Investigating the Effect of the Novel Heparan Sulfate Mimetic, Tet-29, on Homeostatic Immune Cell Trafficking

<p dir="ltr"><b>The central nervous system (CNS) was historically understood to have immunological privilege, isolated from peripheral immune activity. However, recent experimental evidence has revealed the constant bidirectional trafficking of immune cells between the CNS and peripheral circulation, termed immunosurveillance. This routine surveillance of the CNS is primarily facilitated through the choroid plexus (ChP), an epithelial barrier in the brains ventricles, which forms a key immunological interface between peripheral blood and the cerebrospinal fluid, enabling continuous immune cell migration.</b></p><p dir="ltr">The consequences of disrupted CNS homeostatic immune cell trafficking are highlighted in both neuroinflammatory disorders as well as certain therapeutics for these diseases. Multiple sclerosis (MS) is a complex neuroinflammatory disease, characterised by demyelination in the CNS. A key process of MS is the unregulated infiltration of autoreactive immune cells which breach CNS barriers, and attack the myelin sheath enclosing neuronal axons, causing a progressive neurological decline. Current therapeutics for neuroinflammation broadly target immune cell migration in the brain. While these therapeutics are successful at blocking inflammatory leukocyte migration into the CNS to improve disease outcome, they also risk severe side effects by disrupting routine surveillance. This highlights the need for the understanding of therapeutic influence on homeostatic trafficking.</p><p dir="ltr">A current promising therapeutic strategy for inflammatory disorders is the inhibition of heparanase by using heparan sulfate mimetics. Heparanase is a ubiquitously expressed enzyme which cleaves its substrate heparan sulfate, a key component of the extracellular matrix. Heparanase is upregulated in multiple diseases including cancer and neuroinflammation. The inhibition of heparanase by heparan sulfate mimetics, which mimic endogenous heparan sulfate, are found to be promising at preventing heparanase-mediated angiogenesis and metastasis in multiple tumour models. These mimetics have also demonstrated promise in disease reduction in the EAE animal model of MS. Although an attractive therapeutic targeting candidate, the complex synthesis, expensive production, and clinical translation is a major downfall of these mimetics. Tet-29 is a novel heparan sulfate mimetic that has been developed by the Ferrier Research Institute comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of Tet-29 is much shorter due to its smaller size and more economical with cheaper starting materials. Tet-29 was proposed to prevent the migration of autoreactive immune cells through the inhibition of lymphocyte heparanase, and rescue blood brain barrier integrity, reducing overall disease in EAE animals. However, investigations from the La Flamme group demonstrated Tet-29’s potential to enhance homeostatic immune cell trafficking into the CNS of healthy animals. This thesis investigates and further characterises the role of Tet-29 on homeostatic immune cell trafficking in the CNS.</p><p dir="ltr">Treatment with Tet-29 <i>in vivo</i> was first shown to maintain immune cell trafficking into the CNS compared to the well-known therapeutic used in MS, natalizumab, which broadly blocks immune cell migration. Additionally, Tet-29 was shown to enhance the migration of specific immune populations into CNS tissue. To further investigate Tet-29’s mechanism, we assessed its effect in IFN-γ dependent CNS trafficking through its co-administration with the innate immunomodulatory microparticle MIS416 <i>in vivo</i>. This combined treatment resulted in a further increase in CNS immune cell trafficking, suggesting a synergistic effect.</p><p dir="ltr">Following in vivo characterisation, we investigated the influence of Tet-29 independently and in combination with MIS416 on choroid plexus phenotype. Immunohistochemical analysis of brain sections from healthy mice treated with Tet-29, MIS416 or in combination revealed Tet-29 had a modest increase in expression of trafficking molecules including ICAM-1, as well as increased leukocyte presence. Furthermore, the combined treatment of Tet-29 and MIS416 significantly increased both ChP CAM expression and leukocyte abundance. Finally, transmission electron microscopy (TEM) was employed to investigate ultrastructural changes of the ChP following therapeutic administration, where preliminary investigations found no changes in ChP ultrastructure between treatments.</p><p dir="ltr">These findings indicate that Tet-29 preserves homeostatic immune cell migration in a specific manner, which possible implications in IFN-γ dependent migration. In addition, this increased migration is understood to be facilitated by the ChP. The research further contributes to our understanding of Tet-29 in homeostatic migration.</p>