G-CSF drives autoinflammation in APLAID.

Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

mTOR signalling controls the formation of smooth muscle cell-derived luminal myofibroblasts during vasculitis.

The accumulation of myofibroblasts within the intimal layer of inflamed blood vessels is a potentially catastrophic complication of vasculitis, which can lead to arterial stenosis and ischaemia. In this study, we have investigated how these luminal myofibroblasts develop during Kawasaki disease (KD), a paediatric vasculitis typically involving the coronary arteries. By performing lineage tracing studies in a murine model of KD, we reveal that luminal myofibroblasts develop independently of adventitial fibroblasts and endothelial cells, and instead derive from smooth muscle cells (SMCs). Notably, the emergence of SMC-derived luminal myofibroblasts-in both mice and patients with KD, Takayasu's arteritis and Giant Cell arteritis-coincided with activation of the mechanistic target of rapamycin (mTOR) signalling pathway. Moreover, SMC-specific deletion of mTOR signalling, or pharmacological inhibition, abrogated the emergence of luminal myofibroblasts. Thus, mTOR is an intrinsic and essential regulator of luminal myofibroblast formation that is activated in vasculitis patients and therapeutically tractable. These findings provide molecular insight into the pathogenesis of coronary artery stenosis and identify mTOR as a therapeutic target in vasculitis.

G-CSF - A double edge sword in neutrophil mediated immunity.

Neutrophils are vital for the innate immune system's control of pathogens and neutrophil deficiency can render the host susceptible to life-threatening infections. Neutrophil responses must also be tightly regulated because excessive production, recruitment or activation of neutrophils can cause tissue damage in both acute and chronic inflammatory diseases. Granulocyte colony stimulating factor (G-CSF) is a key regulator of neutrophil biology, from production, differentiation, and release of neutrophil precursors in the bone marrow (BM) to modulating the function of mature neutrophils outside of the BM, particularly at sites of inflammation. G-CSF acts by binding to its cognate cell surface receptor on target cells, causing the activation of intracellular signalling pathways mediating the proliferation, differentiation, function, and survival of cells in the neutrophil lineage. Studies in humans and mice demonstrate that G-CSF contributes to protecting the host against infection, but conversely, it can play a deleterious role in inflammatory diseases. As such, neutrophils and the G-CSF pathway may provide novel therapeutic targets. This review will focus on understanding the role G-CSF plays in the balance between effective neutrophil mediated host defence versus neutrophil-mediated inflammation and tissue damage in various inflammatory and infectious diseases.

Muscle biopsy practices in the evaluation of neuromuscular disease: A systematic literature review.

AIMS: Muscle biopsy techniques range from needle muscle biopsy (NMB) and conchotome biopsy to open surgical biopsy. It is unknown whether specific biopsy techniques offer superior diagnostic yield or differ in procedural complication rates. Therefore, we aimed to compare the diagnostic utility of NMB, conchotome and open muscle biopsies in the assessment of neuromuscular disorders. METHODS: A systematic literature review of the EMBASE and Medline (Ovid) databases was performed to identify original, full-length research articles that described the muscle biopsy technique used to diagnose neuromuscular disease in both adult and paediatric patient populations. Studies of any design, excluding case reports, were eligible for inclusion. Data pertaining to biopsy technique, biopsy yield and procedural complications were extracted. RESULTS: Sixty-four studies reporting the yield of a specific muscle biopsy technique and, or procedural complications were identified. Open surgical biopsies provided a larger tissue sample than any type of percutaneous muscle biopsy. Where anaesthetic details were reported, general anaesthesia was required in 60% of studies that reported open surgical biopsies. Percutaneous biopsies were most commonly performed under local anaesthesia and despite the smaller tissue yield, moderate- to large-gauge needle and conchotome muscle biopsies had an equivalent diagnostic utility to that of open surgical muscle biopsy. All types of muscle biopsy procedures were well tolerated with few adverse events and no scarring complications were reported with percutaneous sampling. CONCLUSIONS: When a histological diagnosis of myopathy is required, moderate- to large-gauge NMB and the conchotome technique appear to have an equivalent diagnostic yield to that of an open surgical biopsy.

Retrospective comparison of ultrasound guided percutaneous and open skeletal muscle biopsies.

BACKGROUND: Open skeletal muscle biopsy has been the mainstay of sample retrieval in patients with suspected muscle diseases. However, this technique is limited by surgeon and theatre availability, potentially resulting in delayed diagnosis and increasing hospital stay. AIMS: To compare the effectiveness and timeliness of ultrasound guided 14-gauge needle percutaneous muscle biopsy in comparison with open biopsy. METHODS: We performed a retrospective chart review on 19 inpatients who underwent ultrasound-guided percutaneous muscle biopsy using a 14-gauge needle and 19 consecutive inpatients who underwent open surgical muscle biopsy between January 2017 and June 2019. Patient demographics, length of stay, biopsy sample size and the correlation between histological and clinical diagnosis were compared between groups. RESULTS: The median age of both groups was 64 years. Seventy-nine percent of surgical patients were female compared with 58% who had percutaneous biopsy. Surgical biopsies yielded larger samples (median 864 mm3 vs 17 mm3 , P = 0.03). While there was no difference in the length of inpatient stay (median 8 days), patients who had percutaneous biopsy had a shorter referral to procedure time (median 3 days vs 5 days, P = 0.012). Eighty-four percent of patients underwent MRI prior to percutaneous muscle biopsy, whereas only 16% had imaging before surgical biopsy (P ≤ 0.001). Most surgical biopsies were performed on the quadriceps whereas a wide range of muscles were sampled using the percutaneous technique. Overall, the percutaneous muscle sample was non-diagnostic in five cases (26%) despite a clinical diagnosis of myopathy. By comparison, two surgically obtained samples (11%) were non-diagnostic. CONCLUSION: Ultrasound guided percutaneous muscle biopsies were performed faster and a wider range of muscles were targeted. However, this technique yielded smaller samples, which were non-diagnostic in 26% of cases. Increasing the needle gauge or number of passes may improve the diagnostic yield of this technique.

How toxic is an old friend? A review of the safety of hydroxychloroquine in clinical practice.

Hydroxychloroquine (HCQ) and its close relative chloroquine (CQ) were initially used as antimalarial agents but are now widely prescribed in rheumatology, dermatology and immunology for the management of autoimmune diseases. HCQ is considered to have a better long-term safety profile than CQ and is therefore more commonly used. HCQ has a key role in the treatment of connective tissue diseases including systemic lupus erythematosus (SLE), where it provides beneficial immunomodulation without clinically significant immunosuppression. HCQ can also assist in managing inflammatory arthritis, including rheumatoid arthritis (RA). Debate around toxicity of HCQ in COVID-19 has challenged those who regularly prescribe HCQ to discuss its potential toxicities. Accordingly, we have reviewed the adverse effect profile of HCQ to provide guidance about this therapeutic agent in clinical practice.

Efficacy and safety of mavrilimumab in giant cell arteritis: a phase 2, randomised, double-blind, placebo-controlled trial.

OBJECTIVES: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is implicated in pathogenesis of giant cell arteritis. We evaluated the efficacy of the GM-CSF receptor antagonist mavrilimumab in maintaining disease remission. METHODS: This phase 2, double-blind, placebo-controlled trial enrolled patients with biopsy-confirmed or imaging-confirmed giant cell arteritis in 50 centres (North America, Europe, Australia). Active disease within 6 weeks of baseline was required for inclusion. Patients in glucocorticoid-induced remission were randomly assigned (3:2 ratio) to mavrilimumab 150 mg or placebo injected subcutaneously every 2 weeks. Both groups received a 26-week prednisone taper. The primary outcome was time to adjudicated flare by week 26. A prespecified secondary efficacy outcome was sustained remission at week 26 by Kaplan-Meier estimation. Safety was also assessed. RESULTS: Of 42 mavrilimumab recipients, flare occurred in 19% (n=8). Of 28 placebo recipients, flare occurred in 46% (n=13). Median time to flare (primary outcome) was 25.1 weeks in the placebo group, but the median was not reached in the mavrilimumab group (HR 0.38; 95% CI 0.15 to 0.92; p=0.026). Sustained remission at week 26 was 83% for mavrilimumab and 50% for placebo recipients (p=0.0038). Adverse events occurred in 78.6% (n=33) of mavrilimumab and 89.3% (n=25) of placebo recipients. No deaths or vision loss occurred in either group. CONCLUSIONS: Mavrilimumab plus 26 weeks of prednisone was superior to placebo plus 26 weeks of prednisone for time to flare by week 26 and sustained remission in patients with giant cell arteritis. Longer treatment is needed to determine response durability and quantify the glucocorticoid-sparing potential of mavrilimumab. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT03827018, Europe (EUdraCT number: 2018-001003-36), and Australia (CT-2018-CTN-01 865-1).

Emerging roles for IL-11 in inflammatory diseases.

