Innate immune regulation of infection tolerance

All living organisms have immune responses to protect them from infections. We regard these immune responses as working in two basic ways: "resistance" responses protect the host by killing invaders, while "tolerance" responses protect the host by preventing damage caused by infection. These two types of response must work together: resistance without tolerance cannot keep us alive in the presence of many pathogens, while tolerance without resistance will result in an increase in both the abundance and proliferation of pathogens, leading to infections against which our immune systems will be defenseless . Many resistance mechanisms are relatively well-understood, but we know very little about how infection tolerance works. For example, even though tolerance has been cited as a potential therapeutic intervention, we still don't know whether tolerance is generally activated by immune responses ("inducible" tolerance), or if tolerance is a pre-existing state. We also don't understand how tolerance and resistance work together in controlling infections and keeping the host healthy.

In our lab, we study the immune response of the fruit-fly Drosophila melanogaster to bacterial infection. The fruit-fly's immune response is similar in many ways to those of humans and other mammals, but because flies are small, inexpensive, and have a short generation time, we can study immune responses to infection in the whole animal in ways that are impossible in mammals. We have recently discovered that flies with a genetic mutation in a "resistance" gene also have a serious defect in infection tolerance: when these mutants are infected with pathogenic bacteria, they die significantly sooner than non-mutant flies, but show no difference in terms of resistance (mutants and non-mutants contain the same number of bacteria). Thus, the immune response of these flies suggests that tolerance may be an inducible trait. This is very exciting because it allows us to identify the mechanisms of tolerance that are being activated by this gene.

The experiments we propose will focus on three aspects of this tolerance effect. First, we will measure metabolic and behavioural differences between mutant and non-mutant flies during infection. This is important because metabolic control and behaviour have been suggested to be critical determinants of tolerance, but we don't really have any grasp on how these two things would be different between tolerant and non-tolerant hosts. Second, we will test the roles of related genes in affecting tolerance, and determine which tissues these genes are functioning in to promote infection tolerance. This is important because it will give us insight into the mechanisms of tolerance induction, as well as allow us to determine to what extent tolerance is determined by host genetics. Finally, we will use this information and information on genes turned on by infection, to identify the genetic mechanisms of tolerance induction. This is important because it will let us know the specific mechanisms that can promote tolerance in the host, separate from effects on resistance.

This work may ultimately lead to the ability to promote tolerance in people with infections. Therapies that promote tolerance would be useful to allow the immune system or antibiotics time to work. Thus, we hope that our analyses of infection biology in fruit flies will give us information that will ultimately be useful in the treatment of human disease.

Hosts survive infection by engaging mechanisms that combine resistance and tolerance. Resistance mechanisms include responses to infection that protect the host by killing pathogens or restricting their growth; this is the immune response as we normally envision it. In contrast, tolerance is defined as the ability to maintain health during infection; experimentally, a more tolerant host is one that remains healthy longer at a given pathogen load. Recent years have seen increasing interest in tolerance, driven in part by the idea of developing tolerance-based infection therapies.

We show here that Drosophila melanogaster imd mutants exhibit a strong tolerance defect when infected with two different bacterial pathogens. imd encodes the Drosophila homolog of the TNF pathway component RIPK1 and plays a well-established role in infection resistance; the fact that we observe a tolerance defect in these infections implies that infection-induced imd signalling triggers infection tolerance as well as resistance. The experiments we propose will identify the mechanisms underlying imd-driven infection tolerance.

We propose three aims. First, we will identify the physiological parameters associated with the tolerance difference between imd mutants and wild-type flies. Second, we will determine in which tissues imd is required to promote infection tolerance and which imd signalling mechanisms are relevant to tolerance. Third, we will identify the genetic mechanisms acting downstream of imd by functional screening of imd target genes; we will then identify the specific functional connections between imd targets, the physiological effects identified in the first aim, and realised infection tolerance.

By identifying the genetic and physiological mechanisms behind imd-driven infection tolerance, we will shed light on in vivo mechanisms protecting animals from damage due to infection.

Societal beneficiaries
Because our work is in fruit-flies, it is unlikely to have direct translational effect (though you never know; we will be alert to such potential). However, public interest in infectious disease, and in how we can stay healthy despite infection, is high; we hope to be able to offer factual information on this topic.
We hope that our programme of work will have significant 3R's impact on the large scale (see also "Industrial Interactions"). By showing the use of Drosophila to examine complex issues of immune regulation during infection, 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 experiments on mammals.

Industrial interactions
This research may generate commercially-exploitable intellectual property and research tools. Targeted regulators of immune function are an area of high topical interest in the pharmaceutical industry. 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 Imperial 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 is currently in the lab, working 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. Imperial College London organizes numerous opportunities to lecture on our findings to lay audiences. We will work with the Imperial Press Office and MRC public outreach department to publicize our findings.

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 immunology, microbiology, and physiology; Dr Vincent 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 research in infection and immunity at Imperial is also a critical aspect of this training.
Dr Vincent will also receive training in career skills, such as organization of projects, supervision of students, grantsmanship, manuscript writing, 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 MRC or through the Imperial 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.
As evidence of my prior success in training of this kind, I would point to Rebecca Clark, my first postdoc, who spent four years in a BBSRC-funded position in my laboratory; Rebecca is now a lecturer at the University of Durham.