Disease susceptibility and gut health in the wild: Determining interactions between diet, gut microbiome and immunity.

Parasitic worms are extraordinarily common infections of humans, livestock and wildlife. They cause ill heath, a reduced ability to put on weight, reduced productivity and economic loss. Drugs exist to tackle parasite infections but as parasites develop drug resistance, these sorts of control methods lose effectiveness. Thus, discovering new ways to limit infections is important. The body's immune system is a natural defence system that protects the body from infections and disease. However, it has to contend and adapt to the many environmental variables that an individual is exposed to, such as a changing diet for example. These variables will differ between individuals at any one point in time, and over time in the same individual. Understanding the factors which enable infections, such as parasites, to be cleared efficiently from the body is important. Defending against infections takes energy and therefore one might predict that individuals with good quality diets might be able to deploy more energy in to the immune system and so respond more effectively to infectious threats.

Laboratory studies have shown that changing the diet of laboratory mice in otherwise very controlled systems can change the way the body defends itself against parasites. Further, in wild wood mice, which face many challenges in their natural environment (for example avoiding predation, finding food), a better quality diet improves their ability to control parasites. However, the mechanism by which a good diet helps parasite clearance is unknown, particularly in natural settings. Indeed changes in diet are known to not only alter the body's immune system but also the collection of bacteria living within the intestine, the microbiome. The gut microbiome itself plays an important role in supporting an individual's health. It is therefore important to understand how diet affects several factors at once, including the microbiome and the immune response, to support fighting of parasitic infections.

The observations in wild wood mice are powerful but this study system is limited in the tools available to understand how diet promotes resistance to infection. However, a wild house mouse population - the same species as laboratory mice - offers both the laboratory reagents required to study the body's immune system and a real world context.

We therefore propose to study a wild house mouse population on the Isle of May. We will provide an improved diet to one group of mice and compare their anti-parasite immune response and the collection of bacteria living in their guts to those of mice which do not receive an improved diet. Further, to understand the importance of the microbiome versus the immune system we will also treat groups of mice with an antibody, which will block the immune-based anti-parasite defence. In this way we can tease apart whether the diet-driven changes in parasite clearance are acting via the immune system.

To assess the relevance of diet-driven microbiome changes, we will collect faeces from wild mice on an improved diet versus a normal diet and transfer the faeces to laboratory mice that do not have any bacteria in their intestines. Subsequent infection with a parasite will test if we have transferred an improved ability to clear the worm and additional experimental removal of the anti-parasite immune response will confirm whether the diet-enhanced microbiome operates via the immune system.

Overall, this project will disentangle the ways in which an improved diet supports health and protects against parasitic infection. It will do so by using a novel, interdisciplinary study design bridging between controlled laboratory mouse studies and investigations in wild mouse populations. This allows both detailed and mechanistic investigation as well as taking into account the real-world environment in which an individual's immune system operates.

We will study wild house mice in a multivariate environment. We will trap mice for 2 months/year for 3 years over 2 96-trap grids. We will tag up to 600 mice allowing a cull of >240 mice. At first capture mice (day (d) -4 to -1) will be tagged, sexed and measured, blood and faecal samples collected for immunological, parasitological and microbiome analyses, and released at capture site. At next capture (d0-2) mice will be randomly allocated to 1 of 4 groups: diet supplementation (DS) plus control Ig (cIg), DS plus anti-IL4 treatment (11B11), no diet supplementation (NDS) plus cIg, NDS plus 11B11, intraperitoneally injected with 1.5mg of 11B11 or cIg, and released at their trap site. Food supplementation will be provided continuously via feeding stations, equipped with RFID loggers. Animals recaptured on d5-7 and/or 10-12 will receive further doses of 11B11 or cIg. 2 weeks after treatment start, recaptured animals will be culled.

Tissues will be taken for: endoparasite burden, age, serum antibody/cytokine levels, faecal IgA and inflammatory markers, gut microbiome composition. Proximal colon samples will be stored in:
(a) RNA later for qPCR/RNA-Seq
(b) methacarn for histopathology
(c) snap-frozen in OCT for immunohistochemistry

Each year, caecal contents from the DS and NDS cIg groups will be collected and frozen. Prior to faecal transplant, samples from mice which have entered feeders >8 times will be assessed by 16S rRNA sequencing for exclusion of outliers, and pooled within groups (>15 animals/group). GF mice will be gavaged with pooled faecal slurries selected from DS or NDS wild mice; one group of GF mice will receive a faecal slurry from SPF laboratory mice; faecal transplants will be allowed to stabilise over 3 weeks prior to low dose T.muris infection. Within each of the 3 faecal transplant groups, mice will receive 11B11 or cIg. Mice will be culled 21 days post-infection and analysed as above.