MECHANISM OF INFLAMMATION IN ENVIRONMENTAL ENTERIC DYSFUNCTION

Malnutrition is the greatest barrier to healthy growth and development for children living in poverty. It affects more than 150 million children under 5 years old, and because it dramatically increases vulnerability to severe infection it is indirectly responsible for almost half of all global child deaths - more than 3 million every year. Malnutrition stops children from reaching their potential in growth, development, and education, preventing them from escaping poverty, and increasing the risk that their children, in turn, will grow up in hardship.

Surprisingly, malnutrition often fails to get better even if children are provided with a healthy diet. We do not fully understand the reasons for this, but it may be due to a condition called Environmental Enteric Dysfunction (EED). Children with EED have inflamed intestines, possibly due to living in a unhygienic environments with constant exposure to disease-causing bacteria and other microorganisms. Inflammation prevents them from absorbing nutrients properly and has an energy cost in itself, meaning that nutrients which are absorbed can not be used for growth.

Our understanding of the root cause of EED is remarkably poor. This is partly because it is difficult to know exactly what is happening in children's intestines without taking a biopsy, which would not usually be ethical or practical in resource-limited settings. Recently, my collaborators have found a possible way around this constraint. They have identified that a condition very similar to EED can be induced in laboratory mice by feeding them a malnourished diet and exposing them to a cocktail of bacteria. The objective of this Fellowship is to use this 'mouse model' of EED to try and find out exactly why and how it arises.

1. Millions of bacteria live inside our intestines. They are prevented from travelling into our tissues and causing us harm by the existence of a barrier, which is made by intestinal epithelial cells (IEC). I will use a variety of laboratory techniques to look at how well this barrier is performing in mice with EED and to see whether it is permitting intestinal bacteria to enter our tissues and activate white blood cells (WBC, whose role it is to identify and fight infection). I will induce EED in mice with genetic modifications that alter either IEC's or WBC's capacity to detect bacteria in order to see whether responding to bacteria that have managed to get into our tissues is good or bad. My suspicion (hypothesis) is that bacteria making it across the intestinal barrier lead to sustained activation of WBC, that this activation has unhelpful consequences for IEC, worsening their ability to sustain the intestinal barrier, and creating a vicious cycle.

2. I will develop a test-tube-based system that replicates what is happening in the intestine of a mouse with EED and use it to characterise signalling between WBC and IEC in order to pinpoint chemical signals that might interfere with barrier function. I will use genetic and drug-based approaches to see if these signals can be interrupted both in the test-tube system and in mice. I will assess whether this reduces the severity of EED in mice, and make sure that it does not increase vulnerability to infection.

3. In order to test the applicability of my findings to human populations I will recruit a group of adults who either have EED or are healthy (controls) in Zambia. Volunteers will undergo an endoscopy (under sedation and with fully informed consent) and I will use biopsy samples to replicate the test-tube system established in EED mice. I will look to see if the same chemical signals are present in man as in mouse, and gain the most detailed picture yet of what happens to intestinal WBC in EED.

The long-term aim of my work is to determine how to alter WBC (immune) function to ameliorate the harms caused by EED. By getting rid of EED we may help to support healthy growth of children living in poverty.

BACKGROUND: Malnutrition is responsible for 45% of global child deaths, and via long-term effects on health and development it perpetuates poverty across societies and through generations. Chronic malnutrition (stunting) fails to respond to improvement in diet and may be driven by Environmental Enteric Dysfunction (EED), a highly-prevalent, sub-clinical inflammatory enteropathy. The mechanism of inflammation in EED is undefined.

