TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species

Abstract

Tumor necrosis factor (TNF) constitutes a critical host defense against tuberculosis, but its excess is also implicated in tuberculosis pathogenesis in zebrafish and humans. Using the zebrafish, we elucidate the pathways by which TNF mediates tuberculosis pathogenesis. TNF excess induces mitochondrial reactive oxygen species (ROS) in infected macrophages through RIP1-RIP3-dependent pathways. While initially increasing macrophage microbicidal activity, ROS rapidly induce programmed necrosis (necroptosis) and release mycobacteria into the growth-permissive extracellular milieu. TNF-induced necroptosis occurs through two pathways: modulation of mitochondrial cyclophilin D, implicated in mitochondrial permeability transition pore formation, and acid sphingomyelinase-mediated ceramide production. Combined genetic blockade of cyclophilin D and acid sphingomyelinase renders the high TNF state hyperresistant by preventing macrophage necrosis while preserving increased microbicidal activity. Similarly, the cyclophilin D-inhibiting drug alisporivir and the acid sphingomyelinase-inactivating drug, desipramine, synergize to reverse susceptibility, suggesting the therapeutic potential of these orally active drugs against tuberculosis and possibly other TNF-mediated diseases.

Figure 1. Zebrafish susceptibility phenotypes and assays in LTA4H/TNF-low and -high states

Figure 2. TNF-mediated ROS production kills both mycobacteria and infected macrophages

(A) Mean (±SEM) number of bacteria per infected macrophage in WT and LTA4H-high larvae in presence or absence of 40 μM NAC. ***p < 0.001 (one way ANOVA with Tukey's post-test). (B) Bacterial burden (FPC) in WT and LTA4H-high siblings in presence or absence of 40 μM NAC. **p < 0.01 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (C) Representative fluorescence microscopy images of individual fish in (B) represented by red dots. (D) FPC of 1 dpi larvae injected with TNF or vehicle with or without 40 μM NAC. *p < 0.05 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (E) FPC 3dpi of the same fish in (D). *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (F) Number of yellow fluorescent macrophages or red fluorescent neutrophils in 2dpi fish one day after injection with TNF or vehicle in presence or absence of 40 μM NAC. ***p < 0.001 (one-way ANOVA with Tukey's post-test). (G) Confocal microscopy of granulomas in 3dpi Tg(mpeg1:YFP) larvae injected with TNF or vehicle. White arrowheads show extracellular bacteria. Scale bar 10 μm. (H) Number of yellow fluorescent macrophages in Tg(mpeg1:YFP) uninfected fish 1 day post-injection with TNF or vehicle. Difference not significant by Student's t-test. Representative of 2 independent experiments. (I) Representative fluorescence microscopy images of 4dpi WT and LTA4H-high larva. Scale bar 10 μm. (J) Percentage of animals in (D) and (E) with cording 4dpi. ***p < 0.001 (Fisher's exact test). (K) Representative fluorescence microscopy images of 1dpi LTA4H-high larvae 6 hours after incubation with CM-H₂DCFDA. Arrowheads point to infected macrophages. Scale bar 10 μm. (L) Quantification of ROS production as relative fluorescence units (RFU) (±SEM) in WT siblings infected with Mm or mock-infected (See Experimental Procedures) at the indicated time points after injection of TNF or vehicle. (two-way ANOVA). Representative of 2 independent experiments. (M) Quantification of ROS production as RFU (±SEM) in WT infected siblings at the indicated time points after injection of TNF or vehicle in presence or absence of 40 μM NAC. (two-way ANOVA). (N) Quantification of ROS production as RFU (±SEM) in infected WT or PU.1 morphant siblings at the indicated time points after injection of TNF or vehicle. (two-way ANOVA). (Also see Figures S1, S2 and S3).

Figure 3. TNF excess mediates necrosis through the RIP1-RIP3 kinase pathway

(A) Zebrafish RIP1 and RIP3 amino acid residue analysis for protein domains. RHIM, RIP homotypic interaction motif; aa, amino acid residue. Numbers indicate percent identity between zebrafish and human domains. (B) Mean (±SEM) number of bacteria per infected macrophage in WT and RIP1 morphant siblings on LTA4H-high or WT background. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (C) FPC in WT and RIP1 morphant siblings on LTA4H-high or WT background. **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (D) Percentage of animals in (C) with cording among WT, RIP1 morphants and RIP1 morphants on LTA4H-high background. *p < 0.05; **p < 0.01 (Fisher's exact test). (E) Mean (±SEM) number of bacteria per infected macrophage in WT and RIP3 morphant siblings on LTA4H-high or WT background. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (F) FPC in WT and RIP3 morphant siblings on LTA4H-high or WT background. ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (G) Percentage of animals in (F) with cording among WT, RIP3 morphants and RIP3 morphants on LTA4H-high background. **p < 0.01 (Fisher's exact test). (H) Mean (±SEM) number of bacteria per infected macrophage in WT and RIP1 or RIP3 morphant larvae injected with TNF or vehicle. ***p < 0.001 (one way ANOVA with Tukey's post-test). (I) FPC in infected WT and RIP1 or RIP3 morphant larvae injected with TNF or vehicle. **p < 0.01 (one-way ANOVA with Tukey's post-test). (J) Percentage of animals in (I) with cording among WT, RIP1 and RIP3 morphants injected with TNF or vehicle. *p < 0.05; **p < 0.01 (Fisher's exact test). (K) Mean (±SEM) number of bacteria per infected macrophage in WT and LTA4H-high larvae in presence of 10 μM Necrostatin-1 or 10 μM Necrostatin-1 inactive control. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (L) FPC in WT and LTA4H-high larvae in presence of 10 μM Necrostatin-1 or 10 μM Necrostatin-1 inactive control. *p < 0.05; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (M) Percentage of animals in (L) with cording among WT and LTA4H-high larvae in presence of 10 μM Necrostatin-1 or 10 μM Necrostatin-1 inactive control. *p < 0.05 (Fisher's exact test). (Also see Figures S4 and S5).

