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Glucose Metabolism Linked to Antiviral Responses
Leading Edge
Previews Glucose Metabolism Linked to Antiviral Responses Joshua S. Stoolman1 and Navdeep S. Chandel1,2,* 1Department
of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA of Biochemistry & Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.05.057 2Department
Viral infection causes the host to activate an antiviral response that, in part, is dependent on mitochondrial antiviral signaling protein (MAVS) to stimulate type I interferons. Zhang et al. (2019) demonstrate that glucose-generated lactate interacts with MAVS to suppress type I interferons. This study links glucose metabolism to antiviral responses.
The retinoic-acid-inducible gene I (RIG-I)like receptor family, or RLRs, detect cytosolic viral RNA via their C-terminal repressor containing an RNA-binding domain. After RNA binding, activated RLRs undergo a conformational change that allows for N-terminal caspase recruitment domain (CARD) interactions with other CARD-containing proteins. This includes the mitochondrial antiviral signaling protein (MAVS) (Tan et al., 2018), which is required for activation of interferon regulatory transcription factor 3 (IRF3) that drives type I interferon (IFN) expression. MAVS localization to mitochondria indicates a potential link to metabolism in these organelles. Surprisingly, there have been few clear links of metabolic inputs to the RLR-MAVS-IFN pathway outside of the mitochondrial localization of MAVS. In this issue, Zhang et al. (2019) identify lactate produced during glycolysis as a potent suppressor of RLR-driven antiviral signaling, adding metabolite specificity to our understanding of metabolic control of innate immune responses. MAVS is anchored to the mitochondria via its transmembrane (TM) domain. Activated RLRs interact with MAVS proteins through CARD-CARD interactions, which induce RLR-MAVS protein accumulation into prion-like structures. These prionlike structures are sufficient to induce aggregation of other MAVS proteins not directly bound to RLR sensors (Tan et al., 2018). These aggregates form supramolecular organizing centers (SMOCs), hubs of signal transduction that allow for small amounts of activated RLRs to induce potent antiviral responses. Loss of the SMOC significantly reduces type I IFN mRNA, as observed in Mavs / mice,
which are viable but show significant decreases in type I IFN responses to poly(I:C) and are susceptible to vesicular stomatitis virus (VSV) infection (Sun et al., 2006). Zhang et al. (2019) initially demonstrate that activation of RLR signaling through poly(I:C) transfection decreased levels of glycolytic intermediates downstream of hexokinase-2 (HK-2). They observed that HK-2 normally associates with MAVS in a voltage-dependent anion channel (VDAC)-contingent manner on the outer mitochondrial membrane. However, activation of RLRs diminishes MAVS interaction with HK-2 on the outer mitochondrial membrane, causing a decrease in glycolytic flux. Similarly, the ectopic expression of an active form of RIG-I, an RLR family member, was sufficient to decrease MAVS interaction with HK-2. Additionally, RLR-stimulated IFN-b mRNA was increased in cells cultured in low-glucose conditions or with hexokinase inhibition, suggesting reciprocal feedback between RIG-I and HK-2. Interestingly, induction of IFN-b mRNA by stimulator of interferon genes (STING) and Toll-like receptor (TLR) signaling pathways, which are not MAVSdependent, was not affected by reducing glycolytic flux. Mechanistically, Zhang et al. (2019) show that lactate production by glycolysis was the key metabolite suppressing IFN-b mRNA. Glycolysis generates 2 molecules of pyruvate from glucose, which are converted either to lactate by lactate dehydrogenase A (LDHA) or imported into mitochondria, where pyruvate is oxidized to acetyl-CoA by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle. LDHA and PDH inhibi-
