Testing the inside-out theory of multiple sclerosis aetiology using the cuprizone-induced demyelination model

Abstract

Multiple sclerosis (MS) is a common disabling neurological disorder affecting about 2.5 million people worldwide. There are two competing theoretical aetiologies of the disease: First, an 'outside-in' theory in which peripheral factors (autoreactive T-cells) traverse the blood brain barrier (BBB) leading to central nervous system (CNS) demyelination. Second, an 'inside-out' theory in which central demyelination causes BBB disruption leading to autoimmune response. Cuprizone (CPZ)-feeding in mice induces oligodendrocyte loss (oligodendrocytosis), demyelination and reactive gliosis of astrocytes and microglia in the central nervous system (CNS), similar to that seen in MS; however, the CPZ-induced pathology lacks T-cell involvement in the CNS, a hallmark of MS, in particular by CD8+ T-cells. This thesis explored whether incremental modification of the standard CPZ mouse model would yield a pattern of disease development and progression more closely resembling that seen in human MS. The first modification tested the hypothesis that blood brain barrier (BBB) disruption (using pertussis toxin, PT) would facilitate a T-cell-mediated immune response to the myelin antigens in the CNS of CPZ-fed mice. This was tested by using low (0.1%) and standard (0.2%) CPZ-feeding for 5 weeks (Study I) and also assessing whether similar effects can be initiated by inducing a slow progressive demyelination by prolonged feeding of 0.1% CPZ for 12 weeks (Study II). The use of 0.1% CPZ yields a comparable CNS pathology to 0.2% but less effect on splenic T-cell (CD4 and CD8, CD4/8) levels and no change in spleen mass; thus, only 0.1% CPZ was used in Study II. Although oligodendrocytosis, demyelination and gliosis were evident in Studies I and II, no T-cells were detected in the CNS. Proteomic analysis of whole brain tissue reveals that CPZ has changed the abundance of many proteoforms that are directly involved in metabolism and suppression of T-cell function. Importantly, 0.1% and 0.2% CPZ-feeding lead to a reduction in splenic T-cells, indicating that the peripheral immune system is suppressed due to the effects of CPZ-feeding. In study III, castration (Cx, orchiectomy) is used to protect the peripheral immune system against the dose dependent (0.2%>0.1%) effects of CPZ-feeding on thymus and spleen size (and T-cell levels) yet produces the same amount of demyelination and gliosis in the MCC. Study IV demonstrates that combining Cx induced preservation of the thymus and spleen with 0.1% CPZ-feeding and blood brain barrier disruption result in CD8+ T-cell recruitment into the brain and spinal cord. In study V, 0.1% CPZ-feeding in gonadally intact (Gi) female mice induces peripheral immune organ atrophy, T-cell signal suppression, demyelination and gliosis indistinguishable from that seen in males, but no CD8+ T-cell infiltration into the CNS. Taken together, suppression of the peripheral immune system in male and female mice by CPZ-feeding explains why CPZ-induced demyelination does not result in the recruitment of peripheral immune cells into the CNS. Moreover, these findings indicate that a short period (2 weeks) of 0.1% CPZ-feeding in Cx males (where the peripheral immune structures are preserved), induces both demyelination and glial activation that are sufficient to activate and recruit CD8+ cells to the CNS, in effect initiating an 'inside-out' T-cell mediated immune response. The CD8+ T-cell recruitment into the CNS of CPZ-fed mice is a new variant of the CPZ model that should prove useful to explore the earliest events involved in CNS demyelinating diseases like MS.

Date of Award	2020
Original language	English