Our faculty research broadens the knowledge of rheumatic disease. 

Immunogenicity of a rheumatoid arthritis protective sequence when acquired through microchimerism.

Kanaan SB, Sensoy O, Yan Z, Gadi VK, Richardson ML, Nelson JL.

HLA class II genes provide the strongest genetic contribution to rheumatoid arthritis (RA). HLA-DRB1 alleles encoding the sequence DERAA are RA-protective. Paradoxically, RA risk is increased in women with DERAA+ children born prior to onset. We developed a sensitive qPCR assay specific for DERAA, and found 53% of DERAA-/- women with RA had microchimerism (Mc; pregnancy-derived allogeneic cells) carrying DERAA (DERAA-Mc) vs. 6% of healthy women. DERAA-Mc quantities correlated with an RA-risk genetic background including DERAA-binding HLA-DQ alleles, early RA onset, and aspects of RA severity. CD4+ T cells showed stronger response against DERAA+ vs. DERAA- allogeneic cell lines in vitro, in line with an immunogenic role of allogeneic DERAA. Results indicate a model where DERAA-Mc activates DERAA-directed T cells that are naturally present in DERAA-/- individuals and can have cross-reactivity against joint antigens. Moreover, we provide an explanation for the enigmatic observation that the same HLA sequence differentially affects RA risk through Mendelian inheritance vs. microchimeric cell acquisition.

High TLR7 Expression Drives the Expansion of CD19+CD24hiCD38hi Transitional B Cells and Autoantibody Production in SLE Patients.

Wang T, Marken J, Chen J, Tran VB, Li QZ, Li M, Cerosaletti K, Elkon KB, Zeng X, Giltiay NV.

Signaling through Toll-like receptor 7 (TLR7) drives the production of type I IFN and promotes the activation of autoreactive B cells and is implicated in the pathogenesis of systemic lupus erythematosus (SLE). While TLR7 has been extensively studied in murine lupus, much less is known about its role in the pathogenesis of human SLE. Genetic studies support a link between the TLR7 rs3853839 C/G polymorphism, which affects TLR7 mRNA turnover, and SLE susceptibility; however, the effects of this polymorphism on B cells have not been studied. Here we determined how changes in TLR7 expression affect peripheral B cells and auto-Ab production in SLE patients. High TLR7 expression in SLE patients driven by TLR7 rs3853839 C/G polymorphism was associated with more active disease and upregulation of IFN-responsive genes. TLR7hi SLE patients showed an increase in peripheral B cells. Most notably, the percentage and numbers of CD19+CD24++CD38++ newly-formed transitional (TR) B cells were increased in TLR7hi SLE patients as compared to HCs and TLR7norm/lo SLE patients. Using auto-Ab arrays, we found an increase and enrichment of auto-Ab specificities in the TLR7hi SLE group, including the production of anti-RNA/RNP-Abs. Upon in vitro TLR7 ligand stimulation, TR B cells isolated from TLR7hi but not TLR7norm/lo SLE patients produced anti-nuclear auto-Abs (ANA). Exposure of TR B cells isolated from cord blood to IFNα induced the expression of TLR7 and enabled their activation in response to TLR7 ligation in vitro. Our study shows that overexpression of TLR7 in SLE patients drives the expansion of TR B cells. High TLR7 signaling in TR B cells promotes auto-Ab production, supporting a possible pathogenic role of TR B cells in human SLE.

 

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A Neutrophil Activation Biomarker Panel in Prognosis and Monitoring of Patients with Rheumatoid Arthritis.

Bach M, Moon J, Moore R, Pan T, Nelson JL, Lood C.

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. Here we assess markers of neutrophil activation and NET formation in plasma samples, asking whether they add clinical value in improving on determination of prognosis and monitoring in RA patients.

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Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis.

Duvvuri B, Pachman LM, Morgan G, Khojah AM, Klein-Gitelman M, Curran ML, Doty S, Lood C.

Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). The role of neutrophils in juvenile dermatomyositis (JDM) is not known.

