UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis.

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The Journal of Clinical Investigation

Research article

UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis Jong-Seok Moon,1,2 Seonmin Lee,3,4 Mi-Ae Park,5 Ilias I. Siempos,1,2,6 Maria Haslip,7 Patty J. Lee,7 Mijin Yun,3,8 Chun K. Kim,5 Judie Howrylak,9 Stefan W. Ryter,1,2 Kiichi Nakahira,1,2 and Augustine M.K. Choi1,2 Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medical College, and New York-Presbyterian Hospital, New York, New York, USA. 2Division of Pulmonary and Critical Care Medicine,

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Weill Cornell Medical College, New York, New York, USA. 3Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital (BWH), Harvard Medical School, Boston, Massachusetts, USA. Division of Pulmonary and Critical Care Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 5Department of Radiology, BWH,

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Harvard Medical School, Boston, Massachusetts, USA. 6Department of Critical Care Medicine and Pulmonary Services, Evangelismos Hospital, University of Athens Medical School, Athens, Greece. Department of Internal Medicine, Section of Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, New Haven, Connecticut, USA. 8Department of Nuclear Medicine,

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Yonsei University College of Medicine, Seoul, Republic of Korea. 9Division of Pulmonary and Critical Care Medicine, Penn State College of Medicine, Hershey, Pennsylvania, USA.

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.

Introduction

Sepsis remains the leading cause of death in the medical intensive care unit (MICU) (1). Although inflammatory responses are important for host defense against invading microbes, excessive inflammation in sepsis can cause severe cell and tissue damage and organ dysfunction, leading to death (2). Our recent studies suggest that altered plasma metabolic profiles may reflect disease severity and predict mortality in critical illnesses such as sepsis (3, 4). Furthermore, we have shown that the plasma levels of proinflammatory cytokines (e.g., IL-18) and circulating mitochondrial injury markers, such as mitochondrial DNA, increase in critical illness and are predictive of mortality (5, 6). Emerging evidence suggests the involvement of inflammasomes in the pathogenesis of human diseases, including sepsis (7). Inflammasomes are molecular platforms for the caspase-1– dependent maturation and secretion of proinflammatory cytokines such as IL-1β and IL-18 in immune cells (8). Cytoplasmic receptors of the nucleotide-binding domain leucine-rich repeat–containing (NLR) family are key components of the inflammasome and Conflict of interest: The authors have declared that no conflict of interests exists. Submitted: July 29, 2014; Accepted: December 9, 2014. Reference information: J Clin Invest. 2015;125(2):665–680. doi:10.1172/JCI78253.

interact with apoptosis-associated adaptor protein (ASC), which recruits the precursor form of caspase-1 (7, 8). The NLRP3 (also known as cryopyrin or NALP3) inflammasome is unique in its ability to recognize molecular patterns associated with host-derived metabolites, such as glucose or saturated fatty acids (9–13). Recent studies from our laboratory and others demonstrate that mitochondrial dysfunction involving increased mitochondrial ROS production (12, 13) and the release of mitochondrial DNA into the cytosol (14) are critical events associated with NLRP3 inflammasome activation. However, recent studies have also suggested that NLRP3 inflammasome activation may occur independently of mitochondrial damage and ROS production, a finding which warrants further investigation (15, 16). The integration of metabolic derangements, mitochondrial dysfunction, and inflammasome activation and their contribution to the pathogenesis of sepsis thus remain incompletely understood. We hypothesized that metabolic regulation by mitochondria may represent a critical determinant of the pathogenesis of sepsis. We therefore sought to study the effect of metabolic regulation on inflammatory responses by modulating the expression of mitochondrial uncoupling proteins (UCPs), a superfamily of mitochondrial anion carrier proteins with previously identified roles in metabolic regulation (17–20). UCPs uncouple oxidative phosjci.org Volume 125 Number 2 February 2015

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Figure 1. Deficiency of UCP2 protects against CLP-induced mortality in vivo. (A) Immunoblot analysis of UCP2 and voltage-dependent anion channel (VDAC) in lung tissues from wild-type mice 8 and 24 hours after CLP or sham laparotomy. β-Actin served as the standard. *P < 0.05, ANOVA. (B) ELISA assay for IL-1β, IL-18, and TNF in the sera and (C) in the lung tissues from Ucp2+/+ or Ucp2–/– mice 24 hours after CLP or sham laparotomy. For both Ucp2+/+ and Ucp2–/– mice, sham, n = 2, and CLP, n = 8. *P < 0.05, **P < 0.01, ANOVA. (D) Organ dysfunction of CLP was determined in Ucp2+/+ or Ucp2–/– mice 24 hours after CLP or sham laparotomy. Organ dysfunction biomarkers, including creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), were measured in sera. For both Ucp2+/+ and Ucp2–/– mice, sham, n = 2, and CLP, n = 8. *P < 0.05, ANOVA. (E) Survival curve of CLP was determined in Ucp2+/+ or Ucp2–/– mice. Ucp2+/+, n = 16; Ucp2–/–, n = 29. ***P < 0.0055, log-rank test. (F) Box plots comparing measures of UCP2 mRNA levels in wholeblood samples of sepsis (n = 30) and control (n = 19) patients. The UCP2 mRNA levels are presented as median value (black line), interquartile range (box), and minimum and maximum of all data (whiskers). *P < 0.05, ANOVA.

phorylation from ATP synthesis with energy dissipated as heat, a process referred to as the mitochondrial proton leak (17–20). Of these, UCP1 was originally identified as a protein responsible for thermogenesis in brown adipose tissue; whereas UCP2, its cellular homolog, with weak uncoupling activity, appears predominantly in cells of the innate immune system (17, 18). Although the physiological function of UCP2 remains incompletely understood, a role for UCP2 in regulating cellular energy homeostasis and glucose metabolism has been demonstrated in various cell types (21–29). Modulation of the glycolytic phenotype by UCP2 has been demon666

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strated in various cells in which UCP2 is highly expressed (23–28). Moreover, UCP2 affects the metabolic response to fasting in mice by regulating the expression of lipid regulatory enzymes and transcription factors (29). These observations suggest that UCP2 exerts a central role in cellular metabolism. Among the metabolic processes potentially influenced by mitochondrial dysfunction, de novo fatty acid synthesis is tightly regulated by key enzymes, including fatty acid synthase (FASN) (30). FASN and related enzymes are found upregulated in many cancer cells and associated with oncogenic progression (31–34).



Research article Figure 2. Deficiency of UCP2 inhibits lipid production through glucose uptake in vivo. (A) TG production in the lungs, livers, and sera from LysM-Cre/Ucp2+/+ or LysM-Cre/Ucp2fl/fl mice after injection of LPS for 4 hours (10 mg/kg, i.p.). n = 3 per group, *P < 0.05, ANOVA. (B) Transverse SUV of PET/CT imaging using 18 F-FDG of lungs from LysM-Cre/Ucp2+/+ or LysM-Cre/Ucp2fl/fl mice after injection of LPS for 4 hours (10 mg/kg, i.p.). Representative image of 3 independent experiments. Original magnification, ×2. (C) Quantification of transverse SUV in PET/CT imaging from B. n = 3 per group, **P < 0.01, ANOVA. (D) Ex vivo γ-ray counting using 18F-FDG of lungs and livers from LysM-Cre/Ucp2+/+ or LysM-Cre/Ucp2fl/fl mice after injection of LPS (10 mg/kg, i.p.) for 4 hours. n = 3 per group, **P < 0.01 ANOVA. (E) Luminex assay and ELISA assay for IL-1β, IL-18, and TNF secretion in the serum from B and D. n = 6 per group, *P < 0.05, ANOVA.

