Jun R. Huh, Ph.D. - US grants

DESCRIPTION (provided by applicant): The nuclear hormone receptor ROR3 was recently shown to play crucial roles in the pathogenesis of multiple diseases including inflammatory bowel disease (IBD). However, its regulatory mechanisms remain largely unknown. My goal of this proposal is to identify genes regulating ROR3 activity in mammalian cells and to develop small molecules that inhibit its function. These efforts may eventually lead to potential therapies for IBD and a variety of ROR3-dependent diseases. Since I started a post-doctoral career at the Littman laboratory at NYU medical center, I completed genome- wide RNAi and unbiased small molecule screens with heterologous ROR3 reporter systems that are based on a insect cell line. From these screens, I identified ROR3 regulators in three distinct Drosophila genetic pathways and several classes of antagonists with distinct chemical templates. I also confirmed that these small compound antagonists function not only in Drosophila cells, but also in mouse and human T cells. During the K99 period, I plan to study mammalian homologues of the identified genetic regulators in mouse T cells by taking RNA interference approaches (Aim 1). For one or two genes exhibiting the most specific activities for ROR3, during the R00 period, I will generate conditional knock-out mouse lines and test their effects on mouse models of IBD (Aim 3). In addition, I will try to identify potent ROR3 chemical antagonists with high in vivo efficacy during the K99 and the early R00 stages (Aim 2). I already acquired several chemical derivatives as lead compounds. By synthesizing and testing various chemical derivatives, I anticipate that we will be able to identify compounds that can inhibit ROR3 activity with high efficiency in vivo and to investigate their therapeutic potentials in the mouse models of IBD (Aim 3). My immediate research goal is to explore therapeutic options of IBD by genetically and chemically modulating the function of the nuclear hormone receptor ROR3, which has been shown to be crucial for certain types of IBD. My long-term research goal is to identify genetic targets and their modulators for additional types of IBD, utilizing insights from this study.

ABSTRACT Bile acids are cholesterol-derived, natural surfactants, produced in the liver. They are critical to lipid digestion, antibacterial defense, and cholesterol synthesis, as well as other aspects of human biology. Importantly, bile acid dysregulation has been linked to intestinal bowel disease. Intestinal inflammation is modulated through a fine balance between the intestinal microbiota and the mucosal immune system. Imbalance can activate immune signaling pathways, leading to uncontrolled, pathological immune responses. Gut-residing bacteria in both the small and large intestines are exposed to a significant amount of bile acids and are known to convert host- derived bile acids into more hydrophobic, and thus, more bioavailable derivatives. We hypothesize that bacteria-produced secondary bile acids directly regulate the differentiation and/or function of key immune cells under inflammatory conditions, providing a communications link between commensal bacteria and mucosal immunity. We propose 1) to dissect the mechanisms of bile acid-mediated immune modulation and identify bile acid immune cell targets, 2) to identify bacteria and their enzymes that generate immune-modulatory bile acids and 3) to identify the novel metabolites of bile acids that regulate mucosal inflammation in vivo, at physiological concentrations. Elucidation of these mechanisms will not only provide novel therapeutic options for inflammatory diseases, but also open exciting avenues to study unique regulatory interactions between gut-residing microorganisms, small molecule metabolites and host immune cells.

Abstract Th17 cells are an Interleukin-17-producing subset of CD4+ T effector cells that are centrally implicated in many human inflammatory and autoimmune diseases, from inflammatory bowel disease to multiple sclerosis. The retinoid orphan receptor gamma t (ROR?t), a nuclear hormone receptor, programs Th17 cell development and function. With the ultimate goal of modulating inflammatory T cells in disease settings, we undertook chemical and genetic screening to identify ROR?t target genes that drive human Th17 cell function. Intriguingly, our results implicated a group of genes in a novel pathway that would link lipid metabolism, ROR?t activity and Th17 cell functions. We will thus test the hypothesis that Th17 cell function is controlled in large part, by ROR?t target genes, via lipid droplets (LDs) (cellular organelles associated with lipid metabolism). We will take the following approaches: First, we will determine if LD- related genes ? specifically, HILPDA and BNIP3 ? are necessary, sufficient and specific regulators of human Th17 cell functions. Second, we will elucidate the molecular mechanisms by which LDs modulate Th17 cell differentiation. Third, using autoimmune disease models in mice, we will determine the physiological significance of LDs in inflammation and autoimmune pathologies. Our results may shed a light on novel lipid-regulatory mechanisms that control Th17 cell responses in inflammatory diseases.

