Shedding LIGHT (TNFSF14) on inflammation associated with IBD

The intestinal immune system has to be carefully balanced to tolerate food molecules and the gut- resident bacteria as well as efficiently attack invading pathogens. Therefore, it is not surprising, that imbalances of these regulatory mechanisms manifest in inflammatory bowel disease (IBD), such as Crohns disease and ulcerative colitis in which the mucosal immune system actively destroys the intestinal tissue. Medical treatment consists of anti-inflammatory therapeutics, but these are limited in efficacy and show unwanted side-effects. Given the lack of adequate treatment for IBD, it is important to understand the fundamental regulatory mechanisms of the intestinal immune system. The immune mediator LIGHT helps to activate immune cells and seems to play an important role in IBD as it was found to be present in high levels in the intestine of IBD patients. This study will focus on the examination of the influence of LIGHT related to the pathology of ulcerative colitis, a chronical inflammation of the colonic mucosa. Although LIGHT typically has been associated with promoting inflammation, surprisingly, we found that LIGHT-deficient mice treated with a chemical to induce colitis showed a more severe disease progression, indicating a protective role for LIGHT. LIGHT interacts with two cell membrane-bound receptor proteins, the Herpes Virus Entry Mediator (HVEM) and lymphotoxin ß receptor (LTßR) and this effect was found to be LTßR rather than HVEM-mediated. We found that LTßR-deficiency in specific white blood cells, neutrophils, enhanced disease progression in our colitis model similar to LIGHT-deficient mice. Furthermore, we found that LTßR-deficient neutrophils produced more tissue- destroying reactive oxygen species compared to control mice, even in steady-state, providing a potential link to the colitis protective effect of the LIGHT- LTßR interaction. Neutrophils express both LTßR and HVEM, but it is possible that the two receptors activate different metabolic pathways. Thus, we will compare the outcome of LIGHT stimulation on either receptor on neutrophils from mice. Furthermore, we will examine in steady-state as well as when colitis is induced if neutrophils from LIGHT-deficient mice also produce more reactive oxygen, similar to LTßR-deficient mice. As there are no reliable tools to easily detect LIGHT we have genetically modified mice to generate a fluorescent LIGHT protein which can be visually tracked. In order to identify the cellular source of LIGHT, we will generate mice with LIGHT-deficiency in distinct cell types. The experiments in this proposal will lead towards deeper understanding of the origin of LIGHT and its effects in steady-state as well as chronic inflammatory conditions.

The intestinal immune system has to strike a careful balance between tolerating gut-resident bacteria and efficiently attacking invading pathogens. Not surprisingly, any imbalances in these regulatory mechanisms manifest in chronic inflammation of the intestinal tract. In Inflammatory Bowel Disease (IBD), one of these disorders, the immune system actively destroys intestinal tissue. Ulcerative colitis is a chronic form of IBD, leading to diarrhea, rectal bleeding, severe abdominal pain and consistent weight loss in colitis patients. Given the lack of fully adequate treatments for this disease, it is important to understand in detail the fundamental regulatory mechanisms of the intestinal immune system. LIGHT is present in high levels in the intestine of IBD patients and this study focused on the examination of the influence of TNF superfamily member 14, TNFSF14 (also known as LIGHT), in intestinal inflammation related to the pathology of ulcerative colitis. LIGHT interacts with two cell membrane-bound receptor proteins, the Herpes Virus Entry Mediator (HVEM) and lymphotoxin ß receptor (LTßR). We found that LTßR-deficiency in specific white blood cells, neutrophils, enhanced disease progression in our colitis model similar to LIGHT-deficient mice. Furthermore, we studied LTßR-deficient neutrophils and found that they produce more tissue-destroying reactive oxygen compared to control mice, in healthy state and colitis, providing a potential link to this colitis protective effect of the LIGHT- LTßR interaction. Because of the need for comparing histology samples from mouse colon I taught myself how to efficiently score tissue sections. My method got validated by a pathologist and I performed this analysis for this study. I found that colitis improved when these metabolic changes related to the LTR deletion in neutrophils were specifically targeted by a drug. These results demonstrate that the immune mediator LIGHT binds to the receptor LTßR on neutrophils to suppress activation of related metabolism and this prevents exacerbated colitis pathogenesis. It is still unclear which cell type produces LIGHT and how exactly it affects chronically inflamed mucosa in the colon. As there are no reliable tools to easily detect LIGHT I took on the task of generating a knockout mouse where the Light gene can be turned on in one specific cell type and off in another. This makes tracking the cellular source of this immune modulator possible. I modified mouse embryonic stem cells and we are currently waiting for the genetic verification. The stems cells will then be injected into a mouse blastocyst. Apart from that, I worked towards another mouse model where the LIGHT protein has a small molecule on its surface which should facilitate its detection by commercially available tools. Together, these studies led to a deeper understanding of chronic inflammatory conditions in IBD and towards finding a proper treatment for these patients.