Activation of FASN can promote glucose-dependent fatty acid synthesis, illustrating that glucose utilization is coregulated with lipogenesis (30, 33, 35). Recent studies suggest that cellular fatty acid synthesis (e.g., synthesis of triglycerides [TGs] and cholesteryl esters) is activated during inflammation (36–38). Fatty acid synthesis has also been implicated in the activation of dendritic cells (39) and the differentiation of B lymphocytes and human monocytes (40, 41). Obesityinduced danger signals, such as free fatty acids, can activate the NLRP3 inflammasome and the downstream production of IL-1β in adipose tissue of patients with type 2 diabetes and in mice fed with a high-fat diet (42, 43). The role of cellular fatty acid synthesis in macrophages during inflammation currently remains unclear. We hypo thesized that upregulation of FASNdependent lipid synthesis by UCP2 regulates inflammation in sepsis through NLRP3 inflammasome activation. In the current study, we show that UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis and that UCP2 expression was increased in human sepsis. Consistent with these results, UCP2-deficient mice displayed reduced lipid synthesis and production of IL-1β and IL-18 in vivo and in vitro. Importantly, we demonjci.org Volume 125 Number 2 February 2015

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Figure 3. UCP2 regulates NLRP3-mediated caspase-1 activation by NLRP3 and IL-1β induction in macrophages. (A) Immunoblot analysis for caspase-1 and IL-1β of cell lysates from Ucp2+/+ or Ucp2–/– BMDMs treated with LPS (500 ng/ml) for 4 hours, followed by incubation with ATP (2 mM) for 30 minutes. β-Actin served as the standard. (B) Luminex assay and ELISA assay for IL-1β, IL-18, and TNF secretion in the media from A. **P < 0.01, ANOVA. (C) ELISA assay for IL-1β, IL-18, and TNF secretion in the supernatants from Ucp2+/+ or Ucp2–/– BMDMs treated with LPS (500 ng/ml) for 4 hours, followed by incubation with ATP (2 mM) for 30 minutes or silica (200 mg/ml) for 10 hours. **P < 0.01, ANOVA. (D) Immunoblot analysis for Myc-tagged UCP2 and caspase-1 of cell lysates from mouse J774A.1 macrophages transfected with control vector or Myc-tagged UCP2 expression vector, treated with LPS (500 ng/ml) for 4 hours, followed by followed by incubation with ATP (5 mM) for 30 minutes. β-Actin served as the standard. (E) ELISA assay for IL-1β, IL-18, and TNF secretion in the supernatants from mouse J774A.1 macrophages transfected with control vector or Myc-tagged UCP2 expression vector, treated with LPS (500 ng/ml) for 4 hours, followed by incubation with ATP (5 mM) for 30 minutes. *P < 0.05, ANOVA.

strated that UCP2 regulates NLRP3 inflammasome–mediated caspase-1 activation by upregulating FASN-dependent lipid synthesis in macrophages. The pharmacologic and genetic inhibition of FASN suppressed NLRP3-mediated caspase-1 activation by downregulation of NLRP3 and IL-1β expression. Our results suggest that UCP2 is a propathogenic mediator in sepsis, whereby UCP2 regulates NLRP3-mediated caspase-1 activation through lipid synthesis (see Supplemental Figure 1 for a summary of the overall study; supplemental material available online with this article; doi:10.1172/JCI78253DS1).

Results

UCP2 promotes mortality in experimental sepsis and is upregulated in human sepsis. We first investigated the functional role of UCP2 in the inflammatory response using cecal ligation and puncture (CLP), a clinically relevant murine model of polymicrobial sepsis. The expression of UCP2 was significantly increased in the lungs of mice at 8 hours after CLP relative to that in sham-operated control mice and remained elevated for up to 24 hours after CLP. In contrast, the expression of voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, was not changed 668


(Figure 1A). To evaluate the role of UCP2 in the inflammatory response to CLP, we analyzed the serum levels of the proinflammatory cytokines IL-1β, IL-18, and TNF in Ucp2+/+ and Ucp2–/– mice 24 hours after CLP relative to those in the corresponding control mice subjected to sham surgery. The serum levels of IL-1β, IL-18, and TNF were markedly elevated in Ucp2+/+ mice after CLP compared with those in sham-operated control mice. In contrast, the serum levels of IL-1β and IL-18 were significantly decreased in Ucp2–/– mice after CLP relative to those in Ucp2+/+ mice, whereas TNF levels were comparable between Ucp2+/+ and Ucp2–/– mice (Figure 1B). Similar to serum levels, the levels of IL-1β and IL-18 in lung tissues after CLP were significantly decreased in Ucp2–/– mice compared with those in Ucp2+/+ mice, whereas TNF levels were comparable between Ucp2+/+ and Ucp2–/– mice (Figure 1C). We also analyzed organ dysfunction by measurement of biochemical indicators of organ function in the serum. We observed that the levels of creatinine (an indicator of renal dysfunction) and aspartate aminotransferase and alanine aminotransferase (indicators of hepatic dysfunction) were increased in Ucp2+/+ mice after CLP compared with those in sham-operated control mice. Consistent with cytokine levels, Ucp2–/– mice displayed reduced organ



Research article

cantly decreased in LysM-Cre/Ucp2fl/fl mice relative to those in LysM-Cre/Ucp2+/+ mice (Figure 2A). Next, to confirm that LPSinduced TG synthesis was associated with glucose utilization, we measured glucose uptake using 18F-fluoro-2-deoxyglucose (18F-FDG) in mice treated with LPS. The uptake of 18F-FDG in mouse organs was measured using positron emission tomography/computerized tomography (PET/ CT) imaging and ex vivo γ-ray counting in tissue sections of LysM-Cre/Ucp2+/+ and LysM-Cre/Ucp2fl/fl mice. The uptake of 18F-FDG in mouse organs was imaged using a micro-PET scanner for 20 minutes, and then the anatomical localization of the lung and liver was imaged using a CT scanner. The analysis of 18F-FDG uptake was +/+ –/– Figure 4. UCP2 regulates lipid synthesis via FASN in macrophages. (A) TGs in Ucp2 or Ucp2 performed using a combination of wholeBMDMs treated with LPS (500 ng/ml) for 4 hours. *P < 0.05, ANOVA. (B) 18F-FDG uptake and PEP and body CT images and standard uptake vallactate production in Ucp2+/+ or Ucp2–/– BMDMs treated with LPS (500 ng/ml) for 4 hours. *P < 0.05, ues (SUVs) from PET imaging. Consistent ANOVA. (C) Citrate production in Ucp2+/+ or Ucp2–/– BMDMs treated with LPS (500 ng/ml) for 4 hours. with the observed increases in TG synthe+/+ *P < 0.05, ANOVA. (D) Immunoblot analysis for FASN, ACACA, and ACLY in cell lysates from Ucp2 or sis, LPS treatment increased the uptake of Ucp2–/– BMDMs treated with LPS (500 ng/ml) for 4 hours. β-Actin served as the standard. 18 F-FDG in LysM-Cre/Ucp2+/+ mice 4 hours after LPS injection, as detected in transdysfunction relative to Ucp2+/+ mice (Figure 1D). Importantly, we verse PET/CT imaging of the lungs. In contrast, the lung uptake of 18F-FDG in LysM-Cre/Ucp2fl/fl mice was decreased compared observed that Ucp2–/– mice were more resistant to CLP-induced +/+ +/+ mortality than Ucp2 mice. At 7 days after CLP, 75% of Ucp2 with that in LysM-Cre/Ucp2+/+ mice (Figure 2, B and C). In cor–/– mice had died, whereas 70% of Ucp2 mice survived longer than onal PET/CT imaging, the uptake of 18F-FDG was reduced in 12 days (Figure 1E). These results demonstrate improved survival the lungs and livers in LysM-Cre/Ucp2fl/fl mice relative to that in –/– of Ucp2 mice in the CLP model. Next, to correlate our findings in LysM-Cre/Ucp2+/+ mice after LPS treatment (Supplemental Figmice to patients with sepsis, we analyzed the mRNA level of UCP2 ure 2). To confirm the results of PET/CT imaging, we measured in leukocytes from patients admitted to the MICU. We observed the uptake of 18F-FDG in tissue sections using ex vivo γ-ray countthat UCP2 mRNA levels of leukocytes from the whole blood of ing. Consistent with results observed in PET/CT imaging, ex vivo patients with sepsis were significantly increased (P = 0.029) comγ-ray counting revealed increased uptake of 18F-FDG in the lungs pared with those of control patients admitted to the MICU without and livers of LysM-Cre/Ucp2+/+ mice after LPS treatment (Figure sepsis (see Methods for selection criteria, Figure 1F and Supple2D). The levels of glucose uptake in LysM-Cre/Ucp2fl/fl mice were mental Table 1). Taken together, these results suggest that UCP2 is decreased in the lungs and livers after LPS treatment compared a critical mediator in the pathogenesis of sepsis. with those in LysM-Cre/Ucp2+/+ mice (Figure 2D). These results UCP2 deficiency suppresses lipid synthesis and cytokine producindicate that UCP2 regulates lipid synthesis, which is correlated tion in vivo. The accumulation of lipid metabolites has been assowith glucose utilization in tissues in response to LPS challenge ciated with systemic inflammation in mice and humans (37, 38). in vivo. Next, we investigated whether lipid synthesis was assoMoreover, alterations in glucose metabolism, including glucose ciated with serum levels of IL-1β and IL-18 in LysM-Cre/Ucp2+/+ consumption and glycolysis, have been linked to cytokine producmice. Consistent with reduced TG levels and glucose utilization, tion in immune cells (7, 9, 39). We investigated whether UCP2 can the serum levels of IL-1β and IL-18 were significantly suppressed act as a critical regulator of cellular metabolism as it relates to the in LysM-Cre/Ucp2fl/fl mice relative to those in LysM-Cre/Ucp2+/+ activation of inflammation. Since UCP2 functions in cellular metmice, whereas TNF production was unchanged (Figure 2E). abolic regulation (25–28), we investigated whether UCP2 can regThese results suggest that UCP2 regulates de novo lipid synthesis, ulate lipid synthesis in response to LPS challenge in vivo. We first which was associated with glucose utilization and the production measured the levels of TGs, a final product in the lipid synthesis of IL-1β and IL-18 in vivo. pathway, in the lung and liver tissues of mice with a myeloid cell– UCP2 regulates NLRP3 expression and caspase-1 activation specific deletion of UCP2 (LysM-Cre/Ucp2fl/fl mice) in response in macrophages. UCP2 deficiency and corresponding metabolic changes were associated with altered regulation of inflammasomto LPS challenge. LPS treatment increased TG levels in the lungs e-associated cytokines in vivo. Therefore, we investigated whether and livers of LysM-Cre/Ucp2+/+ mice (Figure 2A). Consistent with UCP2 regulates the production of IL-1β and IL-18 in macrophages elevated TG levels in tissues, LPS treatment also increased TG through NLRP3-dependent caspase-1 activation. We used an levels in the sera of LysM-Cre/Ucp2+/+ mice (Figure 2A). In conestablished model of NLRP3 inflammasome–mediated caspase-1 trast, the levels of TGs in the lungs, livers, and sera were signifijci.org Volume 125 Number 2 February 2015

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Figure 5. FASN regulates NLRP3 and IL-1β expression in macrophages. (A) Immunoblot analysis for FASN, NLRP3, and IL-1β in cell lysates from wild-type mouse peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA (Control shRNA) or 3 independent shRNA for FASN (shFASN #1, #2, and #3) and stimulated with LPS (500 ng/ml) for 4 hours. β-Actin served as the standard. (B) TG production and (C) PEP, citrate, and lactate production in wild-type mouse peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA or 3 independent shRNA for FASN and stimulated with LPS (500 ng/ml) for 4 hours. *P < 0.05, ANOVA.

activation in vitro, the stimulation of LPS-primed macrophages with ATP treatment. We analyzed caspase-1 activation in bone marrow–derived macrophages (BMDMs) isolated from Ucp2–/– mice or corresponding wild-type (Ucp2+/+) mice. Ucp2–/– BMDMs displayed reduced expression of cleaved caspase-1 and cleaved IL-1β in response to LPS and ATP, relative to Ucp2+/+ BMDMs (Figure 3A). Ucp2–/– BMDMs displayed reduced protein induction of pro–IL-1β in response to LPS. Importantly, Ucp2–/– BMDMs also displayed reduced protein expression of NLRP3 in response to LPS compared with Ucp2+/+ BMDMs. Consistent with protein levels, UCP2 deficiency significantly reduced Nlrp3 gene expression (Supplemental Figure 3). In contrast to NLRP3, the expression of ASC and pro–caspase-1 was unchanged in Ucp2–/– BMDMs (Figure 3A). Moreover, IL-1β and IL-18 secretion was significantly decreased in Ucp2–/– BMDMs relative to that in Ucp2+/+ BMDMs, while TNF secretion was comparable in both Ucp2+/+ and Ucp2–/– BMDMs (Figure 3B). Additionally, we tested the role of UCP2 in modulating inflammasome responses to other stimuli. Ucp2–/– BMDMs displayed reduced IL-1β and IL-18 secretion in response to treatment with silica, an alternative NLRP3 inflammasome activator, relative to Ucp2+/+ BMDMs, while the secretion of TNF was comparable in both Ucp2+/+ and Ucp2–/– BMDMs (Figure 3C). Next, we overexpressed UCP2 in J774A.1 macrophages and validated the expression levels by Western immunoblot analysis (Figure 3D). Overexpression of UCP2 in J774A.1 macrophages increased caspase-1 activation in response to LPS and ATP compared with control-transfected cells (Figure 3D). Moreover, overexpression of UCP2 increased IL-1β and IL-18 secretion in response to LPS and ATP, while the secretion of TNF was not changed (Figure 3E). Next, we analyzed whether UCP2 can regulate caspase-1 activation in response to LPS and poly(dA:dT) treatment, which activates the absent in melanoma 2 (AIM2) inflammasome pathway. Unlike NLRP3-mediated cas670


pase-1 activation in Ucp2–/– BMDMs, the expression of AIM2 and cleaved caspase-1 was unchanged in Ucp2–/– and Ucp2+/+ BMDMs in response to LPS and poly(dA:dT) treatment (Supplemental Figure 4), suggesting that the AIM2 inflammasome pathway is not regulated by UCP2. Overall, our results suggest that UCP2 regulates NLRP3-mediated caspase-1 activation as well as the production of NLRP3 and pro–IL-1β in macrophages. UCP2 regulates glucose utilization and de novo lipid synthesis in macrophages. UCP2 deficiency was associated with metabolic changes in vivo, including reduced glucose utilization and TG synthesis (Figure 2). De novo fatty acid synthesis is known to require glucose (30, 32–34). We next evaluated the role of UCP2 in de novo lipid synthesis and associated changes in glucose metabolism in vitro. We first measured TG synthesis in BMDMs isolated from Ucp2–/– or Ucp2+/+ mice. Similar to reduced TG levels observed in the lungs, livers, and sera of Ucp2–/– mice (Figure 2A), Ucp2–/– BMDMs displayed less TG synthesis in response to LPS relative to that of Ucp2+/+ BMDMs (Figure 4A). Next, to examine whether LPS-induced TG synthesis was associated with glucose utilization in vitro, we measured glucose utilization using 18F-FDG and the production of glycolytic metabolites in Ucp2–/– BMDMs in response to LPS. Consistent with reduced TG synthesis, Ucp2–/– BMDMs displayed less 18F-FDG uptake compared with Ucp2+/+ BMDMs (Figure 4B). Furthermore, the production of phosphoenolpyruvate (PEP) and lactate (Figure 4B) and citrate (Figure 4C), representative metabolites in glycolysis and the tricarboxylic acid cycle, respectively, was significantly suppressed in Ucp2–/– BMDMs relative to that in Ucp2+/+ BMDMs. Similar to results obtained in BMDMs, TG synthesis (Supplemental Figure 5A) and the production of PEP, lactate, and citrate (Supplemental Figure 5, B and C) were significantly suppressed in Ucp2–/– peritoneal macrophages in response to LPS compared with Ucp2+/+ peritoneal macrophages.



Research article Figure 6. FASN regulates NLRP3 and IL-1β expression through AKT activation in macrophages. (A) Immunoblot analysis for NLRP3 and IL-1β and (B) measurement of TGs from wild-type BMDMs pretreated with C75 (0, 10, 20, and 50 μM) as well as (C) measurement of PEP, citrate, and lactate production from wild-type BMDMs pretreated with C75 (20 μM) for 2 hours before stimulation with LPS (500 ng/ml, 4 hours). β-Actin served as the standard. **P < 0.01, ANOVA. (D) Immunoblot analysis of AKT Ser473 phosphorylation in wild-type BMDMs pretreated with C75 (20 μM) for 2 hours before stimulation with LPS (500 ng/ml; 0, 10, and 20 minutes). Total AKT served as the standard. (E) Immunoblot analysis of AKT Ser473 phosphorylation in cell lysates from wild-type BMDMs pretreated with C75 (0, 10, 20, and 50 μM) for 2 hours before stimulation with LPS (500 ng/ml, 4 hours). Total AKT served as the standard. (F) Immunoblot analysis of AKT Ser473 phosphorylation in cell lysates from wildtype peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA or 3 independent shRNA for FASN after stimulation with LPS (500 ng/ml, 4 hours). Total AKT served as the standard. (G) Immunoblot analysis for NLRP3, IL-1β, and AKT Ser473 phosphorylation in cell lysates from wild-type BMDMs pretreated with BX795 (10 μM) for 1 hour before stimulation with LPS (500 ng/ml, 4 hours). Total AKT served as the standard.

