Iloprost treatment enhanced IL-4 signalling to reverse trypanosome-induced cognitive deficits and suppressed neuroinflammation in mice

Abstract

Introduction: The development of neurological disorders following trypanosome brain infection has been recognised for over a century. However, an ongoing endeavour persists to comprehensively understand the neurocognitive and neuropathological changes accompanying such infection and the disease progression. Efforts are ongoing to screen for effective new trypanocidal compounds. Therapies now favor activating M2 macrophages via IL-4 or IL-13 anti-inflammatory cytokines. This study aims to investigate whether prostacyclin analogue (Iloprost) treatment against CNS Trypanosoma brucei infection would beneficially affect cognitive and anxiety-like behaviours in IL-4/IL-13-expressing mice, and to determine if trypanosome-induced neurocognitive alterations can be therapeutically reversed and to elucidate the underlying molecular and epigenetic mechanisms. Methods: Sixty-four male albino Swiss mice (8-10 weeks old) were used, divided into two groups (n=32 per group): IL-4Rα-/- and wild-type (WT) littermates. At day 4 post-infection with Trypanosoma brucei brucei, both groups received intraperitoneal Iloprost (200µg/kg) for 3 days. The mice were assessed for locomotion and anxiety-like behavior. Blood samples were collected for parasitaemia and hemogram tests. Target gene transcripts in the prefrontal cortices (PFC) and hippocampal (HPC) tissues were quantified using RT-PCR. Cytokine expression and methylation profiling were assessed using ELISA and EZ DNA methylation kits. Results: IL-4R inhibition induced systemic inflammation in IL-4R-/- mice, whereas Diminazine treatment reversed decreased vertical exploration in the open field and revealed reduced anxiety-like behaviour in these animals. Molecular analysis shows IL-13 gene downregulation in both PFC and HPC. IL-4 and IL-4R genes are only downregulated in the HPC of IL-4R-/- infected mice, indicating different responses to infection in distinct brain regions. Furthermore, Iloprost and Diminazine treatments not only reversed downregulated BDNF gene expression in the PFC but also attenuated the upregulation of TNF-α genes in both brain regions, while simultaneously suppressing the presence of trypanosome parasites in the CNS parenchyma by decreasing Pfr gene expression. We show that mice with hypomethylation of IL-4, IL-4R, IL-13, and BDNF in the PFC and HPC (WT) exhibited these changes. In contrast, iloprost-infected groups (WT and IL-4R-/-) had hypermethylation of TNF-α, IL-4R, IL-4, and IL-13 in the PFC and HPC. Iloprost significantly increased NF-kB levels (P<0.05) and decreased TNF-α in the Hippocampus and PFC, as determined by ELISA. Discussion: This study revealed that Iloprost promotes angiogenesis and suppresses trypanosome-induced inflammation. Knowing that IL-4 has been implicated in cognitive learning and the established anti-inflammatory roles of prostacyclin show that Iloprost treatment can potentially strengthen IL-4/13 signalling via IL-4Rα to reverse anxiety and cognitive deficits associated with CNS trypanosome infection. Conclusion: This study suggests that Iloprost plays a role in suppressing neuroinflammation of Trypanosomiasis in mice. Our findings suggest that Iloprost activates the IL-4Rα pathway by upregulating BDNF and IL-4 signalling, potentially contributing to its protective effects against cognitive impairment in trypanosomiasis.