OBJECTIVE:Patients with systemic lupus erythematosus (SLE) experience photosensitivity, with exposure to UVB light driving lupus flares and triggering symptoms like joint pain, fatigue, and cutaneous lesions. Although the mechanism(s) linking UVB exposure to systemic effects are unclear, type I interferons (IFNs) are known to play a role. Our previous work has shown that TRIM21, an autoantigen in SLE, functions as a negative regulator on the pathways driving IFN expression. Here we explore how TRIM21 functions to regulate both local and systemic inflammation following UVB exposure and how altered expression may drive cutaneous inflammation and photosensitivity in SLE. METHODS:Wild-type (WT; C57BL/6) and Trim21-/- mice were irradiated with UVB (100 mJ/cm2) on the shaved dorsal region on consecutive days for 1 and 3 weeks, and UVB-induced local cutaneous manifestations and systemic inflammation in blood, spleen, and kidney were examined by messenger RNA expression of inflammatory and type I IFN response genes, histology, and flow cytometry. Mechanistic studies were performed in bone marrow-derived macrophages (BMDMs) and murine dermal fibroblasts (MDFs) from WT and Trim21-/- mice and TRIM21-/- THP-1 cells. RESULTS:Infiltration of inflammatory cells and induction of type I IFN developed in UVB-exposed areas in both sets of mice. Most notably after UVB exposure, we observed splenomegaly and enhanced expression of IFN-stimulated genes in the blood and spleen of Trim21-/- mice. Inflammatory chemokines CXCL10 and CXCL12 were also detected at significantly higher levels in serum of Trim21-/- mice after UVB exposure. Trim21-/- mice exposed to UVB also demonstrated enhanced total IgG levels in serum accompanied by increased skin and kidney deposition of IgG and increased glomerular cellularity and size. To determine the mechanism, we assessed UVB- and cyclic GMP-AMP-dependent Ifnb1 expression in Trim21-/- BMDMs and MDFs, noting increased responses compared with WT cells. This effect was lost in BMDMs from Trim21/Sting1 double knockout mice and skin explants, in keeping with the ability of TRIM21 to regulate cytoplasmic DNA sensing. In keeping with previous reports, we found that degradation of both DDX41 and STING levels were affected in stimulated Trim21-/- BMDMs. CONCLUSION:Taken together, our results indicate that TRIM21 protects against IFN induction at both local and systemic levels by restricting STING signaling.

Silica crystals activate the NLRP3 inflammasome in macrophages, resulting in the caspase-1-dependent secretion of the proinflammatory cytokine IL-1β. Caspase-1-mediated cleavage of gasdermin D (GSDMD) triggers the formation of GSDMD pores, which drive pyroptotic cell death and facilitate the rapid release of IL-1β. However, the role of GSDMD in silica-induced lung injury is unclear. In this study, we show that although silica-induced lung injury is dependent on the inflammasome adaptor ASC and IL-1R1 signaling, GSDMD is dispensable for acute lung injury. Although the early rapid secretion of IL-1β in response to ATP and nigericin was GSDMD dependent, GSDMD was not required for IL-1β release at later time points. Similarly, secretion of IL-1β from macrophages in response to silica and alum proceeded in a GSDMD-independent manner. We further found that gasdermin E did not contribute to macrophage IL-1β secretion in the absence of GSDMD in vitro and was also not necessary for silica-induced acute lung injury in vivo. These findings demonstrate that GSDMD and gasdermin E are dispensable for IL-1β secretion in response to silica in vitro and in silica-induced acute lung injury in vivo.

ABSTRACT Background Exposure of systemic lupus erythematosus (SLE) patients to ultraviolet light B (UVB) triggers local and systemic inflammation, with cytosolic DNA sensing and induction of type I interferons (IFNs) known to play a role. We previously identified TRIM21 as a negative regulator of DNA sensing and IFN expression. Here we explore the role of TRIM21 in regulating local and systemic responses following UVB exposure. Methods WT (C57BL/6) and Trim21 -/- mice were irradiated with UVB (100mJ/cm 2 ) daily for 1 and 3 weeks, and UVB-induced inflammation in skin, blood, and spleen were analyzed by qPCR, histology, RNA sequencing and flow cytometry. Mechanistic studies were performed in bone marrow-derived macrophages (BMDMs) and mouse skin fibroblasts (MDF) from WT and Trim21 -/- mice, and TRIM21 -/- THP-1 cells. Results Infiltration of inflammatory cells and induction of type I IFN developed in UVB-exposed areas in both sets of mice, however Trim21 -/- mice developed splenomegaly, enhanced total IgG levels and IFN-stimulated genes (ISG) in the blood and spleen. Enhanced basal and UVB-dependent Ifnb1 expression was observed in Trim21 -/- BMDMs and MDFs, which was dependent on the cytosolic DNA sensing cGAS-STING pathway. Mechanistically, we found both degradation of DDX41 and STING levels were impaired in stimulated Trim21 -/- BMDMs. Conclusion Taken together, our results indicate that TRIM21 protects against IFN induction at local and systemic levels through restricting STING signaling. Our finding that reduced levels of TRIM21 are observed in SLE patients with cutaneous involvement indicates a potential role for TRIM21 in guarding against systemic flare in SLE patients.

Arthritis & RheumatologyAccepted Articles Reply Reply to The limitation of flowcytometry is ignored in the assessment of antimitochondrial antibodies in rheumatoid arthritis Richard Moore MSc, Richard Moore MSc Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorTing Wang MD, PhD, Ting Wang MD, PhD orcid.org/0000-0002-8974-596X Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorMarina Barguil Macêdo MD, MSc, Marina Barguil Macêdo MD, MSc Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorChristian Lood PhD, Corresponding Author Christian Lood PhD [email protected] orcid.org/0000-0002-6171-1952 Division of Rheumatology, University of Washington, Seattle, WA, USACorresponding author: Christian Lood, PhD, University of Washington, Division of Rheumatology, 750 Republican Street, Room E-545, Seattle, WA, 98109, USA. Email: [email protected], Phone: +1 206-221-8446Search for more papers by this author Richard Moore MSc, Richard Moore MSc Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorTing Wang MD, PhD, Ting Wang MD, PhD orcid.org/0000-0002-8974-596X Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorMarina Barguil Macêdo MD, MSc, Marina Barguil Macêdo MD, MSc Division of Rheumatology, University of Washington, Seattle, WA, USASearch for more papers by this authorChristian Lood PhD, Corresponding Author Christian Lood PhD [email protected] orcid.org/0000-0002-6171-1952 Division of Rheumatology, University of Washington, Seattle, WA, USACorresponding author: Christian Lood, PhD, University of Washington, Division of Rheumatology, 750 Republican Street, Room E-545, Seattle, WA, 98109, USA. Email: [email protected], Phone: +1 206-221-8446Search for more papers by this author First published: 13 October 2023 https://doi.org/10.1002/art.42727 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/art.42727. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Accepted ArticlesAccepted, unedited articles published online and citable. The final edited and typeset version of record will appear in the future. RelatedInformation

ObjectiveMitochondria are found in the extracellular space in rheumatoid arthritis (RA). However, whether mitochondria are a source of autoantigens in RA has not been carefully addressed. Thus, we undertook this study to investigate the presence and significance of antimitochondrial antibodies (AMAs) in patients with RA.MethodsAMAs were measured in serum samples from 3 cross‐sectional cohorts of RA patients (n = 95, n = 192, and n = 117) and healthy individuals (n = 38, n = 72, and n = 50) using a flow cytometry–based assay. Further, AMAs were detected using an anti–mitofusin‐1 (anti–MFN‐1) IgG enzyme‐linked immunosorbent assay and Western blot analysis. A longitudinal inception cohort, followed up for a median of 8 years, was used to study disease progression.ResultsAMA levels were elevated in RA patients from all 3 cohorts as compared to healthy individuals (P < 0.001, P < 0.05, and P < 0.01), with a range of 14–26% positivity. Levels of anti–MFN‐1 antibodies correlated with AMA levels (r = 0.31, P = 0.006) and were elevated in RA patients as compared to healthy individuals (P < 0.001). The presence of AMAs was associated with erosive disease (P < 0.05) and interstitial lung disease (P < 0.01). Further, AMA levels were found to predict erosive disease (odds ratio [OR] 4.59, P = 0.006) and joint space narrowing (OR 3.08, P = 0.02) independent of anti–citrullinated protein antibodies. Finally, anti–MFN‐1 antibodies identified seronegative patients developing erosive disease (OR 9.33; P = 0.02).ConclusionOur findings demonstrate the presence of novel autoantibodies targeting mitochondria in the setting of RA. AMAs were used to stratify patients based on disease phenotype and to predict development of erosive disease, including in patients with seronegative disease. Our results highlight the essential role of mitochondria in the pathogenesis of RA and suggest a possible benefit of therapies targeting mitochondrial‐mediated inflammation and clearance in these patients.

Objectives: To elucidate mechanisms contributing to skeletal muscle calcinosis in patients with juvenile dermatomyositis. Methods: A well-characterized cohorts of JDM (n = 68), disease controls (polymyositis, n = 7; juvenile SLE, n = 10, and RNP + overlap syndrome, n = 12), and age-matched health controls (n = 17) were analyzed for circulating levels of mitochondrial (mt) markers including mtDNA, mt-nd6, and anti-mitochondrial antibodies (AMAs) using standard qPCR, ELISA, and novel-in-house assays, respectively. Mitochondrial calcification of affected tissue biopsies was confirmed using electron microscopy and energy dispersive X-ray analysis. A human skeletal muscle cell line, RH30, was used to generate an in vitro calcification model. Intracellular calcification is measured by flow cytometry and microscopy. Mitochondria were assessed for mtROS production and membrane potential by flow cytometry and real-time oxygen consumption rate by Seahorse bioanalyzer. Inflammation (interferon-stimulated genes) was measured by qPCR. Results: In the current study, patients with JDM exhibited elevated levels of mitochondrial markers associated with muscle damage and calcinosis. Of particular interest are AMAs predictive of calcinosis. Human skeletal muscle cells undergo time- and dose-dependent accumulation of calcium phosphate salts with preferential localization to mitochondria. Calcification renders skeletal muscle cells mitochondria stressed, dysfunctional, destabilized, and interferogenic. Further, we report that inflammation induced by interferon-alpha amplifies mitochondrial calcification of human skeletal muscle cells via the generation of mitochondrial reactive oxygen species (mtROS). Conclusions: Overall, our study demonstrates the mitochondrial involvement in the skeletal muscle pathology and calcinosis of JDM and mtROS as a central player in the calcification of human skeletal muscle cells. Therapeutic targeting of mtROS and/or upstream inducers, such as inflammation, may alleviate mitochondrial dysfunction, leading to calcinosis. AMAs can potentially identify patients with JDM at risk for developing calcinosis.

Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.

Type I interferon (IFN-I) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and the closely associated monogenic autoinflammatory disorders termed the "interferonopathies." Recently, the cytosolic DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and its downstream signaling adaptor stimulator of interferon genes (STING) have been identified as having important, if not central, roles in driving IFN-I expression in response to self-DNA. This review highlights the many ways in which this pathway is regulated in order to prevent self-DNA recognition and underlines the importance of maintaining tight control in order to prevent autoimmune disease. We will discuss the murine and human studies that have implicated the cGAS-STING pathway as being an important contributor to breakdown in tolerance in SLE and highlight the potential therapeutic application of this knowledge for the treatment of SLE.

OBJECTIVE:Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are linked to inflammation and autoimmunity, including rheumatoid arthritis (RA). However, whether NETs are present in the circulation of RA patients and contribute to inflammation and disease progression has not been carefully addressed. We undertook this study to assess markers of neutrophil activation and NET formation in plasma samples, investigating whether they add clinical value in improving the determination of prognosis and monitoring in RA patients. METHODS:Markers of neutrophil activation (calprotectin) and cell death (NETs) were analyzed, using enzyme-linked immunosorbent assay, in serum and plasma obtained from patients in 3 cross-sectional RA cohorts and sex-matched healthy controls. A longitudinal inception cohort (n = 247), seen for a median follow-up of 8 years, was used for predictive analyses. RESULTS:Markers of neutrophil activation and cell death were increased in RA patients compared to healthy individuals (P < 0.0001). Calprotectin levels correlated with the Clinical Disease Activity Index (r = 0.53, P < 0.0001) and could be used to distinguish between patients with disease in remission and those with active disease, an observation not seen when examining C-reactive protein levels. A biomarker panel consisting of anti-citrullinated protein antibody and calprotectin could predict erosive disease (odds ratio [OR] 7.5, P < 0.0001) and joint space narrowing (OR 4.9, P = 0.001). NET levels were associated with markers of inflammation (P = 0.0002). Furthermore, NETs and a "neutrophil activation signature" biomarker panel had good predictive value in identifying patients who were developing extraarticular nodules (OR 5.6, P = 0.006). CONCLUSION:Neutrophils undergo marked activation and cell death in RA. Neutrophil biomarkers can provide added clinical value in the monitoring and prognosis of RA patients and may allow for early preventive treatment intervention.

Cardiac fibrosis, denoted by the deposition of extracellular matrix, manifests with a variety of diseases such as hypertension, diabetes, and myocardial infarction. Underlying this pathological extracellular matrix secretion is an expansion of fibroblasts. The mouse is now a common experimental model system for the study of cardiovascular remodeling and elucidation of fibroblast responses to cardiac growth and stress is vital for understanding disease processes. Here, using diverse but fibroblast specific markers, we report murine fibroblast distribution and proliferation in early postnatal, adult, and injured hearts. We find that perinatal fibroblasts and endothelial cells proliferate at similar rates. Furthermore, regardless of the injury model, fibroblast proliferation peaks within the first week after injury, a time window similar to the period of the inflammatory phase. In addition, fibroblast densities remain high weeks after the initial insult. These results provide detailed information regarding fibroblast distribution and proliferation in experimental methods of heart injury.

Background: Infections are a major problem in burns patients. Knowledge of the incidence and antimicrobial sensitivities of the microorganisms commonly encountered within each institution's burns unit is important as it informs and directs empiric antibiotic therapy.Methods: This was a retrospective review of patients admitted from 1 January 2008 to 31 December 2012 to an adult burns intensive care unit. Specimens chosen for analysis were wound swabs, blood cultures, venous catheter tips, tracheal aspirates, sputum, urine and wound tissue. Records were accessed from the admission register and laboratory information system to obtain the relevant data.Results: During the study period, 352 patients were admitted to the adult burns intensive care unit, of which, 341 patients were included. The mortality rate was 446%. Flame burns were the commonest. Mortality rate amongst patients with bacteremia was 46.9%. Acinetobacter baumannii, Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MASA) were found to be the most common organisms cultured in most specimens.Conclusion: The main three organisms identified in specimen cultures in our adult burns intensive care unit were A. baumannii, P. aeruginosa and MRSA. This study has helped establish a better empiric approach to the management of our septic burns patients. (C) 2015 Elsevier Ltd and ISBI. All rights reserved.