The Mechanism for Type I Interferon Induction by <i>Mycobacterium tuberculosis</i> is Bacterial Strain-Dependent

<div><p>Type I interferons (including IFNαβ) are innate cytokines that may contribute to pathogenesis during <i>Mycobacterium tuberculosis</i> (Mtb) infection. To induce IFNβ, Mtb must gain access to the host cytosol and trigger stimulator of interferon genes (STING) signaling. A recently proposed model suggests that Mtb triggers STING signaling through bacterial DNA binding cyclic GMP-AMP synthase (cGAS) in the cytosol. The aim of this study was to test the generalizability of this model using phylogenetically distinct strains of the Mtb complex (MTBC). We infected bone marrow derived macrophages with strains from MTBC Lineages 2, 4 and 6. We found that the Lineage 6 strain induced less IFNβ, and that the Lineage 2 strain induced more IFNβ, than the Lineage 4 strain. The strains did not differ in their access to the host cytosol and IFNβ induction by each strain required both STING and cGAS. We also found that the three strains shed similar amounts of bacterial DNA. Interestingly, we found that the Lineage 6 strain was associated with less mitochondrial stress and less mitochondrial DNA (mtDNA) in the cytosol compared with the Lineage 4 strain. Treating macrophages with a mitochondria-specific antioxidant reduced cytosolic mtDNA and inhibited IFNβ induction by the Lineage 2 and 4 strains. We also found that the Lineage 2 strain did not induce more mitochondrial stress than the Lineage 4 strain, suggesting that additional pathways contribute to higher IFNβ induction. These results indicate that the mechanism for IFNβ by Mtb is more complex than the established model suggests. We show that mitochondrial dynamics and mtDNA contribute to IFNβ induction by Mtb. Moreover, we show that the contribution of mtDNA to the IFNβ response varies by MTBC strain and that additional mechanisms exist for Mtb to induce IFNβ.</p></div

Mitochondrial stress contributes to accumulation of mtDNA in the cytosol during H37Rv/Lineage 4 and 4334/Lineage 2 infections.

<p>A-H) BMDM were treated with MitoQ or control (dTPP) for 4 hours and then infected with the indicated mycobacterial strains at an MOI of 5. A) Uninfected cells were stained with MitoSOX at the time of infection. B) Mean fluorescence intensity (MFI) of MitoSOX was determined using ImageJ. C-H) 24 hr post infection, cells were collected and fractionated. Amount of DNA in cytosolic (C-E) and undisturbed cell (F-H) fractions was determined using gene-specific primers as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005809#ppat.1005809.g004" target="_blank">Fig 4A–4D</a>; amount in ng or μg was determined using standards that were generated independently of experimental samples and that contained abundant levels of each gene. *p<0.05, **p<0.01, ****p<0.0001 by two-way ANOVA with Sidak post-tests; means ± SD (n = 3).</p

New protein synthesis is required for inhibition of CIITA and CXCL11.

<p>BMDM were pretreated with 500 nM cycloheximide or DMSO for 1 h prior to treatment with 10 ng/ml Pam₃CSK₄ for 8 h followed by 20 ng/ml IFN-γ for 4 h in the continued presence or absence of inhibitor. Total RNA was harvested after IFN-γ stimulation. CXCL11 (A), CIITA (B), and NOS2 (C) expression was assayed by qPCR and normalized to GAPDH and untreated samples. The concentration of cycloheximide used inhibited TNF production (as a measure of protein synthesis) by over 90% with minimal cell death. Statistical significance between IFN-γ alone samples and those treated with Pam₃CSK₄ prior to IFNγ was determined by two-tailed t-test.</p

IFNβ induction is bacterial strain-dependent during Mtb infection.

<p>A) BMDM were infected with the indicated mycobacterial strains at an MOI of 5 or stimulated with LPS (10 ng/mL). RNA was harvested for IFNβ mRNA quantification by qRT-PCR at 3, 6, and 24 hr post infection. *p<0.05, **p<0.01, ****p<0.0001 by two-way repeated measures ANOVA with Tukey post-tests; means ± SD (n = 3). B) BMDM were infected with the indicated bacterial strains at MOI of 1, 5, and 10 or stimulated with LPS (10 ng/mL). Supernatants were collected for IFNβ protein quantification by ELISA at 48 hr post infection. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Tukey post-tests for each MOI; means ± SD (n = 3). Results are representative of 3 independent experiments. C-D) Cell lysates from the experiment shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005809#ppat.1005809.g001" target="_blank">Fig 1B</a> were collected at 3, 24, and 48 hr post infection. Colony forming units (CFU) were quantified by serial dilution on 7H11 agar plates. CFU for each MOI at each time point are plotted on the x-axes and the corresponding IFNβ (C) and TNF (D) secretion at 48 hr post infection is plotted on the y-axes.</p

IFNβ induction by each MTBC strain is dependent on STING and cGAS.

<p>A) Wild type and STING^-/- BMDM were infected with the indicated mycobacterial strains at an MOI of 5. Supernatants were collected for IFNβ protein quantification by ELISA at 48 hr post infection. B) Wild type and cGAS^-/- BMDM were infected with the indicated bacterial strains at an MOI of 5. Supernatants were collected for IFNβ protein quantification by ELISA at 48 hr post infection. ****p<0.0001 by two-way ANOVA with Tukey post-tests; means ± SD (n = 3).</p

IFN-γ-induced transcription of CXCL11, but not CIITA, is restored in TLR2 stimulated RelA^−/− macrophages.

