Inflammasome Cytokine Secretion in Heparin-Induced Thrombocytopenia Is Supported By Platelet-Derived Mediators and Complement

Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that is caused by IgG immune complexes that activate platelets and monocytes through Fc Receptor for IgG IIA (FcγRIIA). These immune complexes also fix complement. Stimulated monocytes produce proinflammatory cytokines including inflammasome dependent IL-1β release, which has not been investigated in HIT.

Aims: Platelets have been shown to support monocyte inflammasome cytokine release during inflammation, primarily in microbial infection. This study sought to investigate the role of inflammasome cytokine production in HIT pathology.

Methods: We investigated the role of the NLRP3 inflammasome following immune complex (IC) challenge in healthy donor whole blood or immortalized monocyte-like cells. The contribution of inflammasome assembly and cytokine release on HIT pathology was assessed utilizing NLRP3 inhibitor MCC950 in the triple allele ‘HIT’ mouse model. Plasma IL-1β cytokine production was measured using ELISA, and complete blood counts were taken to assess thrombocytopenia. Furthermore, the contribution of complement was assessed using IC challenge of isolated primary blood mononuclear cells suspended in plasma or heat-inactivated plasma.

Results: Following human whole blood challenged or isolated mononuclear cell/platelet suspensions with two distinct immune complexes (heat aggregated IgG or heparin/PF4 complexes) we observed a significant increase in inflammasome cytokine production at 22 hours. Mononuclear/platelet cell fractions challenged with heparin/PF4 complexes had a significant increase in inflammasome cytokine secretion that was reduced with pretreatment with an FcγRIIA blocking antibody, or removal of complement with heat-inactivated plasma. We observed that administration of heparin and monoclonal αPF4/heparin antibody KKO to HIT mice resulted in thrombocytopenia and significantly increased circulating IL-1β, whereas mice treated with NLRP3 inflammasome inhibitor MCC950 protected mice from a platelet drop and cytokine production in our humanized mouse model of HIT in vivo.

Conclusions: These data demonstrate for the first time that HIT IC mediates the production and secretion of inflammasome cytokines, which is influenced by the presence of platelets in a FcγRIIA dependent manner as well as by complement. Furthermore, the NLRP3 inflammasome is shown in HIT model mice to be a critical component of HIT pathophysiology. These results provide a scientific rationale for exploring mechanisms that drive platelet/monocyte/complement communication and targeting the inflammasome as a possible therapeutic strategy in HIT.