FIGURE SUMMARY
Title

Tumor necrosis factor induces pathogenic mitochondrial ROS in tuberculosis through reverse electron transport

Authors
Roca, F.J., Whitworth, L.J., Prag, H.A., Murphy, M.P., Ramakrishnan, L.
Source
Full text @ Science
Fig. 1

Fig. 1. ETC-derived mROS drive necrosis of Mm-infected macrophages in TNF-high conditions.
(A) Representative pseudocolored confocal images of wild-type or TNFhi larvae with yellow fluorescent protein (YFP)–expressing macrophages (green), 1 day after infection with EBFP2-expresssing Mm (blue), showing MitoTracker Red CM-H2Xros (magenta) fluorescence. White arrowheads, uninfected macrophages; yellow arrowheads, infected macrophages; yellow arrows, infected macrophages positive for mROS. Scale bar, 20 μm. (B) Quantification of mROS in wild-type or TNFhi larvae 9 hours after injection of live or heat-killed Mm. Each point represents the mean maximum intensity fluorescence of MitoTracker Red CM-H2Xros per fish. Black symbols represent macrophages that do not contain bacteria. Red and purple symbols represent Mm-infected and heat-killed Mm-containing macrophages, respectively, in the same animal. Horizontal bars denote means; *P < 0.05 (one-way ANOVA with uncorrected Dunn’s post-test for differences between macrophages in the same animal and with Tukey’s post-test for differences between treatments). Data are representative of two independent experiments. (C to F) Quantification of mROS in larvae 1 day after infection with Mm that are wild-type or TNFhi treated with FCCP (C), DNP (D), nigericin (E), or diazoxide (F), or vehicle. Horizontal bars denote means; ****P < 0.0001 (one-way ANOVA with Tukey’s post-test). Data are representative of two or three independent experiments.
Fig. 2

Fig. 2. TNF induces RET mROS at complex I in mycobacterium-infected macrophages.
(A and B) Illustrations of mROS production at complex I during forward electron transport (A) and reverse electron transport (B). FAD, flavin adenine dinucleotide; ΔΨ, membrane potential; IMM, inner mitochondrial membrane; ADP/ATP, adenosine diphosphate/triphosphate; cytC, cytochrome C; I to V, complexes I to V; zigzag arrows, induction; red blunted arrows, inhibition. (C to H) Quantification of mROS in larvae 1 day after infection with Mm [(C) to (E)] or Mtb [(F) to (H)] that are wild-type treated with rotenone or vehicle (C); wild-type or TNFhi treated with rotenone or vehicle (D); wild-type, TNFhi, or TNFhi expressing AOX (E); wild-type or TNFhi (F); wild-type treated with rotenone or vehicle (G); or wild-type or TNFhi treated with rotenone or vehicle (H). Horizontal bars denote means; *P < 0.05, **P < 0.01, ****P < 0.0001 [one-way ANOVA with Dunn’s post-test in (C), (G), and (H); Tukey’s post-test in (D) and (E); uncorrected Dunn’s post-test in (F)]. Black and red symbols in (C), (F), and (G) represent uninfected (ui) and infected macrophages, respectively, in the same animals. Data are representative of two or three independent experiments [(C to G)] or a single experiment (H).
Fig. 3

Fig. 3. TNF increases succinate-dependent mROS in mycobacterium-infected macrophages.
(A and B) Quantification of mROS in larvae 1 day after infection with Mm that are wild-type or TNFhi treated with atpenin A, TTFA, DM-malonate, or vehicle (A) or wild-type treated with succinate, DEBM, or vehicle. Horizontal bars denote means; *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001 [one-way ANOVA with Tukey’s post-test (A) or Dunn’s post-test (B)]. Black and red symbols in (B) represent uninfected (ui) and Mm-infected (Mm) macrophages, respectively, in the same animal. Data are representative of two or three independent experiments.
Fig. 4

Fig. 4. TNF-induced glutamine cellular uptake and increased glutaminolysis is responsible for RET and mROS production in mycobacterium-infected macrophages.
(A) Illustration of main metabolic pathways fueling the Krebs cycle with inhibitors used (truncated red arrows). GLUD1, glutamate dehydrogenase 1. (B to K) Quantification of mROS in larvae 1 day after infection with Mm that are wild-type or TNFhi treated with GPNA, BPTES, R-162, or vehicle (B); wild-type treated with GPNA, BPTES, R-162, or vehicle (C); wild-type or TNFhi treated with telaglenastat or vehicle (D); wild-type treated with telaglenastat, R-162, or vehicle (E); wild-type or TNFhi treated with vehicle, or GPNA or R-162 alone or in combination with DM-glutamate (F); wild-type or TNFhi treated with UK5099 or vehicle (G); wild-type or TNFhi treated with perhexiline, 4-BrCA, or vehicle (H); wild-type treated with UK5099, perhexiline, 4-BrCA, or vehicle (I); wild-type or TNFhi treated with vehicle, or UK5099 or perhexiline alone or in combination with M-pyruvate (J); or GPNA or R-162 alone or in combination with M-pyruvate (K). Horizontal bars denote means; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [one-way ANOVA with Tukey’s post-test in (B), (D), (F) to (H), (J), and (K); Dunn’s post-test in (C), (E), and (I)]. Black and red symbols in (C), (E), and (I) represent uninfected (ui) and Mm-infected (Mm) macrophages, respectively, in the same animals. Data are representative of two or three independent experiments [(B) to (D), (G) to (I)] or a single experiment [(E), (F), (J), and (K)].
Fig. 5