Although glucose utilization and production of glycolytic metabolites was suppressed in Ucp2–/– BMDMs compared with that in Ucp2+/+ BMDMs, the expression of the glucose transporter-1 (GLUT1) was comparable between Ucp2–/– and Ucp2+/+ BMDMs (Supplemental Figure 6). These results suggest that UCP2 regulates de novo lipid synthesis in macrophages through modulation of glycolysis. UCP2 regulates lipid synthesis through FASN in macrophages. We next investigated the molecular mechanism by which lipid synthesis is regulated by UCP2 in macrophages. Since UCP2 can regulate the lipid metabolic response to fasting in mice through the expression of key lipid regulatory enzymes (29, 33), we analyzed whether UCP2 regulates lipogenic enzymes in the lipid synthesis pathway in macrophages. Lipid synthesis is regulated by the expression of key lipogenic enzymes, including FASN, acetyl-CoA carboxylase (ACACA), and ATP citrate lyase (ACLY). We analyzed the expression of FASN, ACACA, and ACLY in Ucp2–/– BMDMs in response to LPS. The expression of FASN, ACACA, and ACLY was increased in Ucp2+/+ BMDMs in response to LPS (Figure 4D). In contrast, Ucp2–/– BMDMs displayed markedly reduced FASN induction compared with Ucp2+/+ BMDMs, while the expression of ACACA was partially reduced and the expression of ACLY was unchanged (Figure 4D). Consistent with results obtained in BMDMs, the induction of FASN was markedly suppressed in Ucp2–/– peritoneal macro-

phages in response to LPS relative to Ucp2+/+ peritoneal macrophages (Supplemental Figure 5D). These results suggest that UCP2 regulates lipid synthesis via FASN in macrophages. FASN regulates NLRP3 expression in macrophages. FASN, a critical lipogenic enzyme that promotes glucose-dependent fatty acid synthesis (30, 33, 35), was preferentially downregulated in Ucp2–/– BMDMs and peritoneal macrophages, in association with inhibition of the inflammatory response. We therefore sought to investigate the role of FASN in NLRP3 regulation in macrophages. We analyzed whether deficiency of FASN could suppress NLRP3 expression in response to proinflammatory stimuli. Genetic deletion of FASN results in early embryonic lethality (44), precluding the use of Fasn–/– mice in the current study. Hence, we used 3 independent shRNA lentiviral constructs against mouse Fasn to knockdown FASN in wild-type peritoneal macrophages. The protein expression of NLRP3 in response to LPS stimulation was suppressed by Fasn shRNA transduction relative to control shRNA transduction (Figure 5A). Moreover, knockdown of FASN suppressed the protein expression of pro–IL-1β (Figure 5A). Consistent with the results observed in Ucp2–/– BMDMs, knockdown of FASN significantly suppressed the production of TGs compared with control (Figure 5B). Similarly, knockdown of FASN reduced the production of PEP, citrate, and lactate in response to LPS relative to control, as previously reported (ref. 31 and Figure 5C). Consistent with the results in peritoneal macrophages, the protein jci.org Volume 125 Number 2 February 2015

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Figure 7. Deficiency of FASN suppresses the transcription of Nlrp3 and Il1b genes through p38 MAPK in macrophages. (A) Quantitative PCR analysis for Nlrp3, Il1b, Il18, Tnf, and Il12 gene expression in wild-type mouse peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA or shRNA for FASN (shFASN) and stimulated with LPS (500 ng/ml) for 4 hours. **P < 0.01, ANOVA. (B) Immunoblot analysis for activation of p38 MAPK, ERK, and JNK in cell lysates from wild-type mouse peritoneal macrophages transduced with control shRNA or shRNA for FASN and stimulated with LPS (500 ng/ml) for 0, 10, 20, and 30 minutes.

expression of NLRP3 and pro–IL-1β in response to LPS stimulation was suppressed by Fasn-specific siRNA transfection relative to control siRNA transfection in J774A.1 macrophages. The production of TG, PEP, citrate, and lactate in response to LPS was reduced by FASN knockdown in J774A.1 macrophages (Supplemental Figure 7). These results support that FASN promotes glucose-dependent lipid synthesis in macrophages. Taken together, these results suggest that FASN regulates NLRP3 expression in macrophages. FASN activity regulates NLRP3 expression via AKT activation in macrophages. To validate the functional role of FASN in the regulation of NLRP3 expression, we examined whether inhibition of FASN activity by C75, a selective inhibitor of FASN (31, 33), could inhibit the expression of NLRP3 in macrophages in response to LPS. We analyzed the protein induction of NLRP3 in wild-type BMDMs treated with C75. Pretreatment with C75 dose dependently suppressed the protein expression of NLRP3 and pro–IL-1β in wildtype BMDMs in response to LPS (Figure 6A). Consistent with FASN downregulation, C75 dose dependently reduced TG synthesis in wild-type BMDMs (Figure 6B). Similarly, C75 suppressed the production of PEP, citrate, and lactate in response to LPS relative to vehicle control (Figure 6C). Furthermore, C75 suppressed NLRP3 and pro–IL-1β expression in wild-type BMDMs after stimulation with alternate TLR ligands (Supplemental Figure 8A). Consistent with the results in wild-type BMDMs, C75 dose dependently suppressed the protein expression of NLRP3 and pro–IL-1β in wildtype peritoneal macrophages in response to LPS compared with vehicle control (Supplemental Figure 9A). Consistent with C75 treatment, cerulenin, another selective inhibitor of FASN (31, 33), dose dependently suppressed the protein expression of NLRP3 and pro–IL-1β in wild-type peritoneal macrophages in response to LPS compared with vehicle control (Supplemental Figure 9B). Similar to C75 treatment, cerulenin significantly suppressed TG synthesis and the production of PEP, citrate, and lactate in response to LPS relative to vehicle control (Supplemental Figure 9, C and D). 672


Since FASN activity is known to modulate AKT activation in cancer cells (45), we investigated whether inhibition of FASN activity by C75 can suppress NLRP3 expression through AKT in macrophages. First, we analyzed the phosphorylation of AKT on Ser473 in wild-type BMDMs treated with C75 prior to LPS stimulation. C75 suppressed the phosphorylation of AKT on Ser473 in response to LPS treatment, relative to vehicle control (Figure 6D). Similarly, C75 suppressed the phosphorylation of AKT on Ser473 in a dose-dependent manner (Figure 6E). Moreover, TLR agonist-induced AKT activation was suppressed by C75 compared with vehicle control (Supplemental Figure 8B). Consistent with inhibition of AKT activation by C75, knockdown of FASN by Fasn shRNA suppressed AKT activation relative to control shRNA transduction in wild-type peritoneal macrophages (Figure 6F). Similarly, cerulenin suppressed the phosphorylation of AKT on Ser473 in a dose-dependent manner (Supplemental Figure 9E). Consistent with Fasn shRNA transduction, knockdown of FASN by Fasn-specific siRNA suppressed AKT activation relative to control siRNA transfection in J774A.1 macrophages (Supplemental Figure 9F). Next, to evaluate the role of AKT activation in the regulation of NLRP3 expression, we examined whether inhibition of AKT by BX795, a selective AKT inhibitor, could suppress NLRP3 expression in macrophages in response to LPS (Figure 6G). Pretreatment of BMDMs with BX795 prior to LPS stimulation resulted in reduced NLRP3 expression in response to LPS. Moreover, BX795 also suppressed the protein expression of pro–IL-1β and phosphorylation of AKT on Ser473 in response to LPS (Figure 6G). These results suggest that FASN regulates NLRP3 expression via AKT activation. FASN regulates the transcription of NLRP3 and IL-1β in macrophages through p38 MAPK. We investigated the molecular mechanism by which the induction of NLRP3 and pro–IL-1β is regulated by FASN in macrophages. Since AKT signaling is involved in the regulation of NLRP3 by FASN (Figure 6), we examined whether



