Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages

Zhao, X; Khan, N; Gan, H; Tzelepis, F; Nishimura, T; Park, S-Y; Divangahi, M; Remold, H G

doi:10.1038/mi.2017.12

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Published: 12 April 2017

Infectious diseases and host defense

Bcl-x_L mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages

X Zhao¹^na1,
N Khan²^na1,
H Gan¹,
F Tzelepis²,
T Nishimura^1,3,
S-Y Park¹,
M Divangahi² &
…
H G Remold¹

Mucosal Immunology volume 10, pages 1553–1568 (2017)Cite this article

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Abstract

Virulent Mycobacterium tuberculosis (Mtb) triggers necrosis in host Mϕ, which is essential for successful pathogenesis in tuberculosis. Here we demonstrate that necrosis of Mtb-infected Mϕ is dependent on the action of the cytosolic Receptor Interacting Protein Kinase 3 (RIPK3) and the mitochondrial Bcl-2 family member protein B-cell lymphoma—extra large (Bcl-x_L). RIPK3-deficient Mϕ are able to better control bacterial growth in vitro and in vivo. Mechanistically, cytosolic RIPK3 translocates to the mitochondria where it promotes necrosis and blocks caspase 8-activation and apoptosis via Bcl-x_L. Furthermore, necrosis is associated with stabilization of hexokinase II on the mitochondria as well as cyclophilin D-dependent mitochondrial permeability transition. Collectively, these events upregulate the level of reactive oxygen species to induce necrosis. Thus, in Mtb-infected Mϕ, mitochondria are an essential platform for induction of necrosis by activating RIPK3 function and preventing caspase 8-activation.

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Introduction

Cell death is triggered by regulated and highly specific intracellular and extracellular signals. Apoptosis and necrosis are two major forms of cell death that have a critical role in immunity to infection by Mycobacterium tuberculosis (Mtb).¹ These distinct cell death modalities of macrophages (Mϕ) directly affect control of Mtb growth and dissemination^{2, 3, 4} as well as antigen-specific T-cell-mediated immunity via cross-presentation.^{5, 6, 7, 8} Thus it is not surprising that virulent strains of Mtb have developed mechanisms that allow evasion of Mϕ apoptosis by inducing necrosis.¹ Furthermore, we and others have demonstrated that during infection with Mtb, mitochondrial function is critical in determining the cell death modality.^{2, 9, 10, 11} Infection of human Mϕ with the attenuated Mtb H37Ra predominantly induces apoptosis,^{2, 3, 12} which is dependent on mitochondrial outer membrane permeabilization and release of mitochondrial cytochrome c into the cytosol.¹¹ In contrast, infection of Mϕ with the virulent Mtb strain H37Rv induces necrosis^{2, 3} associated with irreversible permeabilization of the mitochondrial inner membrane leading to mitochondrial permeability transition (MPT) and loss of mitochondrial integrity as well as function.¹¹ The MPT is regulated by calcium and occurs after opening of a functional pore, that includes the adenine nucleotide translocator (ANT), and a voltage-dependent anion channel (VDAC), whose functions are regulated by cyclophilin D (CypD).^{13, 14} CypD is a member of the cyclophilin family of peptidyl-prolyl cis–trans isomerases and has been implicated in MPT-dependent necrotic (but not apoptotic) cell death.^{15, 16} We have previously shown that CypD is involved in induction of Mtb-dependent Mϕ necrosis, which allows bacterial growth in virulent Mtb-infected cells.¹⁰ These studies collectively indicate that programmed necrosis is essential for evasion of immunity by Mtb and is regulated at the level of mitochondria but the molecular mechanisms involved in the necrotic pathway remain elusive.

It has recently been shown that inhibitors of apoptosis (e.g., IAP) trigger formation of a complex including RIPK3 and RIPK1, which induces a type of necrosis referred to as necroptosis.¹⁷ RIPK3-dependent necrosis seems to be tightly regulated by caspase 8¹⁸ as it was shown that caspase 8 inactivates RIPK3.¹⁹ Interestingly, an elegant study in a murine tumor model recently has demonstrated that RIPK3-mediated necroptosis is more efficient in cross-priming CD8⁺ T cells than apoptosis and within those dying cells RIPK1 signaling is required for initiating T-cell-mediated immunity.²⁰ Death-associated molecular patterns released from necroptotic cells also provoke a strong inflammatory response.²¹ However, little is known about the role of the RIPK3 mediated cell death program during host interaction with a living pathogen that initiates multiple complex signaling pathways.

NAD(P)H oxidases (NOX) family of enzymes and mitochondria are the major intracellular sources of ROS.²² ROS is produced in the mitochondria predominantly by the complexes I–III of the electron transport chain and by mitochondrial dehydrogenases including glycerol-3-phosphate dehydrogenase and α-ketoglutarate dehydrogenase, a Krebs cycle enzyme,²³ which produces NADH and generates ROS if the NADH/NAD⁺ ratio is high.²⁴ Processing of glucose is initiated by enzymes of the hexokinase (HK) family, which has four isoforms (HK-I to HK-IV). HK-II is critical for mitochondrial energy production since its presence on the outer mitochondrial membrane couples glycolysis with oxidative phosphorylation.²⁵ Augmented levels of HK-II increase ROS accumulation if the normal function of the mitochondrial electron chain is compromised. Interestingly, increased mitochondrial ROS production has been associated with RIPK3-dependent necrosis.²⁶ Moreover, the mitochondrial Bcl-2 family protein B-cell lymphoma-extra large (Bcl-x_L) on the mitochondria is known to block apoptosis by inhibiting the function of Bcl-2 homologous antagonist/killer (BAK)²⁷ via blocking Bcl-2-associated X protein (BAX) recruitment to the mitochondria.^{28, 29}

Here we report that in Mϕ infected with virulent Mtb, RIPK3 translocates to mitochondria as evidenced by its presence in mitochondria along with Bcl-x_L and pro-caspase 8. Mitochondrial pro-caspase 8 in the presence of RIPK3 and Bcl-x_L remains in its inactive zymogen form, suggesting that Bcl-x_L is critical for preventing caspase 8-mediated apoptosis and that the presence of active RIPK3 on the mitochondria is required for induction of necrosis. Furthermore, we identify RIPK3 as a kinase involved in the induction of necrosis impairing host Mϕ immunity both in vitro and in vivo. Interestingly, the enhanced protection of RIPK3-deficient mice was independent of T-cell-mediated immunity to pulmonary Mtb infection. Collectively, our findings demonstrate that following Mtb infection RIPK3 is involved in the activation of several independent mechanisms that ultimately converge at the inhibition of apoptosis and promotion of necrosis in Mϕ, which enhances the susceptibility of the host to infection.

Results

RIPK3 is required for induction of necrosis in Mϕ infected with virulent Mtb

A hallmark of Mϕ infection with virulent Mtb is necrosis, which allows the bacteria to evade the host defense mechanisms.¹ RIPK3 is required for the induction of necrosis in a variety of cell models.¹⁸ To test whether a RIPK3-dependent pathway is directly involved in necrosis and innate control of virulent H37Rv infection, we silenced the RIPK3 gene in human Mϕ using two different RIPK3 siRNAs (Figure 1a and Supplementary Figure 1A online). In the absence of RIPK3, necrosis was remarkably reduced in Mtb-infected Mϕ (Figure 1b and Supplementary Figure 1A). This reduction of necrotic cell death also correlated with decreased bacterial growth in RIPK3-deficient Mϕ (Figure 1c). Because of potential off-target effects of siRNA, we performed similar experiments using RIPK3-deficient mice. Similar to human Mϕ, Mtb-infected RIPK3-deficient murine Mϕ revealed decreased necrosis (Figure 1d), which led to significant reduction of bacterial growth in vitro (Figure 1e). Decreased necrosis in RIPK3^−/− Mϕ was also associated with significant reduction of extracellular bacteria compared with Mtb-infected wild-type (WT) Mϕ (Figure 1f). To determine whether reduced necrosis also occurs in the lungs of RIPK3^−/− mice following Mtb infection, we infected RIPK3^−/− and WT mice via the intratracheal route with H37Rv and collected bronchoalveolar lavage 2 days after infection. The levels of dead cells were significantly lower in the lungs of infected RIPK3^−/− mice than WT mice (Figure 1g). We next investigated the involvement of the mixed lineage kinase domain-like protein (MLKL), which is a substrate for RIPK3 kinase activity to mediate necrosis.³⁰ Interestingly, the levels of MLKL were increased in human Mϕ infected with H37Rv (Figure 1h) and silencing of MLKL significantly decreased necrosis in H37Rv-infected Mϕ (Figure 1i and Supplementary Figure 1B). Collectively, these data from both in vitro human and murine Mϕ as well as the in vivo experiments indicate that RIPK3 plays a critical role in the necrosis pathway during the course of Mtb infection.

RIPK3 and pro-caspase 8 translocate from the cytosol to the mitochondria in Mtb-infected Mϕ

In cells undergoing genotoxic stress pro-caspase 8 forms a cytosolic complex with RIPK1, RIPK3 and FADD termed the “riptoptosome”, which can become a cytosolic cell death signaling platform.^{17, 31} We therefore investigated whether a similar complex exists in human Mϕ infected with Mtb. Here we found that RIPK1, RIPK3, and pro-caspase 8 co-precipitate in the cytosol but not on mitochondria of human Mϕ prior to infection (Figure 2a, Supplementary Figure 3A) similar to the ripoptosome. At 2 h after infection of human Mϕ with virulent H37Rv or avirulent H37Ra, we observed that RIPK3, RIPK1 and pro-caspase 8 translocate from the cytosol to the mitochondria (Figure 2a, right lane). In H37Rv-infected pro-necrotic Mϕ, the levels of pro-caspase 8 and RIPK3 increased on the mitochondria over time (Figure 2b). In contrast, in Mϕ infected with the avirulent H37Ra, the levels of pro-caspase 8 and RIPK3 on the mitochondria were markedly reduced by 24 h, which is consistent with pro-caspase 8 becoming activated at 24 h to induce apoptosis (Figure 2c). The translocation of cytosolic RIPK3 and pro-caspase 8 to the mitochondria cannot be explained by contamination of the mitochondrial fractions with cytosolic proteins, because mitochondrial preparations from uninfected Mϕ did not contain significant amounts of RIPK1, RIPK3 and pro-caspase 8 (Figure 2a). Moreover, using fluorescence confocal microscopy we demonstrated that the levels of pro-caspase 8 and RIPK3 colocalization were significantly increased in Mϕ infected with the virulent H37Rv strain (Figure 2d and e). Additionally, the levels of cleaved caspase 8 was significantly higher in H37Rv-infected RIPK3-deficient Mϕ compared with WT Mϕ (Figure 2f) while there was no difference in Mϕ infected with H37Ra (Supplementary Figure 2A). These data indicate that after infection of Mϕ with virulent Mtb the mitochondria acquire a complex containing RIPK1, RIPK3 and pro-caspase 8 where RIPK3 was required to prevent caspase 8 activation.

RIPK3 and Bcl-x_L prevent activation of pro-caspase 8 on the mitochondria promoting necrosis in Mtb-infected Mϕ

Bcl-x_L protects mitochondria against apoptosis by inhibiting BAK activation.²⁸ RIPK3 and pro-caspase 8 translocate to the mitochondria in H37Rv-infected Mϕ as shown by their presence in mitochondrial fractions after 6 and 24 h of infection (Figure 3a). Bcl-x_L was required for the sequestration of pro-caspase 8 on the mitochondria in its inactive form, as silencing of the Bcl-x_L-gene led to proteolytic processing of pro-caspase 8 and disappearance of RIPK3 from the mitochondria 24 h post-H37Rv-infection (Figure 3a). In contrast, 24 h after infection of Mϕ with avirulent H37Ra (which promotes apoptosis), the Bcl-x_L levels on the mitochondria were significantly diminished, which was also associated with disappearance of pro-caspase 8 and RIPK3 from the mitochondria (Figure 3b). These findings are in agreement with a study demonstrating that by outcompeting Bax via Bcl-x_L, cells are protected against apopotosis.²⁸ Finally, we demonstrated that silencing Bcl-x_L in H37Rv-infected Mϕ significantly reduced necrosis (Figure 3c and Supplementary Figure 1C). These experiments collectively indicate that the presence of RIPK3 and Bcl-x_L on the mitochondria is required to inhibit capsase 8 activation, which leads to increase necrosis in Mtb-infected Mϕ.

The dual function of caspase 8 in initiation of apoptosis and suppression of RIPK3-dependent necrosis,^{32, 33, 34} which has been shown to be required for normal murine embryonic development and survival³⁵ is well documented. Active caspase 8 blocks necrosis by proteolytically inactivating RIPK1 and RIPK3.^{19, 36, 37} Thus we next inhibited the apoptotic pathway in H37Ra-infected Mϕ using the caspase inhibitor z-IETD-FMK and then measured the levels of mitochondria-associated RIPK3. As expected, in Mϕ infected with avirulent H37Ra, pro-caspase 8 (Figure 3b) and both apoptosis-executor caspases 9 and 3 were not activated in the presence of z-IETD-FMK (Figure 3d) confirming an essential role for the caspases 8, 9 and 3 in apoptosis of Mtb-infected Mϕ. Importantly, in H37Ra-infected Mϕ treated with z-IETD-FMK the levels of mitochondria-associated RIPK3, Bcl-x_L, and pro-caspase 8 were all increased to the similar levels found in Mϕ infected with the virulent strain H37Rv (Figure 3b) indicating that inhibition of caspase 8, 9 and 3-activation is required for the RIPK3-dependent necrotic pathway to be initiated.

Finally, to test whether RIPK3 is also required for regulation of Bcl-x_L-mediated pro-caspase 8-persistence on the mitochondria, we next silenced the RIPK3 gene in H37Rv-infected Mϕ. At 24 h after infection, reduction of mitochondrial Bcl-x_L-accumulation correlated with reduced pro-caspase 8-accumulation on the mitochondria (Figure 3e) suggesting that proteolytic processing of pro-caspase 8 initiates apoptosis. In addition, silencing RIPK3 leads to caspase 3-activation, which is a key indicator of apoptosis (Figure 3f). In contrast to H37Rv infection, apoptosis was significantly increased in H37Ra-infected Mϕ as measured by caspase 8 and 3 activation (Supplementary Figure 2A–C) as well as increased apoptotic surface markers (Supplementary Figure 2D). Thus the presence or absence of RIPK3 had no significant effect on the apoptosis of Mϕ infected with H37Ra. Collectively these data indicate that induction of necrosis in Mtb-infected Mϕ is critically dependent on blocking activation of pro-caspases 8-, 9- and 3-mediated apoptosis via RIPK3.

To determine whether caspase 8-activation is part of the intrinsic mitochondria-dependent apoptotic pathway, which includes activation of BID to tBID and depends on BAX,²⁹ we next evaluated activation of BID and BAX in Mϕ infected with H37Ra or H37Rv. After 10 min of infection with H37Ra, pro-caspase 8-activation led to formation of tBID on the mitochondria (Figure 3g, right). However, tBID was not detectable on the mitochondria of H37Rv-infected Mϕ (Figure 3g, left). Most importantly, silencing of the BAX gene in H37Ra-infected Mϕ inhibited apoptosis-executor caspase 3 activation (Figure 3h). This indicates that activation of pro-caspase 8 on the mitochondria is initiated by the intrinsic apoptotic pathway via BAX translocation, which is part of the mitochondrial amplification loop of apoptosis and triggers degradation of RIPK3 from the mitochondria.

RIPK3 mediates hexokinase II translocation to the mitochondria via Bcl-x_L in H37Rv-infected Mϕ

As RIPK3 regulates ROS production following TNF-α treatment and because ROS have an important role in regulation of cell death programs,²⁶ we next studied whether necrosis in Mtb-infected Mϕ is ROS-dependent. We found that ROS accumulation in Mϕ infected with virulent H37Rv was significantly increased in comparison with Mϕ infected with the avirulent strain H37Ra (Figure 4a). RIPK3 was required to increase ROS accumulation in H37Rv-infected Mϕ because silencing of the RIPK3 gene significantly reduced ROS production (Figure 4b). This increased production of ROS was independent of mitochondrial mass (data not shown). Increased ROS accumulation triggers necrosis in H37Rv-infected Mϕ because scavenging of ROS with Tiron significantly reduced necrosis (Figure 4c). As hexokinase II (HKII) binds to the VDAC in the mitochondrial outer membrane and is the limiting enzyme of glucose metabolism,²⁵ we next determined whether HKII contributes to ROS accumulation. Kinetic studies of HKII-translocation to the mitochondria indicated that the levels of HKII were significantly increased and maintained on the mitochondria of H37Rv-infected Mϕ compared with H37Ra-infected Mϕ (Figure 4d). Increased binding of HKII to mitochondria in Mϕ infected with virulent H37Rv correlated with enhanced ROS production, which was reduced after HKII gene silencing (Figure 4e). As NADH accumulation is required for ROS production in a HKII-dependent manner,²⁵ NADH levels were subsequently diminished after silencing of the HKII gene and the RIPK3 gene (Supplementary Figure 3C and D). Further, NADH oxidase inhibitor Diphenyleneiodonium (DPI) abolished ROS production and reduced necrosis in H37Rv-infected Mϕ (Figure 4f and g). Interestingly, in H37Rv-infected Mϕ, silencing of the Bcl-x_L gene diminished HKII recruitment to the mitochondria at 24 h post infection (Figure 4h), while silencing of the RIPK3 gene prevented HKII recruitment to the mitochondria as early as 6 h post infection that persisted up to 24 h post infection (Figure 4i). Collectively, these data indicate that in Mϕ infected with virulent Mtb, HKII translocation to the mitochondria mediates via Bcl-x_L and RIPK3 that leads to increased ROS production.

RIPK3 mediates MPT in Mϕ infected with H37Rv

Increased mitochondrial permeability transition (MPT), a sign of mitochondrial damage, induces pyridine nucleotide release from the mitochondria,³⁸ which decreases mitochondrial respiration and cellular ATP-levels, which leading to necrotic cell death.³⁹ MPT increases electron leakage predominantly from mitochondrial complex I and III and enhances ROS production in the presence of sufficient NADH.³⁹ We have previously shown that virulent Mtb induced MPT-dependent necrosis and that cyclosporin A (CsA), a selective inhibitor of the peptidyl-prolyl cis–trans isomerase activity of CypD, blocks MPT mediated necrosis.^{10, 11, 40, 41} The critical role of CypD in necrosis was demonstrated in Ppif null mice, which lack the gene encoding CypD, as these mice were protected against necrosis.^{15, 16} Thus we wondered whether there is a link between RIPK3, CypD, and MPT in Mtb-infected Mϕ. We first showed that silencing of the RIPK3 gene blocked H37Rv-induced MPT in Mϕ after 48 and 72 h post infection (Figure 5a). Since we demonstrated that induction of MPT by RIPK3 was required for initiation of necrosis, we next sought to determine how virulent Mtb initiates MPT. In H37Rv-infected Mϕ treatment with CsA inhibited ROS accumulation (Figure 5b) and necrosis (Figure 5c). We also found that in CsA-treated Mϕ infected with H37Rv, the levels of mitochondrial RIPK3, pro-caspase 8, Bcl-x_L, and HKII were diminished (Figure 5d). However, in H37Ra-infected Mϕ there was no interaction between mitochondrial CypD and ANT (Figure 5e). Moreover, immunoprecipitation of ANT from the mitochondria of H37Rv-infected Mϕ treated with CsA showed that CypD binding to ANT was completely abolished (Figure 5f). These data suggest that RIPK3 acts as a regulator of CypD function to induce MPT leading to inhibition of ATP generation and contributing to necrosis. To test whether RIPK3 causes MPT by initiating CypD-binding to ANT and MPT pore opening, we silenced RIPK3 in H37Rv-infected Mϕ and assessed the interaction of CypD with ANT. In the absence of RIPK3 in H37Rv-infected Mϕ the CypD/ANT interaction was also diminished (Figure 5g), which correlated with the reduction of MPT (Figure 5a).

Thus RIPK3 induces necrosis in H37Rv-infected Mϕ, by at least two independent, but interrelated mechanisms: (1) RIPK3 increases Bcl-x_L-dependent HKII recruitment to the mitochondria possibly leading to enhanced glucose consumption and increased NADH accumulation as well as ROS generation; and (2) RIPK3 induces MPT in a CypD-dependent manner, which in the presence of abundant NADH boosts accumulation of ROS by uncoupling of the mitochondrial electron chain.

RIPK3 deficient Mϕ mediate resistance to pulmonary Mtb infection

To translate our in vitro observation in vivo, we have utilized two models of Mtb infection. In the first model, mice were intravascularly (IV) infected with a high dose of H37Rv (1 × 10⁶ colony forming units (CFU)) and after 4 weeks of infection, we found a significant reduction in the pulmonary bacterial burden of RIPK3^−/− compared to WT mice (Figure 6a). Interestingly, the number of neutrophils (cellular marker of necrotic lung tissues) was also significantly reduced in Mtb-infected RIPK3^−/− mice (Figure 6b, c and Supplementary Figure 4). However, with the exception of TNFα, which was significantly reduced in RIPK3^−/− mice, the expression levels of other cytokines, including IL-1β, IL-6, and IL-12 were not markedly changed (Figure 6d). In the second model, we aerosolized WT and RIPK3^−/− mice with a low dose (∼50 CFU) of the virulent strain H37Rv. Similar to IV-model, RIPK3^−/− mice had significantly lower lung bacterial burden (∼half log) after 5 weeks of infection (Figure 7a). Considering the important role of T cells in protection against Mtb infection, we next examined the potential contribution of T-cell-mediated immunity in protection of RIPK3^−/− mice. Similar to other studies,^{20, 42} we also found that the presence or absence of RIPK3 had no effect on T cell proliferation in vitro (Supplementary Figure 5A). At 5 weeks post-Mtb infection there was no significant difference in frequency of pulmonary CD4+/CD8+ T cells (Figure 7b) as well as TB10.4-specific CD8+ T cells (Figure 7c) or ESAT6-specific CD4+ T cells between WT and RIPK3^−/− mice (Figure 7d). Moreover, there was no difference in total cell numbers of T cells or antigen-specific T cells (Supplementary Figure 5B and C). Furthermore, ELISPOT experiments revealed that the function of T cells was intact as they produced similar levels of IFN-γ following stimulation with Mtb-antigens recognized by either CD4+ or CD8+ T cells (Supplementary Figure 5D). Thus these data indicate that the quantity and quality of T cells was not affected by RIPK3 and the increased protection of RIPK3^−/− mice after Mtb infection was independent of T-cell-mediated immunity.

To directly evaluate the protective role of RIPK3 in Mϕ innate immunity in the absence of any contribution from adaptive immune responses, we utilized our established adoptive transfer model of Mtb-infected Mϕ.^{3, 5, 43} We infected WT and RIPK3^−/− murine alveolar Mϕ in vitro with a low dose of the virulent strain H37Rv (MOI∼1), then transferred the cells by the intratracheal route into recipient mice deficient in recombinase-activating gene 1 (Rag1^−/−) mice. The number of bacteria in WT and RIPK3^−/− alveolar Mϕ was identical before transfer (Supplementary Figure 5E). However, 4 weeks after adoptive transfer, the pulmonary bacterial burden was significantly lower in Rag1^−/− mice that received Mtb-infected RIPK3^−/− Mϕ than in the recipients of Mtb-infected WT Mϕ (Figure 7e). This experiment demonstrates that transfer of infected RIPK3^−/− Mϕ, which are resistant to necrosis restrict bacterial replication independent of adaptive immunity in vivo. Thus RIPK3 plays an important role in early Mϕ immunity and protection against Mtb infection.

Discussion

Mtb hijacks Mϕ cell death pathways to maintain an environment for propagation within the host. We and others have previously demonstrated that Mtb actively inhibits apoptosis, which has a protective role in both innate and adaptive immunity to Mtb infection.⁴⁴ By inducing necrosis, virulent Mtb is able to exit the Mϕ and escape anti-mycobacterial defense mechanisms.¹ Although we have shown that Mtb induces necrosis by targeting the mitochondrial inner membrane^{10, 11, 40} and plasma membrane repair mechanisms,³ the molecular mechanisms involved in necrosis remain largely unknown. Here we have extended these studies and shown that Bcl-x_L and RIPK3 are involved in triggering Mϕ programmed necrosis during infection with virulent Mtb.

Recently a novel form of necrosis, necroptosis, was described, which depends on the action of a cytosolic complex containing RIPK1 and RIPK3. Formation of this complex is inhibited by the RIPK1 inhibitor necrostatin-1.¹⁷ However, similar to another infection model,⁴⁵ we found no effect of RIPK1 on necrosis either by using a necroptosis inhibitor, necrostatin-1, or silencing RIPK1 (data not shown). As necroptosis seems to be independent of the mitochondria and is a rapid early event,⁴⁶ the mechanisms involved in the Mtb-induced necrosis, which develops slowly and reaches its maximum between 72 and 96 h after infection, might be different from classical necroptosis. Interestingly, a recent study suggests that induction of necroptosis by RIPK3 requires inactivation of caspase-8.⁴⁷ Similarly, our findings indicate that caspase 8 determines the fate of Mϕ infected with virulent Mtb. Following infection with Mtb, RIPK3 translocates from the cytosol to the mitochondria of Mϕ. Bcl-x_L, an inhibitor of BAK^{27, 28} leading to a block of caspase 8 activation and apoptosis,⁴⁸ protects RIPK3 from caspase 8-dependent proteolysis.¹⁹ We also found that mitochondrial Bcl-x_L is required for RIPK3 to prevent caspase 8 activation and inducing necrosis in Mtb-infected Mϕ. Although binding of Bcl-x_L to caspase 8 was reported earlier,⁴⁸ we failed to find any binding of Bcl-x_L to pro-caspase 8 (data not shown).

Although in the current study, we have not demonstrated how caspase 8 translocates into mitochondria, other studies have shown that in human lymphoblastoid cell lines caspase 8 was associated with cardiolipin rafts at contact points of the inner and outer mitochondrial membranes leading to its activation. Cardiolipin was also found to be responsible for the translocation of caspase 8 to the mitochondria as well as caspase 8 activation following death receptor signaling.⁴⁹ Targeting of tBid to the mitochondria and mobilization of cytochrome c are also cardiolipin dependent indicating their importance in the transduction of the apoptotic signaling cascades.⁵⁰

Consistent with these studies we found that in Mϕ infected with an avirulent strain of Mtb, H37Ra, pro-caspase 8 is proteolytically processed, which not only correlates with activation of the effector caspases 9 and 3 leading to apoptosis, but also with preventing necrosis by inactivation of RIPK3. We currently do not know the exact mechanism of mitochondrial caspase 8 activation in Mϕ infected with an avirulent strain of Mtb. Interestingly, Niture and colleagues⁵¹ have reported that the levels of Bcl-x_L on the mitochondria are regulated by a member of the phosphoglycerate mutase family 5 (PGAM5), which forms a bridge between Bcl-x_L and Kelch-like ECH-associated protein-1 (a sensor of chemical and radiation-induced stress) and targets Bcl-x_L degradation by activating an E3 ubiquitin ligase. The involvement of this or additional mechanisms in Bcl-x_L-degradation or the role of signaling platforms, such as cardiolipin that promotes mitochondria-dependent caspase 8 activation remains to be determined. In addition, it is currently unclear whether Bcl-x_L is the only anti-apoptotic component on the mitochondria involved in this mechanism. It is likely that other Bcl-2 family members including Mcl-1 and Bcl-2^{52, 53} may also contribute to RIPK3 mediated caspase 8-inactivation in mitochondria leading to the induction of necrosis.

The mitochondria are a major source of energy and ROS production, and disruption of mitochondrial function significantly contributes to ROS generation and cell death. For example, blocking ROS accumulation in L929 cells prevents necrosis.²⁶ Our results indicate that in Mϕ infected with virulent Mtb, increased ROS accumulation contributed to induction of necrosis. Virulent Mtb may induce necrosis by several mechanisms including inhibition of ATP production, e.g., due to blocking of mitochondrial ADP transport by ANT,⁵⁴ sustained JNK activation⁵⁵ and release of toxic proteins from the permeable mitochondria.⁵⁶

In cells with increased glucose consumption, NADH levels are drastically increased, which can result in increased accumulation of ROS.³⁹ Our results indicate that increased ROS accumulation in H37Rv-infected Mϕ requires activation of two independent mechanisms associated with mitochondria. First, the generation of sufficient amounts of electron donors and increased HKII levels on the mitochondria lead to enhanced production of glucose-6-phosphate, the rate-limiting step of glycolysis.²⁵ HKII is predominantly associated with VDAC on the mitochondria and couples oxidative phosphorylation to glycolysis, which is essential for ROS production. Thus reduction of mitochondrial HKII downregulates glycolysis as well as ROS generation. Interestingly, similar to H37Rv-infected Mϕ, HKII is highly upregulated on the mitochondria of many tumors, which leads to increased aerobic glycolysis known as the Warburg effect.^{25, 57} Second, RIPK3 induces MPT, which interrupts the electron chain in mitochondrial complex I leading to electron-leakage and allows molecules with a size up to ∼1.5 kDa to exit the mitochondrial matrix. MPT is part of a cellular suicide mechanism and its contribution to the pathophysiology of several diseases including heart injury¹⁴ is well documented. Although it was proposed that induction of MPT is a mechanism by which aging mitochondria are earmarked for removal, there is no evidence of this mechanism in studies using CypD knockout mice.¹⁵ Thus the role of MPT under physiological conditions is still unclear. In contrast, under certain pathological conditions including Mϕ infection with virulent Mtb, MPT triggers induction of necrosis, which is essential for the release of the bacilli from the host cell ensuring the vicious cycle of infection. Here we have identified two different complementary mechanisms, MPT and HKII accumulation, which act in concert to increase ROS accumulation triggering a necrotic cell death program in Mtb-infected Mϕ (Figure 8).

How does RIPK3 induce MPT? Virulent Mtb increases HKII binding to VDAC on the outer mitochondrial membrane that enhances the generation of glucose-6-phosphate as well as CypD binding to the ANT⁵⁸ in the inner mitochondrial membrane. This opens the permeability transition pore causing MPT.¹⁵ CypD keeps the MPT pore in the mitochondrial inner membrane in the “open” position by binding to ANT.⁵⁹ Here we show that CypD binds to ANT to induce MPT in virulent Mtb-infected-Mϕ. We furthermore demonstrate that RIPK3 is required for binding of CypD to ANT to initiate ROS-mediated necrosis. Considering that several components of MPT are kinase substrates⁶⁰ it is tempting to speculate that RIPK3 may phosphorylate some components involved in MPT for generating increased levels of ROS.

RIPK3^−/− mice were more resistant than WT mice in both the low-dose aerosol and high-dose intravascular models of Mtb infections. In addition, we observed a significant decrease in the total number of neutrophils in the lungs of Mtb-infected RIPK3^−/− mice. Reduced number of neutrophils further supports the notion of less necrosis in RIPK3^−/− Mtb-infected animals. However, this protection was mainly dependent on Mϕ immunity, considering that there were no qualitative or quantitative differences in T-cell-mediated immunity between Mtb-infected RIPK3^−/− or WT mice. In addition, Mϕ transfer experiments directly demonstrated that the fate of Mtb-infected Mϕ was the key determinant of the relative resistance of these mice. Interestingly, a recent study by Albert’s group has elegantly shown that RIPK3-mediated necroptosis is more effective in cross-priming T cells than apoptosis.²⁰ However, we speculate that during Mtb infection the host may compensate for the lack of necrosis-dependent T-cell-mediated immunity via apoptosis to maintain an adequate T-cell-response to Mtb infection.^{5, 6, 7} The links between apoptotic/necrotic Mϕ, DC, and T-cell immunity in the control of Mtb infection certainly requires further investigation.

The dramatic differences in behavior of avirulent vs. virulent strains of Mtb in induction of apoptosis and necrosis, respectively, suggest the involvement of a specific pathogen-encoded inducer of RIPK3-dependent necrosis, which needs to be elucidated. Several virulent strains of Mtb encode specific genes, such as the nuoG gene, which actively inhibits the apoptotic death pathway in Mϕ.⁶¹ The nuoG gene of Mtb encodes type I NADH dehydrogenase, which neutralizes NOX-2 dependent ROS production in the Mϕ and is required for induction of apoptosis.⁶² It will be interesting to know whether the nuoG gene or other genes are involved in RIPK3-dependent necrosis.

There are several differences between our study and other previous published data using various cell lines including HeLa, 293A, L929, and 3T3-cells.^{26, 46, 63, 64} We envision that these differences are mainly due to using cell lines and soluble ligands vs. primary Mϕ and live-replicating bacteria. For instance, addition of caspase inhibitors to L929 cells induces necrosis due to autocrine production of TNF in the absence of active pro-apoptotic caspases.³⁵ In contrast, addition of caspase inhibitors to primary human Mϕ in the absence of Mtb infection does not induce necrosis as autocrine TNF-production is minimal indicating that the pathways leading to necrosis in certain cell lines and in primary Mϕ are significantly different. Similar to our findings in human Mϕ infected with virulent Mtb, Roca and Ramakrishnan have reported a comparable role of RIPK3 in a zebra fish model of infection with Mycobacterium marinum,⁶⁵ which underscores the evolutionary conservation of the RIPK3-dependent necrosis pathway in Mtb pathogenesis.

Despite the world-wide application of BCG vaccination and other anti-Mtb interventions, Mtb remains one of the most successful human pathogens. More than 2 billion people carry latent tuberculosis infection, and ∼20 million people have currently active tuberculosis, of which 1.8 million people die every year.⁶⁶ The success of this pathogen is closely linked to its ability to alter the intracellular environment of Mϕ including induction of cell death. Thus dissecting the molecular mechanisms involving mitochondria as entities determine whether Mtb-infected Mϕ undergo apoptosis or necrosis is of significant importance in identifying the outcome of host immunity to Mtb infection and might pave the road for novel therapies that target the necrotic death program.

Methods

Materials. Anti-caspase 8 (551242), and anti-RIP1 (551042) antibodies were from BD Biosciences. Anti-caspase 8 (52183, for immunofluorescence), anti-RIPK3 (56164), and anti-adenine nucleotide translocase (ANT, 109864) antibodies were from Abcam. Anti-CypD (AP1035) antibody was from Calbiochem (Merck KGaA, Darmstadt, Germany). Anti-hexokinase II (2106), anti-BAX (2772), anti-Bcl-x_L (2762), anti-caspase 3 (9662), anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8 (Asp387, 14071, for flowcytometry) were from Cell Signaling Technology (Danvers, MA). Anti-VDAC (SP5361P) antibody was from Acris (Herford, Germany). Active caspase 3 for flowcytometry was from BD. Anti-human mitochondria antibody (HMS-0100) was from Immunovision. Dylight 650-conjugated Donkey anti-rabbit IgG antibody and Alexa fluro 488-conjugated Donkey anti-human IgG antibody were from Jackson Immuno Research Laboratory (Bar Harbor, ME, USA). The caspase inhibitor z-IETD-FMK (FMK007) was from R&D Systems (Minneapolis, MN). Gammabind protein G sepharose (17-0885-01) was from GE Health Care (Pittsburgh, PA). Cyclosporin A (30024), Tiron (89460) and Protease Inhibitor Cocktail (P2714) from Calbiochem (Saint Louis, MO) and Phosphatase Inhibitor Cocktail 1 (P2850) was from Sigma-Aldrich (St. Louis, MO). Mitochondria/Cytosol Fractionation Kit (ThermoFisher Scientific Mitochondrial Isolation kit #89874, Rockford, IL) and MitoTracker Red CMXRos (M7512), MitoTracker Red CM-H₂XRos (M7513), and LIVE/DEAD Fixable Dead Cell Stain Kits (MP34955) from Invitrogen (Eugene, OR).

In vivo M. tuberculosis infection. Six- to ten-week-old C57BL/6 or Rag1^−/− mice were obtained from Jackson Labs; RIPK3^−/− mice were obtained from Genentech. Mice were infected with virulent M. tuberculosis (H37Rv) via the aerosol route using a nose-only exposure unit and received ∼50–100 CFU/mouse⁶⁷ or intratracheally (0.5 × 10⁶ CFU) or intravenously (1 × 10⁶ CFU)³. Mice were killed by CO₂ inhalation and the lung, spleen, and lymph node were aseptically removed, minced, and pressed through 70-μl cell strainer followed by red blood cell lysis to obtain single-cell suspension. Bronchoalveolar lavage was performed with phosphate-buffered saline (PBS) in mice infected intratracheally.^{3, 8}

Adoptive transfer models of infection. Alveolar Mϕ were collected from the BAL of naive WT and RIPK3^−/− mice and were immediately suspended at a concentration 0.5 × 10⁶ cells/500 μl RPMI medium with 10% fetal calf serum and infected in suspension using virulent H37Rv (MOI∼1) for 30 minutes. Free bacteria were then removed by 6 washes with cold PBS, each time followed by centrifugation for 10 min at 1000 RPMI at 4 °C. Cells were resuspended in PBS at 0.5 × 10⁶/40 μl and transferred by the intratracheal route into naive C57BL/6 mice.^{3, 5}

CFU determination. Following infection, mice were killed and their lungs removed and individually homogenized in 0.9% NaCl-0.02% Tween 80 with a Mini-Bead-Beater-8 (BioSpec Products) (Bartlesville, OK). Viable bacteria were enumerated by plating 10-fold serial dilutions of organ homogenates onto 7H11 agar plates (Thermo Fisher Scientific Remel Products, Lenexa, KS). Colonies were counted after 3 weeks of incubation at 37 °C.

Bacteria. The virulent Mtb strain H37Rv and attenuated stain H37Ra (American Type Culture) were grown in Middlebrook 7h9 broth (BD Biosiences, Sparks, MD) with BBL Middlebrook OADC Enrichment (Becton Dickson, Sparks, MD) and 0.05% (vol/vol) Tween 80 Difco (Sparks). Aliquots were distributed into small tubes and stored at −80 °C. 100 μl of the aliquot was serially diluted 10-fold with 0.02% Tween 80 in PBS and plated on 7H11 agar plates (Thermo Fisher Scientific Remel Products, Lenexa, KS, USA). The bacterial concentration of each batch was determined by colony counting after 4-weeks.

Cells and cell culture. Human mononuclear leukocytes from buffy coat preparations (Research Blood Component, Boston, MA, USA) were cultured for 7 days in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% (vol/vol) human AB serum (Gemini bio-Products, West Sacramento, CA) at a density of 2.0 × 10⁶ cells per ml in six-well plates and 2.0 × 10⁷ cells per ml in 10 cm plates, and then were changed to IMDM with 2% (vol/vol) human AB serum 1 day before challenged with varying MOI of Mtb. Bone marrow from 8- to 10-week-old mice was harvested from femurs and differentiated into Mϕ for 7 days in RPMI-1640 (Invitrogen) supplemented with 10% L929 cell (ATCC)-conditioned medium, 10% fetal bovine serum, 2 mM L-glutamine and 1 mM sodium pyruvate, 1% essential and non-essential amino acids (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin.

In vitro infection. Adherent human Mϕ were infected with H37Rv or H37Ra at different MOIs. After 4 h the Mϕ were washed three times with HBSS to remove non-adherent bacilli and replaced with fresh IMDM with 2% AB. Adherent murine Mϕ were infected with H37Rv at different MOIs. After 4 h the Mϕ were washed three times with warm PBS to remove non-adherent bacilli and replaced with fresh media containing fetal bovine serum.

FACS analysis. Cells were stained as previously described using antibodies specific for mouse CD3, CD4, CD8, and CD19 (BD Biosciences) Ly6G, Ly6c, CD11b, F4/80, CD11c (San Diego, CA).⁵ Antigen-specific CD4+ and CD8+ T cells were identified using IA^bESAT6_1-20 and H2-K^bTB10.4_4-11 tetramers, respectively (National Institutes of Health Tetramer Core Facility, Emory University Vaccine Centre, Atlanta, GA, USA). Cleaved Caspase 8 Staining was performed in WT and RIPK3−/− Mφ. Cells were washed, collected and suspended in formaldehyde to obtain a final concentration of 4% for 30 min at room temperature. Cells were permeabilized by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol by incubating 30 min on ice. Cells were washed by with buffer and PE-conjugated cleaved caspase 8 Ab was added for 1 h at room temperature. Cells were then washed and resuspended in buffer to be analyzed by flow cytometer. Flow cytometry was performed by using a BD LSR-II (BD Biosciences) and FlowJo analysis software (Tree Star, Ashland, OR).

In vitro T cell proliferation. CD3⁺ T cells were purified from spleens of naive WT and Annex1^−/− mice using MACS column purification procedure by negative selection (Pan T cell kit—MACS Miltenyi). The purity of CD3⁺ T cells was usually between 92–97%. Purified CD8⁺ T cells were stained with 5 μM CFSE in PBS containing 0.1% bovine serum albumin at room temperature. Staining was stopped after 8 min of incubation by addition of RPMI medium containing 10% fetal bovine serum. T cells were then cultured with different concentrations of anti-CD3 and anti-CD28. After 3 days, T cell proliferation was measured by reduction in CFSE expression (CFSE^low) using flow cytometry.

ELISPOT assay. The IFN-γ Elispot assay was conducted according to the manufacturer’s instructions (R&D Sytems). Briefly, isolated cells were seeded into a 96-well plate pre-coated with mouse IFN-γ capturing Abs. Cells were then incubated for 24 h with or without antigenic stimulation. The plate was developed and the spots were enumerated using a series A immunospot plate reader, Image Acquisition version 4.0, and Immunospot version 3.2 analysis software (Cellular Technology, Cleveland, OH).

Isolation of mitochondria from Mϕ. Mitochondria were isolated using the Mitochondria/Cytosol Fractionation Kit (ThermoFisher Scientific Mitochondrial Isolation kit #89874) according to the manufacturer’s recommendations. Briefly, cells were washed twice with ice-cold PBS and then 1 ml of Isotonic Mitochondrial Buffer (containing protease inhibitors) was added. Cells were homogenized on ice by passing through a 25-gauge needle (Becton Dickinson) after detachment from the culture dish by using a cell scraper. The lysate was centrifuged at 600 × g for 10 min at 4 °C to remove the nuclei, debris and intact cells. The supernatants were centrifuged at 10 000 × g for 30 min at 4 °C. The enriched mitochondrial fraction was washed and lysed in 100 μl of a buffer containing Tris-HCl (50 mM, pH7.4), NaCl (150 mM), EDTA (5 mM), Triton X-100 (1%), sodium ortho-vanadate (0.2 mM) and protease inhibitor cocktail (Sigma) at 4 ° C and stored at −80 °C together with the cytosolic fraction. The purity of the mitochondria was determined by taking 10 μg of protein from the mitochondrial and cytosolic fractions and measuring Bcl-x_L and GAPDH levels by Western blotting.

Silencing of the genes encoding human RIPK3, Bcl-x_L, hexokinase II, MLKL and BAX. The target siRNA sequences for human RIPK3 specific siRNA (5′-GGAAUGCCUACCAAAAACU-3′), the Bcl-x_L siRNA (5′-GGAGAUGCAGGUAUUGGUG-3′),⁶⁸ and for hexokinase II (5′-CACGAUGAAAUUGAACCUGGU-3′),⁶⁹ MLKL siRNA is a pool of 3 different duplexes (5′-GGAAUACCGUUUCAGAUGUtt-3′, 3′-ACAUCUGAAACGGUAUUCCtt-5′; 5′-GGAAUAGUGAGGUUCACUUtt-3′, 3′-AAGUGAACCUCACUAUUCCtt-5′; 5′-GAAGGCUGUGAUUCUAAGAtt-3′, 3′-UCUUAGAAUCACAGCCUUCtt-5′) and RIPK3 second siRNA pool (5′-CUGAGUGGCUAAACAAACUtt-3′, 3′-AGUUUGUUUAGCCACUCAtt-5′; 5′-GGAGACAACAACUACUUGtt-3′, 3′-UCAAGUAGUUGUUGUCUCCtt-5′; 5′-CUCCAAGAGUUACGAGUUtt-3′, 3′-UAACUCGUAACUCUUGGAGtt-5′ were generated by IDT. Human BAX-specific siRNA (#6321) was from Cell Signaling Technology. Mϕ were cultured in IMDM with 10% (vol/vol) human AB serum and in IMDM with 2% human AB serum 1 day before transfection. All siRNA was transfected at 37 °C into the cells at a final concentration of 50 nM with Lipofectamine RNAiMAX (13778-150, Invitrogen, Waltham, MA). Mϕ were infected with Mtb 48–72 h after transfection.

Immunoblot analysis. Protein was measured with the Bradford assay. Samples (5 μg) were fractionated by SDS-polyacrylamide gel electrophoresis and protein was transferred to a PVDF membrane at 4 °C. The membrane was blocked with 5% dried milk in Tris HCl buffer (10 mM), pH 8.0 containing NaCl (150 mM) and Tween 20 (0.5%) and was incubated with the appropriate primary antibodies overnight at 4 °C. After three washes blots were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence reagent was added and the blots were exposed to Denville Hyblot CL film (Holliston, MA). Western input of Mϕ infected with H37Rv for RIPK3, RIPK1, procaspase8, and loading control (actin and VDAC) is incorporated in supplementary (Supplementary Figure 3A).

Co-immunoprecipitation. After centrifugation at 18 000 × g for 30 min the Mϕ lysates were immunoprecipitated overnight with immobilized monoclonal antibody under gentle rotation at 4 °C. 50 μl of protein G beads-slurry (50%) was added to the sample and the mixture incubated for another 1 h at 4 °C. To remove non-specific bound material the beads were washed with lysis buffer 3 times. The beads were then gently mixed with lysis buffer, centrifuged at 4 °C and the supernatant removed. Twenty μl of SDS-sample buffer was added to the samples and the samples were then loaded on SDS-polyacrylamide gel electrophoresis gels after boiling.

Assessment of MPT. MPT was measured as described.¹¹ Briefly, human Mϕ were cultured at a density of 1.5 × 10⁶ mononuclear cells per well with 2 ml medium containing IMDM with 2% AB serum in six-well cluster plates (Corning Glass, Corning, New York). After 7 days cells were pre-loaded with 1.5 nM DiOC₆³ in IMDM for 20 min at 37 °C, washed, and incubated at 37 °C for 10 min in medium containing 15 μg/ml digitonin, washed, and fixed with 1% paraformaldehyde for 1 h at r t. Mϕ were then dislodged with a rubber policeman, washed with PBS and resuspended in PBS with 1% (wt/vol) bovine serum albumin. A FACSort flow cytometer (BD Biosciences) was used for flow cytometry.

Assay for apoptosis. Apoptosis was measured by determining caspase 3-activation using western blotting and flow cytometry because the caspases 3/10 are the direct executioners of pro-apoptotic pathways. Apoptosis was also determined by flow cytometry with Annexin V staining. Briefly, RIPK3^−/− Mφ were infected with H37Ra (MOI 5) for 4 h followed by washing and incubation for 96 h in BMDM media. Cells were harvested and incubated with Annexin V dye in buffer recommended by manufacturer for 30 min. Mφ were resuspended with 7AAD for dead cell staining and acquired using flowcytometer within 1 h.

Assay for necrosis. Mϕ necrosis was evaluated in vitro and in vivo by flow cytometry with the LIVE/DEAD fixable Dead Cell Stain kit (Invitrogen, L34955) according to the instructions of the manufacturer. Briefly, adherent infected and uninfected human Mϕ (1.5 × 10⁵ per well) were washed, 0.5 ml of 1% of reconstituted fixable violet fluorescent reactive dye was added and the mixture incubated at room temperature for 30 min in dark. Mϕ were washed and fixed with 3.7% paraformaldehyde for 1 h at room temperature. Mϕ were dislodged with a rubber policeman, washed and resuspended in PBS with 1% bovine serum albumin. A FACSort flow cytometer was used for flow cytometry. In some experiments necrosis was measured with cell death detection ELISA^PLUS photometric enzyme immunoassay (11 920685 001; Roche Applied Science, Sandhofer Strasse, Mannheim, Germany).

ROS assay. ROS accumulation was measured by using reduced rosamine-Mitotracker probes (Invitrogen, M7513). Adherent Mϕ were washed with IMDM medium, stained with 200 nM Mitotracker Red CM-H₂XRos in IMDM without serum at 37 ° C for 30 min. Then the medium was removed, and the cells were washed and fixed with 3.7% paraformaldehyde for 1 hour at room temperature. After fixation, Mϕ were washed and resuspended in 0.5 ml PBS with 1% bovine serum albumin. Fluorescence intensity of ROS was measured using a flow cytometer. To control for possible changes in the mitochondrial mass under the different conditions the cells were tested with the ROS-insensitive mitotracker dye red FM (Invitrogen). No differences in in fluorescence emission were seen indicating that the mitochondrial mass was not different in the Mϕ subjected to the different experimental conditions.

NADH determination. NADH in Mϕ extracts was measured with the NAD+/NADH Quantification Colorimetric Kit from BioVision (Milpitas, CA) according to the recommendation of the manufacturer.

Immunostaining and confocal microscopy. Mϕ were cultured on poly-D lysine-coated cover slips in 24-well plates at a density of 1.5 × 10⁵ cells/well and were infected for 24 h with mCherry labeled H37Rv. Mϕ were then fixed for 1 h with 4% paraformaldehyde and blocked over night at 4 °C with PBS containing 10% horse serum. Cover slips were incubated for 2 h at room temperature with anti-mitochondria, anti-caspase 8 or anti-RIPK3 (dilution, 1:100) ab. Mϕ were then washed and stained for 1 h at room temperature with fluorescent secondary ab. Mϕ were mounted and then subjected to confocal and transmission light microscopy. Microscope images were acquired at the Brigham and Women's Confocal Core Facility with a Nikon TE2000-U inverted microscope, Nikon C1 Plus confocal system, 60 × Nikon Plan Apochromat objective, 10-mW Spectra Physics 488-nm argon laser, Melles Griot Red HeNe 543-nm laser, Chroma 515-nm/30-nm and 543-nm emission filters and a 30-μm pinhole. Images were acquired under identical exposure conditions and micrographs were compiled and analyzed with Nikon EZ-C1 v3.8 (Nikon, Minato, Tokyo, Japan) and Adobe Photoshop v10.0.1. Colocalization of RIPK3 and pro-caspase 8 on the mitochondria was quantified using Metamorph. Each channel (R, G, B) was considered separately with each pixel assigned an intensity value (0–255) with care taken to assure that no pixel was oversaturated in any image used for quantification. The lower threshold is set whereby areas of the image without staining are excluded. The software then calculates that area of caspase 8 or RIPK3 with mitotracker and reports it in terms of percent positive cells. Quantification was done on 100–200 cells for every condition, and representative images are shown from at least three independent experiments.

Quantitative real-time PCR. Total RNA was isolated with Trizol reagent from lungs of Mtb-infected animals. RNA was quantified with the help of a NanoDrop spectrophotometer (Wilmington, DE). The A260/A280 ratio of all samples was in the range of 1.90 to 2.00. DNA contamination from RNA samples was removed by amplification grade DNase (Ambion Invitrogen; according to manufacturer’s instructions). Briefly, RNA samples (1 μg) were incubated with DNase (1 U) for 15 min in the reaction buffer. After the incubation, DNase activity was terminated by stop solution. Further, the samples were heated to 70 °C for 10 min to inactivate DNase activity. Real time PCR was performed using SYBR Green Master Mix Kit. Melting curve analysis was performed to eliminate the possibility of non-specific amplification. Results are represented in arbitrary units. Analysis was done by comparative Ct method, where Ct values were normalized against the house-keeping control GAPDH.

Statistics. Results are expressed as mean±s.d. or s.e.m. The data were analyzed by using Microsoft Excel Statistical Software (Jandel, San Rafael, CA, USA) using the t-test for normally distributed data with equal variances. In some experiments, one-way analysis of variance with Dunnett’s posttest or with Bonferroni’s posttest was performed using Prism version 5 for Windows (Graph-Pad Software, La Jolla, CA).

Ethics statement. All animal experiments were conducted in accordance with Canadian Council on Animal Care guidelines. The animal used protocol was approved by the McGill Animal Care Committee (protocol number: 2010–5860).

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Acknowledgements

We are grateful to V.W. Hsu, E Remold-O’Donnell and C Geadas for helpful discussions and Nancy Kedersha for supplying anti-mitochondrial antibody. This study was supported by the US National Institute of Health Grant AI072143 to H.G.R. and the Canadian Institute of Health Research (CIHR) Foundation Grant (FDN-143273) to M.D and M.D. holds a CIHR New Investigator Award.

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X Zhao and N Khan: These authors equally contributed to this work.

Authors and Affiliations

Division of Rheumatology, Department of Medicine, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
X Zhao, H Gan, T Nishimura, S-Y Park & H G Remold
Department of Medicine, Department of Microbiology and Immunology, McGill International TB Centre, McGill University Health Centre, Meakins-Christie Laboratories, Montreal, Quebec, Canada
N Khan, F Tzelepis & M Divangahi
Health Center, Keio University, Tokyo, Japan
T Nishimura

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X Zhao
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N Khan
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H Gan
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F Tzelepis
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T Nishimura
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S-Y Park
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M Divangahi
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H G Remold
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Correspondence to M Divangahi or H G Remold.

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Zhao, X., Khan, N., Gan, H. et al. Bcl-x_L mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages. Mucosal Immunol 10, 1553–1568 (2017). https://doi.org/10.1038/mi.2017.12

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Received: 05 May 2016
Accepted: 13 January 2017
Published: 12 April 2017
Issue Date: November 2017
DOI: https://doi.org/10.1038/mi.2017.12

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