Fig. 5. TNF-induced glutaminolysis increases succinate levels in mycobacterium-infected macrophages in a RIP3- and PGAM5-dependent manner.
(A and B) Quantification of succinate in zebrafish larvae 1 day after infection with Mm or mock-injected, that are wild-type or TNFhi treated with GPNA, BPTES, or vehicle (A) and TNFhi, TNFhi RIP3 morphants, or TNFhi PGAM5 morphants (B). Each point represents the mean of four independent experiments in (A) and two independent experiments in (B). Vertical bars denote pooled SD. ***P < 0.001, ****P < 0.0001 (one-way ANOVA with Tukey’s post-test); ns, not significant.
Fig. 6

Fig. 6. TNF-mediated increased glutamine cellular uptake in mycobacterium-infected macrophages increases succinate oxidation, mROS, and necrosis.
(A) Representative pseudocolored confocal images of 5-dpi granulomas in wild-type or TNFhi larvae with YFP-expressing macrophages (green) infected with tdTomato-expressing Mm (magenta). Arrowheads, extracellular cording bacteria. Scale bar, 50 μm. (B) Bacterial cording in wild-type larvae 5 days after infection with Mm, treated with vehicle, or succinate or DEBM alone or in combination with diazoxide; **P < 0.01, ***P < 0.001 (Fisher’s exact test). (C) Bacterial cording 5 days after infection with Mm in wild-type and TNFhi larvae and wild-type and TNFhi larvae expressing AOX; **P < 0.01, ****P < 0.0001 (Fisher’s exact test). (D) Bacterial cording 5 days after infection with wild-type or AOX-expressing larvae infected with Mm and treated with succinate, DEBM, or vehicle; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Fisher’s exact test). (E) Number of trunk macrophages in Mm-infected larvae and mock-injected (ui) larvae 1 day after infection. Horizontal bars denote means; ****P < 0.0001 (one-way ANOVA with Dunn’s post-test). (F and G) Percentage of dead THP-1 macrophages 5 hours after addition of TNF, treated with rotenone or vehicle starting 1 hour before TNF addition (F) or MitoParaquat (MitoPQ) or vehicle for 5 hours (G). Black and red symbols represent uninfected (ui) and Mtb-infected macrophages, respectively, within the same treatment well. Horizontal bars denote means; *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way ANOVA with Tukey’s post-test). (H) Schematic diagram showing the role of TNF, mROS, and mycobacterial factor(s) in TNF-mediated necrosis of mycobacterium-infected macrophages. Data are representative of two independent experiments [(C), (D), (E), and (G)] or a single experiment [(B) and (F)].
Fig. 7

Fig. 7. Currently available drugs can intercept TNF-induced mROS production and inhibit necrosis of mycobacterium-infected macrophages.
(A) Representative pseudocolored confocal images of 5-dpi granulomas in larvae with YFP-stained macrophages (green) that are wild-type or TNFhi treated with diazoxide or vehicle, infected with red fluorescent Mm (magenta). Arrowheads, extracellular cording bacteria. Scale bar, 50 μm. (B to E) Bacterial cording in wild-type or TNFhi larvae 5 days after infection with Mm, treated with vehicle or diazoxide (B), DM-malonate (C), telaglenastat (D), or perhexiline (E). *P < 0.05, **P < 0.01, ****P < 0.0001 (Fisher’s exact test). (F) Quantification of mROS in wild-type or TNFhi larvae 1 day after infection with Mm, treated with metformin, phenformin, or vehicle. Horizontal bars denote means; **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post-test). (G) Bacterial cording in wild-type or TNFhi larvae 5 days after infection with Mm, treated with metformin or vehicle. ****P < 0.0001 (Fisher’s exact test). (H) Bacterial cording in wild-type larvae 5 days after infection with Mm, treated with vehicle or with succinate or DEBM alone or in combination with metformin. *P < 0.05, **P < 0.01, ***P < 0.001 (Fisher’s exact test). (I) Quantification of mROS in wild-type or TNFhi 1 day after infection with Mtb, treated with metformin or vehicle. Horizontal bars denote means; *P < 0.05 (one-way ANOVA with Tukey’s post-test). (J) Percentage of dead THP-1 macrophages at 5 hours after addition of TNF, treated with metformin or vehicle starting 1 hour before TNF addition. Black and red symbols represent uninfected (ui) and Mtb-infected macrophages, respectively, within the same treatment well. Horizontal bars denote means; **P < 0.01, ****P < 0.0001 (one-way ANOVA with Tukey’s post-test). Data are representative of two independent experiments [(B) to (G) and (I)] or a single experiment [(H) and (J)].
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Science
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