The ligand-dependent transcription factor ROR?t regulates the development and functions of lymphocytes, including Th17 cells and type 3 innate lymphoid cells (ILC3) that have central roles in mucosal barrier protection and in mediating multiple autoimmune inflammatory diseases. Targeting of ROR?t by small molecule inhibitors is a strategy that has been successfully validated in animal models and is being developed for therapeutic application in human autoimmune diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and, potentially, multiple sclerosis. Although many of the targets of ROR?t and other key transcription factors involved in Th17 cell differentiation have been identified, the roles of ROR?t in the gene regulatory programs of other cell types, e.g. thymocytes, ILC3, and lymphoid tissue inducer cells, have not been studied, nor have the roles of the closely-related ROR? isoform that is expressed outside of the immune system. A better understanding of how ROR?t executes cell type-specific functions may facilitate development of better strategies to selectively inhibit its activities in the desired cells and tissues. We have ued genetic and proteomics screens to identify molecules that contribute to ROR?t-dependent target gene regulation in Th17 cells, either indirectly or directly by associating with ROR?t complexes. Molecules identified in both screens include nuclear pore proteins that have been recently implicated as having roles in the nucleoplasm as well as the nuclear envelope. We propose to study the roles of Nup98 and Nup153, proteins that interact with ROR?t and contribute to the expression of a large number of ROR?t target genes. In preliminary studies, we have identified mutations in ROR?t that abrogate interaction with Nup153. Mice with one of these mutations have impaired Th17 cell induction but normal T cell development. We will extend our studies with Th17 cells, and also study the role of the ROR?t-nucleoporin interactions in differentiation and function of TCR-??-17 cells and ILC3 and in the development of lymphoid tissue inducer cells and thymocytes. For the first aim, we will characterize the biochemical basis of the interaction of ROR?t with Nup153 and Nup98 and will determine how this interaction influences formation of ROR?t transcriptional complexes and the DNA occupancy of each factor in polarized Th17 cells. We will also examine the effect of ligand binding on association of ROR?t with its partners, taking advantage of small molecule antagonists and our finding that Nup98 regulates enzymes involved in cholesterol biosynthesis, which may thus influence endogenous ligand availability.
In Aim 2, we will introduce relevant mutations into the mouse germ line and examine the effect on chromatin accessibility and gene expression programs in Th17 cells generated in vitro and in vivo and in other ROR?t-dependent cells.
In Aim 3, we will use mice with the various point mutations in ROR?t or its partner proteins to determine the effects in models of Th17-mediated autoimmune disease or ILC3-dependent barrier protection. Together, these studies will help in the design of better screens for cell type-selective targeting of ROR?t functions.

Public Health Relevance

We propose to study the mechanism of action of a regulator of genes that are important for the function of white blood cells involved in autoimmune diseases such as Crohn's disease, psoriasis, and rheumatoid arthritis. Inhibitors of this regulator are likely to be effective therapeutics for these diseases, but will also affect other cell types ad may thus result in unwanted toxicity. Our studies are aimed at identifying properties of this regulator that are specific for the cells involved in autoimmunity, and that can thus result in its selective targeting, thus sparing other essential functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI080885-08
Application #
9378417
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Mallia, Conrad M
Project Start
2009-06-15
Project End
2020-10-31
Budget Start
2017-11-01
Budget End
2018-10-31
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Huang, Wendy; Thomas, Benjamin; Flynn, Ryan A et al. (2015) DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature 528:517-22
Santori, Fabio R (2015) Nuclear hormone receptors put immunity on sterols. Eur J Immunol 45:2730-41
Santori, Fabio R; Huang, Pengxiang; van de Pavert, Serge A et al. (2015) Identification of natural ROR? ligands that regulate the development of lymphoid cells. Cell Metab 21:286-97
van de Pavert, Serge A; Ferreira, Manuela; Domingues, Rita G et al. (2014) Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 508:123-7
Huh, Jun R; Littman, Dan R (2012) Small molecule inhibitors of ROR?t: targeting Th17 cells and other applications. Eur J Immunol 42:2232-7
Hooper, Lora V; Littman, Dan R; Macpherson, Andrew J (2012) Interactions between the microbiota and the immune system. Science 336:1268-73
Honda, Kenya; Littman, Dan R (2012) The microbiome in infectious disease and inflammation. Annu Rev Immunol 30:759-95
Huh, Jun R; Leung, Monica W L; Huang, Pengxiang et al. (2011) Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing ROR?t activity. Nature 472:486-90