To investigate the proportion of low-density granulocytes (LDGs), circulating plasma neutrophil extracellular traps (NETs), and serum-induced NET formation in patients with incomplete systemic lupus erythematosus (iSLE) and systemic lupus erythematosus (SLE). LDGs were measured cross-sectionally in 18 iSLE patients, 11 SLE patients and 14 healthy controls (HCs), whereas circulating NETs and serum-induced NET formation were assessed in 35 iSLE patients, 41 SLE patients and 16 HCs. LDGs (CD14^lowCD15⁺) were measured in PBMCs using flow cytometry and circulating plasma NETs were measured using anti-myeloperoxidase-DNA, anti-citrullinated histone H3 and anti-elastase-DNA complex ELISAs. Serum-induced NET formation was assessed by incubating healthy neutrophils with serum from iSLE patients, SLE patients or HCs and visualizing NETs with fluorescence microscopy. Proportions of LDGs and circulating plasma NETs were similarly elevated in iSLE and SLE patients compared with those in HCs. Furthermore, patients under hydroxychloroquine (HCQ) treatment had lower proportions of LDGs than those without. Serum from iSLE and SLE patients similarly induced NET formation in healthy neutrophils. In iSLE patients, myeloperoxidase-DNA complexes were correlated with proportions of age-associated B-cells, memory B-cells and negatively with naïve B-cells, while we did not find associations between measures of NETs or serum-induced NET formation and interferon score or clinical parameters. These results show that neutrophil dysfunction, including higher proportions of LDGs, and increased NET formation, already occur in iSLE, similar to SLE, despite differences in disease manifestations. Thereby, neutrophil dysfunction may contribute to sustained exposure to autoantigens and autoreactivity in early stages of SLE.

Background: Belimumab, an anti-B-cell activating factor antibody, is approved for the treatment of auto-antibody positive systemic lupus erythematosus with a high degree of disease activity. Anti-CD20 B cell depletion with rituximab is used in refractory SLE as well, although with variable responses. We hypothesized that incomplete B cell depletion, related to a surge in BAFF levels following rituximab treatment, can cause ongoing disease activity and flares. The Synbiose 1 study primarily focused on immunological effects and shows the preliminary clinical benefit of combined rituximab and belimumab in SLE. The Synbiose 2 study will evaluate the clinical efficacy of combining belimumab with rituximab in patients with severe SLE, allowing the tapering of prednisolone and mycophenolate.Methods: Synbiose 2 is a phase 3, multicenter, randomized, controlled, open-label 2-year clinical trial. Seventy adults with severe SLE including lupus nephritis will be randomized 1:1 to receive either standard of care consisting of prednisolone and mycophenolate as induction and maintenance treatment, or belimumab and rituximab combined with standard of care as induction treatment, followed by prednisolone and belimumab as maintenance treatment. The primary objective is to assess whether combined B cell therapy will lead to a reduction of treatment failure. Secondary endpoints are complete and partial clinical and renal response and the improvement of SLE-specific autoimmune phenomena. Safety endpoints include the incidence of adverse events, with a special interest in infections.Discussion: The Synbiose 2 trial is the first multicenter phase 3 clinical trial investigating combined B cell targeted therapy in SLE, including lupus nephritis. The outcome of this study will provide further evidence for the clinical efficacy of this new treatment strategy in severe SLE.Trial registration: ClinicalTrials.govNCT03747159. Registered on 20 November 2018.

Oxidative stress plays an important role in systemic lupus erythematosus (SLE) and especially in lupus nephritis (LN). The aim of this study was to compare redox-related biomarkers between patients with active LN, quiescent SLE (Q-SLE) and healthy controls (HC) and to explore their association with clinical characteristics such as disease activity in patients. We investigated levels of plasma free thiols (R-SH, sulfhydryl groups), levels of soluble receptor for advanced glycation end products (sRAGE) and levels of malondialdehyde (MDA) in SLE patients with active LN (n = 23), patients with quiescent SLE (n = 47) and HC (n = 23). Data of LN patients who previously participated in Dutch lupus nephritis studies and longitudinal samples up to 36 months were analyzed. Thiol levels were lower in active LN at baseline and Q-SLE patients compared to HC. In generalized estimating equation (GEE) modelling, free thiol levels were negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) over time (p < 0.001). sRAGE and MDA were positively correlated with the SLEDAI over time (p = 0.035 and p = 0.016, respectively). These results indicate that oxidative stress levels in LN patients are increased compared to HC and associated with SLE disease activity. Therefore, interventional therapy to restore redox homeostasis may be useful as an adjunctive therapy in the treatment of oxidative damage in SLE.

Hydroxychloroquine (HCQ) is obtained by hydroxylation of chloroquine (CQ) and the first indication was malaria. Nowadays, HCQ is commonly used in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) with favorable results. Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity and persistent positivity of antiphospholipid antibodies. Around 20–30% of pregnant women with APS develop adverse pregnancy outcomes despite conventional treatment with aspirin and heparin, called refractory obstetric APS. Interestingly, HCQ has shown positive effects on top of the standard of care in some refractory obstetric APS patients. HCQ mechanisms of action in APS comprise its ability to bind sialic acid present in cell membranes, its capacity to block the binding of antiphospholipid antibodies to the cell and the induced increase of pH in extracellular and intracellular compartments. However, the precise mechanisms of HCQ in the specific situation of refractory APS still need to be fully clarified. Therefore, this review summarizes the known modulating effects of HCQ and CQ, their side effects and use in APS and different pathologies to understand the benefit effects and the mechanism of action of HCQ in refractory obstetric APS.

Purpose: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective long-term protection against COVID-19 is therefore of great importance in these patients, but little is known about the decay of the immune response after primary vaccination. We studied the immune responses 6 months after two mRNA-1273 COVID-19 vaccines in 473 IEI patients and subsequently the response to a third mRNA COVID-19 vaccine in 50 patients with common variable immunodeficiency (CVID). Methods: In a prospective multicenter study, 473 IEI patients (including X-linked agammaglobulinemia (XLA) (N = 18), combined immunodeficiency (CID) (N = 22), CVID (N = 203), isolated or undefined antibody deficiencies (N = 204), and phagocyte defects (N = 16)), and 179 controls were included and followed up to 6 months after two doses of the mRNA-1273 COVID-19 vaccine. Additionally, samples were collected from 50 CVID patients who received a third vaccine 6 months after primary vaccination through the national vaccination program. SARS-CoV-2-specific IgG titers, neutralizing antibodies, and T cell responses were assessed. Results: At 6 months after vaccination, the geometric mean antibody titers (GMT) declined in both IEI patients and healthy controls, when compared to GMT 28 days after vaccination. The trajectory of this decline did not differ between controls and most IEI cohorts; however, antibody titers in CID, CVID, and isolated antibody deficiency patients more often dropped to below the responder cut-off compared to controls. Specific T cell responses were still detectable in 77% of controls and 68% of IEI patients at 6 months post vaccination. A third mRNA vaccine resulted in an antibody response in only two out of 30 CVID patients that did not seroconvert after two mRNA vaccines. Conclusion: A similar decline in IgG titers and T cell responses was observed in patients with IEI when compared to healthy controls 6 months after mRNA-1273 COVID-19 vaccination. The limited beneficial benefit of a third mRNA COVID-19 vaccine in previous non-responder CVID patients implicates that other protective strategies are needed for these vulnerable patients.