, and CXCL2. Our data support the notion that NETs promote atherosclerosis and that the use of specific PAD4 inhibitors may have therapeutic benefits in this potentially devastating condition. [ABSTRACT FROM AUTHOR]

Objectives: To assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects.Materials and Methods: We studied 74 subjects with SLE and 77 healthy controls. Neutrophils and low-density granulocytes were isolated, and NETs were induced. Ubiquitin content was quantified in NETs by western blot analysis, ELISA and immunofluorescence microscopy, while ubiquitination of NET proteins was assessed by immunoprecipitation. Monocyte-derived macrophages from SLE and controls were isolated and stimulated with NETs or ubiquitin. Calcium flux and cytokine synthesis were measured following these stimuli.Results: NETs contain ubiquitinated proteins, with a lower expression of polyubiquitinated proteins in subjects with SLE than in controls. Myeloperoxidase (MPO) is present in ubiquitinated form in NETs. Patients with SLE develop antiubiquitinated MPO antibodies, and titres positively correlate with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (P<0.01), and negatively correlate with complement components (P<0.01). Stimulation of monocyte-derived macrophages with NETs or with ubiquitin led to enhanced calcium flux. In addition, stimulation with NETs led to enhanced cytokine (tumour necrosis factor-α and interleukin-10) production in macrophages from patients with SLE when compared with controls, which was hampered by inhibition of NET internalisation by macrophages.Conclusion: This is the first study to find ubiquitinated proteins in NETs, and evidence for adaptive immune responses directed towards ubiquitinated NET proteins in SLE. The distinct differences in ubiquitin species profile in NETs compared with healthy controls may contribute to dampened anti-inflammatory responses observed in SLE. These results also support a role for extracellular ubiquitin in inflammation in SLE. [ABSTRACT FROM AUTHOR]