Interleukin-11 (IL-11) is a cytokine that has been strongly implicated in the pathogenesis of fibrotic diseases and solid malignancies. Elevated IL-11 expression is also associated with several non-malignant inflammatory diseases where its function remains less well-characterized. Here, we summarize current literature surrounding the contribution of IL-11 to the pathogenesis of autoimmune inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, diabetes and systemic sclerosis, as well as other chronic inflammatory conditions such as periodontitis, asthma, chronic obstructive pulmonary disease, psoriasis and colitis.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

Defining cardiac involvement in idiopathic inflammatory myopathies: a systematic review.

OBJECTIVE: Recent advances in cardiac MRI (CMR) and other diagnostic techniques have made it easier to identify subclinical cardiac inflammation and dysfunction in the idiopathic inflammatory myopathies (IIM). Herein, we systematically review the literature regarding cardiac involvement in IIM. METHODS: We searched Medline and EMBASE from 1990 to 2020 using keywords related to IIM and cardiac disease. We included English language studies in adults with any immune-mediated, inflammatory muscle pathology. RESULTS: We identified 10 425 potentially relevant abstracts, of which 29 were included. Most frequently these included patients with PM or DM without symptomatic myocarditis. Five categories of cardiac investigation were used in these patients: cardiac enzyme testing, ECG, transthoracic echocardiography, CMR and nuclear medicine testing. Patients with clinical myocarditis had universally abnormal cardiac troponin levels and ECG. Elevated cardiac troponin T was more common than cardiac troponin I and may correlate with disease activity, whereas cardiac troponin I was more specific for cardiac involvement. Non-specific ECG changes were common. The major finding on transthoracic echocardiography was abnormal ejection fraction. Gross systolic dysfunction was unusual, but subclinical systolic dysfunction was reported in several studies. Abnormal diastolic function was common and may be associated with disease duration. Late gadolinium enhancement (reflecting regional necrosis or scarring) and abnormal myocardial mapping parameters (reflecting myocardial inflammation, fibrosis and oedema) were frequently identified on CMR, suggesting significant subclinical myocardial pathology (despite typically normal ejection fraction). CONCLUSION: Abnormal cardiac investigations are commonly found in asymptomatic IIM patients, which has potential prognostic and treatment implications.

TNF and IL-1 Play Essential but Temporally Distinct Roles in Driving Cardiac Inflammation in a Murine Model of Kawasaki Disease.

Kawasaki disease (KD) is a leading cause of pediatric heart disease, characterized by the emergence of life-threatening coronary vasculitis. Identifying which cytokines drive KD has been a major research goal, and both TNF and IL-1 have been identified as potential candidates. Using a murine model of KD induced by the injection of the water-soluble component of Candida albicans, we therefore undertook a mechanistic study to determine how and when these two cytokines mediate cardiac inflammation. In this study, we show that TNF signaling is active in the acute phase of cardiac inflammation, which is characterized by a diffuse myocarditis that precedes the development of coronary vasculitis. Mechanistically, TNF is produced by the myeloid cells and triggers acute cardiac inflammation by stimulating both stromal and immune compartments of the heart. In contrast to this early involvement for TNF, IL-1 signaling is dispensable for the development of acute myocarditis. Critically, although mice deficient in IL-1 signaling have extensive acute inflammation following C. albicans water-soluble complex challenge, they do not develop coronary vasculitis. Thus, TNF and IL-1 appear to play temporally distinct roles in KD, with TNF being active in acute cardiac inflammation and IL-1 in the subsequent development of coronary vasculitis. These observations have important implications for understanding the progression of cardiac pathology in KD and the relative therapeutic use of targeting these cytokines.

The Pyroptotic Cell Death Effector Gasdermin D Is Activated by Gout-Associated Uric Acid Crystals but Is Dispensable for Cell Death and IL-1ß Release.

The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1ß-dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1ß activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal-induced cell death, IL-1ß release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal-induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal-mediated release of bioactive IL-1ß. Consistent with in vitro findings, IL-1ß induction and autoinflammation in MSU crystal-induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal-mediated cell death. These findings 1) demonstrate that not all IL-1ß-driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal-induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1ß release.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

The Selective Expansion and Targeted Accumulation of Bone Marrow-Derived Macrophages Drive Cardiac Vasculitis.

