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

Abstract

Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. In zebrafish, we identified the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake leads to an increased concentration of succinate, a Krebs cycle intermediate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor metformin, a widely used antidiabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis-infected zebrafish and human macrophages; metformin may therefore be useful in tuberculosis therapy.

Figure 1:. ETC-derived mROS drive necrosis of Mm-infected macrophages in TNF-high conditions.

(A) Representative pseudocolored confocal images of wild-type (WT) or TNF^hi larvae with YFP-expressing macrophages (green), 1 day post infection (dpi) 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 TNF^hi larvae 9 hours post-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, 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). Representative of two independent experiments. (C to F) Quantification of mROS in larvae 1 dpi with Mm that are wild-type, TNF^hi treated with (C) FCCP, (D) DNP, (E) nigericin, or (F) diazoxide, or vehicle. Horizontal bars represent means; ****P<0.0001 (one-way ANOVA with Tukey’s post-test). Representative of two-to-three independent experiments.

Figure 2:. TNF induces RET mROS at complex I in mycobacterium-infected macrophages.

(A and B) Illustrations of mROS production at complex I during (A) forward electron transport and (B) reverse electron transport. ΔΨ, membrane potential; IMM, inner mitochondrial membrane; I-V, complexes I-V; zigzag arrows, induction; red blunted arrows, inhibition. (C to H) Quantification of mROS in larvae 1 dpi with Mm (C to E) or Mtb (F to H) that are (C) wild-type treated with vehicle or rotenone, (D) wild-type (WT), TNF^hi treated with rotenone or vehicle, (E) wild-type, TNF^hi, or TNF^hi expressing AOX, (F) wild-type or TNF^hi, (G) wild-type treated with rotenone or vehicle, (H) wild-type, or TNF^hi treated with rotenone or vehicle. Horizontal bars represent means; *P<0.05, **P<0.01, ****P<0.0001 (one-way ANOVA with Dunn’s post-test (C, G, and H), Tukey’s post-test (D and E) or uncorrected Dunn’s post-test (F)). Black and red symbols in (C, F, and G) represent uninfected (ui) and infected macrophages, respectively, in the same animals. (C to G) representative of two-to-three independent experiments; (H) data from a single experiment.

Figure 3:. TNF increases succinate in mycobacterium-infected macrophages.

Quantification of mROS in larvae 1 dpi with Mm that are (A) wild-type (WT), or TNF^hi treated with atpenin A, TTFA, DM-malonate, or vehicle (B) wild-type treated with succinate, DEBM, or vehicle. Horizontal bars represent means; *P<0.05; **P<0.01, ****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. (A and B) representative of two-to-three independent experiments.

Figure 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). (B to K) Quantification of mROS in larvae 1 dpi with Mm that are (B) wild-type (WT) or TNF^hi treated with GPNA, BPTES, R-162, or vehicle, (C) wild-type treated with GPNA, BPTES, R-162, or vehicle, (D) wild-type or TNF^hi treated with telaglenastat or vehicle, (E) wild-type treated with telaglenastat, R-162, or vehicle, (F) wild-type or TNF^hi treated with vehicle, or GPNA or R-162 alone or in combination with DM-glutamate, (G) wild-type or TNF^hi treated with UK5099 or vehicle, (H) wild-type, or TNF^hi treated with perhexiline, 4-BrCA, or vehicle, (I) wild-type treated with UK5099, perhexiline, 4-BrCA, or vehicle, (J and K) wild-type or TNF^hi treated with vehicle, or UK5099 or perhexiline (J), or GPNA or R-162 (K) alone or in combination with M-pyruvate. Horizontal bars represent means; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA with Tukey’s post-test (B, D, F to H, J and K), Dunn’s post-test (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. (B to D and G to I), representative of two-to-three independent experiments; (E, F, J, and K); data from a single experiment.

Figure 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 dpi with Mm or mock-injected, that are (A) wild-type (WT) or TNF^hi treated with GPNA, BPTES, or vehicle and (B) TNF^hi, TNF^hi RIP3 morphants, or TNF^hi PGAM5 morphants. Each point represents the mean of four independent experiments in A and two independent experiments in B. Horizontal bars represent pooled SD. ***P<0.001, ****P<0.0001 (one-way ANOVA with Tukey’s post-test).

Figure 6:. TNF-mediated increased glutamine cellular uptake in mycobacterium-infected increases succinate oxidation, mROS and necrosis.

(A) Representative pseudocolored confocal images of 5 dpi granulomas in wild-type (WT) or TNF^hi 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 dpi 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 dpi with Mm in wild-type and TNF^hi larvae and wild-type and TNF^hi larvae expressing AOX; **P<0.01, ****P<0.0001 (Fisher’s exact test). (D) Bacterial cording 5 dpi 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 (Mm) larvae and mock-injected (ui) larvae 1 dpi. Horizontal bars represent means; ****P<0.0001 (one-way ANOVA with Dunn’s post-test). (F and G) Percentage of dead THP-1 macrophages at 5 hours post-TNF, treated with (F) rotenone or vehicle starting 1 hour before TNF addition or (G) MitoParaquat (MitoPQ) or vehicle for 5 hours. Black and red symbols represent uninfected (ui) and Mtb-infected macrophages (Mtb), respectively, within the same treatment well. Horizontal bars represent 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. (C, D to E, and G) representative of two independent experiments; (B and F) data from a single experiment.

Figure 7:. Currently available drugs can intercept TNF-induced mROS production and inhibit necrosis of mycobacterium-infected macrophages.

Representative pseudocolored confocal images of 5 dpi granulomas in larvae with yellow fluorescent macrophages (green) that are wild-type (WT), or TNF^hi 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 TNF^hi larvae 5 dpi with Mm, treated with vehicle or (B) diazoxide, (C) DM-malonate, (D) telaglenastat, or (E) perhexiline. *P<0.05; **P<0.01, ****P<0.0001 (Fisher’s exact test). (F) Quantification of mROS in wild-type or TNF^hi larvae 1dpi with Mm, treated with metformin, phenformin, or vehicle. Horizontal bars represent means; **P<0.01; ***P<0.001 (one-way ANOVA with Tukey’s post-test). (G) Bacterial cording in wild-type or TNF^hi larvae 5 dpi with Mm, treated with metformin or vehicle. ****P<0.0001 (Fisher’s exact test). (H) Bacterial cording in wild-type larvae 5 dpi with Mm, treated with vehicle, or 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 TNF^hi 1 dpi with Mtb, treated with metformin or vehicle. Horizontal bars represent means; *P<0.05 (one-way ANOVA with Tukey’s post-test). (J) Percentage of dead THP-1 macrophages at 5 hours post-TNF, treated with metformin or vehicle starting 1 hour before TNF addition. Black and red symbols represent uninfected (ui) and Mtb-infected macrophages (Mtb), respectively, within the same treatment well. Horizontal bars represent means; **P<0.01, ****P<0.0001 (one-way ANOVA with Tukey’s post-test). (B to G, and I) representative of two independent experiments; (H and J) data from a single experiment.