Infection of alveolar macrophages by Mycobacterium tuberculosis (M tb) is a fundamental step in the progression to disease. Yet little is known about the response of macrophage to M tb, especially with respect to the impact on macrophage secretion of and response to cytokines that mediate interactions with other cells and play a key role in pathogenesis. To maximize relevance to the clinical situation and thus facilitate application of the data obtained in way that will benefit public health, studies will be carried out with clinical isolates of currently prevalent M tb strains, and in part with alveolar macrophages from normal human volunteers. Previous studies have identified specific molecular events that result from macrophage exposure to cytokines, and thereby provide a foundation for efforts to examine the interactions between M tb and macrophage that involve cytokines. The overall goal of the research proposed here is to identify the molecular mechanisms that account for M tb-induced alterations in macrophage response to key cytokines that are believed to contribute to the pathology of human tuberculosis.
The specific aims will thus test the hypothesis that M tb affects macrophage response to cytokines by examining whether: 1. M tb infection of macrophage alters activation or induction of transcription factors by interferons (IFNs) and tumor necrosis factor alpha (TNFalpha); 2. M tb effects on the macrophage response to IFNs and TNFalpha impinge on regulation of gene expression; and 3. expression of downstream gene products implicated in the pathogenesis of M tb or the modulation of macrophage function reflects the effects of M tb on macrophage responses to IFNs and TNFalpha. The DNA-binding activity of specific transcription factors known to be involved in responses to IFNs and TNFalpha, including ISGF-2/IRF-1 and NFkappaB, among others will be determined with the electrophoretic mobility shift assay. Quantitating the expression of marker genes such as inducible nitric oxide synthase, intercellular adhesion molecule, and interleukin-8, among others, implicated in M tb pathogenesis and/or the function of activated macrophage will be done with the run-on assay of transcription rate, and northern blot, RNase protection, primer extension or RT-PCR experiments to determine steady- state mRNA levels. The amount of protein expressed from these and other genes will be determined by assays of enzymatic activity, and with immunochemical methods to detect the intracellular protein present in an extract, or membrane proteins present at the cell surface. Elucidating the point(s) at which macrophage responses to cytokines are altered by M tb infection, and determining the consequences for expression of genes involved in pathogenesis, may provide important information for potential therapeutic interventions.
