The HN878 strain of Mycobacterium tuberculosis is regarded as “hypervirulent” due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-γ. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4+CD25+FoxP3+CD223+IL-10+ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.

The production of immunosuppressive cytokines, such as IL-10 and TGF-β, has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN-γ secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in promoting reactivation tuberculosis.

The production of immunosuppressive cytokines, such as IL-10 and TGF- (cid:1) , has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN- (cid:2) secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in pro-moting reactivation tuberculosis. The Journal of Immunology, 2002, 169: 6343–6351.

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.

The purpose of this brief commentary is to describe the growing effort to discover new drugs that can be used to treat disease caused by Mycobacterium tuberculosis. In this regard, it is estimated that over 8 million people contract tuberculosis each year, and approximately 2 to 3 million people die of this disease (6). In addition, it is thought that as many as 2 billion people have been exposed to the tuberculosis bacillus and are therefore at risk of developing active disease. In certain areas of the world, such as countries within the former Soviet Union, the case rates are climbing alarmingly. This problem is further compounded by a dramatic increase in multidrug-resistant strains of M. tuberculosis (10). An additional factor is human immunodeficiency virus, which has significantly increased the incidence of tuberculosis in sub-Saharan Africa and elsewhere (5). While the long-term solution is a better vaccine (14), for the next several decades there will have to be continued reliance on chemotherapy. In this regard, however, there have been few additions to the existing “main-line” drugs for a considerable time. While there are some promising new agents, such as the longer-acting rifamycins, fluoroquinolones, oxazolidinones, and nitroimidazopyrans (1, 4, 7, 8, 11–13, 15, 17–19), there are as yet no extensive clinical trial data to determine the efficacy of these compounds. Finding new drugs represents a challenge. Therapy currently takes a considerable time, and it is hoped that new compounds can be found that will reduce the total duration of treatment regimens. Drugs are also needed that will be effective against the growing number of drug-resistant strains and against bacilli that may be in a state of latency. As part of the overall research effort at the National Institute of Allergy and Infectious Diseases, the National Institutes of Health has established a screening program at several institutions to efficiently screen large numbers of compounds for possible activity against M. tuberculosis. An acquisitions program receives from both academic and commercial sources compounds which are initially screened for in vitro activity. To date, over 50,000 compounds have been provided to the program (Fig. 1). Each compound is first screened for activity at 6.25 mg/ml (or the molar equivalent of the highest-molecular-weight compounds in a series of congeners) against cultures of M. tuberculosis H37Rv in BACTEC 12B medium using a microplate alamar blue assay (2). Compounds that are active in this assay are reconfirmed using a BACTEC 460 radiometric system. In the next stage of the procedure, the MIC of the compound is determined. In addition, the compound evaluated for cytotoxicity in cultures of Vero cells to determine a 50% inhibitory concentration (IC50). A selectivity index (SI) can then be calculated by dividing the IC50 by the MIC; if the SI is .10, the compound is then evaluated further. The next stage of screening uses the bone marrow macrophage infection assay (16) to determine if the compound can inhibit the growth of M. tuberculosis in its intracellular environment. Cultures of bone marrow-derived macrophages are infected with M. tuberculosis and then cultured for 7 to 8 days in the presence of a range of concentrations of the test compound. The monolayers are then lysed and plated on 7H11 agar to determine the numbers of surviving bacteria. By plotting the bacterial load in each culture against the drug concentration, one can determine whether a compound is bacteriostatic or bactericidal under these relevant physiological conditions. The concentration of drug reducing the bacterial load by 1 log unit (90% reduction [EC90]) or by 2 log units (99% reduction [EC99]) is recorded as a numerical readout (Fig. 2). Compounds that perform well in the macrophage infection assay are then tested in a mouse model if sufficient amounts of the compounds are available. After the maximum tolerated dose for a compound is determined, mice are exposed to a low-dose aerosol infection with M. tuberculosis (9). Twenty days later, as the bacterial load reaches its peak in control animals, the test compound is administered daily at appropriate dosages for 45 days. The route of administration is usually gavage, although subcutaneous or intraperitoneal injection can sometimes be given for a limited time. At 15, 30, and 45 days of therapy, groups of five mice are removed and euthanatized. The lungs and spleen are homogenized and plated on agar to determine the bacterial load. A positive control group of mice is given isoniazid (25 mg/kg of body weight/day) for comparison. The bacterial load for each test group is then plotted against time and compared to the values for untreated control mice (Fig. 3). Under these conditions, a reduction of 0.7 log unit or greater is considered statistically significant, as determined by analysis of variance. * Corresponding author, Ian Orme. Mailing address: Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-5777. Fax: (970) 491-5125. E-mail: iorme@lamar.colostate.edu. Other members are J. Secrist, S. Anathan, C. Kwong, J. Maddry, and R. Reynolds (all at Southern Research Institute, Birmingham, Ala.); A. Poffenberger, M. Michael, and L. Miller (all at Southern Research Institute, Frederick, Md.); J. Krahenbuh, L. Adams, and A. Biswas (all at National Hansen’s Disease Laboratory Research Branch, Baton Rouge, La.); S. Franzblau (at Institute for Tuberculosis Research, University of Illinois); D. Rouse and D. Winfield (at Research Triangle Institute, Research Triangle Park, N.C.); and J. Brooks (at Colorado State University).

