Investigating regulatory mediators during chronic Trichuris muris infection

Abstract

Human trichuriasis is a disease caused by infection with the soil-transmitted gastrointestinal (GI) nematode Trichuris trichiura (T. trichiura), accountable for approximately half a billion infections worldwide. Natural infections in man are often chronic, with the parasite able to regulate the host immune response to facilitate its own survival. Exactly how Trichuris facilitates chronicity is unknown. However, work using the murine colonising species of Trichuris, Trichuris muris (T. muris), a species of Trichuris that is genetically and phylogenetically parallel to T. trichiura, permits laboratory investigations to decipher the immunological responses evoked upon parasite infection. Work within this thesis aimed to investigate the regulatory mediators of chronic T. muris infection in C57BL/6 mice, in an effort to understand regulatory mechanisms that facilitate chronicity. Firstly, it was shown that counter to expectations goblet cell expansion was evoked in C57BL/6 mice following chronic T. muris infection; a Th1 mediated response. Paradoxically, goblet cell hyperplasia was dependent on adaptive derived IL-13 and IL-22, showing that Th2 immune responses can operate within a Th1 setting. These mice also provided an excellent platform to assess the mucus associated proteome during homeostasis and chronic infection, as the mucus gel may provide a source of regulatory proteins that influences parasite and host survival. Initially, it was important to optimise a protocol to extract the mucus gel with minimal disruption to the tissue epithelium. Sequential washes with PBS and 2M urea were utilised to remove the mucus gel of the large intestine and samples were extracted at day 35 from naive and chronically infected mice. After tandem mass spectrometry analysis, several mucus associated proteins found to be exclusive to infected caecal and colonic mucus samples were identified, including Complement 3, Murinoglobulin-1 and Haptoglobin (Hp). The most notable finding was the identification of Hp, a protein that is predominantly expressed by liver hepatocytes and found in serum. Investigations to determine the functional role for Hp following T. muris infection showed that increased levels of Hp were not simply derived from the serum. mRNA and protein levels of Hp were increased during infection, locally within the intestine at the site of infection (i.e. the caecum), but not at non-infected sites (i.e. the colon). Using Hp knock-out (KO) mice and wild-type (WT) counterparts it was demonstrated that Hp deficiency increased Th2 associated responses during a chronic T. muris infection, including an increase in GATA3+CD4+ T cells, total IgE and parasite specific IgG1 production. It was also shown that neutrophils were the cellular source of increased Hp mRNA expression in the caecum following T. muris infection. These results imply that neutrophils can influence the immune response to T. muris and that neutrophil derived Hp may help promote chronic infection. Upon further investigation into the T. muris induced neutrophil response it was demonstrated that the cell surface expression of PD-L1 (a check point inhibitor ligand) was increased on populations of caecal neutrophils. Experiments also showed that caecal neutrophils maintained PD-1 expression within CD4+ T populations within the infected caecum and that neutrophils regulated caecal crypt hyperplasia. Transcriptomic analysis of caecal neutrophils during chronic T. muris supported these observations with significant increase in transcripts for Pd-l1 and Hp and genes associated with intestinal crypt hyperplasia (i.e. cxcl10 and Ido1). Taken together, data from this thesis extends our understanding of immune regulation during chronic T. muris infection and identifies novel roles for intestinal neutrophils in immunity to parasitic infection and the regulation of intestinal inflammation in the wider sense.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Richard Grencis (Supervisor) & David Thornton (Supervisor)