PPAR-Α Activation Mediates Innate Host Defense Through Induction of TFEB and Lipid Catabolism

PPAR-α Activation Mediates Innate Host Defense through Induction of TFEB and Lipid Catabolism

This information is current as Yi Sak Kim, Hye-Mi Lee, Jin Kyung Kim, Chul-Su Yang, of September 28, 2021. Tae Sung Kim, Mingyu Jung, Hyo Sun Jin, Sup Kim, Jichan Jang, Goo Taeg Oh, Jin-Man Kim and Eun-Kyeong Jo J Immunol published online 8 March 2017 http://www.jimmunol.org/content/early/2017/03/08/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 8, 2017, doi:10.4049/jimmunol.1601920 The Journal of Immunology

PPAR-a Activation Mediates Innate Host Defense through Induction of TFEB and Lipid Catabolism

Yi Sak Kim,*,†,1 Hye-Mi Lee,*,1 Jin Kyung Kim,*,† Chul-Su Yang,‡ Tae Sung Kim,*,† Mingyu Jung,†,x Hyo Sun Jin,* Sup Kim,*,† Jichan Jang,{ Goo Taeg Oh,‖ Jin-Man Kim,†,x and Eun-Kyeong Jo*,†

The role of peroxisome proliferator–activated receptor a (PPAR-a) in innate host defense is largely unknown. In this study, we show that PPAR-a is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-a deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-a agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette–Gue´rin. PPAR-a agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow–derived Downloaded from macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-a activation promoted lipid catabolism and fatty acid b-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-a mediates antimicrobial responses to mycobacterial infection by inducing TFEB and lipid catabolism. The Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/

uclear receptor peroxisome proliferator–activated re- retinoids, and cholesterol metabolites, saturated and unsaturated ceptor a (PPAR-a) is a PPAR isoform that regulates the fatty acids, as well as pharmacologic agents (4, 5). PPAR-a is a N expression of genes involved in glucose and lipid transcription factor that binds to cis-acting DNA elements, metabolism as well as inﬂammatory processes (1–3). All PPAR i.e., PPAR response elements in the promoter region of numerous members belong to the adopted orphan nuclear receptor family target genes (6). Through the regulation of these target genes, and are activated by various endogenous ligands, such as steroids, PPAR-a acts as a key regulator of energy metabolism, and mitochondrial and peroxisomal function (2, 4, 7). In addition, PPAR-

a is responsible for activation of fatty acid b-oxidation (FAO) and by guest on September 28, 2021 *Department of Microbiology, Chungnam National University School of Medicine, ketogenesis and simultaneous inhibition of glycolysis and fatty Daejeon 35015, South Korea; †Department of Medical Science, Chungnam National University School of Medicine, Daejeon 35015, South Korea; ‡Department of Mo- acid synthesis (2, 8). However, the function of PPAR-a in innate lecular and Life Science, College of Science and Technology, Hanyang University, host defense during mycobacterial infection is largely unknown. Ansan 15578, South Korea; xDepartment of Pathology, Chungnam National Univer- sity School of Medicine, Daejeon 35015, South Korea; {Molecular Mechanism of Mycobacterium tuberculosis is the major causative pathogen of Antibiotics, Division of Life Science, Research Institute of Life Science, Gyeongsang human tuberculosis (TB), an important infectious disease that ‖ National University, Jinju 52828, South Korea; and Department of Life Science, causes two million deaths per year worldwide (9). M. tuberculosis Ewha Womans University, Seoul 03760, South Korea is a highly successful pathogen that is capable of surviving and 1 Y.S.K. and H.-M.L. are cofirst authors. persisting within host phagocytes by inhibiting phagosomal mat- ORCIDs: 0000-0003-4918-961X (C.