Immunological studies in the brain and signaling pathways in experimental African trypanosomiasis

Sammanfattning: African Trypanosomiasis is a disease prevalent among humans and animals in tropical Africa which is caused by the extracellular hemoflagellate Trypanosoma brucei (T. b.). The infection is initiated when a tsetse fly commences a blood meal, the mammalian infective forms of the parasites (or the metacyclic forms) are inoculated subcutaneously with the tsetse fly saliva. The late phase of the disease is characterised by central nervous system (CNS) involvement. Trypanosomes are highly adapted to life within their host and in particular are capable of evading the host immune response by altering their outer body variant surface glycoprotein (VSG). Moreover, trypanosomes manipulate the host immune system by releasing a specific molecule, trypanosome lymphocyte triggering factor (TLTF), that binds to CD8+ T-cells and activates them to produce IFN-gamma, which in turn triggers proliferation of the parasite. Neuronal IFN-gamma (NIFN-gamma) is a molecule that bears the same biological activities of lymphocyte IFN-gamma but differs in molecular mass. N-IFN-gamma has been shown to support trypanosome growth. Gene transcription for growth and other biological activities in the trypanosome is directed by RNA polymerases but transcription initiation sites or DNA sequences for polymerase binding have not yet been identified. However, the intracellular signaling pathways used by IFN-gamma to induce parasite proliferation and the role of different transcription factors are not known. To further dissect the dynamic relationship between parasite and host immune system, this work aimed to study the induction of inflammatory and proinflammatory cytokines in the brain and to investigate the role and cellular source of chemokines in the brain during early infection. Other important goals of this work were to study the effect of TLTF on N-IFN- gamma production by dorsal root ganglion nerve cells, the downstream signals in the IFN- gamma signaling pathway, and also the effect of IFN-gamma on trypanosome parasite gene transcription. We evaluated the early induction of 5 potential cytokines in the brain, cerebrospinal fluid (CSF) and spleen of T. b. brucei-infected and uninfected Sprague-Dawley rats. IFN- gamma and TGF-beta were found to be highly expressed in all compartments, but low levels of IL-4, IL-10 and TN17-u mRNA were seen when compared to control rats. The pattern of these cytokines reflected the necessity of each of them in this model. Single and double immunohistochemistry was utilized to evaluate (X- and beta-chemokine induction in the brains of Sprague-Dawley rats infected with T. b. brucei and to identify the cellular sources of these chemokines. High production of MIP-2, RANTES and MIP-1alpha and to a lower extent, MCP-1 was found in infected animals compared to controls. MIP-2, RANTES and MIP- 1alpha were produced early by astrocytes and microglia and later by macrophages and T- lymphocytes. These findings suggest that chemokines may be involved in CNS immunopathogenesis during the early phase of experimental African trypanosomiasis. In vitro stimulation of DRG neurons with TLTF activated them to produce N-IFN-gamma. Western blotting of TLTF-stimulated DRG lysates demonstrated that the mechanism of N-IFN-gamma production involves tyrosine phosphorylation activity and translocation of the transcription factor STAT-1 to the nucleus of these neurons. The activation of this pathway was blocked by the addition of tyrphostine A47, a specific tyrosine kinase inhibitor. Using Electophoretic Mobility Shift Assay (EMSA), transcription factors binding to IFN-gamma stimulated trypanosome DNA were investigated. AP-1, E74, hSIE and NF-kappaB expression was investigated in IFN-gamma -stimulated trypanosomes. Transient and prolonged up-regulation of AP-1, E74 and hSIE was observed but not for NF-kappaB, suggesting that IFN-gamma could be strongly involved in activation of developmental gene transcription in trypanosomes.