Dose of BCG does not influence the efficient generation of protective immunity in mice challenged with Mycobacterium tuberculosis.

It is generally agreed that BCG vaccination is relatively ineffective in adults exposed to tuberculosis infection. The reasons for this may well be multiple, and may include the possibility that higher doses of BCG may induce a mixed TH1 and TH2 response, which may lessen the protective effect of the vaccine. To test this hypothesis, mice were vaccinated with a range of doses of BCG and then challenged by the intravenous or aerogenic routes with virulent Mycobacterium tuberculosis. While the data support the hypothesis that a TH2 response is induced by higher doses of BCG, this was found to have no influence whatsoever on the capacity of the vaccinated mouse to express acquired specific resistance to the challenge infection.

Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst.

Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence of Mycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in the sodC gene. In this report, we show that the M. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parental M. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-gamma)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-gamma or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages.

Immunological basis for reactivation of tuberculosis in mice.

In this study different inbred strains of mice appeared to control and contain a low dose aerosol infection with Mycobacterium tuberculosis in a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection with M. tuberculosis may reflect an inability to localize antigen-specific lymphocytes within the lung.

The progression of chronic tuberculosis in the mouse does not require the participation of B lymphocytes or interleukin-4.

The aging process is associated with alterations in the immune system. Some of the changes reported are an increase in the proportion of B lymphocytes, and a shift to a TH2-like cytokine environment. It has been hypothesized that the development of immunopathology within the lung during tuberculosis is linked to increased interleukin-4 (IL-4) production. In addition, a role for B cells in maintaining granuloma integrity has been recently proposed. This study investigated the role of B cells and IL-4 during the long-term course of chronic tuberculosis in mice and showed that the course of Mycobacterium tuberculosis infection in the lungs was not influenced by the absence of B lymphocytes or the TH2 cytokine IL-4.

Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology.

It is well established in animal models that production of the cytokine tumour necrosis factor-alpha (TNF-alpha) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100-200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF-alpha production was harmful during this phase mice were treated with a TNF-alpha inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF-alpha, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.

Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis.

Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-A(b+) and CD11c(+) cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c(+) and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8 alpha antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naïve and immune T cells to produce gamma interferon.

Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis.

The progression of the immune response in the lungs after aerosol infection with Mycobacterium tuberculosis is a complex cellular event dominated by macrophages and lymphocytes. Although the phenotype of lymphocytes participating in this response is becoming increasingly well characterized, the dynamic influx of these cells during the infection and their spatial arrangements within the lung tissue are still poorly understood. This study shows that in the first month after aerosol infection with M. tuberculosis there was a steady increase in the percentages of total CD3+, CD3+ CD4+ and CD3+ CD8+ cells, with consistently larger numbers of CD3+ CD4+ cells than of CD3+ CD8+ cells. As granuloma formation continued, the granuloma was found to consist of macrophages, CD4, and CD8 T cells, as well as a smaller number of B cells. Whereas CD4 T cells formed organized aggregates, CD8 T cells were fewer and more scattered and tended to be more prominent toward the periphery of the granulomas. The possible ramifications of the juxtapositions of these two major T-cell subsets are discussed.

Tuberculosis in aged gammadelta T cell gene disrupted mice.

In young mice exposed to aerosol infection with Mycobacterium tuberculosis removal of the gammadelta T cell population by targeted gene disruption does not affect the expression of host resistance, but does influence the integrity of the early granulomatous response. The current study demonstrates that in aged gammadelta T cell gene disrupted mice similar immunopathologic changes ensued in both gene knockout and wild type control mice. Changes in cell surface marker expression, evident in other gene knockout models, was not observed in the aged gammadelta T cell knockout mice. These data imply that gammadelta T cell functions previously observed in young mice become much less important as the animal ages.

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis.

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.

Expression of the nitric oxide synthase 2 gene is not essential for early control of Mycobacterium tuberculosis in the murine lung.

