Major gaps in our picture of host resistance to M. tuberculosis (Mtb) include a lack of understanding of the biochemical basis of the restriction of Mtb replication by activated macrophages, lack of experimental approaches to the evaluation of an antituberculous contribution by the respiratory epithelium, and ignorance of the genetic and biochemical bases of mycobacterial resistance to host anti-tuberculous defenses. Improved antituberculous therapy might result from better understanding of each of these points, as well as from the development of an otherwise healthy, inexpensive laboratory animal in which Mtb can replicate as quickly as it is intrinsically able. The present experiments are designed to address each Of these points from the perspective of the hypothesis that the inducible form of nitric oxide synthase (iNOS) may play a major anti-tuberculous role. Genetic approaches that knock out iNOS selectively in the whole mouse or mainly in macrophages, or express iNOS mainly in macrophages or respiratory epithelium, will include homologous recombination, adenovirus- assisted transfection, and transgenic expression controlled by a cell type-restricted promoter. Second, we will use iNOS-deficient and wild type mice to screen differentially for Mtb genes, expressed in recombinant E. coli, that confer resistance or sensitivity to host nitroxergic defenses. The level of resistance or sensitivity will be analyzed by comparing the survival of these organisms in mice, in cultures of mammalian cells transfected with iNOS cDNA, and in culture medium containing NO-generating compounds. Third, we will explore the relevance of the murine findings to man by probing pathologic specimens from tubercular and control subjects with a new antibody to human iNOS and with restricted cDNA from human iNOS. These experiments should provide an unambiguous answer to the importance of iNOS in defense of the murine lung against Mtb, and should help differentiate between the contribution of NO arising from macrophages and from respiratory epithelium. We may generate mice whose immune system and overall physiology have undergone, the least perturbation consistent with allowing Mtb to replicate rapidly. Moreover, these experiments will shed light on the genetic basis of mycobacterial virulence, insofar as it relates to NO as a host defense mechanism against TB. Finally, these studies should establish whether iNOS is expressed in the tubercular human lung.
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