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High prevalence and disease correlation of autoantibodies against p40 encoded by long interspersed nuclear elements (LINE-1) in systemic lupus erythematosus.

Carter V, LaCava J, Taylor MS, Liang SY, Mustelin C, Ukadike KC, Bengtsson A, Lood C, Mustelin T.

The long interspersed nuclear element 1 (LINE-1) encodes two proteins, the RNA-binding p40 and the endonuclease and reverse transcriptase (ORF2p); both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with the LINE-1 RNA and additional RNA-binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). We asked if SLE patients also make autoantibodies against the LINE-1 p40.

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Cell-Free DNA as a Biomarker in Autoimmune Rheumatic Diseases.

Duvvuri B, Lood C.

Endogenous DNA is primarily found intracellularly in nuclei and mitochondria. However, extracellular, cell-free (cf) DNA, has been observed in several pathological conditions, including autoimmune diseases, prompting the interest of developing cfDNA as a potential biomarker. There is an upsurge in studies considering cfDNA to stratify patients, monitor the treatment response and predict disease progression, thus evaluating the prognostic potential of cfDNA for autoimmune diseases. Since the discovery of elevated cfDNA levels in lupus patients in the 1960s, cfDNA research in autoimmune diseases has mainly focused on the overall quantification of cfDNA and the association with disease activity. However, with recent technological advancements, including genomic and methylomic sequencing, qualitative changes in cfDNA are being explored in autoimmune diseases, similar to the ones used in molecular profiling of cfDNA in cancer patients. Further, the intracellular origin, e.g., if derived from mitochondrial or nuclear source, as well as the complexing with carrier molecules, including LL-37 and HMGB1, has emerged as important factors to consider when analyzing the quality and inflammatory potential of cfDNA. The clinical relevance of cfDNA in autoimmune rheumatic diseases is strengthened by mechanistic insights into the biological processes that result in an enhanced release of DNA into the circulation during autoimmune and inflammatory conditions. Prior work have established an important role of accelerated apoptosis and impaired clearance in leakage of nucleic acids into the extracellular environment. Findings from more recent studies, including our own investigations, have demonstrated that NETosis, a neutrophil cell death process, can result in a selective extrusion of inflammatory mitochondrial DNA; a process which is enhanced in patients with lupus and rheumatoid arthritis. In this review, we will summarize the evolution of cfDNA, both nuclear and mitochondrial DNA, as biomarkers for autoimmune rheumatic diseases and discuss limitations, challenges and implications to establish cfDNA as a biomarker for clinical use. This review will also highlight recent advancements in mechanistic studies demonstrating mitochondrial DNA as a central component of cfDNA in autoimmune rheumatic diseases.

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Low plasma concentrations of apolipoprotein M are associated with disease activity and endothelial dysfunction in systemic lupus erythematosus.

Tydén H, Lood C, Jönsen A, Gullstrand B, Kahn R, Linge P, Kumaraswamy SB, Dahlbäck B, Bengtsson AA.

Apolipoprotein M (apoM) is a 25-kDa apolipoprotein present in 5% of high-density lipoprotein (HDL) particles. It is suggested to be anti-atherogenic and to play a key role in sustaining endothelial barrier integrity. SLE patients have increased cardiovascular disease risk, and we aimed to investigate if apoM levels reflect endothelial function in SLE. Since apoM plasma levels decrease during inflammatory conditions, our aim was also to determine the impact of SLE disease activity on apoM plasma levels.

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Determinants of Positive Temporal Artery Biopsies in the Veterans Health Administration National Database Cohort.

Chung SH, Morcos MB, Ng B.

This study sought to determine the effect of temporal artery biopsy (TAB) post-fixation length, laterality, age, and prior prednisone exposure on TAB positivity utilizing the Veterans Health Administration national database.

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Sources of Pathogenic Nucleic Acids in Systemic Lupus Erythematosus.

Mustelin T, Lood C, Giltiay NV.

