Dendritic cells (DCs) are multi-potent regulators of the immune system. Many critical DC functions involve calcium (Ca2+) signaling. Ca2+ is the most versatile eukaryotic intracellular messenger that mediates regulation of many processes important to cell life. Ca2+ levels in DCs and many other immune cell types are tightly regulated through multiple mechanisms during the resting state as well as during activation. Chemokine receptor stimulation increases intracellular Ca2+ concentration ([Ca2+]i) and chemotaxis in DCs. Despite important advances in our understanding of Ca2+ signaling in lymphocytes, the molecular players involved in shaping intracellular Ca2+ in DCs remain to be characterized. The current paradigm states that increases in DC cytosolic Ca2+ levels are caused by release from endoplasmic reticulum (ER) Ca2+ stores and/or via influx of extracellular Ca2+ by opening of store-operated Ca2+ channels at the plasma membrane. We propose a novel Ca2+ release mechanism in DCs, which operates via a Ca2+ channel expressed on lysosomal compartments and, we postulate this pathway is essential for Ca2+-dependent DC functions. Preliminary data that support our proposal show: i) Exclusive localization of functional Ca2+-permeant melastatin-related transient receptor potential channel (TRPM2) in endolysosomal compartments of DCs. ii) Both adenosine diphosphoribose (ADPR) and chemokines induce TRPM2 mediated Ca2+ signals in DCs, iii) TRPM2 channel acts as a Ca2+ release channel in DCs. iv) TRPM2-deficient DCs exhibit impaired chemotactic responses to chemokines. v) TRPM2-deficient mice elicit reduced inflammatory recruitment upon infection. Based upon these data, we hypothesize that: TRPM2 functions as an intracellular lysosome- associated calcium release channel that regulates Ca2+-dependent processes in DCs. To critically test our hypothesis, we propose the following specific aims: Aim 1. Identify mechanisms of activation of Ca2+ release via TRPM2 channel, and elucidate how this pathway impacts on DC Ca2+-dependent functions. Aim 2. Define the specific roles for TRPM2-mediated Ca2+ signaling in DCs during inflammation, immunization and cellular inflammatory infiltration to the mucosa using a mouse model of urinary tract infection. The findings from this proposal will provide insights into how the intracellular function of TRPM2 channel affects Ca2+-mediated signal transduction pathways that play a central regulatory role during DC responses. The completion of these studies will unveil the utility of new pharmacological targets (ADPR and/or TRPM2) for manipulation of DCs'Ca2+responses and functions, and will further facilitate the design of new therapeutic strategies for chronic inflammatory or infectious diseases, where DCs may play important pathogenic roles. PUBLIC HEALTH RELEVANCE: Dendritic cells play a critical role in initiating and regulating immunity. This grant proposes to elucidate a novel calcium release mechanism that regulates calcium homeostasis and therefore, calcium-dependent functions in dendritic cells. The successful completion of the experiments proposed here will provide insights into a signaling pathway in dendritic cells, which has strong potential to uncover novel therapeutic targets, and unappreciated approaches for chronic inflammatory diseases.