Multiple sclerosis (MS) was considered a T cell driven auto immune disorder in the past, with adaptive immunity driven neurodegeneration. However, ongoing in-depth characterization of MS pathology has elucidated a much wider concerted involvement of additional immune cell types belonging to both innate and adaptive immune response system, such as B cell lymphocytes, macrophages, microglial cells etc.[1] This expanded understanding of different pathological cellular mechanisms has allowed for the development of several emerging MS therapies. A subset of these therapies utilize small molecules targeted at suppressing MS relevant immunological processes such as containing metabolically active cells using teriflunomide,[2] lymphocyte export prevention at lymph nodes using S1P receptor inhibitors such as fingolimod, siponimod, ozanimod and ponesimode, and targeting of proliferating immune cells using cladribine, among others.[1]
Along these lines, Bruton tyrosine kinase (BTK) is a therapeutically targeted enzyme that is critical for regulation of both B-cell receptor signaling and microglial function.[3] BTK was discovered as an enzyme involved in X linked agammaglobulinemia. Its expression in B cell lineage is up regulated in pre-B cell stage and is sustained up to plasma B cell stage.[4] BTK belongs to tyrosine expressed in hepatocellular carcinoma family and its key function is to initiate signaling cascades i.e. nuclear factor kappa B activation, phosphatidylinositol-4-phosphate5 kinase activation and phosphorylation of phospholipase C-γ2 for production of the secondary messengers diacylglycerol and inositol-1,4,5-triphosphate. These pathways are integral to B cell functions such as survival, expansion, development into progenitor B-cells, formation of germinal center and class switching to generate plasma B cells.[5] In addition to its role in B cell lineage, BTK enzyme function allows microglial cells to activate in response to disease stimuli and aid in clearing of infected cells as well.[6] It aides in microglial functions such as activation, cytokine secretion, cell phagocytosis etc. which are involved in MS tissue pathologies.
Therefore, in the context of autoimmune diseases, BTK enzyme inhibition has been proposed as a targeted treatment strategy,[3] in contrast to immunotherapy-based B cells population depletion strategies. Overall, B cell depletion using immunotherapy-based approaches have been tested with success in MS clinical trials.[7–9] However, in a few pre-clinical studies, B cell depletion was noted to worsen the disease pathology. For example, one study found that TMEV disease pathology was worsened when treated with anti-CD20 antibody.[10] To explain this, it has been suggested that B cells not only help sustain neurodegeneration and demyelination in autoimmune diseases, but also support neurotrophic and remyelination processes. Additionally, cyclical depletion of B cells involved in immunotherapy results in added stress on immune system.[11] Therefore, a controlled suppression of B cell function, such as BTK inhibition has been suggested to be more sustainable therapy for treating autoimmune demyelinating diseases. Hence, BTK enzyme inhibitor such as Ibrutinib (iBTK) which covalently binds with BTK and is blood brain barrier (BBB) permeable, is a promising MS treatment drug.[12, 13]
However, the effect of iBTK treatment on various above immune cell pathways has not been previously characterized in a non-immunization-based model of MS, such as Theiler’s Murine Encephalomyelitis Virus (TMEV). TMEV is an animal model of demyelinating disease, involving a viral infection and it produces an acute state of encephalomyelitis and spinal demyelination, accompanied by neuronal death in chronic phase.[14, 15] During the TMEV disease course, the microglia are capable antigen-presenting cells that play an important role at the onset of clinical disease and contribute to demyelination.[16, 17] Furthermore, B cell activation and secretion of auto antibodies has been shown to participate in the TMEV disease pathology as well. [18] Therefore, in comparison to a T cell driven immunization based preclinical Experimental autoimmune encephalomyelitis (EAE) disease model, TMEV is better suited for characterizing iBTK's role in MS disease pathology.
Against this background, we aimed to investigate the effects of iBTK on the disease pathology and clinical disability of the TMEV mouse model of MS. To this end, SJL mice were intra-cerebrally (IC) injected with TMEV virus to induce TMEV. Half the TMEV injected animals were treated with iBTK and the other half were treated with vehicle solution. To assess the impact of iBTK, blood serum was collected to measure B cell fraction at 2-months post induction (mPI), furthermore, blood serum immunoglobulin G (IgG) concentration levels were measured at 3 and 5 mPI. The animals were weekly monitored to record body weight and assign a clinical disability score. Since spinal cord demyelination drives TMEV induced disability, to assess the impact of the iBTK on the TMEV disease progression at cellular level, animal spinal cord tissue was isolated from half of the study animals at 3 and 5 mPI.