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.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

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 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 2DCFDA. 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

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

Figure 5. Cyclophilin D and ceramide mediate cell necrosis

(A) FPC in WT or CYPD morphant siblings on WT or LTA4H-high background. **p 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 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

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

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)