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The phagocyte NADPH oxidase (the NOX2 complex) generates superoxide, the precursor to reactive oxygen species (ROS). ROS possess both antimicrobial and immunoregulatory function. Inactivating mutations in alleles of the NOX2 complex cause chronic granulomatous disease (CGD), characterized by an enhanced susceptibility to infections and autoimmune diseases such as Systemic lupus erythematosus (SLE). The latter is characterized by insufficient removal of dead cells, resulting in an autoimmune response against components of the cell's nucleus when non-cleared apoptotic cells lose their membrane integrity and present autoantigenic molecules in an inflammatory context. Here we aimed to shed light on the role of the NOX2 complex in handling of secondary necrotic cells (SNECs) and associated consequences for inflammation and autoimmunity during lupus. We show that individuals with SLE and CGD display accumulation of SNECs in blood monocytes and neutrophils. In a CGD phenotypic mouse strain (Ncf1** mice) build-up of SNECs in Ly6CHI blood monocytes was connected with a delayed degradation of the phagosomal cargo and accompanied by production of inflammatory mediators. Treatment with H2O2 or activators of ROS-formation reconstituted phagosomal abundance of SNECs to normal levels. Induction of experimental lupus further induced increased antibody-dependent uptake of SNECs into neutrophils. Lupus-primed Ncf1** neutrophils took up more SNECs than wild type neutrophils, whereas SNEC-accumulation in regulatory Ly6C−/LO monocytes was lower in Ncf1**mice. We deduce that the inflammatory rerouting of immune-stimulatory necrotic material into inflammatory phagocyte subsets contributes to the connection between low ROS production by the NOX2 complex and SLE., Graphical abstract Image 1 Graphical abstract: Inflammatory rerouting of SNECs occurs in individuals with a dysfunctional NOX2 complex.

Systemic lupus erythematosus (SLE) is characterized by autoimmune reactivity against components of the cell's nucleus arising after insufficient removal of dead cells. We used a lupus mouse model induced by the cytotoxic alkane oil pristane to elucidate the impact of ROS on SLE. Pristane-induced lupus was strongly exacerbated in NOX2-deficient (Ncf1**) compared to wild type mice, as seen from elevated levels of antinuclear autoantibodies (ANA), aggravated organ damage, and premature death. We observed a dramatically reduced ability to form neutrophil extracellular traps (NETs) in Ncf1** mice, accompanied by higher levels of inflammatory mediators. Similarly, NET-deficient peptidyl arginine deiminase 4-deficient mice and neutropenic Mcl-1 ΔMyelo mice exhibited higher levels of ANA, which indicates a regulatory function in lupus of NETs and neutrophils, respectively. Furthermore, Ncf1** mice and SLE patients displayed preferential uptake of dead cells into inflammatory monocytes and granulocytes. Treatment of mice with NOX2 activators induced NET formation and ameliorated lupus. Our results show that absence of ROS leads to lupus based on aberrant phagocytosis of dead cell remnants and impaired formation of NETs, which results in uncontrolled release of inflammatory mediators.

The production of reactive oxygen species (ROS) via the oxidative burst has in the traditional view been connected with promotion of inflammation and tissue damage, but has in recent years also been implicated in regulation of inflammation and protection from autoimmunity. We have investigated the effects of ROS on the autoimmunity in a mouse model of lupus and in patients with systemic lupus erythematosus (SLE). Aims The aim of this project was to elucidate the impact of the oxidative burst on the development of lupus-like autoimmunity in wild type and Ncf1** mice which carry a point mutation in one of the subunits of the phagocyte NADPH oxidase 2 complex that abrogates ROS-production. Lupus was induced by intraperitoneal injection of 0.5 ml pristane oil and the clinical course was compared by analysis of serological markers and organ involvement. Ex vivo phagocytosis assays and flow cytometry were deployed to assess uptake of cell debris in ROS deficient mice. The formation of neutrophil extracellular traps (NETs) was analysed by immune fluorescence microscopy. Potential activators of ROS production were injected into mice to investigate the possible beneficial clinical effects on lupus pathology. Results Our results show that the absence of ROS in Ncf1** mice gives rise to dramatically exacerbated lupus. Aberrant phagocytosis in ROS-deficient animals leading to production of inflammatory mediators, accompanied by diminished pristane-induced but higher spontaneous formation of NETs could be responsible for this phenotype. Treatment of pristane-induced lupus with NOX2 agonists ameliorated the clinical course in BALB/c mice, while application of a ROS scavenger worsened disease outcome. Further, downstream nrf2 signalling was decreased in Ncf1** mice. Patients with SLE showed similar aberrant phagocytosis as in murine lupus, and were characterized by higher spontaneous formation of NETs. A fine-tuned balance of ROS-production is necessary to prevent autoimmunity and to avert the development of lupus in mice and men.

The production of reactive oxygen species (ROS) via the oxidative burst has been connected with promotion of inflammation and tissue damage, but in recent years has been implicated in regulation of inflammation. We investigated the effects of ROS on the murine model of lupus and in patients (SLE). Aims The aim of this project was to elucidate the impact of the oxidative burst on the development of lupus-like autoimmunity. Lupus was induced by i.p. injection of pristane oil. Ex vivo phagocytosis assays were deployed to assess the uptake of cell debris in ROS deficient mice. The formation of neutrophil extracellular traps (NETs) was analysed by immune fluorescence microscopy. ROS activators were injected into mice to investigate the possible beneficial clinical effects on lupus pathology. Results The absence of ROS gives rise to dramatically exacerbated lupus. Aberrant phagocytosis in ROS-deficient animals leading to production of inflammatory mediators, accompanied by diminished pristane-induced but higher spontaneous formation of NETs could be responsible for this phenotype. Treatment with NOX2 agonists ameliorated the clinical course in mice, while application of a ROS scavenger worsened disease outcome. A fine-tuned balance of ROS-production is necessary to prevent autoimmunity and to avert the development of lupus in mice and men.

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.