Investigation of the early immune events in African trypanosome infections

Ajibola, Olumide (2015) Investigation of the early immune events in African trypanosome infections. PhD thesis, University of Glasgow.

Abstract

African trypanosomes, the causative agent of sleeping sickness in humans, and nagana in cattle, are typically transmitted by the bite of an infected tsetse fly. The nature of the mammalian innate immune response during and immediately after the bite of an infected tsetse fly remains poorly understood. Previous studies characterising the events occurring in the skin post-infected tsetse fly bite have mainly focussed on the development of the chancre, which occurs from day 5 post-infection. Additionally, most immunopathological studies on trypanosomes have used intravenous or intraperitoneal injections of blood stage parasites, therefore bypassing relevant inoculation routes (tsetse fly), site (skin), and parasite life cycle stages (metacyclics). It is known that following tsetse fly bites, trypanosomes leave the skin via the host lymphatic system in order to initiate a blood stage infection. However, how the host responds to this challenge and how the parasite negotiates the anatomy of the host immune system remains unclear. In the present study, I have built on existing intravital microscopy tools to visualise T. b. brucei infections in the dermis and lymphatics of an infected mouse ear after transmission. I have also characterised by flow cytometry, taqman low density arrays and depletion studies the magnitude and kinetics of the early innate immune response in the skin, as well as the functional role of neutrophils, by examining infections in the context of the natural route of infection- the bite of a tsetse fly. Neutrophils were identified to be the predominant responders at the bite site, the neutrophil response was rapid, and they were recruited independent of the infection status of the tsetse flies. Taqman low-density arrays, which measured expression levels of inflammation-associated genes, suggested that neutrophil recruitment was mediated by CXCL1/CXCL2 release in the skin following mechanical trauma by the tsetse fly, in addition to the release of pro-inflammatory cytokines- IL-1β and IL-6. Following the identification of neutrophils by flow cytometry, I then applied intravital microscopy to visualise influx of neutrophils, which was rapid, directed at the bite site, and did not form dynamic clusters. To further test the functional role of neutrophils very early in infection, neutrophils were depleted using a monoclonal antibody and mice infected via tsetse fly bites. Neutrophil depleted mice had no effect on pathogenesis in vivo. Using Prox-1 mOrange reporter mice, I also examined the interaction of bloodstream trypanosomes with lymphatic vessels in the skin in the period immediately following inoculation using intravital imaging. I imaged metacyclic trypanosomes in situ and demonstrated that they had significantly higher velocity in the extravascular matrix compared to bloodstream forms. Additionally, my data showed bloodstream parasites actively migrating towards lymphatic vessels, and intra- lymphatic T. b. brucei were also observed, enabling comparison of trypanosome motility in the extravascular matrix and lymphatic vessels; in lymph vessels trypanosomes were moving in a more directional and rapid manner. This work revealed the early cellular and molecular responses to T. b. brucei infection and investigated interactions of parasites with the anatomy and cells of the host immune system. These studies demonstrate that furthering our understanding of the very early events in trypanosome infections is essential to understand how a systemic trypanosome infection is established.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: T. b. brucei, tsetse fly, neutrophils, lymphatics
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Parasitology
• Supervisor's Name: Brewer, Prof James
• Date of Award: 2015
• Depositing User: Dr Olumide Ajibola
• Unique ID: glathesis:2015-6776
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Jan 2016 08:46
• Last Modified: 27 Jan 2016 09:53
• URI: https://theses.gla.ac.uk/id/eprint/6776