-S.Y.); 0000-0003-2812-0285 (M.J.). uration and escape from innate immune effectors (10, 11). In Received for publication November 11, 2016. Accepted for publication February 3, addition, pathogenic mycobacteria use host lipids to enter and 2017. multiply within non-maturing parasitophorous vacuoles in host This work was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, Ministry of Health and Welfare, Re- cells (12). Host macrophages use several strategies to clear viru- public of Korea (HI15C0395), by the National Research Foundation grant funded by lent mycobacteria. Autophagy, an intracellular lysosomal degra- the Korean government (MSIP) (NRF-2015M3C9A2054326) at Chungnam National dation process, is an important cell-autonomous defense system University. involved in innate and adaptive immune responses and thus con- Address correspondence and reprint requests to Prof. Eun-Kyeong Jo, Departments of Microbiology and Medical Science, Chungnam National University School of Med- tributes to host defense against various intracellular microbes in- icine, 6 Munhwa-dong, Jungku, Daejeon 301-747, South Korea. E-mail address: cluding M. tuberculosis and M. bovis bacillus Calmette–Gue´rin [email protected] (BCG) (13). Among the transcription factors implicated in auto- The online version of this article contains supplemental material. phagy, transcription factor EB (TFEB) is a critical regulator of Abbreviations used in this article: AHR, aryl hydrocarbon receptor; Atg, autophagy- autophagic activation (14, 15). TFEB transcriptionally regulates related gene; BCG, bacillus Calmette–Gue´rin; BMDM, bone marrow–derived mac- numerous genes involved in multiple steps of autophagy, includ- rophage; COX-2, cyclooxygenase 2; ERFP, enhanced red fluorescent protein; FAO, fatty acid b-oxidation; IHC, immunohistochemistry; Lamp, lysosomal-associated ing autophagosomal and lysosomal biogenesis and substrate tar- membrane protein; mCherry-EGFP-LC3B, tandem fluorescent-tagged LC3; MOI, geting and degradation (15). Furthermore, TFEB is required for multiplicity of infection; OCR, oxygen consumption rate; PBST, PBS plus 0.05% Tween-80; PPAR-a, Ppara, peroxisome proliferator–activated receptor a; qPCR, the expression of genes involved in lipid degradation and FAO in quantitative real-time PCR; Rab, Ras-related protein in brain; RT, room temperature; mitochondria (16). shRNA, short hairpin RNA; shTFEB, lentiviral shRNA specific to Tfeb; TB, tuber- In this study we identified a critical role for PPAR-a in host culosis; TFEB, transcription factor EB. defense against M. tuberculosis and BCG mycobacterial infec- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 tions, mediated by the ability of PPAR-a to directly activate the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601920 2 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY expression of genes involved in autophagosomal and lysosomal histopathological score was assigned to each tissue in each animal based biogenesis and function. In addition, PPAR-a activation led to on the extent of granulomatous inflammation as follows: 0 = no lesion, 1 = upregulation and nuclear translocation of TFEB, which is required minimal lesion (1–10% of tissue in section involved), 2 = mild lesion (11– 30% involved), 3 = moderate lesion (30–50% involved), 4 = marked lesion for lysosomal biogenesis and antimicrobial responses against M. (50–80% involved), and 5 = severe lesion (.80% involved), as described tuberculosis infection. Furthermore, PPAR-a activation inhibited previously (20). lipid body formation and promoted the expression of genes in- For IHC staining, lung paraffin sections (4 mm) were cut and immu- volved in FAO in macrophages, suggesting a role for PPAR-a in nostained with Abs specific for cyclooxygenase 2 (COX-2) (ab15191; Abcam), anti-Ly-6G6C Ab (ab2557, NIMP-R14; Abcam), and TNF-a (sc- lipid catabolism during mycobacterial infection. 