The adult heart contains macrophages derived from both embryonic and adult bone marrow (BM)-derived precursors. This population diversity prompted us to explore how distinct macrophage subsets localize within the heart, and their relative contributions in cardiac disease. In this study, using the reciprocal expression of Lyve-1 and Ccr2 to distinguish macrophages with distinct origins, we show that, in the steady state, both embryonic (Lyvepos) and BM-derived (Ccr2pos) macrophages populate the major vessels of the heart in mice and humans. However, cardiac macrophage populations are markedly perturbed by inflammation. In a mouse model of Kawasaki disease, BM-derived macrophages preferentially increase during acute cardiac inflammation and selectively accumulate around major cardiac vessels. The accumulation of BM-derived macrophages coincides with the loss of their embryonic counterparts and is an initiating, essential step in the emergence of subsequent cardiac vasculitis in this experimental model. Finally, we demonstrate that the accumulation of Ccr2pos macrophages (and the development of vasculitis) occurs in close proximity to a population of Ccr2 chemokine ligand-producing epicardial cells, suggesting that the epicardium may be involved in localizing inflammation to cardiac vessels. Collectively, our findings identify the perivascular accumulation of BM-derived macrophages as pivotal in the pathogenesis of cardiac vasculitis and provide evidence about the mechanisms governing their recruitment to the heart.

Extracellular Vesicles in Synovial Fluid from Rheumatoid Arthritis Patients Contain miRNAs with Capacity to Modulate Inflammation.

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology.

Monosodium Urate Crystals Generate Nuclease-Resistant Neutrophil Extracellular Traps via a Distinct Molecular Pathway.

Neutrophil extracellular traps (NETs) and the cell death associated with it (NETosis) have been implicated in numerous diseases. Mechanistic studies of NETosis have typically relied on nonphysiological stimuli, such as PMA. The human disease of gout is caused by monosodium urate (MSU) crystals. We observed that DNA consistent with NETs is present in fluid from acutely inflamed joints of gout patients. NETs also coat the crystals found in uninflamed tophi of chronic gout patients. We developed a quantitative, live cell imaging assay, which measures the key features of NETosis, namely, cell death and chromatin decondensation. We show that MSU and other physiologically relevant crystals induce NETosis through a molecular pathway that is distinct from PMA and Candida hyphae. Crystals interact with lysosomes to induce NADPH oxidase-independent cell death, with postmortem chromatin decondensation mediated by neutrophil elastase. The resulting MSU-induced NETs are enriched for actin and are resistant to serum and DNase degradation. These findings demonstrate a distinct physiological NETosis pathway in response to MSU crystals, which coats MSU crystals in DNA that persists in tissues as gouty tophi.

Dysregulated IL-1ß-GM-CSF Axis in Acute Rheumatic Fever That Is Limited by Hydroxychloroquine.

BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease are autoimmune consequences of group A streptococcus infection and remain major causes of cardiovascular morbidity and mortality around the world. Improved treatment has been stymied by gaps in understanding key steps in the immunopathogenesis of ARF and rheumatic heart disease. This study aimed to identify (1) effector T cell cytokine(s) that might be dysregulated in the autoimmune response of patients with ARF by group A streptococcus, and (2) an immunomodulatory agent that suppresses this response and could be clinically translatable to high-risk patients with ARF. METHODS: The immune response to group A streptococcus was analyzed in peripheral blood mononuclear cells from an Australian Aboriginal ARF cohort by a combination of multiplex cytokine array, flow cytometric analysis, and global gene expression analysis by RNA sequencing. The immunomodulatory drug hydroxychloroquine was tested for effects on this response. RESULTS: We found a dysregulated interleukin-1ß-granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine axis in ARF peripheral blood mononuclear cells exposed to group A streptococcus in vitro, whereby persistent interleukin-1ß production is coupled to overproduction of GM-CSF and selective expansion of CXCR3+CCR4-CCR6- CD4 T cells. CXCR3+CCR4-CCR6- CD4 T cells are the major source of GM-CSF in human CD4 T cells and CXCL10, a CXCR3 ligand and potent T helper 1 chemoattractant, was elevated in sera from patients with ARF. GM-CSF has recently emerged as a key T cell-derived effector cytokine in numerous autoimmune diseases, including myocarditis, and the production of CXCL10 may explain selective trafficking of these cells to the heart. We provide evidence that interleukin-1ß amplifies the expansion of GM-CSF-expressing CD4 T cells, which is effectively suppressed by hydroxychloroquine. RNA sequencing showed shifts in gene expression profiles and differentially expressed genes in peripheral blood mononuclear cells derived from patients at different clinical stages of ARF. CONCLUSIONS: Given the safety profile of hydroxychloroquine and its clinical pedigree in treating autoimmune diseases such as rheumatoid arthritis, where GM-CSF plays a pivotal role, we propose that hydroxychloroquine could be repurposed to reduce the risk of rheumatic heart disease after ARF.

NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology.

OBJECTIVES: NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. METHODS: Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. RESULTS: The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1ß production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1ß as a pivotal cytokine. CONCLUSIONS: NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1ß over-production.