Role for matrix metalloproteinase 9 in granuloma formation during pulmonary Mycobacterium tuberculosis infection

Abstract

Recent studies have shown that matrix metalloproteinases (MMPs) are induced by Mycobacterium tuberculosis during pulmonary infection. Here, expression of MMP-9 during pulmonary M. tuberculosis infection was characterized to determine whether its production correlated with disease resistance in vivo and to determine what role, if any, MMP-9 might have in granuloma formation. Following aerosol infection with M. tuberculosis, dissemination of bacilli occurred earlier in the C57BL/6 resistant mouse strain than in the susceptible CBA/J strain, as was evident from an increased number of bacteria in the blood, spleen, and liver at day 14 after infection. In addition, early dissemination of the bacilli was associated with early induction of protective immunity as assessed from gamma interferon levels. Nonspecific blocking of MMPs in C57BL/6 mice early during infection reduced hematogenous spread of the bacilli, suggesting that MMPs indeed play a role in facilitating dissemination, likely via extracellular matrix degradation. The concentration of active MMP-9, specifically, was greater in the lungs of C57BL/6 mice than in those of the CBA/J mice at day 28, thereby suggesting that MMP-9 is not one of the MMPs directly involved in promoting early dissemination of M. tuberculosis. Instead, however, histological lung sections and flow cytometric analysis of lung cells from MMP-9-knockout mice showed that MMP-9 is involved in macrophage recruitment and granuloma development. These combined data support the idea that early MMP activity is an essential component of resistance to pulmonary mycobacterial infection and that MMP-9, specifically, is required for recruitment of macrophages and tissue remodeling to allow for the formation of tight, well-organized granulomas.

FIG. 1.

Early dissemination of M. tuberculosis following aerosol infection of C57BL/6 and CBA/J mice. Bacterial counts in the spleen, blood, and liver were higher at day 14 in the C57BL/6 mice than in the CBA/J mice but not in the lung or lung-associated lymph nodes (data not shown). As the infection progressed, the number of bacteria in the lungs and spleen increased in the susceptible CBA/J strain. Circles, CBA/J mice; squares, C57BL/6 mice. Data are expressed as the mean (n = 5 mice) ± standard deviation. P values were calculated using Student's t test comparing C57BL/6 and CBA/J mice at each time point; *, P < 0.05, and **, P < 0.01.

FIG. 2.

MMPs are involved in early dissemination of M. tuberculosis. C57BL/6 mice were treated with MMP inhibitor BB-94 at the time of pulmonary infection with 500 CFU of M. tuberculosis. At day 14 postinfection, dissemination of the bacilli from the lungs to the spleen and blood was decreased in mice given BB-94 compared to the mice that received the vehicle control. Black bars, BB-94-treated mice; white bars, vehicle control-treated mice. Data are expressed as the mean (n = 3 mice) ± standard deviation. P values were calculated using Student's t test comparing BB-94-treated mice and vehicle control-treated mice; *, P < 0.05.

FIG. 3.

Production of proinflammatory cytokines in the lungs of susceptible versus resistant mice. Lungs were harvested over a period of 120 days of pulmonary infection with M. tuberculosis and were analyzed for the production of IFN-γ (A), TNF-α (B), and MCP-1 (C) proteins. Open circles, CBA/J mice; closed circles, C57BL/6 mice. Data are expressed as the mean (n = 5 mice) ± standard error of the mean. P values were calculated using Student's t test comparing C57BL/6 with CBA/J mice at each time point; *, P < 0.05.

FIG. 4.

Level of active MMP-9 in the lungs following aerosol infection with M. tuberculosis. The level of active MMP-9 was measured in lung homogenates from infected C57BL/6 and CBA/J mice. The amount of MMP-9 was consistently higher in the resistant C57BL/6 mice. Open circles, CBA/J mice; closed circles, C57BL/6 mice. Data are expressed as the mean (n = 5 mice) ± standard error of the mean and are representative of two similar experiments. P values were calculated using Student's t test comparing C57BL/6 with CBA/J mice at each time point; *, P < 0.05.

FIG. 5.

Bacterial load in the lungs of MMP-9KO mice. By day 30 and continuing throughout the course of infection, bacterial counts in the lung were decreased in the MMP-9KO mice compared to the wild-type FVB mice. Circles, MMP-9KO mice; squares, FVB mice. Data are expressed as the mean (n = 5 mice) ± standard deviation. P values were calculated using Student's t test comparing FVB with MMP-9KO mice at each time point; *, P < 0.05, and **, P < 0.01.

FIG. 6.

Differences in granuloma organization and structure between mouse strains. At day 28 after aerosol infection, lung sections from C57BL/6 mice (A) showed granulomatous lesions with a marked decrease in alveolar septal wall integrity compared to the CBA/J mice (B). The arrows in panel B depict keratin staining within the granuloma where the septa are still present, and the total magnification of sections A and B is ×400. At day 40 after aerosol infection, granulomas in the C57BL/6 mice were well organized with F4/80-positive macrophages bordering focused lymphocytes and macrophages (C) while granulomas in CBA/J mice were loosely organized with F4/80-positive macrophages distributed randomly throughout the granuloma (D). Sections in panels C and D were stained with anti-F4/80 primary antibody, and the total magnification is ×200. Each section was counterstained with hematoxylin. Photomicrographs are representative of five mice per group.

FIG. 7.

Representative hematoxylin-and-eosin-stained lung sections from MMP-9KO (A to C) and FVB wild-type (D to F) mice at day 60 of infection with M. tuberculosis. In panel A, arrows point to perivascular accumulations of cells that are predominately lymphocytes. There is trapping of cells in the perivascular connective tissue that compresses the surrounding parenchyma with intact septal walls. The leading edge of the lesion is sharply demarcated from the parenchyma (panel B, arrow) where there is infiltration of intact interalveolar septa by lymphocytes (panel C, arrows). In contrast, the FVB wild-type lesion in panel D is expansive and effaces the architecture to the parenchyma (arrow in panel D) such that epithelioid macrophages, fewer lymphocytes, granulocytes, and cells consistent with dendritic cells replace alveolar septal walls and alveolar spaces. The leading edge of the lesion (panel E, arrow) is composed predominately of lymphocytes that are shown at higher magnification in panel F (arrow). The perilesional parenchyma is attenuated such that alveolar septal walls and alveolar spaces are ill defined. The total magnification of panels A and D is ×89, of panels B and E is ×178, and of panels C and F is ×356.

FIG. 8.

MMP-9 is required for macrophage recruitment in the lungs of M. tuberculosis-infected mice. (A) By day 30 of the infection and throughout the course of infection, the total number of macrophages was decreased in the lungs of the MMP-9KO mice compared to the wild-type FVB mice. (B) There was no difference in T-cell numbers over the course of infection between the two mouse strains. (C) The level of MCP-1 in the lungs of the FVB mice was significantly increased at days 40 and 90. Circles, MMP-9KO mice; squares, FVB mice. Data are expressed as the mean (n = 5 mice) ± standard deviation. P values were calculated using Student's t test comparing FVB with MMP-9KO mice at each time point; **, P < 0.01.