10 Cell 178, June 27, 2019 ª 2019 Elsevier Inc.
tion increased and decreased, respectively, IFN-b mRNA levels after RLR stimulation. Pharmacologic inhibition of LDHA also increased IFN-b mRNA expression in response to poly(I:C) transfection, whereas lactate supplementation was sufficient to reverse the effects of LDHA inhibition. Importantly, Zhang et al. (2019) demonstrate that lactate binds MAVS directly to inhibit type I IFN signaling, as in vitro biotin-lactate pull-down assays showed that lactate binds the MAVS TM domain. This was confirmed by mass spectrometry analysis of the biotin-lactate pull-down product. A key experiment was the use of a cellpermeable MAVS TM-Tat fusion protein to demonstrate that cells incubated with MAVS TM show a dose-dependent increase in type I IFN responses after RLR activation. Next, they assessed whether modulation of lactate production in vivo significantly alters responses to RNA viruses. Fasted mice supplemented with low glucose had increased type I IFN responses compared to mice supplemented with high glucose. Ldha / mice, or mice treated with an LDHA inhibitor, also demonstrated increased type I IFN responses and better clearance of VSV from the lung. Conversely, supplementation with sodium lactate decreased type I IFN responses in VSV-infected, low-glucose mice to control levels, indicating that lactate is sufficient to dampen systemic type I IFN responses to VSV. Going forward, it will be important to decipher the residues within MAVS that are necessary for lactate binding. The generation of mice bearing mutations of these residues would rigorously test whether
Figure 1. Lactate Produced by Aerobic Glycolysis Suppresses RIG-I-MAVS-Driven IFN-b Transcription Increased glycolytic flux is associated with lower IFN-b mRNA expression in response to RIG-I activation. Hexokinase-2 (HK-2) interacts with MAVS in the absence of viral RNA. After exposure to viral RNA, activated RLR family members bind MAVS and decrease MAVS:HK-2 association, which diminish glycolytic flux and subsequent lactate production. Lactate inhibits MAVS aggregation, which is necessary to promote transcription of IFN-b. Thus, viral RNA promotes type I IFN responses by limiting lactate production.
lactate binding to MAVS inhibits antiviral responses in vivo. A caveat to this experiment would be that the mutation of these residues should not disrupt normal MAVS function. These findings establish a clear mechanistic link between lactate and MAVS-
induced type I IFN production (Figure 1), which leads to new questions about the role of lactate in other innate immune responses. Type I IFN production driven by TLR or STING pathways is unaffected by lactate; however, MAVS are required for fulminant IL-1b release after lipopolysaccharides (LPS) and NLR family pyrin domain containing 3 (NLRP3) activation (Subramanian et al., 2013). The role of lactate in this pathway remains unclear, as a previous study found that lactate suppressed TLR4-NLRP3 inflammasome-driven IL-1b release in vitro and in vivo via lactate receptor G proteincoupled receptor 81 (GPR81) signaling (Hoque et al., 2014), while another study showed that lactate promotes TLR4 signaling through interactions with TLR4 co-receptor MD-2 (Samuvel et al., 2009). Determining how lactate affects IL-1b release downstream of NLRP3 may provide insight into MAVS function in the assembly of the inflammasome and progression of IL-1b-dependent immune responses. Historically, lactate has been viewed as a waste product. However, recent studies in macrophages and T cells indicate that lactate can be immunomodulatory (Colegio et al., 2014; Haas et al., 2015). Moreover, lactate has emerged as a major fuel source for a wide range of normal tissues and cancers in mice (Hui et al., 2017) as well as human lung cancers (Faubert et al., 2017). The current study by Zhang et al. (2019) provides further evidence that lactate has evolved to be a key metabolite controlling physiological and pathological responses. It will be of interest to assess whether manipulation of lactate levels in humans alters immune responses to viral infections. REFERENCES Colegio, O.R., Chu, N.-Q., Szabo, A.L., Chu, T., Rhebergen, A., Jairam, V., Cyrus, N., Brokowski, C.E., Eisenbarth, S.C., Phillips, G.M., et al. (2014).
Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 513, 559–563. Faubert, B., Li, K.Y., Cai, L., Hensley, C.T., Kim, J., Zacharias, L.G., Yang, C., Do, Q.N., Doucette, S., Burguete, D., et al. (2017). Lactate Metabolism in Human Lung Tumors. Cell 171, 358–371.e9. Haas, R., Smith, J., Rocher-Ros, V., Nadkarni, S., Montero-Melendez, T., D’Acquisto, F., Bland, E.J., Bombardieri, M., Pitzalis, C., Perretti, M., et al. (2015). Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol. 13, e1002202. Hoque, R., Farooq, A., Ghani, A., Gorelick, F., and Mehal, W.Z. (2014). Lactate reduces liver and pancreatic injury in Toll-like receptor- and inflammasome-mediated inflammation via GPR81-mediated suppression of innate immunity. Gastroenterology 146, 1763–1774. Hui, S., Ghergurovich, J.M., Morscher, R.J., Jang, C., Teng, X., Lu, W., Esparza, L.A., Reya, T., Le Zhan, Yanxiang Guo, J., et al. (2017). Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115–118. Samuvel, D.J., Sundararaj, K.P., Nareika, A., Lopes-Virella, M.F., and Huang, Y. (2009). Lactate boosts TLR4 signaling and NF-kappaB pathwaymediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. J. Immunol. 182, 2476–2484. Subramanian, N., Natarajan, K., Clatworthy, M.R., Wang, Z., and Germain, R.N. (2013). The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation. Cell 153, 348–361. Sun, Q., Sun, L., Liu, H.-H., Chen, X., Seth, R.B., Forman, J., and Chen, Z.J. (2006). The specific and essential role of MAVS in antiviral innate immune responses. Immunity 24, 633–642. Tan, X., Sun, L., Chen, J., and Chen, Z.J. (2018). Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids. Annu. Rev. Microbiol. 72, 447–478. Zhang, W., Wang, G., Xu, Z.-G., Tu, H., Hu, F., Dai, J., Yan, C., Chen, Y., Lu, Y., Zeng, H., et al. (2019). Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS. Cell, this issue, 176–189.
Cell 178, June 27, 2019 11
Previews Glucose Metabolism Linked to Antiviral Responses Joshua S. Stoolman1 and Navdeep S. Chandel1,2,* 1Department
of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA of Biochemistry & Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.05.057 2Department
Viral infection causes the host to activate an antiviral response that, in part, is dependent on mitochondrial antiviral signaling protein (MAVS) to stimulate type I interferons. Zhang et al. (2019) demonstrate that glucose-generated lactate interacts with MAVS to suppress type I interferons. This study links glucose metabolism to antiviral responses.
The retinoic-acid-inducible gene I (RIG-I)like receptor family, or RLRs, detect cytosolic viral RNA via their C-terminal repressor containing an RNA-binding domain. After RNA binding, activated RLRs undergo a conformational change that allows for N-terminal caspase recruitment domain (CARD) interactions with other CARD-containing proteins. This includes the mitochondrial antiviral signaling protein (MAVS) (Tan et al., 2018), which is required for activation of interferon regulatory transcription factor 3 (IRF3) that drives type I interferon (IFN) expression. MAVS localization to mitochondria indicates a potential link to metabolism in these organelles. Surprisingly, there have been few clear links of metabolic inputs to the RLR-MAVS-IFN pathway outside of the mitochondrial localization of MAVS. In this issue, Zhang et al. (2019) identify lactate produced during glycolysis as a potent suppressor of RLR-driven antiviral signaling, adding metabolite specificity to our understanding of metabolic control of innate immune responses. MAVS is anchored to the mitochondria via its transmembrane (TM) domain. Activated RLRs interact with MAVS proteins through CARD-CARD interactions, which induce RLR-MAVS protein accumulation into prion-like structures. These prionlike structures are sufficient to induce aggregation of other MAVS proteins not directly bound to RLR sensors (Tan et al., 2018). These aggregates form supramolecular organizing centers (SMOCs), hubs of signal transduction that allow for small amounts of activated RLRs to induce potent antiviral responses. Loss of the SMOC significantly reduces type I IFN mRNA, as observed in Mavs / mice,