OBJECTIVES:
1. To characterise intestinal epithelial cell (IEC)-dependent barrier (dys)function in a murine model of EED.
2. To test the hypothesis that endogenous inflammatory signals from lamina propria leukocytes (LPL) are induced by bacterial translocation and exacerbate IEC-dependent barrier dysfunction, thereby establishing a vicious cycle in EED.
3. To identify endogenous inflammatory pathways and mediators contributing to IEC-dependent barrier dysfunction in EED and to prove the concept that interruption of these pathways improves mucosal immune homeostasis in EED.
4. To assess applicability of findings from the murine model of EED to human populations by extending the IEC monolayer system to investigate consequences of signalling by LPL isolated from patients with EED on IEC-dependent barrier function.

METHODOLOGY: Murine work will centre around a model of 'EED' driven by provision of a moderately protein- and lipid deficiency diet with induction of dysbiosis. The same conditions will be applied to strains with cell type-specific modulation of microbial recognition and response pathways. Complementary work with IEC monolayers will allow elucidation of the endogenous pathways and mediators responsible for impeding IEC-dependent barrier function

OPPORTUNITIES: The results will help to generate adjunctive immunomodulatory approaches that could be subjected to clinical trials. By targeting redundant inflammatory activation I aim to develop strategies to help support healthy growth of children living in poverty.

IMPACT IN THE ACADEMIC COMMUNITY

Enhancing Knowledge: This Fellowship will enhance our understanding of Environmental Enteric Dysfunction (EED) and the pathogenesis of malnutrition and stunting in resource-limited settings. It has the potential to provide direct mechanistic data that could dramatically shift the prevention/treatment landscape and open up a new area of clinical investigation (immunomodulation) within 5 years. Knowledge and conceptual impacts will be delivered by peer-reviewed publication, presentation of research outputs at International conferences, and via press releases.

New Methodologies: The Fellowship will involve the development and refinement of a new method of studying intestinal barrier function in vitro (using intestinal epithelial monolayers with autologous/homologous lamina propria leukocytes and microbiota). This method has potential impact far beyond EED, because intestinal barrier dysfunction has been implicated (with varying levels of evidence) in the pathogenesis of a wide array of intestinal and systemic inflammatory diseases, allergy, and gastrointestinal neoplasia. The method will be written up as a stand-alone open access methods paper and published with raw participant-level data where appropriate alongside full laboratory protocols in order to encourage and enhance uptake of the techniques by other researchers.

Creation of Resources: While this Fellowship is not designed or expected to produce large datasets that would be subject to mandatory deposition, I understand the value of publishing raw data as a resource for other scientists to use, for example to conduct sample size analyses. I am committed to full scientific transparency and where possible will publish all relevant raw data (appropriately anonymised in the case of human work) as supplementary materials to research outputs (and published simultaneously) in order to allow free access for secondary analyses.

Individuals and skills. Experience and training during the Fellowship will increase academic competitiveness of myself and collaborating researchers. The long-term appointment of a Zambian national as research assistant (and hopefully PhD candidate) will enhance scientific capacity in Zambia.

ECONOMIC AND SOCIETAL IMPACTS

Changing policy: As described above, this research could have significant implications for our understanding of conditions with high prevalence and of great importance to health (particularly child health) in resource-limited settings. An open approach to data deposition and dissemination will help in the development of clinical trials based on my findings that could have health policy implications. Over the period of the Fellowship and beyond (2-10 years) I will engage with global health organisations like WHO and UNICEF to ensure that policy implications of my work are fully explored.

Health and wellbeing: I hope this research can be used to support the development of new strategies to prevent and treat EED and malnutrition. Clinically-applicable outputs may be available within 10 years and might help to address a number of UN Sustainable Development Goals, most notably the commitments to end poverty, hunger, and to improve health and wellbeing of populations around the world.

Ethical society: The development of an in vitro system to conduct mechanism-directed work on intestinal barrier function with human samples will help towards the aim of reducing, refining and replacing the use of animals in biomedical research.

Wealth creation: The research will showcase internationally competitive and innovative science originating from a UK centre, encouraging investment from academic, charitable and commercial sectors. The international collaborations developed will help to position UK researchers for investment opportunities from non-UK funding agencies within 5 years.