52746; Santa Cruz). IHC staining tissue slides for COX-2 and neutrophils were imaged using an Aperio ScanScope CS System and confocal images Materials and Methods were taken with a Leica TCS SP8 confocal system and processed with the Mice Leica LAS AF Lite program. The histopathological score data used for this study were provided by the Biobank of Chungnam University Hospital, a Ppara2/2 mice were kindly provided by Dr. Gonzalez (17). We back- member of the Korea Biobank Network. crossed with C57BL/6 mice more than 10 times to establish C57BL/6 congenic Ppara2/2 line. The littermate control mice were used for all CFU assays +/+ animal experiments (control groups; Ppara ). Mice were housed in For quantification of intracellular bacteria, CFU assays were performed as specific pathogen free conditions, and maintained on a 12 h light/dark described previously (21). M. tuberculosis– or BCG-infected BMDM were cycle. The littermate control mice used for experiments (control groups; +/+ then lysed with 0.3% saponin to release intracellular bacteria. The infected Ppara ) were obtained from our inbred C57BL/6 strains. The mice used lysates were then resuspended vigorously, transferred to screw-capped tubes, in the experiments were all 8–10 wk old males. Animal-related procedures

and sonicated in a preheated 37˚C water bath sonicator (Elma) for 5 min. Downloaded from were reviewed and approved by the Institutional Animal Care and Use Aliquots of the sonicates were diluted and each sample was plated separately Committee, Chungnam National University School of Medicine (CNUH- on 7H10 agar plates and incubated at 37˚C in 5% CO for 14 to 21 d. 014-A0006-1; Daejeon, Korea). 2 Bone marrow–derived macrophage culture Immunoﬂuorescence of autophagy analysis Autophagosome formation was assayed by LC3 punctate staining, as de- Bone marrow–derived macrophages (BMDM) were isolated and differ- scribed previously (21). For endogenous LC3 puncta or phagosomal entiated after culture for 4–5 d in medium containing 25 ng/ml M-CSF

maturation analysis, the stimulated cells on coverslips were washed twice http://www.jimmunol.org/ (416-ML; R&D Systems), as described previously (18). The culture me- with PBS, and fixed with 4% paraformaldehyde for 15 min after per- dium consisted of DMEM (12-604F; Lonza) supplemented with 10% meabilization with 0.25% Triton X-100 for 10 min. The cells were incu- heat-inactivated FBS (35-015-CV; Lonza), and penicillin–streptomycin- bated with primary Abs [anti-LC3; MBL International, PM036, anti- amphotericin B (17-745E; Lonza). LAMP2; Santa Cruz, sc-5571, and anti- Ras-related proteins in brain Mycobacterial strains (Rab) 7; Santa Cruz, sc-6563] at 4˚C overnight. Cells were washed twice with PBS to remove excess primary Abs and incubated with fluorescently M. tuberculosis H37Rv was kindly provided by R.L. Friedmann (Univer- labeled secondary Abs at room temperature (RT) for 2 h. Nuclei were sity of Arizona, Tucson, AZ). M. bovis BCG was obtained from the Korean stained with DAPI (Sigma) for 1 min. After mounting, fluorescence images Institute of Tuberculosis (Osong, Korea). All mycobacterial strain cultures were acquired using a Leica TCS SP8 confocal system, and MetaMorph were performed as described previously (18). M. tuberculosis or BCG were Advanced Imaging acquisition software version 7.8 (Molecular Devices). grown in Middlebrook 7H9 (Difco, 271310) medium supplemented with by guest on September 28, 2021 0.5% glycerol, 0.05% Tween-80 (Sigma), and 10% oleic acid, albumin, Autophagic flux analysis using a tandem fluorescent-tagged LC3 dextrose, and catalase (Difco, 212240). For M. tuberculosis– or BCG-expressing enhanced red fluorescent For autophagic flux analysis, tandem-tagged tandem fluorescent-tagged protein (ERFP) strains, we used Escherichia coli–BCG shuttle expression LC3 (mCherry-EGFP-LC3) retrovirus was generated. Phoenix ampho- plasmid pMV262-RFP, under the control of the heat shock protein 60 tropic cells were seeded at 70–80% confluence into a six-well plate and promoter. BCG was transformed with the recombinant plasmid