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Tumor necrosis factor (TNF) is a prototypic proinflammatory cytokine that contributes significantly to the development of immunopathology in various disease states. A complication of TNF blockade therapy, which is used increasingly for the treatment of chronic inflammatory diseases, is the reactivation of latent tuberculosis. This study used a low-dose aerogenic model of murine tuberculosis to analyze the effect of TNF neutralization on disease progression in mice with chronic tuberculous infections. Histological, immunohistochemical, and flow cytometric analyses ofMycobacterium tuberculosis-infected lung tissues revealed that the neutralization of TNF results in marked disorganization of the tuberculous granuloma, as demonstrated by the dissolution of the previously described B-cell-macrophage unit in granulomatous tissues as well as by increased inflammatory cell infiltration. Quantitative gene expression studies using laser capture microdissected granulomatous lung tissues revealed that TNF blockade in mice chronically infected withM. tuberculosisleads to the enhanced expression of specific proinflammatory molecules. Collectively, these studies have provided evidence suggesting that in the chronic phase ofM. tuberculosisinfection, TNF is essential for maintaining the structure of the tuberculous granuloma and may regulate the granulomatous response by exerting an anti-inflammatory effect through modulation of the expression of proinflammatory mediators.

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

Members of the Mycobacterium tuberculosis group synthesize a family of long-chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non-oxygenated α-mycolic acid and the oxygenated keto- and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) againstMycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense againstM. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosisinfection of mice. Furthermore, in vitro studies have demonstrated that different strains ofM. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates ofM. tuberculosisfollowing both aerogenic and intravenous challenges. Three recently isolated and uniqueM. tuberculosisstrains were used to infect both wild-type (wt) C57BL/6 andNOS2gene-disrupted mice. Regardless of the route of infection, NOS2−/−mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain ofM. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/−mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/−mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains ofM. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused byMycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-α), we examined the effects of TNF-α on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-α in the containment of persistent tuberculosis. TNF-α neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-α-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-α-neutralized mice. In summary, results of this study suggest that TNF-α plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Tuberculosis is a major cause of death in much of the world. Current estimates are that one-third of the world's population is infected with Mycobacterium tuberculosis. Most infected persons control the infection but in many cases may not eliminate the organism. Reactivation of this clinically latent infection is responsible for a large proportion of active tuberculosis cases. A major risk factor for reactivation of latent tuberculosis is HIV infection, suggesting a role for the CD4(+) T cell subset in maintaining the latent persistent infection. In this study, we tested the requirement for CD4(+) T cells in preventing reactivation in a murine model of latent tuberculosis. Antibody-mediated depletion of CD4(+) T cells resulted in rapid reactivation of a persistent infection, with dramatically increased bacterial numbers in the organs, increased pathology in the lungs, and decreased survival. Although CD4(+) T cells are believed to be a major source of interferon (IFN)-gamma, expression of the gene for IFN-gamma in the lungs of CD4(+) T cell-depleted mice was similar to that in control mice. In addition, inducible nitric oxide synthase production and activity was unimpaired after CD4(+) T cell depletion, indicating that macrophage activation was present even during CD4(+) T cell deficiency. These data indicate that CD4(+) T cells are necessary to prevent reactivation but may have roles in addition to IFN-gamma production and macrophage activation in controlling a persistent tuberculous infection.

Summary Nitric oxide (NO) and related reactive nitrogen intermediates (RNI) are effective antimycobacterial agents and signal-transducing molecules. The present study uses microarray analysis to examine the effects of RNI on Mycobacterium tuberculosis gene expression. A common set of 53 genes was regulated by two chemically distinct nitric oxide donors. For a subset of the RNI-inducible genes, evidence exists suggesting that they may play a role in promoting survival of the tubercle bacillus in the host. Results obtained from studies based on a murine experimental tuberculosis model involving nos2-deficient mice suggest that RNI could regulate M. tuberculosis gene expression in vivo. Finally, there is a remarkable overlap between the RNI-inducible regulon and that previously reported to be regulated by hypoxia; and both reactive nitrogen species and anaerobicity upregulate the expression of one and the same putative two-component regulatory response system. Together, the results of this study provide evidence suggesting that (i) RNI play a role in regulating M. tuberculosis gene expression in vivo; (ii) the reactive nitrogen species upregulate genes that may be conducive to the survival of the tubercle bacillus in the infected host; and (iii) RNI and hypoxia may regulate mycobacterial gene expression via overlapping signal transduction pathways.