TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe pathogenic life cycle of Mycobacterium tuberculosis (Mtb), the agent of human tuberculosis (TB), is fueled by its multiple interactions with host macrophages (, ). Mycobacteria use macrophages to traverse host epithelial barriers to enter deeper tissues, where they recruit additional macrophages to form granulomas, pathognomonic structures that can serve as intracellular bacterial growth niches (, ). Granuloma macrophages can undergo necrosis, a key pathogenic event that further increases bacterial growth in the more permissive extracellular milieu (, ), thereby increasing disease morbidity and transmission (, , ).Mycobacterium-macrophage interactions and resultant macrophage fates can be detailed in the optically transparent zebrafish larva infected with Mycobacterium marinum (Mm), a close genetic relative of Mtb (, ). In this model, distinct host genetic mutations that increase macrophage necrosis render the host hypersusceptible by promoting unrestricted extracellular mycobacterial growth (, , , ). One genetic perturbation that produces hypersusceptibility through macrophage necrosis increases expression of leukotriene A4 hydrolase (LTA4H), which catalyzes the final step in the synthesis of the inflammatory lipid mediator leukotriene B4 (LTB4) (href="#bib91" rid="bib91" class=" bibr popnode">Tobin et al., 2012). Humans with a functional LTA4H promoter variant that increases LTA4H expression are also hypersusceptible to TB (href="#bib89" rid="bib89" class=" bibr popnode">Thuong et al., 2017, href="#bib91" rid="bib91" class=" bibr popnode">Tobin et al., 2012). Among cases of tuberculous meningitis, the severest form of TB, LTA4H-high individuals had increased risk of death. Consistent with inflammation-induced mortality, survival was dramatically increased among patients who received adjunctive anti-inflammatory therapy with glucocorticoids (href="#bib89" rid="bib89" class=" bibr popnode">Thuong et al., 2017, href="#bib91" rid="bib91" class=" bibr popnode">Tobin et al., 2012).The human relevance of the zebrafish findings provided the impetus to carry out a detailed mechanistic dissection of the necrosis pathway. In the zebrafish, we showed that susceptibility of the LTA4H-high state is due to the excessive production of the pro-inflammatory cytokine tumor necrosis factor (TNF) that, at optimal levels, is host protective (href="#bib91" rid="bib91" class=" bibr popnode">Tobin et al., 2012). Excess TNF triggers RIPK1- and RIPK3-dependent programmed necrosis of mycobacterium-infected macrophages, but not uninfected macrophages in the same animal (href="#bib74" rid="bib74" class=" bibr popnode">Roca and Ramakrishnan, 2013). TNF-RIPK1-RIPK3 interactions increase mitochondrial reactive oxygen species (ROS) production, which are required for necrosis along with cyclophilin D, a mitochondrial matrix protein (href="#bib74" rid="bib74" class=" bibr popnode">Roca and Ramakrishnan, 2013). Oxidative stress can activate cyclophilin D, which promotes sustained opening of the mitochondrial permeability transition pore complex (mPTP); this leads to disruption of the membrane potential and ATP depletion (href="#bib7" rid="bib7" class=" bibr popnode">Baines, 2010, href="#bib13" rid="bib13" class=" bibr popnode">Bernardi, 2013, href="#bib41" rid="bib41" class=" bibr popnode">Halestrap et al., 2004, href="#bib65" rid="bib65" class=" bibr popnode">Nakagawa et al., 2005). The dual requirement for mitochondrial ROS and cyclophilin D would be consistent with a mitochondrion-intrinsic necrosis pathway where TNF-induced mitochondrial ROS activate cyclophilin D (href="/pmc/articles/PMC6736209/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figure 1A).href="/pmc/articles/PMC6736209/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=6736209_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC6736209/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC6736209/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Ceramide Causes Necrosis through Cathepsin D, BID, and BAX(A) Cartoon of TNF-mediated necrosis pathway components. CYPD, cyclophilin D; M, mitochondrion; L, lysosome.(B) Confocal images of granulomas in 3 or 5 dpi TNF-high or control larvae with yellow fluorescent macrophages infected with red fluorescent Mm. Arrowheads, extracellular bacteria; arrows, extracellular, cording bacteria. Scale bar, 100 μm.(C) Cording in 5 dpi TNF-high and control larvae.(D) Cording in 5 dpi TNF-high or control larvae treated with pepstatin A.(E) Cording in 5 dpi TNF-high or control larvae treated with E64d.(F) Cording in 5 dpi TNF-high and control larvae that are wild-type (WT) or cathepsin D morphant.(G) Cording in 5 dpi TNF-high and control larvae that are WT or BID morphant.(H) Cording in 5dpi TNF-high and control larvae treated with BI-6C9.(I) Cording in 5 dpi TNF-high or control larvae that are WT or BAXA mutant.(J) Cording in 5 dpi TNF-high and control larvae that are WT or BAXB mutant.(C–J) ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 (Fisher’s exact test). Each panel representative of 3–6 independent experiments.See also href="/pmc/articles/PMC6736209/figure/figs1/" target="figure" class="fig-table-link figpopup" rid-figpopup="figs1" rid-ob="ob-figs1" co-legend-rid="lgnd_figs1">Figure S1.