MicroRNAs as a novel approach to parasitic nematode control (PhD)

The Challenge

Gastro-intestinal (gi) nematodes of ruminants are a major financial and welfare issue world-wide, including the UK. At present, control is based upon the use of anthelmintics, a situation that is threatened by the continued spread of anthelmintic resistance. We have adopted a novel approach to the control of H. contortus by focusing on defining the function of microRNAs in this important parasite. miRNAs are small non-coding RNAs that regulate the expression of other genes and are essential for the correct development of free-living nematodes such as Caenorhabditis elegans. Our recent results suggest that, in addition to functioning in development, parasite miRNAs may play key roles in modulating host immune responses. We have cloned multiple miRNAs from H. contortus (1) and shown that one of these is abundantly expressed in adult worms, is highly conserved in other gi nematodes and is predicted to regulate the expression of a mammalian gene involved in the activation of T cells. We will investigate the potential of parasite miRNAs to influence infection outcome, specifically by altering the balance of T cell responses.

Many parasitic nematodes produce excretory-secretory (ES) products that suppress host immune responses and that likely interfere with the ability to vaccinate (2). Almost all studies to date have characterised protein components of the ES, but as miRNAs are released by eukaryotic cells and are able to traffic from one cell to another (3), we propose that secreted miRNAs may represent a novel mechanism by which parasitic nematodes down-regulate potentially protective immune responses. Thus the aim of this project will be to characterise miRNAs in the ES of the ovine abomasal nematodes H. contortus and T. circumcincta and determine how these small molecules interact with components of the host immune response to regulate the development of immunity. The outcomes will have important relevance to improving parasite control not only in sheep but also in other ruminant hosts.

The Project

This proposal will adopt a novel approach to the problem of parasitic nematode control in ruminants. Specifically, we will study the role of microRNAs (miRNAs) from the gastro-intestinal parasites Haemonchus contortus and Teladorsagia circumcincta in modulating host immune responses. We will investigate whether inhibiting parasite miRNA function enhances protective immune responses and may offer a novel strategy for nematode control

Adult parasites recovered from donor sheep at post-mortem will be cultured in vitro and ES collected. miRNAs will be precipitated from spent culture medium using standard conditions and characterised by probing a microarray (already available) containing all 192 H. contortus miRNAs identified to date. This analysis will give an overview of all miRNAs secreted by H. contortus. Based on our initial analysis, we anticipate that at least some of the key miRNAs will be conserved in T. circumcincta and identified by our microarray approach. In addition, we will identify selected miRNAs in spent tissue culture medium by RT-PCR using gene-specific primers. 

Subsequent experiments will investigate the interaction of miRNAs with components of the host immune response, starting initially with T cells. We will address the following questions: do parasite miRNAs interfere with activation, proliferation or cytokine production by T cells? Do specific miRNAs regulate the expression of mammalian immune molecules? What is the potential outcome of this regulation on immune responses to the parasite?

These initial experiments will be carried out in vitro using well-characterised approaches and are important to define the experimental parameters for subsequent studies aimed at determining whether miRNAs released in vivo at the mucosal interface affect T cell function. This part of the project will be carried out with Dr Tom McNeilly at the Moredun Research Institute (MRI) with whom we have excellent collaborations. Here we will study the effect of gi nematode infection on aspects of the ruminant immune response using the natural host, the sheep. We will examine whether immune molecules shown to be regulated by miRNAs in vitro are similarly regulated by H. contortus and T. circumcincta infection in vivo, by analysis of archived tissue and abomasal T cells from infected sheep using immunochemistry, qRT-PCR and FACS analysis with appropriate markers/probes; we will seek to ascertain the effect of this immune regulation on the outcome of infection by studying the polarisation of T cell responses in the abomasum; finally we will investigate the potential of locked nucleic acid inhibitors (LNA-miRNAs) to neutralise the activity of secreted miRNAs both in vitro and in vivo. These small molecules are very stable in serum and have been shown to inhibit miRNA function in vivo in other disease models (4), indicating their potential as novel therapeutic agents. Blocking miRNA function may allow a protective immune response to develop and improve vaccination and infection outcome. Thus this project has the potential to shed new light upon the mechanisms by which parasitic nematodes modulate immune responses and offer a novel treatment strategy to control these important parasites.

Student

Henry Gu