Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis

Abstract

Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

Figure 1. Enhanced acute control of M. tuberculosis replication in TNFRp75^–/– mice.

WT, TNFRp75^–/–, TNFRp55^–/–, and TNFRp55/75^–/– mice were infected with 50–100 CFU M. tuberculosis. Mice were monitored for survival (A), and body weight changes were measured (B) (n = 10 per group). Pulmonary bacilli burden (C) was determined at the indicated time points by colony enumeration assay. Data (mean ± SEM of the CFU of 4 mice per time point) are representative of 3 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 2. Reduced pulmonary pathology and inflammation in M. tuberculosis–infected TNFRp75^–/– mice.

WT, TNFRp75^–/–, TNFRp55^–/–, and TNFRp55/75^–/– mice were infected by aerosol inhalation with 50–100 CFU M. tuberculosis. Mice were sacrificed at the indicated time points, pulmonary pathology was recorded 35 days after infection (A), lung weights were assessed (B), and total pulmonary cell numbers at day 35 after infection were determined (C). Values are mean ± SEM of 4 mice per group. (D–K) Lungs were removed on days 28 (D–G) and 35 (H–K), fixed in formalin, embedded in wax, and sectioned, after which H&E staining was performed. Arrows denote the presence of granuloma structures; × symbols denote tissue necrosis. Original magnification, ×40. Data represent 1 of 3 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 3. Increased protection of TNFRp75^–/– mice during chronic M. tuberculosis infection.

WT and TNFRp75^–/– mice were infected by aerosol inhalation with 50–100 CFU M. tuberculosis. (A and B) Survival was monitored (A; n = 10 per group; P < 0.0005, log-rank test), and pulmonary bacilli burdens were determined at the indicated time points by colony enumeration assay (B). (C) Bacilli burden in lung sections was confirmed by Ziehl-Neelsen staining. (D and E) Chronic inflammation was quantified at 4.5 months after infection (D) and confirmed by analysis of H&E-stained lungs sections at 4.5 and 7.5 months after infection (E). Original magnification, ×1,000 (C); ×40 (E). Values in B and D are mean ± SEM of 4 mice per group. Data are representative of 4 similar experiments (A) or 1 of 3 similar experiments (B–E). *P < 0.05, **P < 0.01, ANOVA.

Figure 4. Increased recruitment of activated DCs to the lungs of M. tuberculosis–infected TNFRp75^–/– mice.

WT and TNFRp75^–/– mice were infected at 50–100 CFU with M. tuberculosis. Lungs were harvested at 7, 14, and 21 days after infection, and CD11b⁺ versus CD11c⁺ cell distribution (A) and total number of CD11c⁺ (B), CD11c⁺MHCII⁺ (C), CD11c⁺CD80⁺ (D), and CD11c⁺CD86⁺ (E) cells was assessed by flow cytometry. Data (mean ± SEM of 4 mice per group) are representative of 1 of 2 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 5. Increased activated DCs induces enhanced M. tuberculosis–specific IFN-γ production by activated CD4⁺ T cells in M. tuberculosis–infected TNFRp75^–/– mice.

(A) BMDCs from WT and TNFRp75^–/– mice were infected at an MOI of 5:1 with M. tuberculosis, and IL-12p40 expression in culture supernatants was analyzed by ELISA. (B–E) WT and TNFRp75^–/– mice were infected at 50–100 CFU with M. tuberculosis, and lungs and LNs were harvested 14 and 21 days after infection. The total number of LN CD11c⁺ cells (B), CD11c⁺CD86⁺ cells (C), CD11c⁺MHCII⁺ cells (D), and CD4⁺CD44⁺ T cells (E) was analyzed by flow cytometry. Data (mean ± SEM of 4 mice per group) are representative of 3 similar experiments. (F) IFN-γ expression by CD4⁺ T cells in isolated pulmonary cell cultures restimulated with ESAT6 or M. tuberculosis H37Rv (M.tb). Data (mean ± SEM of triplicate values of pooled samples from 4 mice) are representative of 3 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 6. M. tuberculosis induces TNFR shedding.

(A and B) WT and TNFRp75^–/– BMDCs were infected with M. tuberculosis at an MOI of 5:1, and TNFRp75 surface expression (A) and soluble TNFRp55 (B) were measured. Data (mean ± SEM of quadruplicate experiments) are representative of 1 of 2 experiments. (C–E) WT, TNFRp75^–/–, and TNFRp55^–/– mice were infected with 50–100 CFU M. tuberculosis. Surface TNFRp75 (C) was measured by flow cytometry in BAL cells, and soluble TNFRp55 (D) and TNFRp75 (E) were measured in lung homogenates by ELISA. ND, not detectable. Data (mean ± SEM of 4–5 mice per group) are representative of 1 of 2 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 7. Soluble TNFRp75 inhibits DC activation during M. tuberculosis infection.

(A and B) WT and TNFRp75^–/– DCs were infected with M. tuberculosis at an MOI of 5:1, and Tnf mRNA (A) or bioactive TNF (B) concentrations were measured. (C and D) WT and TNFRp75^–/– mice were infected with 50–100 CFU M. tuberculosis. Total (C) and bioactive (D) TNF was measured in BALF 1, 3, and 6 days after infection. (E and F) In vitro surface expression of TNFRp55 (E) was measured in WT and TNFRp75^–/– BMDCs, and in vivo surface TNFRp55 (F) was measured by flow cytometry in BAL cells. (G–I) The effect of soluble TNFRp75 on M. tuberculosis–infected BMDC cultures from WT and TNFRp75^–/– mice were assessed by measuring bioactive TNF (G), IL-12p40 (H), and MHCII⁺ expression (I) in the presence or absence of anti-TNFRp75. Note the increased expression induced by anti-TNFRp75 in WT mice compared with M. tuberculosis infection alone (dashed lines). Data (mean ± SEM of quadruplicate experiments) are representative of 1 of 2 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 8. DC activation is dependent on soluble TNFRp75 inactivation of bioactive TNF.

(A–D) WT, TNFRp75^–/–, and TNFRp55/75^–/– BMDCs were infected with M. tuberculosis or M. bovis BCG at an MOI of 5:1. Surface expression of TNFRp75 (A), soluble TNFRp75 (B), bioactive TNF (C), and IL-12p40 (D) was measured. (E) The effect of recombinant TNFRp75 on M. tuberculosis–infected BMDC cultures from WT and TNFRp75^–/– mice were assessed by measuring IL-12p40 in the presence or absence of recombinant TNFRp75. Whereas anti-TNFRp75 in WT mice increased IL-12p40 expression, addition of recombinant TNFRp75 dose-dependently reduced expression in both WT and TNFRp75^–/– mice, compared with M. tuberculosis infection alone (dashed lines). Data (mean ± SEM of quadruplicate experiments) are representative of 1 of 2 similar experiments. *P < 0.05, **P < 0.01, ANOVA.

Figure 9. DC activation is primarily mediated by TNF-TNFRp55 signaling and regulated by soluble TNFRp75.

WT, TNFRp75^–/–, TNFRp55^–/–, and TNFRp55/75^–/– DCs were infected with M. tuberculosis at an MOI of 5:1. (A) Bioactive TNF was determined using WEHI assays. (B) IL-12p40 was analyzed by ELISA. Data (mean ± SEM of quadruplicate values) are representative of 1 of 2 similar experiments. **P < 0.01, ANOVA.