The interleukin-12 and gamma interferon (IFN-gamma) pathway of macrophage activation plays a pivotal role in controlling tuberculosis. In the murine model, the generation of supplementary nitric oxide by the induction of the nitric oxide synthase 2 (NOS2) gene product is considered the principal antimicrobial mechanism of IFN-gamma-activated macrophages. Using a low-dose aerosol-mediated infection model in the mouse, we have investigated the role of nitric oxide in controlling Mycobacterium tuberculosis in the lung. In contrast to the consequences of a systemic infection, a low dose of bacteria introduced directly into the lungs of mice lacking the NOS2 gene is controlled almost as well as in intact animals. This is in contrast to the rapid progression of disease in mice lacking IFN-gamma or a key member of the IFN signaling pathway, interferon regulatory factor 1. Thus while IFN-gamma is pivotal in early control of bacterial growth in the lung, this control does not completely depend upon the expression of the NOS2 gene. The absence of inducible nitric oxide in the lung does, however, result in increased polymorphonuclear cell involvement and eventual necrosis in the pulmonary granulomas of the infected mice lacking the NOS2 gene.

A novel nonclassic beta2-microglobulin-restricted mechanism influencing early lymphocyte accumulation and subsequent resistance to tuberculosis in the lung.

In this study, we compared the course of a low-dose aerosol Mycobacterium tuberculosis infection in mice bearing gene disruptions for the beta2-microglobulin molecule, the CD8 molecule, and the CD1 molecule. Over the first 50 d of infection, the CD8- and CD1-disrupted mice were no more susceptible to infection than were the control mice. In contrast, the bacterial load in beta2-microglobulin gene-disrupted mice increased rapidly and attained much higher levels than that observed in the other gene-disrupted mice and in control mice. A second major difference between the beta2-microglobulin gene-disrupted mice and the other animals was the development of lung granulomas; both the CD8- and CD1-disrupted mice developed essentially normal granulomas except for an apparent increased lymphocyte influx in the CD8-disrupted mice. The beta2-microglobulin gene-disrupted mice, on the other hand, developed granulomas virtually devoid of lymphocytes, with these cells instead localized within prominent perivascular cuffing adjacent to the lesions. These data support the hypothesis that a beta2-microglobulin-dependent, non-CD8- and non-CD1-dependent mechanism controls the early and efficient influx of protective lymphocytes into infected lesions, and that the absence of this mechanism decreases the capacity of the animal to initially deal with pulmonary tuberculosis.

Interleukin-6 induces early gamma interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection.

Immunity to Mycobacterium tuberculosis is dependent upon the generation of a protective gamma interferon (IFN-gamma)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-gamma. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity to M. tuberculosis, IL-6 and IL-4 knockout mice were infected with M. tuberculosis via aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-gamma. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-gamma production, it is not essential for the development of protective immunity against M. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-gamma production, this had no significant effect upon bacterial growth.

Tuberculosis: recent progress in basic immunity and vaccine development.

Tuberculosis continues to be the most prevalent cause of death from an infectious agent globally, and its interaction with HIV is having devastating effects, particularly in Sub-Saharan Africa. Over the past decade, my laboratory has developed small animal models of pulmonary infection, which have revealed new information regarding the nature of acquired immunity, and subsequent immunopathology, in the lungs. We propose that cell mediated immunity comprises two separate elements; protective immunity, driven by IL-12 and IFN; and DTH, mediated by TNF and driven by chemokines. The generation of a CD4 response is critical to both processes, but other cells are also involved in the overall control of the infection. These include gamma delta T cells, which we believe control the inflammatory influx of cells; CD4+ NK cells, which may play a role in focussing lymphocytes into lung granulomas; and CD8 T cells, which play a currently undefined role after initial expression of immunity and establishment of chronic disease in the lungs has ensued. Complex interactions between these populations of cells appear to control the influx of mediator cells into the lungs and then focus them at sites of infection. Prior to adequate expression of protective immunity the correct expression of chemokine and adhesion molecules is critical. A better understanding of these processes will hopefully in turn lead to better vaccine design, a topic which is also addressed in this paper.

Transient loss of resistance to pulmonary tuberculosis in p47(phox-/-) mice.

Mycobacterium tuberculosis is an important respiratory pathogen the growth of which is controlled primarily by cytokine-activated macrophages. One of the principal mediators of this control is nitric oxide; however, superoxide has recently been shown to be protective in systemic mycobacterial infections. To determine whether superoxide is important in controlling M. tuberculosis during primary pulmonary infection, mice lacking the cytosolic p47(phox) gene (which is essential for effective superoxide production by the NADPH oxidase) were infected aerogenically. The lack of superoxide during an aerosol infection with M. tuberculosis resulted in a significant increase in bacterial growth over the early period of infection. Once antigen-specific gamma interferon-producing lymphocytes were detected in the draining lymph nodes, however, bacterial growth in the lung stopped. One interesting consequence of the lack of superoxide was an increase in neutrophilic infiltrates within the granuloma. This may be a consequence of increased tissue damage due to more rapid bacterial growth or may reflect a role for superoxide in controlling inflammation.

