Signaling pathways that regulate IFN-g production in human tuberculosis.

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DESCRIPTION (provided by applicant): Because Tuberculosis (TB) causes 1.7 million deaths/year; it urges to better understand the human immune response to Mycobacterium tuberculosis (Mtb). Protective immunity to TB requires IFN-3. Thus, the general goal of this proposal is to understand the signaling pathways that control IFN-3 secretion in TB. Specifically, we propose to use primary T-cells from blood and pleural fluids in a physiologically relevant model to study this process. We reported that SLAM enhances IFN-3 production to Mtb, although SLAM expression is abnormal in TB patients. Our collaborators found reduced CREB expression in TB, but a positive regulation of IFN-3 by CREB. Our preliminary data shows that SLAM/IL-17 affects CREB activation and IFN-3 production in TB. Therefore, we hypothesize that: (1) SLAM-induced-IFN-3 involves CREB activation; (2) low IFN-3 produced by LR (low responder) TB patients (with weak response to Mtb) is caused by aberrant SLAM signaling and CREB activation; (3) Th17 cells regulate SLAM expression modulating Th1 responses in TB. Thus, we propose the following aims: 1. Determine how the SLAM/SAP (SLAM-associated protein) pathway modulates CREB in healthy individuals. 1.1. CREB activation by SLAM signaling. We will determine if SLAM ligation after Mtb-stimulation induces CREB activation/binding to the IFN-3 promoter. We will use CREB siRNA to measure IFN-3 mRNA/protein in cells stimulated with Mtb and SLAM. 1.2. Signaling pathways activated by SLAM. We will determine the activity of PI3K, Akt, ERK and MAPK proteins in cells stimulated with Mtb and SLAM. Because SAP inhibits IFN-3 in TB, we will perform the studies described in SAP deficient patients. 1.3. Do the SLAM-stimulated PI3K, Akt, MAPK, ERK pathways activate CREB enhancing IFN-3? By using specific inhibitors and siRNA to these molecules we will analyze CREB function on IFN-3 production. 2. Characterize the abnormalities in the SLAM/CREB pathway in TB patients. 2.1. Determine the effects of increased SAP expression on CREB/IFN-3 levels in TB. We will perform the studies of the aim 1.1 in TB patients' PBMC/PFMC. We will introduce SAP siRNA in LR's cells to determine if CREB/IFN-3 levels are restored. Longitudinal studies will determine if TB treatment ameliorates abnormalities in the SAP/CREB expression. 2.2. Does SAP interfere with the SLAM-mediated signaling? We will determine in LR patients' PBMC/PFMC the expression/function of the identified molecules linking SLAM to CREB. Longitudinal studies of TB patients will be performed to investigate the effect of TB treatment on abnormalities in the SLAM/SAP pathway. 2.3. Investigate the mechanisms leading to increased SAP in LR patients. We will analyze the Ets expression (controls SAP promoter activity) and AUF1/HuR and mRNA stability. 3. Does IL-17 modulate SLAM/CREB activation during IFN-3 regulation in TB? 3.1. We will determine the effect of IL-17 on SLAM/IFN-3 expression and CREB activation in Mtb-stimulated TB patients' PBMC/PFMC and controls. 3.2. Does enhanced IL-17 production in TB reduce SLAM-induced CREB activation and IFN-3 expression? We will measure IL-17 production by SLAM and Mtb-stimulated cells from patients and controls. We will neutralize IL-17 production and analyze the effects on SLAM-induced CREB activation, IFN-3 expression, and the molecules linking SLAM to CREB. These studies will provide insight into signaling pathways that control IFN-3 in TB, which will be critical for development of vaccines that maximize immune responses. PUBLIC HEALTH RELEVANCE: Tuberculosis, an infectious disease produced by the bacteria Mycobacterium tuberculosis, is responsible of almost 2 million deaths worldwide annually, making the development of an effective vaccine an urgent public health priority. This proposal will provide new insights about the immune mechanisms involved in the development of a protective response of the host against the pathogen. Therefore, this information will enhance the knowledge about the immunopathogenesis of tuberculosis, contributing with new information on the mechanisms that control the production of IFN-3, a key cytokine in the immune response to intracellular pathogens, including viruses, fungi and parasites.