The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas
Bidisha Bhattacharya, … , William Bishai, Igor Kramnik
Bidisha Bhattacharya, … , William Bishai, Igor Kramnik
Published December 10, 2020
Citation Information: J Clin Invest. 2021;131(3):e130319. https://doi.org/10.1172/JCI130319.
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The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas

Abstract

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-β/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis–infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.

Authors

Bidisha Bhattacharya, Shiqi Xiao, Sujoy Chatterjee, Michael Urbanowski, Alvaro Ordonez, Elizabeth A. Ihms, Garima Agrahari, Shichun Lun, Robert Berland, Alexander Pichugin, Yuanwei Gao, John Connor, Alexander R. Ivanov, Bo-Shiun Yan, Lester Kobzik, Bang-Bon Koo, Sanjay Jain, William Bishai, Igor Kramnik

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Figure 2

TNF treatment leads to biphasic upregulation of the ISR in B6.Sst1S BMDMs.

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TNF treatment leads to biphasic upregulation of the ISR in B6.Sst1S BMDM...
(A) Kinetics of integrated stress response (ISR) gene expression in B6.Sst1S and WT B6 BMDMs treated with TNF (10 ng/mL). (B) Kinetics of ISR proteins in TNF-stimulated WT B6 and B6.Sst1S BMDMs (representative of 2 biological replicates). (C) Kinetics of IFN-β and ISR gene expression in B6.Sst1S BMDMs 8–14 hours after stimulation with TNF. (D) Effects of TNF and IFNAR1 blockade on Chac1 and Trb3 mRNA expression in B6.Sst1S BMDMs stimulated with TNF. The neutralizing anti-IFNAR1 and anti–TNF-α or isotype control antibodies were added 2–12 hours after TNF. The mRNA expression was measured at 16 hours of TNF stimulation and percentage inhibition by the neutralizing antibodies added at each time point was calculated with respect to cells treated with TNF alone. (E) Effect of the ER stress (PBA), PKR (C16), ISR (ISRIB), and TBK1 (BX795) inhibitors on late-phase ISR gene expression in TNF-stimulated B6.Sst1S BMDMs. The inhibitors were added 12 hours after TNF stimulation, and the mRNA levels were measured at 16 hours. (F) Kinetics of PKR protein expression and phosphorylation (p-PKR) in B6.Sst1S and WT B6 BMDMs treated with TNF. Ratios of p-PKR to those in unstimulated BMDMs are presented for each time point. The Western blot is representative of 2 independent experiments. In panels A and C–E, 2-way ANOVA with Tukey’s post hoc test was used on combined data of 3 independent experiments (*P = 0.01–0.05; ***P < 0.001). The qRT-PCR data were normalized to 18S rRNA and are presented in panels A and C relative to expression in untreated cells (set to 1). Percentage inhibition in panels D and E was calculated as compared to fold induction by TNF in the absence of inhibitors. NS, not significant.