SETTINGWhile culture filtrate proteins (CFPs) of Mycobacterium tuberculosis appear to be good vaccine candidates for tuberculosis, only CFPs derived from certain popular laboratory strains of M. tuberculosis have been studied for this purpose.OBJECTIVETo compare the relative efficacies of CFP preparations from two laboratory strains and four contemporary clinical isolates of M. tuberculosis to induce T-cell activation.DESIGNCFPs were isolated from six strains of M. tuberculosis and were used to induce 1) T-cell proliferation, 2) IFN-gamma secretion, and 3) IL-12 secretion from peripheral blood derived mononuclear cell (PBMC) preparations from 33 healthy donors.RESULTSSignificant amounts of IL-12 were spontaneously secreted by PBMC preparations; CFP preparations from two clinical isolates (JNU-7 and JNU-51) significantly boosted this response. All six CFP preparations induced IFN-gamma secretion by PBMCs, but those from two contemporary strains of M. tuberculosis (JNU-7 and JNU-22) were most effective in this regard. The effect of CFPs from JNU-7 and JNU-22 was significantly better than those from the laboratory strains (H37Ra and Erdman). Similar results were obtained with the T-cell proliferation parameter.CONCLUSIONThese results suggest that CFPs derived from selected clinical isolates of M. tuberculosis may outperform those of standard laboratory strains, and may therefore be a better source of potential candidates for a tuberculosis vaccine.

Objective: To determine whether synthetic peptides containing an amino terminal formyl-methionine residue and corresponding to the sequence of several proteins produced by Mycobacterium tuberculosis, would elicit an immune response in mice. Design: Peptides corresponding to the amino termini of 8 M. tuberculosis proteins and initiating with formyl methionine residues were synthesized. The ability of these peptides to bind to the mouse non-classical MHC class I molecule H-2M3awas determined by flow microfluorimetry. These peptides were used to pulse dendritic cells that were then injected into normal mice. These mice were subsequently challenged with aerosolized M. tuberculosis and, 30 days later, the number of viable bacteria in the lungs was determined. Results: Four of the 8 synthetic peptides bound to H-2M3aand stabilized its expression on the cell surface. Injection of mice with dendritic cells pulsed with H-2M3abinding peptides elicited non-MHC restricted cytotoxic T lymphocytes that killed peptide pulsed target cells and macrophages infected with M. tuberculosis. Immunization of mice with syngeneic dendritic cells pulsed in vitro with 2 of these peptides led to retardation of the growth of M. tuberculosis following aerosol challenge. Conclusion: Peptides that bind to non-polymorphic class I molecules can elicit immune reactivity directed towards M. tuberculosis.

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l‐N6‐(1‐imino‐ethyl)‐lysine (L‐NIL), a selective NOS2‐inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection‐induced nitrate/nitrite to background levels in mycobacteria‐infected BALB/c mice. Oral treatment with 5 mm L‐NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony‐forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria‐induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium‐infected mice were treated with 5 mm L‐NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse‐transcription–polymerase chain reaction (RT‐PCR). Computer‐ and video‐assisted morphometry performed at 4 and 7 weeks post‐infection showed that treatment with L‐NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L‐NIL, the numbers of CD3+ T cells, CD21/35+ B cells, CD11b+ macrophages and RB6‐8C5+ granulocytes associated with granulomatous lesions was increased. RT‐PCR of the liver showed that in L‐NIL‐treated mice infected with M. avium, mRNA levels of tumour necrosis factor, interleukin‐12p40, interferon‐γ, interleukin‐10 and interferon‐γ‐inducible protein‐10 (IP‐10) were up‐regulated, while mRNA levels of interleukin‐4, monocyte chemotactic protein‐1 (MCP‐1) and MCP‐5 were similar to those in untreated control infected mice. When M. avium‐infected mice were treated with 5 mm L‐NIL between the 5th and 12th weeks of infection, similar changes in granuloma number and size were found in the absence of any effect on the bacterial load. These findings demonstrate that nitric oxide regulates the number, size and cellular composition of M. avium‐induced granulomas independently of antibacterial effects by modulating the cytokine profile within infected tissues.

The murine immune response to a pulmonary mycobacterial infection is slow to develop, allowing bacterial numbers to increase in the lung for several weeks after infection. We sought to enhance the protective immune response induced during Mycobacterium bovis BCG infection by administering an antibody that blocks the interaction of CTLA-4 with its ligands, CD80 and CD86. We found that injection of anti-CTLA-4 monoclonal antibody (MAb) greatly enhanced and accelerated the immune response, as measured by increased cellularity of the draining mediastinal lymph nodes, and enhanced antigen-inducible proliferation and gamma interferon production by mediastinal lymphocytes in vitro. However, despite the apparently enhanced immune response in the mediastinal lymph node following treatment with anti-CTLA-4 MAb, there was no improvement in clearance of mycobacteria in the lungs, liver, or spleen. Examination of the primary site of infection, the lung, revealed that CTLA-4 blockade had no effect on the number or function of lymphocytes infiltrating the infected lung tissue. Taken together, these data suggest that in vivo CTLA-4 blockade enhances mycobacterial-infection-induced lymphocyte expansion and effector cell cytokine production in the draining lymph node but does not alter the number or function of lymphocytes at the primary site of infection and therefore does not lead to enhanced clearance of the infection.

