Development of transgenic mice to determine the role of intelectins as effectors of gastrointestinal nematode expulsion

Parasitic nematode worms that infest the gastrointestinal tract are an important human health concern in developing countries. Nematodes are a major problem for sheep and cattle farming in the UK, and world-wide, as they are increasingly resistant to anti-parasitic drugs. An alternative approach to drug-induced parasite control is to boost the natural immune reaction to parasites. Parasitism and allergy induce the same type of immune response, which has been studied extensively in rodents. The cells and signals involved are well known, but the way that these change the environment of the parasite and cause it to be expelled is less clear. Our recent work shows that the immune response to intestinal parasites results in levels of a protein, intelectin-2, which is produced by gut epithelial cells in greater amounts than any other protein in the vicinity of the parasite. In contrast, the protein intelectin-1 is present both before and after infection. We wish to test the hypothesis that intelectins, and particularly intelectin-2, contribute significantly to the immune expulsion of gut nematodes, while, at the same time, being aware of and testing alternative hypotheses on the function of this abundant protein. The project will aim to answer the following questions: 1. Does the increase in intelectin protein at the site of worm infestation influence the rate at which the parasite is expelled? 2. How does intelectin interact with other substances, such as mucus, in a way that could enhance rejection of parasites? 3. Do intelectins have anti-bacterial properties? We will use genetic engineering to create three new mouse lines in which to investigate the role of intelectins. Firstly, we will create a mutant of the 'C57BL/6' mouse strain, which naturally lacks the intelectin-2 gene, and make the mutant express the intelectin-2 protein. Furthermore, we will make 'knockout' mice on the 129 strain, which normally express intelectin-1 and intelectin-2, and make one line which is no longer able to make intelectin-2, and a second line that is unable to make either intelectin. By infecting these mice with worms and studying the rate of expulsion of the worms from the intestine of the mouse, we will be able to determine whether the ability to make intelectin proteins affects parasite rejection. We will also examine the properties of purified intelectins to see how they might relate to possible parasite rejection mechanisms. For example, does intelectin bind to the parasite or to bacteria? Does intelectin bind to mucus to make it thicker and help expel the parasite by engulfing it? At the end of the project, we will be in a position to say whether or not intelectins contribute significantly to parasite expulsion, and will know more of their properties. If intelectin does indeed help to expel parasites, then understanding how it works will help in the future design of vaccines to boost its parasite expelling effect. In terms of allergy, if intelectin causes mucus thickening, then it may similarly contribute to the life-threatening mucus plugging of the airways in acute asthma, and will therefore become an important new drug target.

Intelectins are novel, evolutionarily conserved molecules, the biology of which is largely unexplored. The intelectins are secreted at mucosal surfaces and as such may represent innate effector molecules produced in defence against intestinal pathogens. Our own recent published data has implicated the intelectins as components of the protective immune response to intestinal nematode parasites. Specifically, in mice, the small intestine constitutively expresses intelectin-1 in Paneth cells, and intelectin-2 is induced in goblet cells in response to parasite infection. We note that the C57BL/6 strain, which naturally lacks the intelectin-2 gene, expels parasites poorly compared to strains which express intelectin-2, such as BALB/c and 129. We propose to explore the biology of the intelectins through the use of transgenic mouse lines, and to investigate their properties with respect to putative anti-microbial, anti-parasitic and mucus-cross linking functions. Our main objectives are to: 1) Determine the sequence of intelectin genes in the 129 strain 2) Create intelectin-2 transgenic mice on the C57BL/6 background 3) Create intelectin-2 knockout mice on the 129 strain 4) Create intelectin-1/intelectin-2 double knockout mice on the 129 strain 5) Analyse the parasite expulsion kinetics and bacteriology of the above transgenic vs wild type strains of mice during infection with Trichinella spiralis. 6) Define physicochemical properties of intelectins, including i) expression / purification of functionally active intelectins, and ii) determining antibacterial, mucin binding and nematode binding properties of these products. 7) Disseminate the results and conclusions of the study By these approaches, we will be able to answer key questions regarding the regulation and function of intelectins during intestinal nematode infections. Understanding the basic biology of the intelectins will impact on our understanding of other diseases such as allergic asthma.