Reactivation of M. tuberculosis Infection in Trans-Membrane Tumour Necrosis Factor Mice

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

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis

BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF / ) mice and compared outcomes of disease against TNF f/f control and global TNF / mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF / mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF / mice were resistant to infection and presented with a phenotype similar to that in TNF f/f control mice. Impaired immunity in TNF / mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB

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

<p>WT mice (A,C) and Tm-TNF mice (B,D) were infected by aerosol inhalation with 100–200 CFUs/mouse <i>M. tuberculosis</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025121#s2" target="_blank">Materials and methods</a>). Micrographs represent 4 animals/group and are shown at ×32 magnification.</p

Tm-TNF contributes to protective bactericidal granuloma formation during <i>M. tuberculosis</i> reactivation but is insufficient to sustain structural integrity and bactericidal efficacy.

<p>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 <i>M. tuberculosis</i> 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.</p

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

<p>WT mice (A,D), TNF^−/− mice (B) and Tm-TNF mice (C,E) were infected by aerosol inhalation with 100–200 CFUs/mouse <i>M. tuberculosis</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025121#s2" target="_blank"><i>Materials and methods</i></a>). Brown stain represents iNOS expression by activated macrophages. Micrographs represent 4 animals/group and are shown at ×32 magnification.</p

Induction of excessive inflammation in the absence of solTNF during reactivation of <i>M. tuberculosis</i>.

<p>WT (black circles), TNF^−/− (white diamonds) and Tm-TNF (white squares) mice were exposed by aerosol inhalation infection to 100–200 CFUs/mouse of <i>M. tuberculosis</i> 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 (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001) were determined by Student's <i>t</i> test for comparisons between two groups and ANOVA for comparisons between three groups.</p

Effect of Tm-TNF on <i>M. tuberculosis</i> replication during reactivation.

<p>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 <i>M. tuberculosis</i> 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 (*<i>p</i><0.05; **<i>p</i><0.01) were determined by Student's <i>t</i> test for comparisons between two groups and ANOVA for comparisons between three groups.</p

Graphic presentation of drug-based <i>M. tuberculosis</i> reactivation mouse model.

<p>Line A: Preimmune phase, short bacterial replication period post-infection with low dose <i>M. tuberculosis</i>; Line B: Steady state phase: Control of <i>M. tuberculosis</i> 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.</p