MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival
C. Korin Bullen, … , Geetha Srikrishna, William R. Bishai
C. Korin Bullen, … , Geetha Srikrishna, William R. Bishai
Published September 19, 2023
Citation Information: JCI Insight. 2023;8(20):e166242. https://doi.org/10.1172/jci.insight.166242.
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Research Article Immunology Infectious disease

MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival

Abstract

Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I–like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5–/– macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5–/– macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ–primed Mda5–/– macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5–/– macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1β production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.

Authors

C. Korin Bullen, Alok K. Singh, Stefanie Krug, Shichun Lun, Preeti Thakur, Geetha Srikrishna, William R. Bishai

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Figure 1

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

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Cytosolic RNA sensor MDA5 modulates host cytokine response to M. tubercu...
(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.