Research article

p38α MAPK in wild-type peritoneal macrophages. The activation of p38 MAPK in response to LPS and p38 MAPK expression was markedly suppressed by p38α shRNA compared with control shRNA transduction in wild-type peritoneal macrophages (Figure 8A). Consistent with knockdown of FASN, transduction with p38α shRNA markedly reduced the protein expression of NLRP3 and pro–IL-1β relative to control shRNA Figure 8. p38 MAPK regulates NLRP3 and IL-1β expression in macrophages. (A) Immunoblot analysis for activation of transduction (Figure 8B). p38 MAPK in cell lysates from wild-type mouse peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA or 2 independent shRNA for p38 (shp38 #1 and #2) and stimulated with LPS (500 ng/ml) for 0, 10, or 20 Next, to examine the role minutes. β-Actin served as the standard. (B) Immunoblot analysis for expression of NLRP3 and IL-1β in cell lysates from of p38 MAPK in the gene wild-type mouse peritoneal macrophages transduced with lentiviruses expressing control shRNA or 2 independent shRNA expression of NLRP3 for p38 and stimulated with LPS (500 ng/ml) for 4 hours. β-Actin served as the standard. (C) Quantitative PCR analysis for and pro–IL-1β, we anaNlrp3, Il1b, and Il18 gene expression in wild-type mouse peritoneal macrophages transduced with lentiviruses expressing lyzed the mRNA levels control shRNA or shRNA for p38 (shp38) and stimulated with LPS (500 ng/ml) for 4 hours. **P < 0.01, ANOVA. of Nlrp3, Il1b, and Il18 in wild-type peritoneal deficiency of FASN can regulate the expression of NLRP3 and macrophages. Similar to protein expression, knockdown of p38 pro–IL-1β through AKT-dependent transcription in macrophages. MAPK markedly reduced Nlrp3, Il1b, and Il18 gene expression in We first analyzed the mRNA levels of Nlrp3, Il1b, Il18, Tnf, and Il12 response to LPS compared with control shRNA transduction (Figin wild-type peritoneal macrophages in response to LPS. Transure 8C). Unlike Il1b and Il18 gene expression, gene expression of duction with Fasn shRNA markedly reduced Nlrp3, Il1b, and Il18 Tnf was significantly reduced by knockdown of p38 MAPK during gene expression in response to LPS compared with control shRNA the early response to LPS stimulation, whereas it was comparable transduction, while gene expression of Tnf and Il12 was unchanged with control conditions at 4 hours after LPS stimulation (Supple(Figure 7A). Similar to transduction with Fasn shRNA, knockdown mental Figure 12, A and B). In contrast, gene expression of Il12 was of FASN by Fasn-specific siRNA markedly reduced Nlrp3, Il1b, unchanged by knockdown of p38 MAPK (Supplemental Figure and Il18 gene expression in response to LPS compared with con12B). Moreover, we evaluated the role of LPS-induced p38 MAPK trol siRNA transfection in J774A.1 macrophages, while Tnf gene activation in the transcription of NLRP3, pro–IL-1β, and other expression was unchanged (Supplemental Figure 10A). cytokine genes. We analyzed the mRNA levels of Nlrp3, Il1b, Il18, Next, to examine whether deficiency of FASN can affect sigand Tnf in wild-type BMDMs using LPS in the absence or presence naling pathways downstream of AKT, we analyzed the effects of SB203580, a potent inhibitor of p38 MAPK activation; U0126, a of FASN knockdown on the activation of MAPKs, including p38 potent inhibitor of ERK activation; and SP600125, a potent inhibMAPK, ERK, and JNK in wild-type peritoneal macrophages. Fasn itor of JNK activation. SB203580 significantly suppressed Nlrp3, shRNA transduction suppressed the activation of p38 MAPK Il1b, and Il18 gene expression in response to LPS compared with and ERK relative to control shRNA, while the activation of JNK vehicle control, while gene expression of Tnf was unchanged. In was unchanged (Figure 7B). Similarly, knockdown of FASN by contrast, U0126 partially suppressed Il1b gene expression relative Fasn-specific siRNA markedly suppressed the activation of p38 to vehicle control, while Nlrp3, Il18, and Tnf mRNA levels were MAPK compared with control siRNA transfection in J774A.1 unchanged. SP600125 did not affect the transcription of Nlrp3, macrophages (Supplemental Figure 10B). Consistent with FASN Il1b, Il18, and Tnf genes in response to LPS compared with vehiknockdown, inhibition of AKT by BX795 treatment suppressed cle control (Figure 9A). Consistent with mRNA levels, SB203580 the activation of p38 MAPK compared with vehicle control (Supmarkedly suppressed the protein expression of NLRP3 and pro– plemental Figure 11). Since the activation of p38 MAPK was markIL-1β (Figure 9B). In contrast, U0126 partially reduced protein edly suppressed by FASN knockdown (Figure 7B), we analyzed the expression of pro–IL-1β, while that of NLRP3 was comparable. role of p38 MAPK in the transcription of NLRP3 and pro–IL-1β in SP600125 did not inhibit the protein expression of NLRP3 and wild-type peritoneal macrophages. pro–IL-1β (Figure 9B). These results suggest that FASN regulates Since the p38α isoform, among the p38 MAPK isoforms, is the the transcription of NLRP3 through the p38 MAPK signaling pathmost abundant in inflammatory cells (46), we used 2 independent way and FASN regulates the transcription of pro–IL-1β through the shRNA lentiviral constructs against mouse p38α to knockdown p38 MAPK and ERK1/2 MAPK signaling pathway in macrophages. jci.org Volume 125 Number 2 February 2015

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The Journal of Clinical Investigation Figure 9. Inhibition of p38 MAPK activation suppresses the transcription of Nlrp3 and Il1b genes in macrophages. (A) Quantitative PCR analysis for Nlrp3, Il1b, Il18, and Tnf gene expression in cell lysates and (B) immunoblot analysis for expression of NLRP3 and IL-1β in cell lysates from wild-type BMDMs pretreated with SB203580 (10 μM), U0126 (10 μM), and SP600125 (10 μM) for 1 hour before stimulation with LPS (500 ng/ml) for 4 hours. *P < 0.05, **P < 0.01, ANOVA. β-Actin served as the standard.

FASN regulates caspase-1 activation and associated cytokine production in macrophages. Based on our results that FASN expression and activity are critical for NLRP3 expression, we therefore investigated the role of FASN in NLRP3-mediated caspase-1 activation in macrophages. Similar to the result observed in Ucp2–/– BMDMs, FASN knockdown by Fasn shRNA suppressed the expression of cleaved caspase-1 and cleaved IL-1β in wild-type peritoneal macrophages in response to LPS and ATP compared with control shRNA-transduced cells (Figure 10A). Moreover, FASN knockdown reduced the protein expression of NLRP3 and pro–IL-1β in response to LPS. In contrast to NLRP3, the expression of ASC and caspase-1 was unchanged in Fasn shRNA-transduced cells (Figure 10A). IL-1β and IL-18 secretion was also significantly decreased in Fasn shRNA-transduced cells relative to control cells, while TNF secretion was comparable between cells (Figure 10B and Supplemental Figure 14A). Similarly, FASN knockdown by Fasn-specific siRNA suppressed the expression of cleaved caspase-1 and cleaved IL-1β in mouse J774A.1 macrophages in response to LPS and ATP compared with control siRNA-transfected cells (Supplemental Figure 13A). Moreover, IL-1β and IL-18 secretion was also significantly decreased (Supplemental Figure 13B), while TNF secretion was comparable (Supplemental Figure 14B). Consistent with FASN knockdown, inhibition of FASN activity by C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation in response to LPS and ATP relative to vehicle treatment in wild-type peritoneal macrophages (Figure 10C). C75 and cerulenin also suppressed the protein induction of NLRP3 and pro–IL-1β in response to LPS, while the expression of ASC and caspase-1 was unchanged (Figure 10C). Moreover, IL-1β and IL-18 secretion was also significantly decreased by C75 and cerulenin treatment (Figure 10D). Similar to results in peritoneal macrophages, inhibition of FASN activity by C75 suppressed NLRP3-mediated caspase-1 activation in response to LPS and ATP and the protein induction of NLRP3 and pro–IL-1β 674


in response to LPS relative to vehicle control in wild-type BMDMs (Supplemental Figure 13C). Moreover, IL-1β and IL-18 secretion was also significantly decreased by C75 in response to LPS and ATP relative to vehicle control (Supplemental Figure 13D). Collectively, these results suggest that FASN is required for NLRP3 inflammasome– dependent caspase-1 activation as well as IL-1β and IL-18 secretion. Inhibition of FASN by C75 suppresses the production of IL-1β and IL-18 in vivo. Finally, we investigated the role of FASN activity in the production of IL-1β and IL-18 in vivo. We measured the sera levels of IL-1β and IL-18 in wild-type mice treated with C75 prior to LPS injection. C75 significantly reduced the sera levels of IL-1β and IL-18 (Figure 11A) and TGs (Figure 11B) in response to LPS, relative to vehicle control. Similarly, cerulenin also reduced the sera levels of IL-1β and IL-18 (Figure 11C) and TGs (Figure 11D) in response to LPS, relative to vehicle control. Consistently, LPS induced lipid accumulation in the liver was inhibited by C75 (Figure 11E). These results suggest that the activity of FASN is required for IL-1β and IL-18 production in vivo.