Figure 4. The TNF-RIP1-RIP3 axis mediates necrosis of infected macrophages through mitochondrial ROS production

(A) FPC in WT and LTA4H-high larvae in presence or absence of 10 μM Necrox-5. **p < 0.01 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (B) Percentage of animals in (A) with cording among WT and LTA4H-high larvae in presence or absence of 10 μM Necrox-5. *p < 0.05; ***p < 0.001 (Fisher's exact test). (C) FPC in WT and LTA4H-high larvae injected with TNF or vehicle in presence or absence of 10 μM Necrox-5. *p < 0.05 (one-way ANOVA with Tukey's post-test). (D) Percentage of animals in (C) with cording among WT and LTA4H-high larvae injected with TNF or vehicle in presence or absence of 10 μM Necrox-5. *p < 0.05 (Fisher's exact test). (E) FPC in WT and LTA4H-high larvae in presence or absence of 10 μM Necrosulfonamide (NSA). **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (F) Mean (±SEM) number of bacteria per infected macrophage in WT, LTA4H-high and PGAM5 morphant siblings on WT or LTA4H-high background. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (G) FPC in WT, LTA4H-high and PGAM5 morphant siblings on WT or LTA4H-high background. ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (H) Percentage of animals in (G) with cording among PGAM5 morphants on WT or LTA4H-high background. **p < 0.01 (Fisher's exact test). (I-K) Confocal and bright field images of different infected macrophages in 1dpi larvae 3 (I), 5 (J) or 6 (K) hours post-TNF injection. Scale bars 1 μ m. (Also see Figure S6).

Figure 5. Cyclophilin D and ceramide mediate cell necrosis

(A) FPC in WT or CYPD morphant siblings on WT or LTA4H-high background. **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (B) Mean (±SEM) number of bacteria per infected macrophage in WT, LTA4H-high and CYPD morphants siblings on LTA4H-high background. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (C) Quantification of ROS production as RFU (±SEM) in infected WT, RIP1, RIP3, PGAM5 and CYPD morphant siblings at indicated time points after TNF or vehicle injection. (two-way ANOVA). (D) Number of yellow fluorescent macrophages in 2 dpi Tg(mpeg1:YFP) RIP1 or CYPD morphant siblings 1 day post-injection with TNF or vehicle. **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (E) Mean (±SEM) number of bacteria per infected macrophage in WT, LTA4H-high and aSMase morphants and acid ceramidase-overexpressing (acer) siblings on WT or LTA4H-high background. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (F) FPC in WT, LTA4H-high, aSMase morphants and acid ceramidase-overexpressing siblings on WT or LTA4H-high background.*p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (G) Number of yellow fluorescent macrophages in 2 dpi Tg(mpeg1:YFP) CYPD morphants and ceramidase-overexpressing siblings 1 day post-injection with TNF or vehicle. **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Dashed line, control group mean. (H) FPC in WT, LTA4H-high, CYPD morphants, double CYPD/aSMase morphants and CYPD morphants-overexpressing simultaneously acid ceramidase on LTA4H-high background.*p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. Dashed line, control group mean. (I) FPC in WT, LTA4H-high, CYPD morphants and CYPD morphants-overexpressing simultaneously acid ceramidase on LTA4H-high background in presence or absence of 40 μM NAC.*p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. Dashed line, control group mean.

Figure 6. Drugs blocking cyclophilin D- and ceramide-mediated necrosis synergize as host-targeting therapies for high-TNF mediated TB

(A) FPC in WT and LTA4H-high larvae treated with Alisporivir or left untreated. *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (B) Mean (±SEM) number of bacteria per infected macrophage in WT and LTA4H-high siblings in presence or absence of Alisporivir. ***p < 0.001 (one way ANOVA with Tukey's post-test). Representative of 2 independent experiments. (C) FPC in WT siblings injected or not with TNF in presence or absence of Alisporivir. **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). (D) FPC in WT and LTA4H-low siblings in presence or absence of Alisporivir. *p < 0.05 (oneway ANOVA with Tukey's post-test). (E) FPC in WT and LTA4H-high larvae in presence or absence of Desipramine. *p < 0.05; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 3 independent experiments. (F) FPC in WT and LTA4H-high larvae in presence or absence of Desipramine, Alisporivir or the combination of both. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post-test). Representative of 2 independent experiments. Dashed line, control group mean. (G) FPC in WT, LTA4H-high and LTA4H-high/CYPD morphant larvae treated with Alisporivir, Desipramine or left untreated. ***p < 0.001 (one-way ANOVA with Dunnett's post-test). Representative of 2 independent experiments. Dashed line, control group mean. (H) FPC in WT, LTA4H-high and LTA4H-high/aSMase morphant larvae treated with Alisporivir, Desipramine or left untreated. *p < 0.05; **p < 0.01 (one-way ANOVA with Dunnett's post-test). Dashed line, control group mean.

Figure 7. TNF-dependent programmed necrosis pathways showing genetic and chemical interventions used in this study

Δψm, dissipation of mitochondrial membrane potential (also see Table S3)