A hallmark of systemic lupus erythematosus (SLE), and several related autoimmune diseases, is the presence of autoantibodies against nucleic acids and nucleic acid-binding proteins, as well as elevated type I interferons (IFNs), which appear to be instrumental in disease pathogenesis. Here we discuss the sources and proposed mechanisms by which a range of cellular RNA and DNA species can become pathogenic and trigger the nucleic acid sensors that drive type I interferon production. Potentially SLE-promoting DNA may originate from pieces of chromatin, from mitochondria, or from reverse-transcribed cellular RNA, while pathogenic RNA may arise from mis-localized, mis-processed, ancient retroviral, or transposable element-derived transcripts. These nucleic acids may leak out from dying cells to be internalized and reacted to by immune cells or they may be generated and remain to be sensed intracellularly in immune or non-immune cells. The presence of aberrant DNA or RNA is normally counteracted by effective counter-mechanisms, the loss of which result in a serious type I IFN-driven disease called Aicardi-Goutières Syndrome. However, in SLE it remains unclear which mechanisms are most critical in precipitating disease: aberrant RNA or DNA, overly sensitive sensor mechanisms, or faulty counter-acting defenses. We propose that the clinical heterogeneity of SLE may be reflected, in part, by heterogeneity in which pathogenic nucleic acid molecules are present and which sensors and pathways they trigger in individual patients. Elucidation of these events may result in the recognition of distinct "endotypes" of SLE, each with its distinct therapeutic choices.

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Pharmacist-managed titration of urate-lowering therapy to streamline gout management.

Huang IJ, Liew JW, Morcos MB, Zuo S, Crawford C, Bays AM.

The treat-to-target approach for serum uric acid is the recommended model in gout management according to the 2012 American College of Rheumatology (ACR) guidelines. Adherence to urate-lowering therapy (ULT) can be difficult for patients due to barriers, which include medication burden, financial hardship, and lack of medical literacy. Our aim was to create a pharmacist-managed referral for the titration of ULT to target serum uric acid (sUA) levels in a complex patient population. We utilized a clinical database to query patients seen at a rheumatology clinic over a 12-month period with an ICD-10 diagnosis for gout. The referral criteria were indications for ULT per the 2012 ACR guidelines. Rheumatology providers, consisting of attendings, fellows, and a physician assistant, were asked to refer the identified patients to the pharmacist-managed titration program. The intervention group consisted of 19 referred patients and the control group consisted of 28 non-referred patients. The baseline sUA (median (IQR)) at the time of referral was 8.8 (2) mg/dL for the intervention group and 7.6 (2.8) mg/dL for the control group (p = 0.2). At the end of the study period, the sUA was 6.1 (1.4) mg/dL for the intervention group and 6.8 (3.2) mg/dL for the control group (p = 0.08). At the end of the study period, 6 of 19 (32%) intervention group and 7 of 28 (25%) control group were at goal (p = 0.3). A newly instituted pharmacist-managed titration program was able to achieve lower average sUA levels in referred patients compared to demographically similar individuals who received standard gout management.

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Corrigendum: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis.

Dam EM, Maier AC, Hocking AM, Carlin J, Ng B, Buckner JH.

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Demographic, Clinical, and Immunologic Correlates among a Cohort of 50 Cocaine Users Demonstrating Antineutrophil Cytoplasmic Antibodies.

Morcos MB, Lood C, Hughes GC.

Cocain/Levamisole-associated Autoimmunity Syndrome (CLAAS) is a poorly understood form of drug-induced autoimunity. Our goals were to better characterize the spectrum of clinical and immunologic features of CLAAS, to identify demographic risk factors, and to generate new hypotheses regarding pathogenesis. 

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Sources of Pathogenic Nucleic Acids in Systemic Lupus Erythematosus.

Mustelin T, Lood C, Giltiay NV.

