Increased Plasma Heparanase Activity in COVID-19 Patients.

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

Cleaved N-terminal histone tails distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation.

OBJECTIVES: Neutrophil extracellular traps (NETs) act in various rheumatic diseases. Although NET formation was originally described as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent pathway, it appears that there are also NOX-independent pathways of NET release. Currently, no tools are available that can discriminate between both NET-forming pathways. We aimed to develop a serological method allowing the discrimination between NETs generated through NOX-dependent or NOX-independent pathways. METHODS: Histones from in vitro generated NOX-dependent and NOX-independent NETs were characterised with a panel of lupus-derived antibodies against N-terminal histone tails using immunofluorescence microscopy, western blot and ELISA. NETs in patients with NET-associated diseases, that is, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriatic arthritis (PsA) and sepsis, were characterised in sandwich ELISAs employing antibodies against myeloperoxidase (MPO) and N-terminal histone tails as detecting and capturing antibodies, respectively. Functional responses of endothelial cells to NOX-dependent and NOX-independent NETs were assessed as well. RESULTS: Neutrophil elastase cleaves the N-terminal tails of core histones during NOX-dependent, but not during NOX-independent NET formation. Consequently, the detection of MPO-histone complexes with antibodies against N-terminal histone tails allows discrimination between NETs formed through a NOX-dependent or NOX-independent manner. Characterisation of in vivo circulating NETs revealed the presence of NOX-independent NETs in RA, SLE and sepsis, but NOX-dependent NETs in PsA. NOX-independent NETs displayed an increased capacity to activate endothelial cells when compared with NOX-dependent NETs. CONCLUSIONS: These results indicate heterogeneity in NET-forming pathways in vivo and highlight the need for disease-specific strategies to prevent NET-mediated pathology.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Interleukin-6 is essential for glomerular immunoglobulin A deposition and the development of renal pathology in Cd37-deficient mice.

Immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is characterized by IgA depositions in the kidney. Deficiency of CD37, a leukocyte-specific tetraspanin, leads to spontaneous development of renal pathology resembling IgAN. However, the underlying molecular mechanism has not been resolved. Here we found that CD37 expression on B cells of patients with IgAN was significantly decreased compared to B cells of healthy donors. Circulating interleukin (IL)-6 levels, but not tumor necrosis factor-α or IL-10, were elevated in Cd37-/- mice compared to wild-type mice after lipopolysaccharide treatment. Cd37-/- mice displayed increased glomerular neutrophil influx, immune complex deposition, and worse renal function. To evaluate the role of IL-6 in the pathogenesis of accelerated renal pathology in Cd37-/-mice, we generated Cd37xIl6 double-knockout mice. These double-knockout and Il6-/- mice displayed no glomerular IgA deposition and were protected from exacerbated renal failure following lipopolysaccharide treatment. Moreover, kidneys of Cd37-/- mice showed more mesangial proliferation, endothelial cell activation, podocyte activation, and segmental podocyte foot process effacement compared to the double-knockout mice, emphasizing that IL-6 mediates renal pathology in Cd37-/- mice. Thus, our study indicates that CD37 may protect against IgA nephropathy by inhibition of the IL-6 pathway.

Acetylated Histones in Apoptotic Microparticles Drive the Formation of Neutrophil Extracellular Traps in Active Lupus Nephritis.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nuclear components. Lupus nephritis (LN) is the major cause of morbidity and mortality in patients with SLE. Central to the pathogenesis of SLE is the accumulation of cellular waste, especially apoptotic microparticles (MPs), which stimulates diverse immune reactions including the formation of neutrophil extracellular traps (NETs). In this study, we investigated the content of MPs from SLE patients with and without (active) LN, their capacity to stimulate NET release, and assessed the molecular mechanisms underlying MP-induced NETosis. METHODS: MPs from SLE patients with biopsy-proven active LN, remissive LN, without LN, and healthy controls were characterized by flow cytometry. Isolated neutrophils were exposed to MPs derived from either patient plasma or apoptotic human umbilical vein endothelial cells, and NET release was quantified by immunofluorescence imaging, spectrofluorometry or an in-house developed NET ELISA. RESULTS: MPs from SLE patients with active LN contain higher levels of acetylated chromatin compared to MPs from those with remissive LN, without LN, or healthy controls. MPs enriched in hyperacetylated chromatin are more potent in inducing NETosis when compared to MPs containing moderate acetylated chromatin. The release of NETs in response to MPs occurs rapidly in a concentration-dependent manner and proceeds independent from the formation of reactive oxygen species (ROS). CONCLUSION: Our data suggest that MPs containing acetylated chromatin drive ROS-independent NET release in SLE patients with active LN, which may lead to the glomerular deposition of NETs and subsequent NET-driven LN.

Circulating Apoptotic Microparticles in Systemic Lupus Erythematosus Patients Drive the Activation of Dendritic Cell Subsets and Prime Neutrophils for NETosis.

OBJECTIVE: Circulating chromatin-containing apoptotic material and/or neutrophil extracellular traps (NETs) have been proposed to be an important driving force for the antichromatin autoimmune response in patients with systemic lupus erythematosus (SLE). The aim of this study was to determine the exact nature of microparticles in the circulation of SLE patients and to assess the effects of the microparticles on the immune system. METHODS: We analyzed microparticles isolated from the plasma of patients with SLE, rheumatoid arthritis (RA), and systemic sclerosis (SSc), as well as from healthy subjects. The effects of the microparticles on blood-derived dendritic cells (DCs) and neutrophils were assessed by flow cytometry, enzyme-linked immunosorbent assay, and immunofluorescence microscopy. RESULTS: In SLE patients, we identified microparticles that were highly positive for annexin V and apoptosis-modified chromatin that were not present in healthy subjects or in RA or SSc patients. These microparticles were mostly CD31+/CD45- (endothelial), partly CD45+/CD66b+ (granulocyte), and negative for B and T cell markers. Microparticles isolated from the plasma of SLE patients increased the expression of the costimulatory surface molecules CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines interleukin-6, tumor necrosis factor, and interferon-α by blood-derived plasmacytoid DCs (PDCs) and myeloid DCs (MDCs). SLE microparticles also primed blood-derived neutrophils for NETosis. Microparticles from healthy subjects and from RA or SSc patients exhibited no significant effects on MDCs, PDCs, and NETosis. CONCLUSION: Circulating microparticles in SLE patients include a population of apoptotic cell-derived microparticles that has proinflammatory effects on PDCs and MDCs and enhances NETosis. These results underline the important role of apoptotic microparticles in driving the autoimmune response in SLE patients.