Investigating the effects of kappa opioid receptor agonists on CD4+ T cell subsets

Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system (CNS), characterised by the infiltration of autoreactive T cells into the brain and spinal cord. These cells drive immune-mediated damage to the myelin sheath, resulting in inflammation and nerve damage within the CNS and subsequent neurological symptoms. CD4+ T cells play a key role in MS pathogenesis with the Th1 subset activating pro-inflammatory CNS resident immune cells, and the Th17 subset promoting disruption of the blood-brain barrier. Regulatory T cells (Tregs), which typically downregulate immune responses are often reduced in number and less suppressive in patients with MS. Despite the availability of several disease-modifying treatments, there is an ongoing need for remyelinating and neuroprotective therapies to complement the current immune-targeting options.  The kappa opioid receptor (KOR) is a G-protein coupled receptor widely expressed throughout the CNS and on peripheral cell populations. The KOR is involved in various signalling pathways, including the stress response, pain, reward and inflammation. KOR agonists were initially investigated for their potential as non-addictive pain medications due to their reduced risk of dependency compared to other opioids like morphine. KOR agonists have also shown promise for driving repair in models of demyelinating disease. KOR knockout in mice worsens the progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS while KOR agonist U50,488 was shown to decrease disease severity and demyelination in the model. However, many KOR agonists induce dysphoria and aversive side effects which have limited their use in the clinic.  Nalfurafine, a selective, KOR agonist, has been investigated by our lab group for its potential remyelinating properties in the EAE model. Nalfurafine has previously been approved for the treatment of pruritus in kidney dialysis patients and unlike most KOR agonists exhibits very few side effects, making it a promising candidate for further translation into the clinic.   In studies by the La Flamme group, nalfurafine treatment was shown to reduce disease severity and promote remyelination in two MS models, (EAE and cuprizone-induced demyelination). Importantly, nalfurafine also prevented the infiltration of T cells into the CNS and altered myelin-specific T cell responses, highlighting potential immune modifying abilities. Specifically, nalfurafine treatment in vitro and in vivo reduced levels of INFγ and IL-17, cytokines associated with the subsets Th1 and Th17, respectively.  This thesis further investigates the immunomodulatory effects of KOR agonists, in particular their influence on the differentiation and expansion of CD4+ T cell subsets under healthy and disease conditions.  Naïve CD4+ T cells were successfully differentiated and expanded into effector subsets using anti-CD3/CD28 activation beads. However, KOR agonist treatment did not show significant effects on this expansion. In splenocyte cultures, nalfurafine treatment resulted in a modest reduction in RORγT expression, suggesting a decrease in Th17 responses. We also observed an increase in the ratio of Treg to Th17 cells following nalfurafine treatment, indicating a shift towards a more regulatory immune environment.  To explore the effects of in vivo KOR agonist treatment on peripheral T cell populations, a shortened version of the EAE model was used. Nalfurafine reduced the proportion of RORγT+ CD4+ cells in both the blood and spleen, suggesting that nalfurafine may influence T cell subsets in peripheral tissues before their migration into the CNS.   Additionally, bulk RNA sequencing of spinal cord samples from healthy and EAE mice treated with nalfurafine was used to assess the effect of nalfurafine treatment on gene expression. Gene ontology analysis revealed that many genes downregulated following nalfurafine treatment were involved in immune-related pathways, further supporting the immune modifying potential of this treatment. Downregulated genes include STAT molecules associated with transcriptional regulation of CD4+ subsets, as well as numerous genes known to be involved in Th17 function and pathogenesis during EAE.  Overall, this thesis provides evidence that nalfurafine treatment can modulate CD4+ T cell subsets. These findings suggest that nalfurafine may provide a promising strategy for the treatment of MS by modifying immune responses alongside addressing the need for remyelination and neuroprotection. Further research is needed to determine the specific mechanism through which KOR agonists can influence CD4+ T cells and how modulating immune responses may contribute to enhanced repair and recovery in the EAE model following KOR agonist treatment.