Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Abstract

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.

Figure 1. Graphic presentation of drug-based M. tuberculosis reactivation mouse model.

Line A: Preimmune phase, short bacterial replication period post-infection with low dose M. tuberculosis; Line B: Steady state phase: Control of M. tuberculosis growth through host immunity and establishment of chronic infection. Line D: Drug treatment phase: Reduction of bacilli replication through INH-RIF chemotherapy. Line E: Reactivation phase: Reactivation of infection upon cessation of antibiotics.

Figure 2. Effect of Tm-TNF on M. tuberculosis replication during reactivation.

WT mice (black circles), TNF^−/− mice (white diamonds) and Tm-TNF mice (white squares) were treated for 6 weeks with 25 mg/Kg INH-RIF in drinking water subsequent to 3 weeks aerosol infection with 100–200 CFU's of M. tuberculosis H37Rv. (A) Body weights were recorded throughout the course of the infection period and bacterial burdens in lungs (B) and spleens (C) were enumerated at time points indicated. Data are representative of two experiments and data points are expressed as the mean ± SD of 5 mice/group (for CFUs). The body weight study consisted of between 6–16 mice/group where the data represents weights for remaining mice in each group. The red “T” in the figure corresponds to the drug treatment phase. Significant differences (*p<0.05; **p<0.01) were determined by Student's t test for comparisons between two groups and ANOVA for comparisons between three groups.

Figure 3. Induction of excessive inflammation in the absence of solTNF during reactivation of M. tuberculosis.

WT (black circles), TNF^−/− (white diamonds) and Tm-TNF (white squares) mice were exposed by aerosol inhalation infection to 100–200 CFUs/mouse of M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. (A) Lung weights were measured at specific time points and BAL derived cell numbers were determined 77 days (B) and 378 days (C) post-infection. The red “T” in the figure corresponds to the drug treatment phase. Data are representative of 1 of 2 experiments performed and are expressed as mean ± SD of 5 mice/group. Significant differences (*p<0.05; **p<0.01; ***p<0.001) were determined by Student's t test for comparisons between two groups and ANOVA for comparisons between three groups.

Figure 4. Tm-TNF contributes to protective bactericidal granuloma formation during M. tuberculosis reactivation but is insufficient to sustain structural integrity and bactericidal efficacy.

WT mice (A,D,G,J), TNF^−/− mice (B, E, H) and Tm-TNF mice (C,F,I,K) were infected by aerosol inhalation with 100–200 CFUs/mouse M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed at the indicated timepoints and tissue sections stained with haematoxylin and eosin to determine the granulomatous response.

Figure 5. Extra-granulomatous pulmonary expression of INOS is associated with susceptibility in Tm-TNF reactivating mice.

WT mice (A,D), TNF^−/− mice (B) and Tm-TNF mice (C,E) were infected by aerosol inhalation with 100–200 CFUs/mouse M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed at 133 and 322 days post infection and tissue sections were stained with polyclonal rabbit anti-mouse antibody (see Materials and methods ). Brown stain represents iNOS expression by activated macrophages. Micrographs represent 4 animals/group and are shown at ×32 magnification.

Figure 6. Macrophage (CD11b⁺ cells) and lymphocyte (CD3⁺ cells) recruitment in WT and Tm-TNF mice.

WT mice (A,C) and Tm-TNF mice (B,D) were infected by aerosol inhalation with 100–200 CFUs/mouse M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed 322 days post infection and tissue sections were stained with either anti-CD11b anti-mouse (A,B) antibody or anti-CD3 anti-mouse antibody (C,D) (see Materials and methods). Micrographs represent 4 animals/group and are shown at ×32 magnification.

Figure 7. Reduced pulmonary cytokine expression is associated with increased susceptibility in Tm-TNF mice during M. tuberculosis reactivation.

WT (closed bars) and Tm-TNF mice (open bars) were exposed by aerosol inhalation to 100–200 CFUs/mouse of M. tuberculosis H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were obtained and homogenized at 133 days and 322 days post-infection and the levels of IL-12p70, IFNγ and IL-10 present in cell supernatants determined by ELISA. Data represent 1 of 2 experiments performed and values are expressed as mean ± SD of 5 animals/group. Significant differences (*p<0.05; **p<0.01) were determined by Student's t test.