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BACKGROUND: Patients with rheumatoid arthritis (RA) have frequent thrombotic events with endothelial dysfunction. Von Willebrand factor (VWF) has been shown to bind neutrophil extracellular traps (NETs) and NETs are part of RA etiology. OBJECTIVES: This study aims to elucidate whether this prothrombotic status exacerbates inflammation in arthritis. Here we focus on the involvement of A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving VWF and its effect on NET deposition and RA development. METHODS: We evaluated the influence of the Adamts13 gene and recombinant human ADAMTS-13 (rhADAMTS-13) on arthritis in the mouse models of collagen-induced arthritis (CIA). We also assessed VWF and NETs in synovial tissue. RESULTS: Several Adamts13(−/−) mice developed arthritis, while Adamts13(+/+) siblings did not. Synovial tissue from Adamts13(−/−) showed accumulation of NETs. Treatment of DBA/1 J mice, an arthritis-susceptible strain, with well-tolerated doses of rhADAMT13 reduced arthritis incidence and alleviated the severity of arthritis. Mice treated with rhADAMT13 presented less serum interleukin 6 and less bone erosion determined by micro-computed tomography. The effects on arthritis severity were observed both when administering rhADAMTS-13 prophylactically and also when given after arthritis has developed. In both conditions, rhADAMTS-13 reduced VWF and NET deposition on proliferated synovial tissue evaluated by immunoblotting. CONCLUSIONS: Our results demonstrate the inhibitory role of Adamts13 in murine arthritis and the effectiveness of rhADAMTS-13 treatment. Additionally, this study suggests that deposition of VWF in the synovium and subsequent pathogenic NET retention promotes arthritis. Treatment with rhADAMTS-13 provides a potential therapeutic approach targeting inflammation and pro-thrombotic state in arthritis.

Neutrophils with intact multilobulated nuclei show ASC speck formation and high histone H3 citrullination in patients with severe COVID-19. In murine neutrophils, ASC speck forms transiently at the microtubule organizing center, before nuclear rounding, early in NETosis. Abstract Infection by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-COV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation, (i.e. ASC speck assembly), and timing relative to NETosis in stimulated neutrophils by real time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that approximately 2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in LPS-stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center, long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

Acute myeloid leukemia (AML) is the most common kind of acute leukemia and the second most common type of leukemia in adults. Poor outcomes resulting from AML are thought to occur due to the inability to target the small pool of cancer-initiating cells that develop from cells within hematopoietic stem (HSC) and progenitor (HPC) cell compartments in bone marrow. DEK, a nuclear protein that can be secreted under stress conditions, plays a role in regulating HSC and HPC function. Extracellular DEK has been found to improve functional HSC expansion in in-vivo and ex-vivo mouse studies. Moreover, RNAseq experiments suggest that recombinant human DEK treatment causes the upregulation of the antioxidant gene programs. Indeed, DEK treatment reduces total reactive oxygen species (ROS) in human umbilical cord blood HSCs and HPCs. Thus, extracellular DEK enhances normal HSC function through antioxidant programs, but the role of the extracellular DEK in AML is unclear. We hypothesized that recombinant mouse (rm)DEK treatment of the mouse-derived AML cell line MLL-AF9 would affect mitochondrial function since mitochondria are an important source of ROS production. Since ROS production can contribute to mitochondrial dysfunction by causing damage to the organelles, we investigated the effects of DEK signaling on mitochondrial metabolism in MLL-AF9 cells using the Seahorse XF instrument, which can measure changes in metabolic flux. Compared to vehicle-treated control, cells treated with DEK demonstrated a decrease in basal and maximal mitochondrial respiration, proton leak, and non-mitochondrial oxygen consumption. Experiments to explore the effects of DEK treatment on the glycolytic function of MLL-AF9 cells are ongoing. Our data shows DEK treatment of MLL-AF9 cells alters mitochondrial function. In the future, we wish to investigate DEK’s effect on proliferation and colony formation.

OBJECTIVES: Genome-wide association studies have connected PADI4, encoding peptidylarginine deiminase 4 (PAD4), with rheumatoid arthritis (RA). PAD4 promotes neutrophil extracellular trap (NET) formation. We studied Padi4 origin and NETs in an arthritis model in C57BL/6 mice. METHODS: To permit the effective use of C57BL/6 mice in the collagen-induced arthritis (CIA) model, we introduced the administration of granulocyte colony-stimulating factor (G-CSF) for four consecutive days in conjunction with the booster immunization on day 21. The model evaluated global (Padi4(−/−)) and hematopoietic lineage-specific (Padi4(Vav1Cre/+)) Padi4-deficient mice. RESULTS: G-CSF significantly increased the incidence and severity of arthritis in CIA. G-CSF-treated mice showed elevated citrullinated histone H3 (H3Cit) in plasma while vehicle-treated mice did not. Immunofluorescent microscopy revealed deposition of H3Cit in synovial tissue in G-CSF-treated mice. Padi4(−/−) mice developed less arthritis, demonstrating lower serum interleukin 6 and plasma H3Cit, less citrullinated histone H4 in synovial tissue, and less bone erosion observed by micro-computed tomography than Padi4(+/+) mice in the G-CSF-modified CIA model. Similarly, Padi4(Vav1Cre/+) mice developed less arthritis compared with Padi4(fl/fl) mice, and presented the same phenotype as Padi4(−/−) mice. CONCLUSIONS: We succeeded in developing an arthritis model suitable for use in C57BL/6 mice that was fully compliant with high animal welfare standards. We observed an over 90% incidence of arthritis in male mice and detectable NET markers. This model, with some futures consistent with human RA, demonstrates that hematopoietic PAD4 is an important contributor to arthritis development and may prove useful in future RA research.

Summary Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.