Discussion

In this study, we demonstrate the physiological relevance of the mitochondrial protein UCP2 in mediating inflammatory responses in two animal models of sepsis: the endotoxin shock model and the CLP model of polymicrobial sepsis. Furthermore, increased UCP2 mRNA expression was observed in the blood of human sepsis patients. We have shown that UCP2 acts as a critical regulator of glucose-dependent de novo lipid synthesis in vivo and in vitro. We describe a molecular mechanism whereby FASN, a critical lipogenic enzyme that converts glycolytic carbon flux into lipid biosynthesis, serves as a downstream target molecule of UCP2-dependent glucose regulation. We show that UCP2dependent lipid synthesis under the regulation of FASN is critical for both NLRP3-mediated caspase-1 activation and the regulation of NLRP3 and pro–IL-1β expression. Similar to observations in UCP2-deficient mice, genetic and pharmacologic inhibition of FASN suppressed NLRP3-mediated caspase-1 activation and the synthesis of NLRP3 and pro–IL-1β in vitro as well as IL-1β and IL-18 production in vivo. In the current study, we describe a molecular mechanism by which UCP2 and FASN regulate the NLRP3 inflammasome and associated caspase-1 activation through the regulation of lipid synthesis. Our results suggest that UCP2- and FASN-mediated lipid synthesis is linked to inflammatory responses. A summary of the proposed role of UCP2 and FASN in the regulation of metabolism and inflammation is shown in Figure 12 and Supplemental Figure 1. Among the known UCPs, UCP2 is highly expressed in immune cells from humans and mice and has been implicated in the pathogenesis of type 2 diabetes and obesity (18, 47). Recently, UCP2 has



Research article

Figure 10. FASN regulates NLRP3-mediated caspase-1 activation in macrophages. (A) Immunoblot analysis for caspase-1 and IL-1β of cell lysates from wild-type mouse peritoneal macrophages transduced with lentiviruses expressing nontarget shRNA or shRNA for FASN and stimulated with LPS (500 ng/ml) for 4 hours, followed by incubation with ATP (5 mM) for 30 minutes. β-Actin served as the standard. (B) ELISA assay for IL-1β and IL-18 in the media from A. **P < 0.01, ANOVA. (C) Immunoblot analysis for IL-1β and NLRP3 of cell lysates from wild-type mouse peritoneal macrophages pretreated with C75 (20 μM) and cerulenin (2 g/ml) for 2 hours before stimulation with LPS (500 ng/ml) for 4 hours, followed by incubation with ATP (5 mM) for 30 minutes. β-Actin served as the standard. (D) ELISA assay for IL-1β and IL-18 in the media from C. **P < 0.01, ANOVA.

also been shown to be involved in numerous pathophysiological conditions, including metabolic disorders, inflammation, ischemic shock, cancer, and aging. UCP2 decreases glucose-derived pyruvate oxidation in mitochondria and enhances aerobic glycolysis (23, 25, 26, 48). Metformin, a drug used in the treatment of type 2 diabetes, can induce UCP2 expression and increase glucose utilization in peripheral tissues (24). UCP2 mediates the shift from mitochondrial respiration to aerobic glycolysis (Warburg effect) as well as tumorigenic properties associated with cancer cells (25– 27). Furthermore, UCP2 can facilitate the transport of 4-carbon (C4) tricarboxylic acid cycle metabolites (e.g., malate, oxaloacetate) from the mitochondria to the cytosol, which supports a role for UCP2 in the reprograming of glucose utilization (28). These studies provide evidence that UCP2 has a significant regulatory role in cellular metabolism and bioenergetics. Our results show that UCP2 regulates cellular glucose utilization and glycolysis during inflammation. Furthermore, our data show that the UCP2-mediated glycolytic phenotype is regulated by FASN-dependent lipid synthesis. Ucp2–/– BMDMs displayed

reduced TG synthesis and production of glycolytic metabolites as the result of FASN downregulation. Consistent with the results observed in Ucp2–/– cells, inhibition of FASN also suppressed the production of glycolytic metabolites. Glucose utilization is regulated by activation of the glucose transporter or by changes in glycolysis (49). GLUT1, which is the predominant glucose transporter in macrophages, mediates high glucose uptake and the glycolytic phenotype in macrophages (50). Our results suggest that UCP2 affects glucose utilization independently of the regulation of GLUT1. Although the function of UCP2 in different tissue types remains controversial (51, 52), our results suggest that UCP2 has a significant impact on metabolic reprograming processes in macrophages. Furthermore, our human data suggest that UCP2 expression was increased in myeloid cells of patients with sepsis, compared with patients with systemic inflammatory response syndrome, suggesting that UCP2 was regulated in patients with systemic inflammation and infection. Our prior studies demonstrate that changes in plasma metabolic profiles, including changes jci.org Volume 125 Number 2 February 2015

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standard care (target glucose range, 180–200 mg/dl) (57). However, most of subsequent trials did not confirm such a benefit, while they also raised the concern of hypoglycemia as a risk factor of death (58). For years, NLRP3 has been exclusively studied as a critical component of the NLRP3 inflammasome, which promotes the cleavage of caspase-1. While previous reports suggest that activation of NF-κB by pattern recognition receptors such as TLR4 is necessary for NLRP3 expression (3), the precise molecular mechanism remains unclear. We show that FASN-dependent lipid synthesis acts as an upstream signal for NLRP3 expression. Consistent with these results, inhibition of fatty acid synthesis by FASN deficiency and inhibition by C75 suppressed NLRP3 expression involving AKT activation in macrophages. FASN is a multifunctional metabolic enzyme that catalyzes the terminal steps in the synthesis of long-chain saturated fatty acids (33). The production of fatty acids supports membrane synthesis in proliferating cells (33). Furthermore, FASN expression and activity are critical for AKT activation, which promotes cell survival and enhances tumor cell growth and invasiveness (45). We show here that FASN deficiency suppressed NLRP3 gene expression involving the inhibition of AKT activation in macrophages. We also demonstrate that inhibition of Figure 11. Inhibition of FASN activity suppresses production of IL-1β and IL-18 in vivo. (A) ELISA p38 MAPK activation as a downstream signal of assay for IL-1β and IL-18 and (B) TG assay in the sera of wild-type mice after injection of C75 AKT activation by FASN deficiency reduced Il1b (10 mg/kg, i.p.) or DMSO for 16 hours, followed by LPS (10 mg/kg) or PBS for 8 hours. n = 3 per and Il18 gene expression, consistent with a pregroup, *P < 0.05, **P < 0.01, ANOVA. (C) ELISA assay for IL-1β and IL-18 and (D) TG assay in the vious report (59). In summary, our data demonsera of wild-type mice after injection of cerulenin (5 mg/kg, i.p.) or DMSO for 16 hours, followed by LPS (10 mg/kg) or PBS for 8 hours. n = 3 per group, **P < 0.01, ANOVA. (E) Mice were injected strate that FASN regulates NLRP3 and pro–IL-1β with C75 (10 mg/kg, i.p.) or DMSO. After 16 hours, mice were injected with LPS (10 mg/kg, i.p.) or expression via AKT and downstream p38 MAPK PBS. Livers were harvested after an additional 8 hours. The image depicts Oil Red O (ORO) stainactivation. Although p38 MAPK suppression ing for neutral lipids and lipid droplet morphology in sections from frozen liver tissues. Scale bar: resulted in inhibition of pro–IL-1β expression, 100 μm; original magnification, ×40. n = 3 per group, **P < 0.01, ANOVA. we cannot exclude the partial involvement of the ERK1/2 MAPK signaling pathway. in glucose and lipid metabolites, are associated with mortality We also identify here a role of FASN-dependent lipid synthesis in patients with pneumonia and sepsis (13–15). Importantly, the in NLRP3-mediated caspase-1 activation. Fatty acids for cellular association of high levels of blood glucose and mortality in critmetabolism have two sources, exogenously derived (dietary) fatty ical care illness, including sepsis, has been described (53–55). acids and de novo synthesized fatty acids. Recently, it has been These observations suggest that cellular glycolysis is upregushown that exogenously derived fatty acids, such as palmitate, lated during septic conditions. While glycolysis is a vital metone of the most abundant fatty acids in plasma that is substantially abolic process for cellular homeostasis, its excess activation elevated following a high fat diet, induces NLRP3 inflammasome induced by pathological conditions such as infection may be activation (43). We showed that the inhibition of lipid synthesis clinically deleterious for septic patients. It is generally accepted by FASN knockdown, and by C75 treatment, suppressed NLRP3that hyperglycemia is associated with worse outcomes in nondimediated caspase-1 activation and NLRP3 gene expression, withabetic patients who are admitted to the intensive care unit (ICU), out changes in the expression of other components of the NLRP3 and therefore it should not been neglected (56). However, the inflammasome (i.e., ASC and pro–caspase-1). optimal target level of blood glucose in patients who are admitIn conclusion, our data suggest that UCP2 regulates NLRP3ted to the ICU is a highly debated topic. A single-center randommediated caspase-1 activation through lipid synthesis mediated ized controlled trial showed that a tight glucose control (target by FASN. Also, our data suggest that the regulation of AKT and glucose range, 80–110 mg/dl) is linked with better survival of downstream p38 MAPK activation by FASN are critical for NLRP3 patients who are admitted to the surgical ICU compared with expression and caspase-1 activation (Figure 12 and Supplemental 676




Research article Figure 12. Schematic of the proposed relationships between UCP2FASN–regulated lipid synthesis and NLRP3-mediated caspase-1 activation. Glu-6-P, glucose-6-phosphate; TCA, tricarboxylic acid.