which are viable but show significant decreases in type I IFN responses to poly(I:C) and are susceptible to vesicular stomatitis virus (VSV) infection (Sun et al., 2006). Zhang et al. (2019) initially demonstrate that activation of RLR signaling through poly(I:C) transfection decreased levels of glycolytic intermediates downstream of hexokinase-2 (HK-2). They observed that HK-2 normally associates with MAVS in a voltage-dependent anion channel (VDAC)-contingent manner on the outer mitochondrial membrane. However, activation of RLRs diminishes MAVS interaction with HK-2 on the outer mitochondrial membrane, causing a decrease in glycolytic flux. Similarly, the ectopic expression of an active form of RIG-I, an RLR family member, was sufficient to decrease MAVS interaction with HK-2. Additionally, RLR-stimulated IFN-b mRNA was increased in cells cultured in low-glucose conditions or with hexokinase inhibition, suggesting reciprocal feedback between RIG-I and HK-2. Interestingly, induction of IFN-b mRNA by stimulator of interferon genes (STING) and Toll-like receptor (TLR) signaling pathways, which are not MAVSdependent, was not affected by reducing glycolytic flux. Mechanistically, Zhang et al. (2019) show that lactate production by glycolysis was the key metabolite suppressing IFN-b mRNA. Glycolysis generates 2 molecules of pyruvate from glucose, which are converted either to lactate by lactate dehydrogenase A (LDHA) or imported into mitochondria, where pyruvate is oxidized to acetyl-CoA by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle. LDHA and PDH inhibi-
10 Cell 178, June 27, 2019 ª 2019 Elsevier Inc.
tion increased and decreased, respectively, IFN-b mRNA levels after RLR stimulation. Pharmacologic inhibition of LDHA also increased IFN-b mRNA expression in response to poly(I:C) transfection, whereas lactate supplementation was sufficient to reverse the effects of LDHA inhibition. Importantly, Zhang et al. (2019) demonstrate that lactate binds MAVS directly to inhibit type I IFN signaling, as in vitro biotin-lactate pull-down assays showed that lactate binds the MAVS TM domain. This was confirmed by mass spectrometry analysis of the biotin-lactate pull-down product. A key experiment was the use of a cellpermeable MAVS TM-Tat fusion protein to demonstrate that cells incubated with MAVS TM show a dose-dependent increase in type I IFN responses after RLR activation. Next, they assessed whether modulation of lactate production in vivo significantly alters responses to RNA viruses. Fasted mice supplemented with low glucose had increased type I IFN responses compared to mice supplemented with high glucose. Ldha / mice, or mice treated with an LDHA inhibitor, also demonstrated increased type I IFN responses and better clearance of VSV from the lung. Conversely, supplementation with sodium lactate decreased type I IFN responses in VSV-infected, low-glucose mice to control levels, indicating that lactate is sufficient to dampen systemic type I IFN responses to VSV. Going forward, it will be important to decipher the residues within MAVS that are necessary for lactate binding. The generation of mice bearing mutations of these residues would rigorously test whether
Figure 1. Lactate Produced by Aerobic Glycolysis Suppresses RIG-I-MAVS-Driven IFN-b Transcription Increased glycolytic flux is associated with lower IFN-b mRNA expression in response to RIG-I activation. Hexokinase-2 (HK-2) interacts with MAVS in the absence of viral RNA. After exposure to viral RNA, activated RLR family members bind MAVS and decrease MAVS:HK-2 association, which diminish glycolytic flux and subsequent lactate production. Lactate inhibits MAVS aggregation, which is necessary to promote transcription of IFN-b. Thus, viral RNA promotes type I IFN responses by limiting lactate production.