pMV262- transfected with 1 mg of pBabe-mCherry-GFP-LC3 (22418; Addgene), RFP by electroporation, then kanamycin-resistant clones were selected in 0.75 mg of pCL-Eco (12371; Addgene), and 0.25 mg of pDM2.G (12259; 7H9 broth supplemented with kanamycin (50 mg/ml) for further experi- Addgene) using Lipofectamine 2000 (Invitrogen). After 18 h, transfection ments (19). M. tuberculosis– or BCG-ERFP strains were grown in Mid- cell culture medium was removed and replaced with fresh medium. The dlebrook 7H9 medium supplemented with OADC and kanamycin. All retrovirus-containing medium was harvested at 60 h posttransfection and mycobacterial suspensions were aliquoted and stored at 280˚C. filtered through a 0.45 mm syringe filter. For autophagic flux analysis, Midlogarithmic-phase bacteria (OD = 0.6) were used in all assays. The BMDM were transduced with tandem fluorescent-tagged LC3 (mCherry- CFU were enumerated on Middlebrook 7H10 agar (BD Biosciences). EGFP-LC3B) retrovirus for 24 h and stimulated with GW7647 (1008613; Cayman Chemical) or Wy14643 (C7081; Sigma). The stimulated cells Mycobacterial infection in vivo were placed on coverslips, washed twice with PBS, and fixed with 4% paraformaldehyde for 15 min. After mounting, fluorescence images were Mycobacterial infection in vivo was performed as described previously (18). acquired using a confocal laser scanning microscope. Frozen M. tuberculosis and BCG were thawed and centrifuged, and then their pellets were resuspended in PBS plus 0.05% Tween-80 (PBST) be- Flow cytometry fore infection. Ppara+/+ and Ppara2/2 mice were intravenously injected with M. tuberculosis (1 3 106 CFU per mouse) or BCG (1 3 107 CFU per Flow cytometry analysis of LC3B was performed as described previously mouse) for 14 or 21 d. Mice were maintained in biosafety level 3 labo- (21). GW7647- or Wy14643- stimulated BMDM were washed in fixation ratory facilities. The experimental procedures were reviewed and approved and permeabilization solution (BD Biosciences) for 10 min. Cells were by the Institutional Animal Care and Use Committee of Bioleaders incubated with an anti-LC3B Ab (2775; Cell Signaling) for 1 h and a (Daejeon, Korea). At 14 or 21 d, mice were euthanized and the lungs, secondary anti-rabbit IgG-Alexa 488 Ab (A11034; Invitrogen) for 30 min spleens, and livers harvested for CFU assay, immunohistochemistry (IHC) on ice. Cells were then washed in PBS, resuspended in FACS buffer and staining, and quantitative real-time PCR (qPCR) analysis. analyzed immediately. The samples were examined using a FACSCanto II For measurement of the bacterial burden in the lung, spleen, and liver of flow cytometer (Becton Dickinson). Flow cytometry data were analyzed mice sacrificed at 14 or 21 d after M. tuberculosis or BCG infection, tissues using the FlowJo software (Tree Star). were homogenized in PBST, and serial dilutions of the homogenates were plated on 7H10 agar plates, with colonies counted 14 d later. Immunoblotting Histology and immunohistochemistry Immunoblotting was performed as described previously (22). For immunoblotting, cells were lysed in RIPA (10 mM Tris-HCl at pH 8, 1 mM Lung samples were fixed in 10% formalin and embedded in paraffin wax. EDTA, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Paraffin sections (4 mm) were then cut and stained with H&E. Inflam- Triton X-100) and a protease inhibitor mixture (Roche). The cell suspen- mation in lung sections was graded for severity by scanning multiple sion was incubation and centrifuged at 14,000 3 g for 10 min at 4˚C. The random fields in 10 sections of each tissue per mouse. An overall protein supernatant was collected and concentration was measured by The Journal of Immunology 3

BCA assay (Pierce). The protein extracts were boiled in 13 SDS sample dehyde in PBS for 10 min. Cells were permeabilized with 0.25% Triton buffer, loaded onto SDS-PAGE, and then transferred to polyvinylidene X-100 in PBS for 10 min and stained with anti-TFEB (A303-673A; Bethyl difluoride membranes (IPVH00010; Millipore). Specific Abs were used in Laboratories) at 4˚C overnight. The cells were then washed in PBS, and this study, as follows: LC3 (L8918; Sigma), PPAR-a (ab45859; Abcam), incubated with anti-rabbit Alexa Fluro 488 (A-11008, for 2 h; Invitrogen) UVRAG (ab70807; Abcam), TFEB (A303-673A; Bethyl Laboratories), at RT. Nuclei were stained by incubation with DAPI for 1 min. After and Actin (sc-1616-R; Santa Cruz). Protein signals were visualized using mounting, fluorescence images were acquired using a confocal laser- ECL solution (WBKL S0500; Millipore) and detected with an UVitec scanning microscope. Alliance mini-chemiluminescence device (UVitec, Rugby, U.K.). Lipid body staining RNA extraction, semiquantitative RT-PCR, and qPCR Ppara+/+ and Ppara2/2 BMDM were infected with M. tuberculosis–or RNA extraction, semiquantitative RT-PCR, and qPCR were performed as BCG-ERFP and stimulated with GW7647 or Wy14643 for 24 h. For described previously (18). Briefly, total RNA from cells was isolated using neutral lipids were stained using BODIPY 493/503, as previously de- TRIzol reagent (15596-026; Thermo Fisher Scientific) and used for syn- scribed (23). Cells were fixed in 4% (v) paraformaldehyde and neutral thesis of cDNA with Superscript II reverse transcriptase (Invitrogen). For lipids were stained using 5 mg/ml of BODIPY 493/503 (D-3922; Molec- semiquantitative RT-PCR reactions involved 30 cycles of annealing, ex- ular Probes) for 30 min at RT. Nuclei were stained by incubation with tension, and denaturation as described previously (22). Reactions were run DAPI for 1 min. After mounting, fluorescence images were acquired using on a Veriti 96-Well Thermal Cycler (Thermo Fisher). The PCR products a confocal laser–scanning microscope. were analyzed by electrophoresis on 1.5% agarose gel. qPCR was carried out using cDNA, primers, and SYBR Green master mix Real-time measurement of oxygen consumption rate assay (Qiagen). Reactions were run on a Rotor-Gene Q 2plex system (Qiagen, Real-time analysis of the oxygen consumption rate (OCR) was performed as Germany). To analyze qPCR data, we performed relative quantification using DD described previously (22). Briefly, BMDM were plated in XF-24 cell culture the 2 threshold cycle (Ct) method with Gapdh as an internal control gene. 5 microplates (2–3 3 10 cells per well in 200 ml) and then stimulated with Downloaded from Data were expressed as relative fold changes. The following primers were GW7647 or Wy14643. After 18 h, the medium was removed and cells were used: Tnf forward 59-GGTGCCTATGTCTCAGCCTCTT-39, reverse 59- washed then analyzed in XF Running Buffer according to the manufacturer’s GCCATAGAACTGATGAGAGGGAG-39; Il6 59-TACCACTTCACAAGT- instructions to analyze real-time values of OCR. Where indicated, OCR was CGGAGGC-39,reverse59-CTGCAAGTGCATCATCGTTGTTC-39; Il1b forward analyzed in response to 2 mg/ml oligomycin A (75351; Sigma), 5 mM 59-TGGACCTTCCAGGATGAGGACA-39, reverse 59-GTTCATCTCGG- carbonyl cyanide 3-chlorophenylhydrazone (C2759; Sigma), and 2 mM ro- AGCCTGTAGTG-39; Becn1 forward 59-AGGCGAAACCAGGAGAGAC-39, tenone (R8875; Sigma). The cells were analyzed using an XF-24 Extracel- reverse 59-CCTCCCCGATCAGAGTGAA-39; Atg7 forward 59-CCTGTGA- lular Flux Analyzer (Seahorse Bioscience). GCTTGGATCAAAGGC-39,reverse59-GAGCAAGGAGACCAGAACAGTG-39; http://www.jimmunol.org/ Lc3b forward 59-TTATAGAGCGATACAAGGGGGAG-39, reverse 59-CG- NF-kB–luciferase reporter assays CCGTCTGATTATCTTGATGAG-39; Lamp1 forward 59-CAGCACTCTTT- GAGGTGAAAAAC-39, reverse 59-CCATTCGCAGTCTCGTAGGTG-39; NF-kB–luciferase reporter assays were performed as described previously Lamp2 forward 59-GAGCAGGTGCTTTCTGTGTCTAG-39,reverse (24). Briefly, BMDM were transduced with NF-kB–luciferase adenovirus 59-GCCTGAAAGACCAGCACCAACT-39; Uvrag forward 59-GACTTTGG- (Genetransfer Vector Core) for 36 h, and infected with