Monica Purmalek

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity

Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This studys objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage.

Anti–Citrullinated Protein Antibodies Are Associated With Neutrophil Extracellular Traps in the Sputum in Relatives of Rheumatoid Arthritis Patients

Studies suggest that rheumatoid arthritis (RA)–related autoimmunity is initiated at a mucosal site. However, the factors associated with the mucosal generation of this autoimmunity are unknown, especially in individuals who are at risk of future RA. Therefore, we tested anti–cyclic citrullinated peptide (anti‐CCP) antibodies in the sputum of RA‐free first‐degree relatives (FDRs) of RA patients and patients with classifiable RA.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-&bgr;, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Haploinsufficiency of NADPH Oxidase Subunit Neutrophil Cytosolic Factor 2 Is Sufficient to Accelerate Full-Blown Lupus in NZM 2328 Mice: ACCELERATED LUPUS IN NCF-2-DEFICIENT MICE

We have previously established that the gene for neutrophil cytosolic factor 2 (NCF‐2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE).

Antibody Responses to Citrullinated and Noncitrullinated Antigens in the Sputum of Subjects With Rheumatoid Arthritis and Subjects at Risk for Development of Rheumatoid Arthritis

The location and mechanisms involved in the initial generation of autoantibodies to citrullinated and noncitrullinated proteins/peptides during the natural history of rheumatoid arthritis (RA) development is incompletely understood. This study sought to explore individual antibody responses to citrullinated and noncitrullinated proteins/peptides in the sputum and associations with neutrophil extracellular traps (NETs) in subjects at risk for the future development of RA.

A Novel ELANE Mutation Associated with Inflammatory Arthritis, Defective NETosis, and Recurrent Parvoviral Infection

We describe a 38‐year‐old woman who presented with a history of inflammatory arthritis, rash, and daily fevers. She was noted to have chronic parvovirus infection with persistently detectable viral titers and a novel mutation in the ELANE gene. ELANE encodes neutrophil elastase, a neutrophil serine protease with important antimicrobial effects, and is found as part of neutrophil extracellular trap (NET) complexes. Pathogenic ELANE mutations have been identified in forms of hereditary neutropenia. However, our patient never had neutropenia. Because of the striking clinical scenario, we investigated this mutation functionally.

OP0054 Neuroimmunomodulation of Neutrophils as A Novel Mechanism of Levamisole-Induced Vasculitis

Background Levamisole, an anti-helminth drug and known cholinergic agonist, has been implicated in cases of drug-induced autoimmunity in humans exposed to cocaine adulterated with levamisole for profit. Clinical manifestations of levamisole-induced autoimmunity include cutaneous vasculitis and specific autoantibodies (e.g. anti neutrophil cytoplasmic antibodies, ANCA; antinuclear antibodies, ANA). Neutrophil extracellular trap (NET) formation is a novel cell death mechanism implicated in certain idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity. Objectives The objective of this study was to determine the role of NET formation in the pathogenesis of levamisole-induced autoimmunity. Methods Human and mouse neutrophils were exposed to graded concentrations of levamisole. NET formation was visualized and quantified by fluorescence microscopy. To assess the pathways involved in levamisole-induced NETosis, the following inhibitors were tested: diphenylene iodonium (DPI), an inhibitor of reactive oxygen species, and Cl-amidine, an inhibitor of peptidylarginine deiminase (PAD) enzymes. Involvement of Akt and Raf-MEK-ERK kinase pathways was assessed by Western blot. The role of Toll-like receptors (TLRs) and muscarinic receptors (MR) in levamisole-induced NETosis was investigated. Pharmacologic inhibitors and MR knockout mice were used to study specific MR subtypes implicated in NETosis. MR subtype receptors on neutrophils from disease comparators and healthy controls were assessed by flow cytometry. Autoantibodies against specific NET-components were examined in cocaine users exposed to levamisole. Results Levamisole induced prominent NETs in human and murine neutrophils that contained the antigenic targets of ANCA. NETs were seen in vasculitic lesions from cocaine/levamisole users. NET formation induced by levamisole was dependent on phosphorylation of Akt and Raf-MEK-ERK kinase pathways. DPI and Cl-amidine significantly abrogated levamisole-induced NETosis, highlighting that levamisole stimulates NETs via generation of reactive oxygen species and activation of PAD enzymes. Both levamisole and acetylcholine induced NETs through MR engagement on the surface of neutrophils and not through stimulation of TLRs. Atropine, a non-selective MR antagonist, blocked levamisole mediated NETosis. Screening experiments with pharmacologic inhibitors and neutrophils from MR knockout mice demonstrated that levamisole-induced NETosis was mediated through engagement of the M3 muscarinic receptor. Increased M3 receptors were seen on neutrophils from patients with systemic lupus erythematosus compared to controls. In a cohort of 21 cocaine users, 100% had quantifiable levamisole in urine, and novel autoantibodies against NET components were detectable in sera. In longitudinal observations, ex-vivo spontaneous NET formation correlated with periods of active cocaine use. Conclusions Levamisole induces NETosis through engagement of M3 muscarinic receptors on the surface of neutrophils. These findings implicate a novel interaction between the cholinergic nervous system and innate immunity in the pathogenesis of levamisole-induced autoimmunity. Disclosure of Interest None declared

Neutrophil subsets and their gene signature associate with vascular inflammation and coronary atherosclerosis in lupus

BACKGROUND Systemic lupus erythematosus (SLE) is associated with enhanced risk of atherosclerotic cardiovascular disease not explained by Framingham risk score (FRS). Immune dysregulation associated to a distinct subset of lupus proinflammatory neutrophils (low density granulocytes; LDGs) may play key roles in conferring enhanced CV risk. This study assessed if lupus LDGs are associated with in vivo vascular dysfunction and inflammation and coronary plaque. METHODS SLE subjects and healthy controls underwent multimodal phenotyping of vascular disease by quantifying vascular inflammation (18F-fluorodeoxyglucose-PET/CT [18F-FDG-PET/CT]), arterial dysfunction (EndoPAT and cardio-ankle vascular index), and coronary plaque burden (coronary CT angiography). LDGs were quantified by flow cytometry. Cholesterol efflux capacity was measured in high-density lipoprotein-exposed (HDL-exposed) radioactively labeled cell lines. Whole blood RNA sequencing was performed to assess associations between transcriptomic profiles and vascular phenotype. RESULTS Vascular inflammation, arterial stiffness, and noncalcified plaque burden (NCB) were increased in SLE compared with controls even after adjustment for traditional risk factors. In SLE, NCB directly associated with LDGs and associated negatively with cholesterol efflux capacity in fully adjusted models. A neutrophil gene signature reflective of the most upregulated genes in lupus LDGs associated with vascular inflammation and NCB. CONCLUSION Individuals with SLE demonstrate vascular inflammation, arterial dysfunction, and NCB, which may explain the higher reported risk for acute coronary syndromes. The association of LDGs and neutrophil genes with vascular disease supports the hypothesis that distinct neutrophil subsets contribute to vascular damage and unstable coronary plaque in SLE. Results also support previous observations that neutrophils may disrupt HDL function and thereby promote atherogenesis. TRIAL REGISTRATION Clinicaltrials.gov NCT00001372FUNDING. Intramural Research Program NIAMS/NIH (ZIA AR041199) and Lupus Research Institute.

Haploinsufficiency of NADPH oxidase subunit NCF2 is sufficient to accelerate full-blown lupus in NZM.2328 mice

We have previously established that the gene for neutrophil cytosolic factor 2 (NCF‐2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE).