cGAS-STING-TBK1-IRF3/7 induced interferon-β contributes to the clearing of non tuberculous mycobacterial infection in mice

Abstract

Type I interferons (IFN-I), such as IFN-α and IFN-β are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-β than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-β in macrophages. Both bacteria induced IFN-β via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-β activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-β impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.

Keywords: STING; Type I interferon; cGAS; extracellular DNA; mycobacterium paratuberculosis; mycobacterium smegmatis; non tuberculous mycobacteria.

Figure 1.

Ifnb induction by NTM mycobacteria in macrophages. (A) qPCR for Ifnb in RAW cells infected with viable MAP 6783, MAP K10, MAH 1287, MAA 44156, MAA 44158, M. bovis BCG Pasteur or M. smegmatis mc (MSM) (MOI 5) for 5 h. (B) IFN-β ELISA of supernatant from RAW cells infected with MAP or MSM. (C) qPCR for Ifnb in RAW cells and (D) BMDM infected with viable (V) or heat inactivated (HI) MAP or MSM (MOI 5) for 30 min (’), 2 h and 5 h. (E) RAW cells and (F) BMDM were infected with MAP or MSM (MOIs 2.5, 5.0, 7.5, 10) for 5 h. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey post test, means ± SEM. (G) RAW cells and (H) BMDM were pre-treated with 5 µM latrunculin B (Lat) for 1 h and subsequently infected with MAP or MSM for 5 h. Data are representative of 3 independent experiments, *p<0.05, **p<0.01 by 2 tailed student's t-test means ± SEM.

Figure 2.

MAP and M. smegmatis (MSM) induce Ifnb expression via the cGAS-STING-TBK1-IRF3/7 signaling pathway. (A) qPCR for Ifnb in BMDM from WT, UNC93B^−/−, MyD88^−/−, TRIF^−/−, (B) IRF3^−/−, IFNAR^/−, IRF7^−/- mice infected with MAP or MSM. *p<0.05, **p<0.01, ns (non-significant) by one-way ANOVA with Tukey post test, means ± SEM. (C) Immunoblot of phosphoTBK1 in RAW cells infected with MAP or MSM for 5 h. (D) qPCR for Ifnb in WT, STING^−/− and (E) cGAS^−/− mice infected with MAP or MSM for 5 h. (F) qPCR analysis of eDNA in RAW cells infected with viable (V) or heat inactivated (HI) MAP or MSM for 5 h. eDNA was extracted and shown as percentage of the input control. (G) qPCR for Ifnb in RAW cells infected with MSM WT or MSM ΔESX-1 for 5 h. Data are representative of 3 independent experiments, *p<0.05, **p<0.01 by 2 tailed student's t-test means ± SEM.

Figure 3.

Pathology of MAP and M. smegmatis (MSM) infected mice. C57BL/6 mice (n = 3–6/group) were infected MAP or MSM. At day 21 post infection, (A) livers (B) spleens and (C) mesentery were plated. *p<0.05, ** p<0.01 by Mann-Whitney U test, means ± SEM. (D) spleen weight was determined. (E) Representative HE stained liver sections. Arrows heads point at granulomas. (F) Numbers of granulomas in livers. Data are representative of 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey post test, means ± SEM.

Figure 4.

Induction of IFN-β after MAP or M. smegmatis (MSM) infection in vivo. (A) IFN-β^+/Δβ-luc (n = 6/group) were infected with MAP or MSM. At day 1, 3, 7, and 14 post infection, mice were injected with D-luciferin and visualized for luciferase activity. (B) Body weights of mice infected with MAP or MSM. **p<0.01 by Two way ANOVA with Bonferroni post test, means ± SEM. (C) C57BL/6 mice (n = 3–10/group) were infected with MAP or MSM. Numbers of CFU in liver were determined. Data are representative of 2 independent experiments. *p<0.05 by Mann-Whitney U test, means ± SEM.

Figure 5.

Clearance of M. smegmatis (MSM) depends on IFN-I signaling. C57BL/6 mice and IFNAR^−/− mice (n = 5–6/group) were injected with PBS, MAP and MSM. (A and B) body weight was monitored. ***p<0.001 by Two way ANOVA with Bonferroni post test, means ± SEM. (C) spleen weights at 21 dpi. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey post test, means ± SEM. (D) Numbers of CFU in liver, (E) spleen and (F) mesentery were determined by plating 21 dpi. Data are representative of 2 independent experiments, *p<0.05, ** p<0.01, ns (non-significant), by Mann-Whitney U test, means ± SEM.

Figure 6.

IFN-β supports MAP clearance. C57BL/6 (n = 3–10/group) were infected with PBS, MAP, MAP + 100 µg poly(I:C)/mouse, or MAP + 1000 units rIFN-β. Mice were killed at 14 dpi. (A) Schematic representation of the experiment. (B) spleen weight was determined. (C) Numbers of CFU in liver, (D) spleen and (E) mesentery were determined by plating. Data are representative of 2 independent experiments, *p<0.05, **p<0.01, ***p<0.001, ns (non-significant), by one-way ANOVA with Tukey post test, means ± SEM.