IL12R?1?TM (Isoform 2) is an alternative IL12 receptor isoform that is expressed by mouse leukocytes following exposure to M. tuberculosis (Mtb). Isoform 2 lacks the transmembrane domain of IL12R?1 (Isoform 1), has a localization pattern that is distinct from that of Isoform 1, and in contrast to the initial predictions, enhancs IL12-dependent activities in vitro. While it is clear than the activation of human leukocytes with Mtb also stimulates Isoform 2 production via alternative splicing / polyadenylation, there is a gap in our understanding of the factors required for Isoform 2 alternative RNA processing and the significance of this pathway for controlling Mtb infection in vivo. This knowledge gap is important because until filled, avenues for therapeutic manipulation of IL12 activity will remain out of reach. Our central hypothesis is that immunogenic signaling induces alternative splicing/polyA via cis elements in the mRNA to generate Isoform 2, a protein that regulates TB control in vivo by promoting IL12-dependent TH1 differentiation. Our long-term goal is to understand how Mtb stimulates signaling to induce IL12RB1 alternative mRNA processing, and to manipulate Isoform 2 production to influence T cell biology and Mtb control. The objectives in this application are to identify the cis-elements in IL12RB1 pre-mRNA that are the targets of signaling and mediate Isoform 2 production and to test, using human and mouse systems, the significance of Isoform 2 to TB control. The rationale for the proposal is that identification of signal-responsive cis elements in IL12RB1 pre-mRNA that mediate alternative RNA processing, as well as the function of Isoform 2 in the context of TB, will lead to therapeutic targets to modiy pathway activity. The hypothesis will be tested through two specific aims: 1) Identify the cis-elements and trans-factors that mediate Isoform 2 RNA alternative processing and 2) determine the extent to which IL12RB1 Isoform 2 influences Mtb immunity.
Aim 1 will utilize IL12R?1 minigenes that will be nucleofected into T cells to identify cis-elements within the IL12RB1 mRNA that mediate pathogen-induced RNA alternative processing. Roles for candidate processing factors (based on potential binding sites near the regulated polyA site) will be assessed for Isoform 2 alternative processing.
Aim 2 will use an IL12-dependent bioassay to test the affect of Isoform 2 silencing in human lymphocytes, as well use a newly generated mouse strain to determine the outcome of experimental TB in the absence of Isoform 2. The work is innovative because it focuses on a completely different level of IL12R?1 activity regulation - signal-mediated post-transcriptional alternative RNA splicing / polyadenylation - in humans. This work is significant because it is the initial effort towards understanding the earlies events in Isoform 2 production in human cells, which should reveal downstream signaling pathways and lead to strategies for manipulating Isoform 2 levels and thus improving T cell function.
Tuberculosis (TB) is a public health concern in many nations, including the United States of America. IL12RB1 is well established as being critical for human immunity to Mycobacterium tuberculosis, the etiological agent of TB. Here, we propose to (1) examine how two major IL12RB1 mRNA isoforms are differentially generated, and (2) test their relative contributions to controlling experimental M. tuberculosis infection. The proposed research is relevant to public health because an increased understanding of IL12RB1 biology will, by extension, improve our understanding of how tuberculosis is normally controlled. The work also relates to the NIH mission of supporting basic research into the understanding and treatment of infectious diseases.