<p>BMDM from TNF^−/−/RelA^+/+ and TNF^−/−/RelA^−/− mice were treated with 10 ng/ml Pam₃CSK₄ for 8 h followed by 20 ng/ml IFN-γ for 4 h. Total RNA was harvested, reverse transcribed, and CXCL11 (A), CIITA (B), and NOS2 (C) expression analyzed by qPCR. All values were normalized to GAPDH and shown as fold induction compared to untreated samples without Pam₃CSK₄ or IFN-γ. Statistical significance was determined by two-tailed t-test between IFN-γ alone with Pam₃CSK₄ prior to IFN-γ samples. C57BL/6 BMDM showed similar results to those obtained with TNF^−/−/RelA^+/+ BMDM.</p

Access to the host cytosol does not vary by mycobacterial strain.

<p>A-B) BMDM were infected with the indicated dsRed-expressing mycobacterial strains at an MOI of 1. BMDM were fixed overnight and stained for FK2 and Galectin-3 at 3, 12, 24, and 48 hr post infection. Representative images are shown for FK2 colocalization at 48 hr post infection (A) and Galectin-3 colocalization at 24 hr post infection (B), with bacteria shown in magenta and FK2 and Galectin-3 shown in green. C-D) 10 images were captured at 100x magnification for each chamber well and the percent of bacteria that colocalized with FK2 (C) and Galectin-3 (D) was calculated for each strain. E-F) Mean fluorescence intensity (MFI) of the FK2 (E) and Galectin-3 (F) staining directly surrounding each colocalized bacterium was calculated using ImageJ. Results are representative of 1–3 independent experiments. All differences in percent colocalization and MFI between 1182, H37Rv, and 4334 were not significant (p>0.05) by two-way repeated measures ANOVA with Tukey post-tests; means ± SD (n = 4).</p

Mitochondrial stress contributes to IFNβ induction by MTBC strains.

<p>A-F) BMDM were treated with MitoQ or control (dTPP) for 4 hours and then infected with the indicated bacterial strains at an MOI of 5. A-B) 48 hr post infection supernatants were collected for IFNβ (A) and TNF (B) protein quantification by ELISA. *p<0.05, ****p<0.0001 by two-way ANOVA with Sidak post-tests; means ± SD (n = 3). IFNβ results are representative of 3 independent experiments. TNF comparison between control and MitoQ treatment is representative of 3 independent experiments, but the differences in TNF between Mtb strains varied between experiments. C-D) Percent inhibition of IFNβ (C) or TNF (D) induction during MitoQ treatment was calculated for each replicate (n = 3) during infection with each strain. Pearson correlation coefficient (r) of percent inhibition (y-axis) and cytokine secretion (x-axis) is shown. G) Percent inhibition of IFNβ during MitoQ treatment. *p<0.05, **p<0.01 by one-way ANOVA with Tukey post-tests; means ± SD (n = 3). F) Lysates from replicates (n = 3) of the experiment shown in Fig 6A–6E were pooled and CFU were quantified by serial dilution on 7H11 agar plates.</p

TLR2 stimulation prevents binding of general transcriptional machinery to the CIITA and CXCL11 promoters.

<p>BMDM were treated with 10 ng/ml Pam₃CSK₄ for 8–9 h, then 20 ng/ml IFN-γ for 4 h. Cross-linked DNA was sheared and immunoprecipitated with anti-PolII (A) or anti-TBP (B) antibodies. Precipitated and input DNA for each sample were assayed by qPCR with primers specific for the transcriptional start site in the promoters of CXCL11, CIITA, and NOS2 (A) or the TATA box of the CXCL11 promoter (B). All values were normalized to GAPDH. Results are expressed as fold increase over untreated controls and are the mean of triplicate samples±SD. Statistical significance between IFN-γ alone samples and Pam₃CSK₄ prior to IFN-γ treated samples was determined by two-tailed t-test. Results are representative of at least two independent experiments.</p

TLR2-mediated inhibition of IFN-γ induction of CXCL11 and CIITA decreases expression of protein products.

<p>A. BALB/c BMDM were treated with 10 ng/ml Pam₃CSK₄ for 8 h followed by 20 ng/ml IFN-γ for 4, 8, and 12 h. Culture supernatants were collected and assayed for CXCL11 protein by ELISA. *, p<0.01, **, p<0.05 comparing IFN-γ alone with Pam₃CSK₄ and IFN-γ treated samples (as determined by two-tailed t-test). B and C. BMDM were treated with 10 ng/ml Pam₃CSK₄ for 12–15 h prior to stimulation with 20 ng/ml IFN-γ for 24 h. Cells were stained with Alexa 647-conjugated anti-mouse I-A/I-E and analyzed by flow cytometry. Data shown are fluorescence intensity vs. cell number (B) and mean I-A/I-E fluorescence (C). Results are expressed as means±SEM from two independent experiments (A) and are representative of at least five independent experiments (B and C).</p