A hallmark of systemic lupus erythematosus (SLE), and several related autoimmune diseases, is the presence of autoantibodies against nucleic acids and nucleic acid-binding proteins, as well as elevated type I interferons (IFNs), which appear to be instrumental in disease pathogenesis. Here we discuss the sources and proposed mechanisms by which a range of cellular RNA and DNA species can become pathogenic and trigger the nucleic acid sensors that drive type I interferon production. Potentially SLE-promoting DNA may originate from pieces of chromatin, from mitochondria, or from reverse-transcribed cellular RNA, while pathogenic RNA may arise from mis-localized, mis-processed, ancient retroviral, or transposable element-derived transcripts. These nucleic acids may leak out from dying cells to be internalized and reacted to by immune cells or they may be generated and remain to be sensed intracellularly in immune or non-immune cells. The presence of aberrant DNA or RNA is normally counteracted by effective counter-mechanisms, the loss of which result in a serious type I IFN-driven disease called Aicardi-Goutières Syndrome. However, in SLE it remains unclear which mechanisms are most critical in precipitating disease: aberrant RNA or DNA, overly sensitive sensor mechanisms, or faulty counter-acting defenses. We propose that the clinical heterogeneity of SLE may be reflected, in part, by heterogeneity in which pathogenic nucleic acid molecules are present and which sensors and pathways they trigger in individual patients. Elucidation of these events may result in the recognition of distinct "endotypes" of SLE, each with its distinct therapeutic choices.

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Aspirin meets cGAS.

Elkon KB.

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The Contribution of PTPN22 to Rheumatic Disease.

Mustelin T, Bottini N, Stanford SM.

One of the unresolved questions in modern medicine is why certain individuals develop a disorder such as rheumatoid arthritis (RA) or lupus, while others do not. Contemporary science indicates that genetics is partly responsible for disease development, while environmental and stochastic factors also play a role. Among the many genes that increase the risk of autoimmune conditions, the risk allele encoding the W620 variant of protein tyrosine phosphatase N22 (PTPN22) is shared between multiple rheumatic diseases, suggesting that it plays a fundamental role in the development of immune dysfunction. Herein, we discuss how the presence of the PTPN22 risk allele may shape the signs and symptoms of these diseases. Besides the emerging clarity regarding how PTPN22 tunes T and B cell antigen receptor signaling, we discuss recent discoveries of important functions of PTPN22 in myeloid cell lineages. Taken together, these new insights reveal important clues to the molecular mechanisms of prevalent diseases like RA and lupus and may open new avenues for the development of personalized therapies that spare the normal function of the immune system.

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Interactions between cadherin-11 and platelet-derived growth factor receptor-alpha signaling link cell adhesion and proliferation.

Madarampalli BWatts GFMPanipinto PMNguygen HNBrenner MBNoss EH.

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.

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Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes.

Akilesh HMBuechler MBDuggan JMHahn WOMatta BSun XGessay GWhalen EMason MPresnell SRElkon KBLacy-Hulbert ABarnes BJPepper MHamerman JA.

Cytopenias are an important clinical problem associated with inflammatory disease and infection. We show that specialized phagocytes that internalize red blood cells develop in Toll-like receptor 7 (TLR7)-driven inflammation. TLR7 signaling caused the development of inflammatory hemophagocytes (iHPCs), which resemble splenic red pulp macrophages but are a distinct population derived from Ly6Chi monocytes. iHPCs were responsible for anemia and thrombocytopenia in TLR7-overexpressing mice, which have a macrophage activation syndrome (MAS)-like disease. Interferon regulatory factor 5 (IRF5), associated with MAS, participated in TLR7-driven iHPC differentiation. We also found iHPCs during experimental malarial anemia, in which they required endosomal TLR and MyD88 signaling for differentiation. Our findings uncover a mechanism by which TLR7 and TLR9 specify monocyte fate and identify a specialized population of phagocytes responsible for anemia and thrombocytopenia associated with inflammation and infection.

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Autoimmunity to bactericidal/permeability-increasing protein in bronchiectasis exhibits a requirement for Pseudomonas aeruginosa IgG response.

Skopelja-Gardner STheprungsirikul JMeagher REBeliveau CMBradley KEAvery MHenkle ESiegel SGifford AHWinthrop KLRigby WFC.

