Contribution of transmembrane tumor necrosis factor to host defense against Mycobacterium bovis bacillus Calmette-guerin and Mycobacterium tuberculosis infections

Abstract

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

Figures

Figure 1

Lung granulomas expressing TNF mRNA and protein at 4 weeks of BCG infection. A–C: Histological sections from lung (stained with H&E) show well-differentiated granulomas in wild-type (A) and TmTNF tg (B) mice but disorganized granulomas and necrotic lesions (arrow) in TNF/LT-α−/− mice (C). D–F: In situ hybridization with 35S-labeled TNF RNA probes of lung sections. TNF mRNA-positive cells can be identified by dark silver grains on the lung sections from wild-type (D), TmTNF tg (E), but not from TNF/LT-α−/− mice (F). TNF protein expression was detected by immunohistochemistry in lung granuloma and multinucleated giant cells of wild-type (G) and TmTNF tg mice (H). These data are representative of two experiments (n = 6 mice per group, A–C; n = 3, D–H). Original magnifications: ×200 (A–H); ×400 (insets in G and H).

Figure 2

Dysregulated systemic and local chemokine release in TNF/LT-α−/− mice and effect of TmTNF on BCG infection. Amounts of MCP-1 (A and B) and RANTES (C and D) were measured in serum (A and C) at different time points, and in BAL (B) and in lung proteins (D) at 4 weeks of infection in wild-type, TmTNF tg, and TNF/LT-α−/− mice. Data are represented as means pg of protein per ml of fluid ± SEM of 10 mice per group in A and C, three to five mice in B, or in D per g of lung, five mice per group. These results are representative of two independent experiments. Asterisks indicate statistically significant differences between wild-type and indicated group (*, P < 0.01; **, P < 0.005).

Figure 3

Alteration of Th1-type cytokine release in TNF/LT-α−/− mice and effect of TmTNF 4 weeks after BCG infection. Amounts of IFN-γ (A and B), IL-12p40 (C and D), and IL-12p70 (E and F) were measured in serum (A, C, and E) and BAL (B, D, and F) of wild-type (wt), TmTNF tg (TmTNF), and TNF/LT-α−/− (TNF/LT−/−) mice. Results are expressed as mean pg of protein per ml of fluid ± SEM (n = 10 mice per group for serum cytokines and n = 4 mice per group for BAL cytokines). These results are representative of two or three independent experiments. Asterisks indicate statistically significant differences between wild-type and indicated group (IFN-γ: *, P < 0.05 and **, P < 0.009; IL-12p40: *, P < 0.02 and **, P < 0.0001; IL-12p70: *, P < 0.03 and **, P < 0.0007).

Figure 4

iNOS activity is reduced in the lungs and spleen of TNF/LT-α−/− mice but not in those of TmTNF tg mice. iNOS activity was determined in crude lung (A) and spleen (B) extracts containing the same protein amounts. Data are represented as means ± SEM of cpm/mg of lung proteins (n = 3) or spleen proteins (n = 5 to 6). Asterisks indicate significant differences between wild-type and TNF/LT-α−/− lungs (*, P < 0.02) and spleen (*, P < 0.03). Experiment has been repeated twice with similar results.

Figure 5

TmTNF tg mice are sensitive to M. tuberculosis infection and show enhanced cellular recruitment to the lung. A: Bacterial loads were determined in lungs, liver, and spleen 4 weeks after intravenous inoculation with 105 CFU of virulent M. tuberculosis strain H37Rv. Data represent individual values of infected mice and horizontal bars indicate means. A statistically significant increase of CFU in lungs from TmTNF tg mice (P < 0.01) when compared to wild-type mice was observed. Each group represents 6 to 10 mice, except TNF/LT-α−/− mouse (n = 1). Data from two experiments are shown. B to F: Enhanced cellular recruitment to the lung of M. tuberculosis-infected TmTNF tg mice despite moderate bacillus proliferation. Histological lung sections at 4 weeks after M. tuberculosis infection stained with H&E (B, D, and F) and Ziehl-Neelsen (C, E, and G). B: Granuloma from wild-type mice with very low number of acid fast bacilli (AFB) (C). D: TmTNF tg mouse lung granuloma reveals a substantial number of lymphocytes and inflammatory cells, with limited number of AFB(E). F: TNF/LT-α−/− mouse lung lesion depicts an dramatic cell recruitment and tissue necrosis, associated with an enormous amount of AFB (G). These data are representative of two independent experiments, with 8 to 10 mice per group, except TNF/LT-α−/− mice (n = 1), which most of them died before 4 weeks of infection. Original magnifications: ×200 (A, C, E); ×400 (B, D, F).

Figure 6

Survival curve of M. tuberculosis-infected mice and differential lung pathologies of moribund TmTNF tg and TNF/LT-α−/− mice. A: Survival curve of wild-type, TmTNF tg, and TNF/LT-α−/− mice after 105 CFU of virulent M. tuberculosis strain H37Rv. Each group represents 10 mice, except TNF/LT-α−/− mice (n = 8). Data from two experiments are shown. The increase in survival time for TmTNF tg mice was statistically significant when compared to TNF/LT-α−/− mice (P < 0.0001). Histological lung sections from moribund TmTNF tg mice (13 weeks after infection) (B and C) and TNF/LT-α−/− mouse (4 weeks after infection) (D and E). Sections were stained with H&E (B and D) or with Ziehl-Neelsen (C and E). Sick TmTNF tg mouse lung sections reveal a marked inflammatory cell infiltration as main lung pathology (B) with a low number of AFB (C). In contrast, moribund TNF/LT-α−/− mouse lung pathology was mainly caseous necrotic lesions (arrows) (D), with a very high number of AFB (E). Original magnifications: ×25 (B, D); ×400 (C and E).

Figure 7

During BCG infection TmTNF leads to efficient iNOS expression but not during M. tuberculosis infection. iNOS immunostaining of spleen (A to F) and lung sections (G, H) at 4 weeks of M. tuberculosis (A–C) or BCG (D–F) infections. M. tuberculosis spleen from wild-type (A), TmTNF tg (B), and TNF/LT-α−/− mice (C). BCG-infected spleen from wild-type (D), TmTNF tg (E), and TNF/LT-α−/− (F). Multinucleated giant cells expressing iNOS were identified in lung sections of wild-type (G) and TmTNF tg mice (H) after BCG infection. Pictures in this figure are representative of four mice per group. Original magnifications: ×200 (A to F); ×400 (G to H).

Figure 8

TmTNF tg mice are deficient in iNOS-expressing cells in the lung after M. tuberculosis but not after BCG infection. Quantification of iNOS immunostaining in the lung (A) and spleen (B) sections from mice at 4 weeks of M. tuberculosis and BCG infection. Data are represented as means of iNOS-positive pixels (±SEM) of three to four mice per group, except TNF/LT-α−/− mouse during M. tuberculosis infection (n = 1). These results are representative of two independent experiments. Asterisks indicate statistically significant differences between the indicated group and wild-type mice (*, P < 0.01).