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. 2023 Oct 23;8(20):e166242. doi: 10.1172/jci.insight.166242

Figure 1. Cytosolic RNA sensor MDA5 modulates host cytokine response to M. tuberculosis–derived RNA and promotes M. tuberculosis intracellular growth in macrophages.

Figure 1

(A) Schematic diagram of experimental design to identify host factors involved in cytosolic surveillance of M. tuberculosis RNA in macrophages. (B) Resting primary human monocyte-derived macrophages were transfected for 24 hours with total M. tuberculosis RNA, total M. tuberculosis RNA treated with RNaseV (+RNase), mock transfection of total M. tuberculosis RNA (–TxRngt), or total human RNA. IFN-β, IL-1β, IL-6, and TNF-α RNA levels were assessed by reverse transcriptase qPCR. RNA levels were normalized to PolR2A. Data are presented as fold-change relative to no-transfection control (mean ± SD, n = 2 biological replicates, 2 independent experiments). (C) CRISPR/Cas9–mediated Mda5-knockout (Mda5-KO) or CRISPR/Cas9 nontarget control (NTC) J774.1 cells were IFN-γ–primed and transfected with total M. tuberculosis RNA for 24 hours. IFN-β, IL-1β, IL-6, and TNF-α levels in the culture medium were quantified by multiplex immunoassay (Luminex) (mean ± SD, n = 6 biological replicates from 3 independent experiments; “<” indicates below limit of quantification). ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey’s posttest. (D) Schematic diagram of experimental design to evaluate the role of Mda5 in innate immune responses to M. tuberculosis infection. (E) MDA5 and β-actin protein levels in WT J774.1 cells at 24 hours after infection with M. tuberculosis at the indicated MOI were detected by immunoblot analysis and quantified by densitometry (mean ± SD, n = 3 biological replicates, 3 independent experiments). ***P < 0.001 by 1-way ANOVA with Tukey’s posttest. (F and G) Growth kinetics of M. tuberculosis (MOI of 1:5) in IFN-γ–primed (F) and resting (G) WT, NTC, and Mda5-KO J774.1 cells. Data are mean CFU ± SEM for each time point (n = 4 biological replicates, 2 independent experiments). *P < 0.05, **P < 0.01, ****P < 0.0001 by 2-way ANOVA with Tukey’s posttest.