Title: Single cell definition of the mechanistic role of kynurenine monooxygenase at the innate immune interface in acute pancreatitis
Funder: Guts UK

Abstract: Background: Acute pancreatitis (AP) initiates a systemic inflammatory response which can lead to multi-organ dysfunction (MODS) and death. The molecular mechanisms underpinning the pathogenesis of AP-MODS remain incompletely understood, but kynurenine monooxygenase (KMO), a critical enzyme in tryptophan metabolism, is important. KMO generates 3-hydroxykynurenine, which causes cellular injury. KMO blockade protects against AP-MODS in experimental models of AP and KMO inhibitors are entering the clinic as novel treatments for AP-MODS. The group I am joining has shown that KMO is highly-expressed in dendritic cells located at the innate-adaptive interface of the immune system, and that genetically-altered mice with elevated 3-hydroxykynurenine show constitutively activated inflammatory signalling pathways. Together, therefore, there is a strong rationale to support the concept that 3-hydroxykynurenine is acting as a damage-associated molecular pattern (DAMP), signalling through pattern-recognition receptors to regulate the innate adaptive immune interface during AP Hypothesis: The KMO product 3-hydroxykynurenine is a damage-associated molecular pattern that signals at the innate-adaptive immune interface to regulate organ dysfunction in acute pancreatitis. Aim: To define the mechanistic role of KMO and 3-hydroxykynurenine as a DAMP at the innate immune interface in AP. My objectives are, specifically: • To test whether 3-hydroxykynurenine is a DAMP, and how it signals. • To define the transcriptomic phenotype at single cell resolution in key immune tissues in relation to KMO activity and 3-hydroxykynurenine levels – in health and in experimental AP in mice. • To explore the clinical correlate of these findings by single cell sequencing of peripheral whole blood from humans with AP. Methods: I will define 3-hydroxykynurenine DAMP-signalling using genetically-engineered reporter cell lines that detect TLR3, 7 and 9, using control cell lines with knockdown of the adapter molecule MyD88 to distinguish TL3 activity, and reporter cell lines for the intracellular receptor, STING, with STING knockdown controls. Positive and negative DAMP controls will be used. If 3- hydroxykynurenine does not signal through TLR3, 7, 9 or STING, we will evaluate other TLRs and PRRs sequentially, in a logical fashion. Next, I will use single cell RNASeq (Chromium 10X platform) to define the immune cell transcriptome in peripheral blood, and key tissues at single cell resolution, using genetically-altered mouse lines which have baseline, absent, elevated and dendritic-cell specific KMO and 3-hydroxykynurenine aberrations, in healthy mice and in experimental AP. If time permits, I will explore the single cell transcriptome in human peripheral blood in patients with AP. Clinical and scientific impact My project will provide a first-class research training, and, if successful, I will have defined the molecular mechanisms that underpin how 3-hydroxykynurenine activates and regulates the immune system in AP. My project is timely and will be highly clinically- and translationally-relevant to patients with AP, especially given the advance of KMO inhibitors into the clinic.

Lay abstract: Background Acute pancreatitis (AP) is a disease where the pancreas gland becomes inflamed and damaged, normally caused by excess alcohol consumption or gallstones. It is a common cause for emergency surgical admission to hospital, and when severe will kill 1 in 5 patients affected. AP not only damages the pancreas gland, but can inappropriately activate inflammation in other organs, most importantly the liver, lung and kidneys. The body’s own immune system damages and destroys these organs. This can make patients extremely unwell, it can kill them, and if they do recover will shorten their life as a result. Our research team has discovered that this process involves a part of metabolism called KMO. In the body KMO produces a chemical called 3HK which directly damages organs. We also have evidence that 3HK prepares the body to launch an inflammatory reaction. We think that it may act on special immune cells that are essential for how the body controls inflammation. Questions and Objectives I want to do this research to discover how 3HK directly activates the immune system in AP. I think it is especially important to discover how 3HK affects the immune cells that control inflammation, and I want to find out how 3HK alters the way genes in our immune cells are controlled. How it will be done I am going to do a number of experiments in the laboratory, then I will see if whatever I discover in the laboratory is also happening in patients with AP. First, I will add 3HK to specially-designed cells to discover how and when inflammatory processes are switched on and off. We will then see how 3HK and KMO changes the number and type of immune cells in organs as they prepare to launch an inflammatory reaction in AP, using laboratory models. When we have mapped that out, we will see whether equivalent processes happen in human patients with AP. Potential Impact: I am doing this scientific research in order to discover how inflammation caused by acute pancreatitis (AP) is regulated by the body’s immune system, and to find out how new medicines that are being developed for AP work, at the molecular level. This will help us to make better new medicines, improve health, and save lives in AP.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL