Mycobacteria lacking the RD1 region do not induce necrosis in the lungs of mice lacking interferon-gamma

Abstract

The genetic region of difference 1 (RD1) in Mycobacterium tuberculosis has recently been hypothesized to encode for proteins that are cytotoxic to the host cell in nature. We demonstrate here that while M. tuberculosis grew progressively in the lungs of gene disrupted mice (GKO) unable to produce interferon-gamma (IFN-gamma), similar mice infected instead with M. bovis bacillus Calmette-Guérin (BCG) reproducibly exhibited an obvious slowing of the disease after about 20 days. Closer examination of BCG-infected GKO mice showed a florid granulomatous inflammation in the lungs, whereas similar mice infected with M. tuberculosis exhibited wholesale progressive necrosis. In the BCG-infected GKO mice large numbers of activated effector T cells, some strongly positive for the cytokine tumour necrosis factor, as well as activated natural killer cells accumulated in the lungs. To further test the hypothesis that the differences observed were directly associated with the loss of the RD1 region, it was then shown that a mutant of M. tuberculosis lacking RD1 grew progressively in both normal and GKO mice but failed to induce any degree of necrosis in either animal despite reaching similar levels in the lungs. However, when mice were infected with this mutant, in which the RD1 region had been restored by complementation, wholesale necrosis of the lungs again occurred. These data support the hypothesis that proteins encoded in the RD1 region are a major cause of necrosis and contribute significantly to the pathogenesis of the disease.

Figure 1

IFN-γ knockout mice control BCG infection in the lung. (a) Growth kinetics in the lungs of C57BL/6 mice infected with M. tuberculosis (▪) or M. bovis BCG (•). (b) Growth kinetics in the lungs of GKO mice infected with M. tuberculosis or M. bovis BCG (symbols as above). Data represent the mean ±SEM from six mice per time-point. The results are representative of three independent experiments.

Figure 2

Mycobacterium tuberculosis induces necrosis in GKO mice while BCG infection confers a florid inflammatory response without necrosis. Mice were aerosol infected with M. bovis BCG or M. tuberculosis (TB). (a, c) Histological pattern seen in wild-type mice (WT), (b, d) histological pattern in GKO mice (100 ×), (e, f) higher magnification (400 ×) of the inflammatory cells characteristic of the lesion seen in (b); arrow in (e) indicates large epithelioid macrophages with a fibrillar hypereosinophilic cytoplasm, arrow in (f) indicates eosinophils; asterisk indicates necrosis. The examples shown here are from mice harvested at 62 days post infection. Haematoxylin & eosin stain. The lesions are representative of one out of three independent experiments with similar results.

Figure 3

Diffuse distribution of CD4⁺ and CD8⁺ T cells in GKO BCG-infected mice. Mice were infected with M. bovis BCG or M. tuberculosis and lungs were collected at 62 days post infection. Frozen sections of lung tissue were prepared and stained for CD4 or CD8 using monoclonal antibodies. Lesions from wild-type mice (WT) and IFN-γ knockout mice (GKO) are shown. The top panel represents mice infected with BCG while the bottom panel represents the lesions found among the M. tuberculosis-infected mice. The dashed bars were added to delineate the borders of the necrotic lesions. Note the diffuse distribution of CD4 and CD8 cells in GKO mice infected with BCG when compared to wild-type mice. CD4 and CD8 cells could be detected in M. tuberculosis-infected GKO mice, but only beyond the necrotic areas (delimited by dashed lines).

Figure 4

BCG infection confers early activation/migration of effector CD4⁺ T cells and natural killer (NK) cells in the lung of infected GKO mice. GKO and wild-type mice were infected with M. bovis BCG. Lung single cell suspensions were characterized by flow cytometry. CD4⁺ cells were gated and analysed for surface molecule characteristics of the effector cell (CD44^high CD62L^low CD45RB^low) phenotype in (a). (b) CD4⁺ cells were gated and analysed for the expression of TNF-α after 4 hr of incubation with anti-CD3/CD28 and monensin. (c,d) NK cells (NK1.1⁺, CD3^–) were gated and analysed for early activating factor expression (CD69). In (c) the dot plot shows the activation of NK cells after M. bovis infection of GKO mice at 40 days post infection. (d) Kinetics of CD69⁺ NK cells in the lung of infected mice during 40 days post infection. Data shown are the mean ± SEM of six mice per time-point/group. Three independent experiments were performed with similar results. *Statistical differences between wild-type and GKO infected mice, P < 0·05.

Figure 5

Growth of an M. tuberculosis H37Rv mutant lacking the RD1 gene region in GKO mice (▪) or in normal C57BL/6 mice (•), compared to the growth of the RD1 re-complemented mutant in GKO mice (▴). Data are given as mean values ± SEM for five mice.

Figure 6

Histological appearance of fixed lung tissues from C57BL/6 (a, b) and GKO mice (c, d) 50 days after aerosol infection with M. tuberculosis RD1 gene KO mutant. In both groups lesions were perivascular and peribronchiolar. In the C57BL/6 mice lesions were focal and compact composed of small mature lymphocytes (L) mixed with macrophages (M) and occasional neutrophils (arrows). Lesions in GKO mice were usually larger and multifocal, containing intra-alveolar accumulations of macrophages, compact foci of lymphocytes, and neutrophils. Some multinucleated giant cells were seen. Haematoxylin & eosin staining; (a, c) 40 ×; (b, d) 400 × magnification.

Figure 7

Comparison of lungs tissues harvested from GKO mice 34 days after infection with the RD1-KO mutant (top panels) or this mutant re-complemented with RD1 (bottom panels). The top left (magnification 100 ×) shows periarteriolar infiltrates of mild mixed inflammatory cells that are composed of primarily lymphocytes and fewer neutrophils. The peribronchiolar alveoli have similar infiltrates in addition to multiple multinucleated giant cells. The top right panel (200 ×) shows sheets of lymphocytes, macrophages and fewer granulocytes with no evidence of necrosis. In comparison, the bottom left panel (100 ×) shows necrosis and mixed inflammation in which neutrophils are a predominant cell type, whereas the bottom right (200 ×) shows increased fibrous connective tissue forming an incomplete capsule (arrow) that delineates necrosis, and neutrophilic inflammation from more mixed mononuclear cell infiltrate.