Several studies have shown that gamma delta T cells influence granuloma development after infection with intracellular pathogens. The role of gamma delta T cells in controlling the influx of inflammatory cells into the lung after Mycobacterium avium infection was therefore examined with gene-disrupted mice (K/O). The mice were infected with either M. avium 724, a progressively replicating highly virulent strain of M. avium, or with M. avium 2-151 SmT, a virulent strain that induces a chronic infection. gamma delta-K/O mice infected with M. avium 2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. gamma delta-K/O mice infected with M. avium 724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates of M. avium that induce protective T-cell-specific immunity are largely unaffected by the absence of gammadelta T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of gamma delta T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.

DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.

General Discussion I: Endocytic Trafficking and the Mycobacterial Vacuole Book Editor(s):Derek J. Chadwick, Derek J. ChadwickSearch for more papers by this authorGail Cardew, Gail CardewSearch for more papers by this author First published: 09 October 1998 https://doi.org/10.1002/0470846526.ch10Book Series:Novartis Foundation Symposia Series Editor(s): Novartis Foundation, Novartis FoundationSearch for more papers by this author AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Genetics and Tuberculosis: Novartis Foundation Symposium 217 RelatedInformation

Mice, guinea pigs and rabbits differ in terms of the severity of lung pathology following exposure to aerosol infection with virulent strains of Mycobacterium tuberculosis or Mycobacterium bovis. Histologically, guinea pigs and rabbits both develop `classical' granulomas similar to those seen in humans with active tuberculosis[ 1 Dannenberg A.M. Bacteriol. Rev. 1968; 32: 85-102 PubMed Google Scholar , 2 Dannenberg A.M. Rev. Infect. Dis. 1989; 11: S369-S378 Crossref PubMed Scopus (136) Google Scholar , 3 Dannenberg A.M. Hosp. Pract. 1993; 28: 51-58 Google Scholar ]. These structures consist of large numbers of epithelioid macrophages surrounded by a mantle of lymphocytes and monocytes. Such lesions can be very large (easily visible to the naked eye), causing disruption to the lung architecture, and are often surrounded by further areas of interstitial pneumonia. Gradually, over several weeks, epithelioid macrophages in the center of the lesion begin to degenerate, leaving behind free nuclei floating in a sea of coagulative cytoplasmic debris (Fig. 1). These events appear to be completely necrotic rather than apoptotic (there is no evidence of dead, but otherwise intact, cells with shrunken nuclei, condensed cytoplasm, etc.). As this necrosis increases, the animal begins to dramatically lose weight [tumor necrosis factor (TNF) release by local macrophages is probably a major culprit] and then dies. A similar picture is seen in the rabbit, the only difference being that the necrotic lesions often progress rapidly to fully liquefied cavities, which are comparable with those seen in humans with advanced disease[ 4 Dannenberg, A.M. and Rook, G.A.W. (1994) in Tuberculosis: Pathogenesis, Protection and Control (Bloom, B.R., ed.), pp. 459–483, ASM Press Google Scholar ].

Murine bone marrow-derived macrophages were infected with a panel of virulent isolates of Mycobacterium tuberculosis including laboratory strains Erdman and H37Rv and various clinical isolates in order to determine the sensitivity of each of these strains to the antimycobacterial activities of macrophage-generated reactive nitrogen intermediates (RNI). All of the M. tuberculosis strains grew in murine bone marrow-derived macrophages; however, gamma interferon-primed macrophages limited the initial growth of intracellular bacilli. Some of the mycobacterial strains, including Erdman, were killed over the first 4 days of infection, as evidenced by significant decreases in the number of viable intracellular bacilli determined by a CFU assay. Other mycobacterial strains were not killed during this same period, and some isolates, including CSU 24 and CSU 31, grew steadily in activated macrophages. The accumulation of nitrite on infected monolayers was measured, and it was found that inhibitory levels of RNI did not vary among infections with the different strains. Nitrite tolerance was determined in a cell-free system for each of the strains in order to compare susceptibilities of the strains to RNI. All of the strains tested were killed by levels of RNI generated by the acidification of 10 mM NaNO2 to pH 6.5 or 5.5, and the strains exhibited a range of tolerance to lower concentrations of RNI. No correlations were observed between such cell-free RNI tolerances and the capacity of bacilli to resist macrophage RNI-mediated killing. These results indicate that under stringent conditions, RNI can kill M. tuberculosis, but that under less harsh, more physiological conditions, the effects of RNI range from partial to negligible inhibition.