All animals exhibit behavioral changes due to infection. Conspicuously, some of these changes are quite general: for example, people and other animals with a very wide variety of different infections tend to show reduced appetite and disruptions to circadian behaviors (like sleep). These general effects of infections on behavior are broadly called "sickness behaviors". Sickness behaviors have significant effects on human health and well-being, but they also have real costs in other animals. For example, reduced feeding due to infections in cattle reduces beef quality and quantity, and similar effects exist in other livestock.

The fact that sickness behaviors are so widespread among animals suggests that they must be an important part of the immune response, but we know relatively little about their actual function. We also know relatively little about their cause, in most cases: many different signals are produced by the immune system in response to infection, and most of these signals have effects on the nervous system, but the actual specific immune signals responsible for any particular sickness behavior tend to be difficult to pin down.

The experiments we propose to perform here focus on two sickness behaviors-reduced feeding and circadian disruption-in a fruit-fly. We have found a specific gene that is turned on in the fruit-fly in response to infections. This gene produces a signal that is received by brain cells. We have found that this signal makes the fruit-fly eat less when it has an infection. One aspect of the work we propose here is to understand how infections turn on this signal, and where the signal is coming from-for example, is it coming from blood cells or brain cells? This is important for the fly because flies that cannot receive this signal have reduced ability to fight infections; it is important for people and other mammals because we also produce the same signal in response to infections. Understanding how this signal is turned on, what cells produce it, and its functions in the fruit-fly will help us understand what kinds of changes can be generated by the same signals in humans and how we can manipulate these signals to promote better immune competence or less disease pathology.

We also would like to understand why it is important that we eat less when we are sick. Our laboratory has previously shown that fruit-flies, like people, alter their metabolic activity in response to infections; we have recently discovered that the fruit-fly must shut down critical energy-storage functions in order to raise a productive immune response. One way to shut down energy storage is to eat less. Thus, our prior work suggests that sickness behaviors might be directly required to produce an immune response. The work we propose to do here will test this: we will learn how important reduced feeding actually is in generating a functional immune response. This kind of experiment is difficult and expensive in mammals, but it is comparatively easy and quick in flies, and it will allow us to ask more-focused questions in experiments on humans and mice.

Technical Abstract:
This proposal focuses on the role of a neuropeptide, Dh44, and one of its receptors, Dh44-R1, in disruption of feeding and circadian behaviours by infection in Drosophila.

All animals exhibit behavioural changes due to infection. Some of these changes are infection-specific, but most infections cause a common suite of behavioural changes, collectively called "sickness behaviours". Two well-documented aspects of sickness behaviour are decreased feeding and disruption of sleep/wake activity patterns. Infection-induced behavioural changes have significant effects on human health and well-being, but they also have real costs in other animals. For example, infection-induced anorexia in cattle reduces beef quality and quantity, and similar effects exist in other livestock.

Two strands of the work in my laboratory have recently come together to focus our attention on sickness behaviour, especially feeding suppression, and its connection to immune function. First, we have demonstrated that the nutrient-regulated kinase p70S6K is shut down in infections, and that this is critical for the function of the inducible immune response in Drosophila. Second, we have identified a mutant in the neuropeptide receptor Dh44-R1 as immunocompromised in a forward genetic screen. We have subsequently shown that Dh44, the Dh44-R1 ligand, is induced by infection and is required for infection-induced feeding suppression. Others have shown that anorexia regulates immune competence in flies, but the underlying mechanism is unknown. Our observations suggest that suppression of feeding may promote immune function by reducing S6K activity.

The work proposed here addresses these questions:

1. Where is infection-induced Dh44 expressed? What signals drive its expression?
2. What cells receive the Dh44 signal to inhibit feeding and drive other sickness behaviours?
3. Is Dh44-driven feeding suppression a key driver in shutting down systemic nutrient signalling to promote immune function?

Potential Impact:
Societal beneficiaries
Direct medical and veterinary implications of this work include clearer identification of mechanisms of sickness behaviours. Sickness behaviours can help immune function or drive pathology; deciphering both aspects of their biology will help identify targets for intervention that may open up new avenues of therapy. Drugs targeted to these systems may be useful in humans or other animals.
Indirect scientific impacts may also produce significant feed-through into societal impact. By improving understanding of Drosophila inflammation/immunity and sickness behaviour, we will expand scientific knowledge of how Drosophila can be applied to human and animal health and disease. This will facilitate the use of this system by others to address other medically-relevant issues.
This work will have significant 3R's impact (see also "Industrial Interactions"). By showing the use of Drosophila to examine complex issues in inflammation and immunity, we will popularize the use of this system alongside existing tissue-culture and whole-animal models. With support, this work will permit refinement of genetic hypotheses in whole-animal infection experiments.

Industrial interactions
This research may generate commercially-exploitable intellectual property and research tools. Targeted regulators of sickness behaviour are an unexplored avenue in pharmaceutical research, in part because of the remarkable difficulty in screening directly for regulators in whole mammals. By demonstrating the utility of Drosophila, we will attract industrial attention to this field as a whole. We will use existing industrial outreach structures at KCL to identify these opportunities and we will forge interactions that may result, for example, in CASE awards for researchers at the academic/industrial interface.
We have already had success in this regard: a GSK/BBSRC-funded CASE PhD student will start next month on a project extending from in vivo analysis in Drosophila to in vitro analysis in primary human cells.

Outreach and education
To reach as many researchers as possible, we will present our work at multiple conferences. All research findings funded by this grant will be published using open-access mechanisms (such as Europe PMC). This allows free access to our work by the scientific community and the public.
We make every effort to promulgate our findings to the public. King's College London organizes numerous opportunities to lecture on our findings to lay audiences. We will work with the KCL Press Office and BBSRC public outreach department to publicize our findings.
Within the KCL School of Medicine, I have worked to improve knowledge among medical students and working clinicians of the medical consequences of biological findings. Scientist-clinician contact is also a goal of the Centre for Molecular and Cellular Biology of Inflammation.

Training
This project will provide useful training for the postdoc as well as for the PhD students in the laboratory. This work is at the intersection of behaviour, metabolism, genetics, and immunology; the postdoc will learn techniques lying in all of these disparate areas. This training is critical for future scientists, who will need diverse skills to address complex problems. The exposure to first-rate immunology and autoimmune disease research in CMCBI is also critical training aspect.
The postdoc will also receive training in career skills, such as organization of projects, supervision of students, grantsmanship, manuscript writing, interpersonal skills and presentation skills. This training will be undertaken informally within the lab as well as formally. For example, postdocs will be encouraged to attend media training, either through BBSRC or through the KCL press office. Postdocs and PhD students present their work at internal seminars (at least once per year) and at at least one external conference per year. Informal presentations within the group take place weekly.