Background/Purpose:

Certain medications are known to induce autoimmune disease in humans, triggering clinical features and autoantibody profiles that mirror idiopathic systemic lupus erythematosus (SLE) and/or ANCA-associated vasculitis (AAV). Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of both SLE and AAV, and NETs contain the antigenic targets (double-stranded DNA, MPO and PR3) of autoantibodies typically identified in cases of drug-induced autoimmunity. A subset of patients with SLE have impaired NET degradation likely due to presence of DNase I inhibitors or anti-NET antibodies in serum. These findings suggest that impaired regulation of NETosis triggers an autoimmune response against components of NETs and induces autoimmune diseases, including AAV and SLE. This study explored whether medications commonly implicated in cases of drug-induced autoimmunity (hydralazine and procainamide) and medications less commonly implicated in drug-induced autoimmunity, but more strongly associated with agranulocytosis (minocycline and clozapine), induce NET formation.

Methods: Human and murine neutrophils (PMNs) were incubated with  medications of interest for one and four hours, respectively, at varying concentrations to mimic physiologic conditions. To address potential induction of impairments in NET degradation, drug-induced NETs were exposed to 10% serum from healthy control patients for 6 hours. NETs were visualized and quantified using fluorescence microscopy. To investigate whether drugs modulate NETosis through canonical pathways, NET inhibition was quantified when PMNs exposed to medications were incubated with Diphenyleneiodonium chloride (DPI; NADPH-oxidase inhibitor), BB-Cl-amidine (peptidylarginine deiminase (PAD) inhibitor) and specific muscarinic receptors antagonists. Release of hydrogen peroxide was measured by Amplex-Red Hydrogen Peroxide/Peroxidase Assay Kit. Whether these drugs stimulate various toll-like receptors (TLRs) was assessed using a human ligand TLR screening platform.

Results: Hydralazine and procainamide induced NETs in human and murine PMNs, while minocycline and clozapine did not. NET degradation was not impaired by these drugs. Hydralazine, but not procainamide, induced significant release of hydrogen peroxide in PMNs and triggered NET formation through canonical pathways (NADPH and PAD4). In addition, induction of NETs by procainamide was inhibited by addition of the muscarinic antagonists atropine, scopolamine, and specific M1/M3 inhibitors. Neither hydralazine nor procainamide stimulated TLRs in PMNs.

Conclusion: Certain medications implicated in drug-induced autoimmunity trigger formation of NETs via different pathways. Induction of NETs by hydralazine is dependent on PADs and NADPH oxidase. Procainamide induces NETs via stimulation of specific muscarinic receptors on PMNs. NETs  may play a causal role in drug-induced autoimmunity.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-mediated-mechanisms-of-drug-induced-autoimmunity/