lactate binding to MAVS inhibits antiviral responses in vivo. A caveat to this experiment would be that the mutation of these residues should not disrupt normal MAVS function. These findings establish a clear mechanistic link between lactate and MAVS-
induced type I IFN production (Figure 1), which leads to new questions about the role of lactate in other innate immune responses. Type I IFN production driven by TLR or STING pathways is unaffected by lactate; however, MAVS are required for fulminant IL-1b release after lipopolysaccharides (LPS) and NLR family pyrin domain containing 3 (NLRP3) activation (Subramanian et al., 2013). The role of lactate in this pathway remains unclear, as a previous study found that lactate suppressed TLR4-NLRP3 inflammasome-driven IL-1b release in vitro and in vivo via lactate receptor G proteincoupled receptor 81 (GPR81) signaling (Hoque et al., 2014), while another study showed that lactate promotes TLR4 signaling through interactions with TLR4 co-receptor MD-2 (Samuvel et al., 2009). Determining how lactate affects IL-1b release downstream of NLRP3 may provide insight into MAVS function in the assembly of the inflammasome and progression of IL-1b-dependent immune responses. Historically, lactate has been viewed as a waste product. However, recent studies in macrophages and T cells indicate that lactate can be immunomodulatory (Colegio et al., 2014; Haas et al., 2015). Moreover, lactate has emerged as a major fuel source for a wide range of normal tissues and cancers in mice (Hui et al., 2017) as well as human lung cancers (Faubert et al., 2017). The current study by Zhang et al. (2019) provides further evidence that lactate has evolved to be a key metabolite controlling physiological and pathological responses. It will be of interest to assess whether manipulation of lactate levels in humans alters immune responses to viral infections. REFERENCES Colegio, O.R., Chu, N.-Q., Szabo, A.L., Chu, T., Rhebergen, A., Jairam, V., Cyrus, N., Brokowski, C.E., Eisenbarth, S.C., Phillips, G.M., et al. (2014).
Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 513, 559–563. Faubert, B., Li, K.Y., Cai, L., Hensley, C.T., Kim, J., Zacharias, L.G., Yang, C., Do, Q.N., Doucette, S., Burguete, D., et al. (2017). Lactate Metabolism in Human Lung Tumors. Cell 171, 358–371.e9. Haas, R., Smith, J., Rocher-Ros, V., Nadkarni, S., Montero-Melendez, T., D’Acquisto, F., Bland, E.J., Bombardieri, M., Pitzalis, C., Perretti, M., et al. (2015). Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol. 13, e1002202. Hoque, R., Farooq, A., Ghani, A., Gorelick, F., and Mehal, W.Z. (2014). Lactate reduces liver and pancreatic injury in Toll-like receptor- and inflammasome-mediated inflammation via GPR81-mediated suppression of innate immunity. Gastroenterology 146, 1763–1774. Hui, S., Ghergurovich, J.M., Morscher, R.J., Jang, C., Teng, X., Lu, W., Esparza, L.A., Reya, T., Le Zhan, Yanxiang Guo, J., et al. (2017). Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115–118. Samuvel, D.J., Sundararaj, K.P., Nareika, A., Lopes-Virella, M.F., and Huang, Y. (2009). Lactate boosts TLR4 signaling and NF-kappaB pathwaymediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. J. Immunol. 182, 2476–2484. Subramanian, N., Natarajan, K., Clatworthy, M.R., Wang, Z., and Germain, R.N. (2013). The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation. Cell 153, 348–361. Sun, Q., Sun, L., Liu, H.-H., Chen, X., Seth, R.B., Forman, J., and Chen, Z.J. (2006). The specific and essential role of MAVS in antiviral innate immune responses. Immunity 24, 633–642. Tan, X., Sun, L., Chen, J., and Chen, Z.J. (2018). Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids. Annu. Rev. Microbiol. 72, 447–478. Zhang, W., Wang, G., Xu, Z.-G., Tu, H., Hu, F., Dai, J., Yan, C., Chen, Y., Lu, Y., Zeng, H., et al. (2019). Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS. Cell, this issue, 176–189.
Cell 178, June 27, 2019 11