The importance of Th17 cells, and their potent inflammatory cytokines (IL-17A and GM-CSF), in multiple sclerosis (MS) and other autoimmune diseases is established. In animal MS models, Th17 cells are recruited and localized to the CNS through ?2 integrin LFA-1-dependent adhesion and transendothelial migration. Although transcriptional regulation of the IL-17A and GM-CSF genes has been well characterized, the mRNAs encoding these cytokines are highly labile and must be dynamically regulated to allow significant gene expression. We have demonstrated that T cell adhesion through ?2 integrin engagement results in marked stabilization of mRNAs encoding TNF-? and IFN-?, through modulation and nuclear-to-cytosolic translocation of the RNA-binding protein (RBP) HuR. Our preliminary data support an equally remarkable extension of the IL-17A and GM-CSF transcript half-lives through an LFA-stimulated, HuR-dependent mechanism. When attempting to characterize potential competitive microRNA (miRNA)- HuR interactions on the IL-17A 3'- untranslated region (3'-UTR), we unexpectedly detected a cooperative, interdependent RNA- stabilizing interaction between miR-466l-3p and HuR. We mapped the miR-466l-3p target site within the IL-17A 3'-UTR. An oligonucleotide preventing this interaction (target site blocker [TSB]) inhibits LFA-1-induced, HuR- dependent IL-17A mRNA stabilization, and enhanced IL-17A production, in a cytokine-specific manner. We intend to define the same for GM-CSF, as its mRNA's 3'-UTR contains a highly conserved AU-rich element which includes 4 potential miR-466l-3p target sites. Our previously published and new data, and the pathogenic importance of IL-17A and GM-CSF in neuroinflammation, have led to our hypothesis, that leukocyte integrin engagement promotes Th17 cell IL-17A and GM-CSF expression via enhanced cooperative binding of HuR and miR-466l-3p to their 3'-UTRs, and that this potent pro-inflammatory switch is amenable to novel therapeutic targeting. Specific proposals now are to: (1) map the miR-466l-3p target site in the GM-CSF 3'-UTR, and generate an effective, specific TSB, using complementary molecular approaches including (a) MS2-TRAP 3'-UTR/miRNA pulldowns, and (b) pBBB ? globin RNA reporter stability assays; and (2) determine the impact of selectively blocking miR-466l-3p's interaction with the IL-17A and GM- CSF transcripts on immunopathology in a chronic, myelin oligodendrocyte glycoprotein (MOG)-specific 2D2 transgenic EAE model, and a relapsing, remitting, proteolipid protein peptide (PLP)-immunized EAE model, evaluating EAE clinical scores, as well as CNS IL-17A and GM-CSF mRNA and protein levels. The novelty of this newly described cooperative miRNA-RBP interaction, and our ability to test inhibitors directed at this cooperativity in neuroinflammation disease models, makes this both a molecular and a highly translational exploratory R21 project. We hope this work directs more extensive efforts in posttranscriptional regulation of pathogenic cytokine expression, and defines a novel therapeutic targeting opportunity.

Public Health Relevance

A significant component of multiple sclerosis (MS), as well as other autoimmune diseases, is the accumulation of immune cells in the target tissue (the spinal cord in MS), and the production of 'inflammatory cytokines', proteins made by these accumulated immune cells that contribute to disease. Such cytokines, important in MS, are IL-17A and GM-CSF, and we have identified a control mechanism in their expression which we believe creates a novel therapeutic targeting opportunity. In this project, we will precisely determine the molecular details of this control mechanism, and attempt to interfere with it in an animal model of MS, hopefully validating the search for a new class of therapeutics in MS and other autoimmune diseases.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Clinical Neuroimmunology and Brain Tumors Study Section (CNBT)
Program Officer
Esch, Thomas R
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Yale University
Internal Medicine/Medicine
Schools of Medicine
New Haven
United States
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