Figure 6. STING signaling is upstream of mitochondrial damage during Mtb infection.

(A) Proposed model of positive feedback loop: IFNβ signaling through IFNAR causes mitochondrial damage, which could release mtDNA to be sensed by the cGAS-STING signaling pathway, leading to induction of Ifnb1 expression and IFNβ secretion.

(B) Quantification of glycolytic parameters in BMDMs infected with live H37Rv (MOI of 10). The ECAR representing glycolytic capacity or the glycolytic reserve of infected WT, infected, untreated STING KO, or infected, IFNβ-treated STING KO BMDMs was normalized to mock-infected controls. Each point represents a single well and the bars are the mean from six plates from two independent experiments. See also Figure S6.

(C) Quantification of mitochondrial parameters in BMDMs infected with live H37Rv (MOI of 10). The OCR dedicated to ATP production (ATP) or at maximal respiration (Max) was normalized to mock-infected, untreated controls. Each point represents a single well and the bars are the mean from six plates from two independent experiments.

(D) Quantification of mROS in WT or STING KO BMDMs infected with an MOI of 10 live H37Rv or HK H37Rv for the indicated time. BMDMs were either untreated or treated with 500 U/mL IFNβ. mROS measured with MSR MFI (flow cytometry) normalized to untreated, mock-infected controls at each time point. A representative experiment of two independent experiments is shown.

(E) Principal-component analysis of WT or STING KO BMDMs at 24 h post-infection with an MOI of 10 live H37Rv or HK H37Rv. BMDMs infected with live H37Rv were left untreated or treated with 500 U/mL IFNβ. The percent of variance explained by the top two principal components is indicated.

(F) Log₂FC in expression (RNA-seq) of the 13 protein coding genes encoded on mtDNA in BMDMs (either WT or STING KO) infected with live H37Rv at an MOI of 10 for 24 h compared to mock infection. STING KO BMDMs were either left untreated or treated with 500 U/mL IFNβ in addition to the infection.

(G) Expression of Ifnb1 in WT, IFNAR KO, or STING KO BMDMs either mock infected (open symbols and dashed line) or infected with an MOI of 10 live H37Rv (filled symbols and solid line) for 4 h. WT and STING KO BMDMs were either left untreated or treated with 500 U/mL IFNβ. Ifnb1 expression was quantified by qRT-PCR. Each point represents a technical replicate and the bars are the mean from two independent experiments from each KO genotype. Statistics shown for comparisons of Ifnb1 expression after H37Rv infection.