Interferon-gamma (IFNgamma) is a cytokine released by TH1 lymphocytes in tuberculosis and is an essential mediator of the cellular immune response, producing macrophage activation. Signaling is transduced through activation of STAT-1, a latent transcription factor, and leads to induced expression of other transcription factors. STAT-1 and the other transcription factors like IRF-1 together increase expression of effector genes such as iNOS. We have been conducting a clinical trial of aerosolized IFNgamma as treatment for pulmonary tuberculosis and have reported that patients with multi-drug resistant tuberculosis improved on this regimen. We now report that aerosolized IFNgamma induces STAT-1 and IRF transcription factors in lung cells. Treatment also markedly reduces HIV-1 levels in the lungs of AIDS patients with tuberculosis, providing further evidence of enhanced lung immunity produced by pharmacological doses of IFNgamma. THP-1 macrophages and alveolar macrophages are similar with regard to induction of STAT-1, IRF-1 or IFR-9 (also called p48 and ISGF3-gamma) after infection with M. tuberculosis (M.tb) or IFNgamma treatment. THP-1 macrophages respond to IFNgamma with increased IRF-9 mRNA but do not change its transcription, demonstrating that IRF-9 is up-regulated by post-transcriptional mechanisms such as splicing or message stability. We used functional genomics to identify other IFNgamma responsive genes. Of 4375 genes present on a high density filter array, 48 were induced and 13 were repressed after 4 hours of IFNgamma stimulation. Only 15 of these 61 genes have been reported to be responsive to IFNgamma Finally, with the goal of assaying genome wide transcription rates, we have developed a novel assay for transcription using Br-UTP labeled nascent RNA. Our goal is to identify genes unique to the cellular immune response during tuberculosis in vivo and genes induced or repressed by IFNgamma and/or M.tb. ex vivo and in vivo. We propose to use functional genomics to identify the genes induced and repressed in: 1) THP-1 macrophages infected with M.tb and/or treated with IFN-gamma in vitro 2) Alveolar macrophages infected with M.tb and/or treated with IFNgamma ex vivo and 3) Bronchoalveolar lavage cells from tuberculosis patients before and after aerosolized IFNgamma. We will then assay transcription rates of the genes that are IFN responsive to define the contribution of transcriptional and post transcriptional regulation to mRNA abundance. We hypothesize that like IRF-9, other genes important to the IFNgamma response are post-transcriptionally regulated in macrophages. This analysis will further the understanding of cellular immunity in tuberculosis and gene expression regulated by IFNgamma.