In non-cystic fibrosis bronchiectasis (BE), idiopathic, genetic, and environmental factors alter the airway landscape and immune responses, rendering the patients susceptible to infection, with the gram-negative bacterium (GNB) Pseudomonas aeruginosa as a major contributor to mortality. The high prevalence of P. aeruginosa in BE patients cannot be explained by a single genetic or environmental influence, and immunologic permissiveness of BE airways to this colonization remains unexplained. We ask two critical questions regarding the immunologic interactions that shape the BE airway permissiveness to P. aeruginosa infection in two BE cohorts from the United States: one from Dartmouth Hitchcock Medical Center (DHMC) in Lebanon, New Hampshire (n=16): i) Do BE patients develop autoimmunity to BPI? and ii) What is the relationship between autoreactivity to BPI and chronic infection by P. aeruginosa? BPI autoreactivity was identified at nearly identical frequencies in the DHMC (56%) and OHSU (52%) cohorts. Autoreactivity to BPI in both BE cohorts was strongly associated with the existence of an antibody response to P. aeruginosa and was absent in patients colonized with Nontuberculous Mycobacterium. Retrospective longitudinal study of sera from 34 BE patients demonstrated that the autoimmune response to BPI follows the same temporal pattern as the humoral response to P. aeruginosa. Our study suggests that rather than viewing anti-BPI as a highly linked epiphenomenon of P. aeruginosa infection, these autoantibodies may play a causal role in the perpetuation of infection. In this model, strategies which eliminate anti-BPI reactivity may enhance clearance of P. aeruginosa leading to improved airway function and clinical outcomes. 

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Inhibition of Cyclic GMP-AMP Synthase Using a Novel Antimalarial Drug Derivative in Trex1-Deficient Mice.

An JWoodward JJLai WMinie MSun XTanaka LSnyder JMSasaki TElkon KB.

Type I interferon (IFN) is strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE) as well as rare monogenic interferonopathies such as Aicardi-Goutières syndrome (AGS), a disease attributed to mutations in the DNA exonuclease TREX1. The DNA-activated type I IFN pathway cyclic GMP-AMP (cGAMP) synthase (cGAS) is linked to subsets of AGS and lupus. This study was undertaken to identify inhibitors of the DNA-cGAS interaction, and to test the lead candidate drug, X6, in a mouse model of AGS.

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Neutrophil extracellular traps (NETs) are increased in the alveolar spaces of patients with ventilator-associated pneumonia.

Mikacenic CMoore RDmyterko VWest TEAltemeier WALiles WCLood C.

Neutrophils release neutrophil extracellular traps (NETs) in response to invading pathogens. Although NETs play an important role in host defense against microbial pathogens, they have also been shown to play a contributing mechanistic role in pathologic inflammation in the absence of infection. Although a role for NETs in bacterial pneumonia and acute respiratory distress syndrome (ARDS) is emerging, a comprehensive evaluation of NETs in the alveolar space of critically ill patients has yet to be reported. In this study, we evaluated whether markers of NET formation in mechanically ventilated patients are associated with ventilator-associated pneumonia (VAP).

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Association of High Anti-Cyclic Citrullinated Peptide Seropositivity and Lean Mass Index With Low Bone Mineral Density in Rheumatoid Arthritis.

Wysham KDShoback DMImboden JB JrKatz PP.

Osteoporotic fractures are associated with high morbidity and mortality. Persons with rheumatoid arthritis (RA) have twice the risk of osteoporosis-related fracture than age-matched controls, the causes for which remain unknown. We investigated contributions of RA characteristics, medication use, and body composition to low bone mineral density (BMD) in patients with RA.

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Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in humanneutrophils.

Zhou YAn LLChaerkady RMittereder NClarke LCohen TSChen BHess SSims GPMustelin T.

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

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Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation.

Davis MAFairgrieve MRDen Hartigh AYakovenko ODuvvuri BLood C, Thomas WEFink SLGale M Jr.

Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result, cells were susceptible to rupture by extrinsic forces, such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpain-dependent and linked with cleavage of vimentin and loss of intermediatefilaments. Moreover, while release of lactate dehydrogenase (LDH), HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli-ASC specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from nonruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.

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