CD95 signaling is not required for the down regulation of cellular responses to systemic Mycobacterium tuberculosis infection.

There is a tendency among tuberculosis patients to have reduced cellular responses to mycobacterial antigens and this loss has been associated with apoptosis of CD4 T cells. In order to determine the role of CD95 in mediating apoptosis of antigen-specific lymphocytes in tuberculosis, mice with a mutated CD95L molecule were infected systemically with virulent Mycobacterium tuberculosis. Both control and CD95L mutant mice exhibited the expected loss of response to mycobacterial antigens, with the only difference being a slight delay in the loss of the response in the mutant mice. The limited persistence of the response in the mutant mice suggests that, while antigen-specific cellular responses do decline in mice infected with mycobacteria, this decline is not dependent upon CD95L.

Granuloma formation is required to contain bacillus growth and delay mortality in mice chronically infected with Mycobacterium tuberculosis.

Previous studies in this laboratory have shown that mice with a gene disruption to the intracellular adhesion molecule-1 (ICAM-K/O) express normal cell-mediated immunity but cannot mount delayed-type hypersensitivity reactions following Mycobacterium tuberculosis infection. However, even in the absence of any appreciable granuloma formation, these mice control bacterial growth for at least 90 days. While not required to control the infection initially, we hypothesized that granuloma formation was required to control chronic infection, acting by surrounding infected cells to prevent bacterial dissemination. To test this, ICAM-1 knockout mice were infected with a low dose aerosol of M. tuberculosis Erdman and were found to succumb to infection 136+/-30 days later, displaying highly elevated bacterial loads compared to wild-type mice. Lung tissue from ICAM-K/O mice displayed extensive cellular infiltration and widespread tissue necrosis, but no organized granulomatous lesions were evident, whereas the control mice displayed organized compact granulomas. These data demonstrate that while a granulomatous response is not required initially to control M. tuberculosis infection, absence of granulomas during chronic infection leads to increased bacterial growth and host death. Thus these data support the hypothesis that granuloma formation is required to control chronic infection, acting by surrounding and walling off sites of infection to prevent bacterial dissemination and maintain a state of chronic infection.

Metronidazole therapy in mice infected with tuberculosis.

The capacity of metronidazole to inhibit the growth of Mycobacterium tuberculosis was tested in in vitro and in vivo mouse models. In vitro addition of metronidazole to cultures of infected bone marrow-derived macrophages had no effect, nor did it increase the reduction in bacterial load due to isoniazid. In vivo, metronidazole did not reduce bacterial numbers in the lungs of aerosol-infected mice during the active stage of the disease, during a phase of containment, or after prolonged isoniazid therapy (Cornell model). A small but significant reduction was seen if metronidazole therapy was given during an established chronic disease state 100 days after aerosol administration. These data indicate that under most conditions M. tuberculosis organisms are not in a metabolic state in which they are susceptible to the action of metronidazole and, hence, that this drug would be of limited clinical value.

The intravenous model of murine tuberculosis is less pathogenic than the aerogenic model owing to a more rapid induction of systemic immunity.

The detection of mRNA in the murine model of tuberculosis for key cytokines involved in protective immunity in the lung tissues revealed a much faster emergence of the interferon (IFN)-gamma response in the intravenous route than in the aerosol route of inoculation. This slower response in the lungs was associated with a stronger inflammatory response, resulting in large granulomatous structures and eventual tissue damage.

Role of gamma delta T cells in immunopathology of pulmonary Mycobacterium avium infection in mice.

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.

CD95 expression in aged mice infected with tuberculosis.

The interaction between CD95 and its ligand is an important homeostatic mechanism that leads to the induction of apoptosis in activated T cells. In view of recent evidence that this pathway might be defective in aged mice, this study investigated CD95 expression on T cells in old mice activated by infection with Mycobacterium tuberculosis. The results of the study do not support the hypothesis that CD95 is poorly expressed on CD4 T cells from old mice; instead, it was found that similar numbers of T cells from young and old mice expressed CD95, with the intensity of expression if anything higher on the cells from the old mice. In addition, the study demonstrated that changes in CD44 and CD45RB expression previously observed in young infected mice proceeded in a similar fashion in old animals and, as would be predicted, that CD95(hi) expression was primarily associated with CD4 T cells expressing the activated CD44(hi) CD45RBhi phenotype.