Investigating the mechanism by which the atypical antipsychotic clozapine reduces disease in experimental autoimmune encephalomyelitis
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by damage to the myelin sheaths that surround nerve axons. Inflammatory damage to the myelin sheath leads to severe physical disability in patients. Whereas approved disease modifying treatments are available for relapsing-remitting forms of MS, there are no approved treatments for the progressive stages, leaving approximately 50% of MS sufferers without treatment. Therefore, there is an urgent need for development of effective alternatives. Atypical antipsychotic agents used for treating schizophrenia have recently been recognized for their immune-modifying properties and our laboratory has shown previously that treating mice with risperidone or clozapine reduces the severity of disease in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Although atypical antipsychotic agents like clozapine have been used in the clinic for almost 60 years, there is very little experimental data that describes the mechanism by which atypical antipsychotic agents like clozapine are able to modify the immune response. This thesis aimed to describe the immunological mechanisms by which clozapine is able to reduce EAE disease and to determine the underlying cellular signalling alterations that occur during treatment to facilitate immune modifying properties. In vitro experiments showed that clozapine can impair induction of Th1 and Th17 cells while promoting the differentiation of iTreg and increasing Foxp3 expression. However, although clozapine effectively delayed disease onset and reduced the severity of EAE, the therapeutic effect of clozapine was not associated with impaired capacity to induce antigen specific Th1 or Th17 responses in the periphery. Moreover, Treg function was dispensable for disease protection by clozapine. Instead, disease protection by clozapine was associated with a suppressed state of activation in CNS resident microglia and infiltrating monocytes assessed by flow cytometric measurement of activation associated receptor expression. In vitro experiments using primary macrophage cell culture revealed that clozapine can alter the activation of activated macrophages towards a less inflammatory state directly. Interestingly, the altered state of activation in primary macrophages was not associated with detectable changes in cell signalling pathways known to mediate activation. This thesis demonstrated that clozapine treatment protects from EAE by a multi-faceted immunological mechanism that likely involves modifying multiple pathways and cell types during EAE and may be of therapeutic benefit to MS patients in the progressive stages of disease. Finally, this thesis also has relevance to psychiatry as it demonstrates that clozapine has potential to alter cellular immune responses.