Figure 1). Given the roles of UCP2 in metabolic changes triggered by the immune response, further investigation of UCP2 in the context of cellular metabolism may be useful for elucidating mechanisms of human diseases, including, not just metabolic diseases, but diseases in which inflammation plays a key role.

Methods

Mice. The Ucp2–/– mice have been described previously (60). The loxUCP2 mice were generated as described previously (60). The LysMCre knockin allele has a nuclear-localized Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene (Lyz2), which abolishes endogenous Lyz2 gene function and places NLS-Cre expression under the control of the endogenous Lyz2 promoter/enhancer elements. When crossed with a strain containing a loxP site–flanked sequence of interest, Cre-mediated recombination results in deletion of the targeted gene in the myeloid cell lineage, including monocytes, mature macrophages, and granulocytes. Macrophage-specific UCP2 deletion was accomplished by crossing loxUCP2 mice with LysM-Cre mice (The Jackson Laboratory). Mice were genotyped using standard PCR of tail DNA. Reagents. LPS (E. coli) (tlrl-pelps), LAM-MS (Mycobacterium smegmatis) (tlrl-lams), poly(I:C) (tlrl-pic), FLA-ST (Salmonella typhimurium) (tlrl-pstfa), CpG oligonucleotide (tlrl-1826), and SB203580 (tlrl-sb20) were from InvivoGen. C75 (C5490), Cerulenin (C2389), U0126 (U120), SP600125 (S5567), and ATP (A2383) were from SigmaAldrich. The following antibodies were used: polyclonal rabbit antiUCP2 for mouse UCP2 (662047, EMD Millipore), monoclonal mouse anti-NLRP3 for mouse NLRP3 (ALX-804-881-C100, Enzo Life Sciences), polyclonal rabbit anti-ASC for mouse ASC (AP07815PU-N, Acris Antibodies), polyclonal goat anti–IL-1β for mouse IL-1β (AF-401-NA; R&D Systems), Lipid Metabolism Antibody Sampler Kit for anti-lipogenic enzymes (8337, Cell Signaling Technology), polyclonal rabbit anti-GLUT1 (ab32551; Abcam), polyclonal rabbit anti–caspase-1 for mouse caspase-1 (SC-514; Santa Cruz Biotechnology), Phospho-AKT Pathway Antibody Sampler Kit for anti-AKT and anti–phospho-AKT (9916S, Cell Signaling Technology), monoclonal rabbit anti–Myc-Tag (2278, Cell Signaling Technology), polyclonal rabbit anti–p38 MAPK antibody for mouse p38 MAPK (9218, Cell Signaling Technology), polyclonal rabbit anti–p38 MAPK antibody for mouse p38 MAPK (9212, Cell Signaling Technology), monoclonal rabbit anti–phospho-p38 MAPK antibody (4511, Cell Signaling Tech-

nology), monoclonal rabbit anti–p44/42 MAPK (ERK1/2) antibody for mouse ERK1/2 (4695, Cell Signaling Technology), monoclonal rabbit anti–phospho-p44/42 MAPK (ERK1/2) antibody (4370, Cell Signaling Technology), monoclonal rabbit anti-SAPK/JNK antibody for mouse JNK (9258, Cell Signaling Technology), monoclonal rabbit anti–phospho-SAPK/JNK antibody (4668, Cell Signaling Technology), and monoclonal mouse anti–β-actin antibody (A5316, Sigma-Aldrich). Cell culture. BMDMs were prepared as described previously (3). Bone marrow collected from mouse femurs and tibias (male, 8–10 weeks old) was plated on sterile petri dishes and incubated for 7 days in DMEM medium (Invitrogen) containing 10% (vol/vol) heat-inactivated FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, and 25% (vol/vol) conditioned medium from mouse L929 fibroblasts (CCL-1, ATCC). Cells were incubated for 4 hours with LPS (500 ng/ml) and then were treated with ATP (2 mM) for 30 minutes as described previously. Peritoneal macrophages were prepared as described previously (61). Mice were injected with thioglycollate broth medium (1.5 ml, i.p.). After 4 days, peritoneal cells were collected from mice with cold PBS. Cells were incubated for 2 hours with DMEM medium containing 10% (vol/vol) heat-inactivated FBS, penicillin, and streptomycin in 6-well tissue culture plates. Nonadherent cells are removed by gently washing with PBS. After cells were cultured for overnight, cells were incubated for 4 hours with LPS (500 ng/ml) and then were treated with ATP (5 mM) for 30 minutes as described previously. Mouse J774A.1 macrophages (TIB-67, ATCC) were cultured in DMEM medium containing 10% (vol/vol) FBS, penicillin, and streptomycin. Immunoblot analysis. Cells or tissues were harvested and lysed in 2 × SDS loading buffer or NP40 Cell Lysis Buffer (FNN0021, Invitrogen) and then briefly sonicated. Lysates were centrifuged at 15,300 g for 10 minutes at 4°C, and the supernatants were obtained. The protein concentrations of the supernatants were determined using the Bradford assay (BioRad Laboratories). Proteins were subjected to NuPAGE 4–12% Bis-Tris gels (Invitrogen) and transferred to Protran nitrocellulose membranes (10600001, GE Healthcare Life Science). Cytokine analysis. Cell culture supernatants, lung tissues, and sera were measured for mouse IL-1β and IL-18 with ELISA (R&D Systems). Cell culture supernatants, lung tissues, and sera samples were measured for mouse tumor necrosis factor with ELISA (R&D Systems). Quantitative real-time PCR. Total RNA was isolated from cultured cells and tissues using the TRIzol Reagent (15596-018, Invitrogen) according to the manufacturer’s instructions. For the quantitative jci.org Volume 125 Number 2 February 2015

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RT-PCR, cDNA was synthesized from 4 μg of total RNA using random hexamers and SuperScript Reverse Transcriptase II (18064014, Invitrogen) according to the manufacturer’s instructions. A 10 μl mixture containing the diluted cDNA and a set of gene-specific primers was mixed with 10 μl of 2 x SYBR Green PCR Master Mix (4309155, Applied Biosystems) and then subjected to RT-PCR quantification using the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems). The following primers were used: mouse Il1b forward, 5′-ATGAGAGCATCCAGCTTCAA-3′, and reverse, 5′-TGAAGGAAAA GAAGGTGCTC-3′; mouse Il18 forward, 5′-TCAAAGTGCCAGTGAACCCC-3′, and reverse, 5′-GGTCACAGCCAGTCCTCTTAC-3′; mouse Il12 forward, 5′-AAATGAAGCTCTGCATCCTGC-3′, and reverse, 5′-TCACCCTGTTGATGGTCACG-3′; mouse Tnf forward, 5′-TACTGAACTTCGGGGTGATTGGTCC-3′, and reverse, 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′; mouse Nlrp3 forward, 5′-GACGAGTGTCCGTTGCAAGCTG-3′, and reverse, 5′-TGTGGCT AGATCCAAGTGATCTGCC-3′; mouse Gapdh forward, 5′-GGTGAAG GTCGGTGTGAACGGA-3′, and reverse, 5′-CCAAAGTTGTCATGGATGACCTTGG-3′. Transduction of shRNA and transfection of siRNA. For stable knockdown of mouse Fasn, 3 independent shRNA (TRCN0000075704, TRCN0000075705, and TRCN0000075706, Sigma-Aldrich) were used. For stable knockdown of mouse p38 MAPK, 2 independent shRNA (TRCN0000310885 and TRCN0000304327, Sigma-Aldrich) were used. Mouse peritoneal macrophages (5 × 105 cells per well) were seeded in 6-well plates and were transduced with shRNA lentiviral constructs against mouse Fasn, mouse p38 MAPK, or nontarget shRNA (SHC016, Sigma-Aldrich) for control. For transient knockdown of mouse Fasn, siRNA (SASI_Mm01_00177854, Sigma-Aldrich) was used. Mouse J774A.1 macrophages (2 × 105 cells per well) were seeded in 6-well plates and were transfected with siRNA for mouse Fasn or siRNA Universal Negative Control (SIC001, Sigma-Aldrich) (200 ng per well) using Lipofectamine RNAiMAX Reagent (13778075, Invitrogen) according to the manufacturer’s instructions. For overexpression of UCP2, mouse Ucp2 cDNA ORF clone (Myc-DDKtagged) (MR204388, OriGene Technologies) was used. Mouse J774A.1 macrophages (2 × 105 cells per well) were seeded in 6-well plates and were transfected with Ucp2 cDNA ORF clone or control vector using Lipofectamine LTX Reagent with PLUS Reagent (15338-100, Invitrogen) according to the manufacturer’s instructions. For AIM2 inflammasome activation, LPS-primed macrophages were transfected with poly(dA:dT) (1 μg/ml) (P0883, Sigma-Aldrich) using Lipofectamine LTX Reagent with PLUS Reagent (15338-100, Invitrogen) according to the manufacturer’s instructions. Glucose uptake, PEP, citrate, and lactate production assays. For a glucose uptake assay, cells (5 × 105 cells per well) were plated on 6-well plates. Cells were then incubated in a glucose-free DMEM media (Invitrogen) for 1 hour. After adding 18F-FDG (0.3 MBq), the cells were incubated at 37°C for 30 minutes. The cells were washed twice with PBS. Cells were harvested in PBS for radioactivity measurement using a 1480 Wizard 3 γ-counter (Perkin Elmer). Data shown are the mean ± SD of triplicate samples from a representative experiment. For in vivo uptake of 18F-FDG (62), male mice (8–10 weeks old) were starved overnight. Mice were injected i.p. with E. coli LPS (10 mg/kg) (L4130, Sigma-Aldrich). After LPS injection, 18F-FDG (16.7 ± 1.3 MBq) was administered intravenously via the tail vein. Mice were kept conscious for 1 hour during 18F-FDG uptake. 18F-FDG injection and PET/ 678


