Targeting P2Y12 limits TH17 accumulation in the CNS and reduces EAE progression

Abstract

Platelets are increasingly recognized as active amplifiers of neuroinflammation in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). P2Y12 is an essential ADP receptor that regulates platelet activation and is upregulated in neuroinflammatory lesions. However, its contribution across different effector populations and therapeutic potential in autoimmune diseases of the central nervous system (CNS) remain incompletely defined. Therefore, we characterized P2ry12 expression across different immune and CNS-resident effector populations involved in EAE. To define P2Y12-dependent mechanisms in autoimmune neuroinflammation, we targeted the P2Y12 receptor both genetically and pharmacologically in MOG35–55-induced EAE and monitored disease course and immunopathology. The neuroinflammatory response was profiled by flow cytometry, histology, and cytokine analyses. Pharmacological inhibition with elinogrel or clopidogrel was evaluated in prophylactic and therapeutic treatment regimens. Additionally, prophylactic elinogrel treatment was combined with antibody-mediated platelet depletion during the effector phase of EAE to define the contribution of platelets to the observed effects. Among the immune and CNS-resident effector populations examined, platelets exhibited the highest abundance of P2ry12 transcripts, and platelet P2ry12 expression increased following EAE induction. Consistent with a proinflammatory role of the P2Y12 receptor in CNS autoimmunity, its genetic deficiency was associated with decreased disease progression, as indicated by lower peak and cumulative scores, while disease onset remained unchanged compared to wild-type mice. Pharmacological inhibition of the P2Y12 receptor reproduced this protective effect. Importantly, treatment with elinogrel did not affect the number of infiltrated platelets, but markedly reduced cellular infiltration and demyelination. Furthermore, the protective effect was associated with a significant reduction in infiltrating CD4+ T cells, particularly pro-inflammatory T helper 17 (TH17) cells. Of note, elinogrel provided no additional benefit beyond platelet depletion, a finding compatible with a platelet-mediated contribution to the observed effect. Clopidogrel, another P2Y12 inhibitor, similarly reduced disease severity when administered prophylactically. Of note, both elinogrel and clopidogrel remained effective when treatment was initiated after clinical onset. These data identify platelet-associated P2Y12 signaling as a functionally relevant amplifier of EAE pathology. P2Y12 blockade limits CNS immune infiltration and neuroinflammation-induced tissue injury, exerts an impact on the TH17 axis, and retains efficacy when administered therapeutically. Collectively, these findings support P2Y12 as a mechanistically grounded and translationally relevant target in CNS autoimmunity.