Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Abstract

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-β signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.