The Journal of Clinical Investigation CT imaging were performed under 1.5% isoflurane general anesthesia. PET/CT imaging was performed for 20 minutes (10 minutes per bed) using a commercial micro-PET/CT scanner (eXplore Vista, GE healthcare), follow by a CT scan. PET images were corrected for random and scatter coincidence events and reconstructed with the ordered-subsets expectation-maximization algorithm using 16 subsets and 2 iterations. No attenuation correction was applied. SUVs were calculated according to the formula (SUV = a / [A/W]), where a represents the activity concentration in the organ region of interest measured using PET images (Bq/ml), A represents the injected activity (Bq), and W represents the mouse body weight in grams. Blood and several organs were harvested for radioactivity measurement using a 1480 Wizard 3 γ-counter (Perkin Elmer). The PEP production was measured using a PEP Fluorometric Assay Kit according to the manufacturer’s instructions (700780, Cayman Chemical Company). The citrate (K655-100, BioVision) and lactate (K607-100, BioVision) production was measured using Colorimetric Assay Kits (BioVision) according to the manufacturer’s instructions. In vivo experiments. The male mice (8–10 weeks old) were injected i.p. with E. coli LPS (10 mg/kg) (L4130, Sigma-Aldrich). CLP was performed as previously described (63). The mice were cohoused in the same cages before and after CLP. The male mouse (8–10 weeks old) cecum was exposed through a 1.5-cm incision. The cecum was ligated below the ileocecal valve with a 6-0 silk suture without bowel obstruction and was then punctured with a 19-gauge needle (1-hole injury). Sham operation consisted of the same procedure without ligation and perforation of the cecum. After surgery, mice were injected with 1 ml physiological saline solution subcutaneously. The organ dysfunction was measured using the Creatinine Parameter Assay Kit (KGE005, R&D Systems), Aspartate Aminotransferase Activity Assay Kit (MAK055, Sigma-Aldrich), and Alanine Transaminase Colorimetric Activity Assay Kit (700260, Cayman Chemical Company). Oil Red O staining. Neutral lipids and lipid droplet morphology in sections from frozen liver tissues from mice was measured by Oil Red O (O1391, Sigma-Aldrich) staining as described previously (64). The sections were incubated with the Oil Red O solution for 5 minutes and then were counterstained with Mayer’s hematoxylin (MHS16, Sigma-Aldrich) for 15 seconds for visualization of the cell nuclei and tissue morphology. The sections were washed under running tap water for 30 minutes. RNA extraction and microarray. We used PaxGene tubes (761115, QIAGEN) to collect 2.5 ml whole-blood samples from hospitalized patients that consented for the study. Samples were collected within 48 hours of MICU admission (day 1). Total RNA was extracted in random batches from all available samples using the RNeasy Mini Protocol (74104, QIAGEN). Arrays were read using the Illumina BeadArray scanner and analyzed using BeadStudio (version 3.1.7) without background correction. Raw expression intensities were processed using the Lumi package of Bioconductor, with background adjustment with RMA convolution, and log2 transformation of each array. The combined samples were quantile normalized. The complete raw and normalized microarray data are available through the Gene Expression Omnibus of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/, accession no. GSE32707). Patient samples. The BWH Research Registry and Human Sample Repository for the Study of the Biology of Critical Illness (BWH RoCI) is an ongoing registry that collects demographic, clinical information, and


Research article

The Journal of Clinical Investigation blood specimens from patients admitted in the MICU (11, 13, 14). All adults (age 18 and older) admitted to the MICU of BWH are considered for enrollment. Exclusion criteria included participants (a) with mental handicaps, (b) who are unable to provide consent or for whom an appropriate legal representative could not be found to provide consent, (c) who had previously indicated that they did not wish to be enrolled in this study (e.g., during a prior admission to the MICU), (d) who were admitted for comfort measures and, (e) for whom there was concern about nonmedically indicated blood draws (e.g., Jehovah’s Witnesses). ICU control and sepsis was identified according to the 2001 SCCM/ESICM/ ACCP/ATS/SIS International Sepsis Definitions Conference guidelines (65, 66). ICU control (systemic inflammatory response syndrome) was defined as having 2 or more of the following criteria: temperature 38°C; heart rate >90; RR interval >20 or PaCO2 1,2000 or 10% immature (band) forms. Sepsis was defined as a documented infection (culture positive or strong clinical suspicion) together with 2 or more ICU control criteria above. Total blood RNA was isolated from patients of the BWH RoCI cohort. Gene expression profiles were generated using Human HT-12 v4 BeadChip arrays (Illumina) according to the manufacturer’s protocol (15). The microarray data are available through the Gene Expression Omnibus of the National Center for Biotechnology Information (accession no. GSE32707). Statistics. All data are mean ± SD, combined from 3 independent experiments. All statistical tests were analyzed by 2-tailed Student’s t test for comparison of 2 groups and ANOVA (with post-hoc comparisons using Dunnett’s test) using a statistical software package (GraphPad Prism version 4.0) for comparison of multiple groups. P values of less than 0.05 were considered statistically significant. Survival was 1. Angus DC, van der Poll T. Severe sepsis and septic shock. N Engl J Med. 2013;369(9):840–851. 2. Hotchkiss RS, Karl IE. The pathology and treatment of sepsis. N Engl J Med. 2003;348(2):138–150. 3. Rogers AJ, et al. Metabolomic derangements are associated with mortality in critically ill adult patients. PLoS One. 2014;9(1):e87538. 4. Langley RJ, et al. An integrated clinico-metabolomic model improves prediction of death in sepsis. Sci Transl Med. 2013;5(195):195ra95. 5. Dolinay T, et al. Inflammasome-regulated cyto kines are critical mediators of acute lung injury. Am J Respir Crit Care Med. 2012;185(11):1225–1234. 6. Nakahira K, et al. Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation. PLoS Med. 2013;10(12):e1001577. 7. Lamkanfi M, Dixit VM. Inflammasomes and their roles in health and disease. Annu Rev Cell Dev Biol. 2012;28:137–161. 8. Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140(6): 821–832. 9. Sutterwala FS, et al. Critical role for NALP3/ CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 2006;24(3):317–327. 10. Jin C, Flavell RA. Molecular mechanism of NLRP3 inflammasome activation. J Clin Immunol. 2010;30(5):628–631. 11. Masters SL, et al. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1β in type 2 diabe-

analyzed with the log-rank test. P values of less than 0.05 were considered statistically significant. Study approval. All animal experimental protocols were approved by the Harvard Standing Committee for Animal Welfare (protocol no. 04435; Harvard Medical School) and the Institutional Animal Care and Use Committee of Weill Cornell Medical College (New York, New York, USA) (protocol no. 2013-0108). All analysis of human tissue was conducted according to the guidelines of and with the approval of the BWH Institutional Review Board. BWH RoCI is approved by the Partners Human Research Committee and operates under protocol 2008-P-000495. Informed consent was obtained directly from each subject and documented in writing before the start of study-related procedures. The written and oral information provided to subjects was approved by the Partners Human Research Committee (Boston, Massachusetts, USA) before and during the informed consent process.

Acknowledgments

We thank E. Ifedigbo for technical assistance. We thank Bradford B. Lowell and Kong Dong for supervising the generation of loxUCP2 mice. This work was supported by the National Institutes of Health (grants P01 HL108801, R01 HL079904, and R01 HL055330 to A.M.K. Choi). Address correspondence to: Augustine M.K. Choi, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medical Center, 525 East 68th Street, Room M-522, Box 130, New York, New York 10065, USA. Phone: 212.746.4720; E-mail: amc2056@ med.cornell.edu.

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NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.

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