ABSTRACT Human epidemiological studies suggest that fetuses exposed to maternal inflammation during the late first or the second trimester have an increased likelihood of neurodevelopmental disorders. Studies are needed to define the molecular and cellular mechanisms by which immune activation during pregnancy translates into neurodevelopmental and behavioral abnormalities in children. Using a mouse model of maternal immune activation (MIA), we demonstrated that Th17 cells are critical mediators that induce neurodevelopmental disorder-like phenotypes in the affected offspring exposed to prenatal inflammation. We also demonstrated that inflammation-induced neurodevelopmental disorder phenotypes in the offspring require maternal intestinal bacteria such as segmented filamentous bacteria (SFB) that promote Th17 cell differentiation. Moreover, we have spatially and functionally mapped the brain regions that mediate behavioral abnormalities. Inflammation during pregnancy in humans, however, does not always lead to the birth of children with neurodevelopmental disorders, suggesting that there are factors that suppress maternal Th17 cell-dependent, neurodevelopmental disorder-like phenotypes in the affected offspring. We hypothesize that pregnancy- associated changes in immune cell function and the composition of commensal bacteria favor anti- inflammatory responses that dictate both the amplitude and specificity of immune responses against infection and other inflammatory conditions. In the proposed application, we will first determine the pregnancy-induced changes in immune cell function and their impact on MIA-like phenotypes in offspring. Secondly, we will investigate if pregnancy-associated changes in the bacterial community of the maternal guts contribute to anti-inflammatory responses. Lastly, we propose that by harnessing pregnancy-associated anti-inflammatory responses we can develop preventive ways to suppress neuronal and behavioral changes in the MIA-affected offspring.

Abstract Maintaining an equilibrium between inflammatory Th17 cells and anti-inflammatory Treg cells is critical to support intestinal barrier function and tissue homeostasis. Nuclear hormone receptors (NhRs) have been shown to play crucial roles in the development and function of key immune cells, including Th17 and Treg cells. Based on our prior work, we hypothesize that host-produced, bacterially modified steroids bind to host NhRs and modulate T cell differentiation and function. Specifically, we posit that secondary bile acids (microbial metabolites of host-produced primary bile acids) bind to host NhRs and modulate T cell differentiation and function. We previously demonstrated that two bile acid metabolites modulate T cell differentiation: 3-oxo- lithocholic acid (3oxoLCA) inhibits the differentiation of naïve T cells into inflammatory Th17 cells, and isoallo- lithocholic acid (isoalloLCA) enhances the differentiation of naïve T cells into anti-inflammatory Treg cells. Although we determined that 3oxoLCA inhibited Th17 cells by acting as a ligand for ROR?t (retinoic acid receptor-related orphan nuclear receptor ? t), it is unknown whether isoalloLCA exerts its Treg cell-modulating activity by acting through NhR(s). In addition, it is likely that there are additional bile acids that modulate T cell responses. In preliminary work, we have identified an abundant bile acid metabolite, iso-lithocholic acid (isoLCA) that inhibits Th17 cell differentiation and function, as well as human gut bacteria that produce isoLCA and isoalloLCA. We have found that the levels of these metabolites are significantly decreased in the feces of human patients with Crohn?s disease compared to healthy controls. We propose to (1) identify human gut bacteria and bacterial genes responsible for the production of isoLCA and isoalloLCA, (2) determine the molecular mechanisms by which these compounds influence Th17 and Treg differentiation and function, and (3) investigate whether gut bacteria producing isoLCA and isoalloLCA affect host immune responses in vivo. Elucidating the pathways that produce immunomodulatory bile acids and their mechanisms of action will open up exciting avenues to study unique regulatory interactions between gut-residing microorganisms and host immune cells. This research will lay the groundwork for the development of new therapies to treat autoimmune diseases, including inflammatory bowel disease.