M. tuberculosis for AATAATGCCGGATCG-39,reverse59-CAGCCCATCCAGGTAGACTTT-39, 6h.M. tuberculosis–infected cells were washed three times in PBS and Vps11 forward 59-AAAAGAGAGACGGTGGCAATC-39,reverse59-AG- cell extracts were prepared by adding 100 mlof13 Passive Reporter Lysis CCCAGTAACGGGATAGTTG-39; Vps34 forward 59-CCTGGACATCA- Buffer (Promega, Madison, WI). Luciferase activity was measured using ACGTGCAG-39,reverse59-TGTCTCTTGGTATAGCCCAGAAA-39; Tfeb the Luciferase Assay System (Promega), according to the manufacturer’s forward 59-CGCCTGGAGATGACTAACAAGC-39,reverse59-GGCAACTC- instructions. TTGCTTCACCACCT-39; Lipa forward 59-TGTTCGTTTTCACCATTGG- by guest on September 28, 2021 GA-39,reverse59-CGCATGATTATCTCGGTCACA-39; Cd36 forward 59- Statistical analysis CCCTTGGCAACCAACCACAA-39,reverse59-AACCATCCACCAGTTG- Data are represented as the mean 6 SD. All analyses were performed using CTCC-39; Cpt1a forward 59-CTCCGCTCGCTCATTCCG-39,reverse59-CA- SPSS 22.0 and GraphPad Prism 5.0. Statistical analysis was performed CACCCACCACCACGATAA-39; Acadl forward 59-CATCGCAGAGAAAC- using an unpaired two-tailed Student t test indicated for comparison be- ATGGCG-39,reverse59-TGGCTATGGCACCGATACAC-39; Actb forward 59- tween groups. A p value ,0.05 was considered significant. CATTGCTGACAGGATGCAGAAGG-39 reverse 59-TGCTGGAAGGTGG- ACAGTGAGG-39; Gapdh forward 59-CATCACTGCCACCCAGAAGACTG- 39,reverse59-ATGCCAGTGAGCTTCCCGTTCAG-39. Results PPAR-a is essential for antimicrobial responses and inhibition ELISA analysis of excessive inflammatory responses to mycobacterial infection BMDM were treated as indicated and processed for analysis by sandwich in macrophages and in vivo ELISA. Cell culture supernatants were analyzed using a Mouse BD OptEIA Set ELISA Kit (BD Biosciences) to detect TNF-a (558534), IL-6 (555240), The function of PPAR-a in innate host defense is largely unknown. and IL-1b (559603). All assays were performed as recommended by the To evaluate its role in host defense against mycobacterial infec- manufacturers. tion, we first investigated whether PPAR-a is essential for anti- Lentiviral short hairpin RNA generation and transduction microbial effects against M. tuberculosis and BCG infection in BMDM. To this end, wild-type (Ppara+/+) and PPAR-a–knockout Generation of lentiviral short hairpin RNA (shRNA) and titration was 2/2 performed as described previously (22). For silencing target genes in (Ppara ) BMDM were infected with M. tuberculosis or BCG at BMDM, the pLKO.1-based lentiviral shRNA construct, TFEB (sc-38510- various multiplicities of infection (MOIs). Macrophage PPAR-a SH; Santa Cruz), was used. Briefly, the lentivirus was generated by gene expression was induced after M. tuberculosis infection (data transfection of HEK293T cells with pLKO puro.1 or target shRNA plas- not shown). As shown in Fig. 1A and 1B, the intracellular sur- mids and packaging plasmids (pMDLg/pRRE, pRSV-REV, and pMD2. vivals of M. tuberculosis and BCG were significantly increased in VSV-g; Addgene) using Lipofectamine 2000 (Invitrogen). After 72 h, 2/2 +/+ lentivirus-containing supernatants were collected and a number of lenti- BMDM from Ppara mice, compared with those from Ppara viruses titrated. For lentivirus transduction, BMDM in medium containing mice, in an MOI-dependent manner. 10% FBS were infected with lentiviral vectors in the presence of 8 mg/ml To further examine the effect of PPAR-a deficiency on anti- polybrene (Sigma). After 2 d, cells were harvested and target gene mycobacterial immunity in vivo, Ppara+/+ and Ppara2/2 mice were knockdown analyzed. infected intravenously with M. tuberculosis or BCG. As shown in Immunofluorescence microscopy of nuclear translocation of Fig. 1C and 1D, the mycobacterial loads were significantly in- TFEB creased in the lung (Fig. 1D), liver, and spleen in Ppara2/2 mice relative to Ppara+/+ mice at days 14 and 21 after M. tuberculosis or Translocation of TFEB into the nucleus was detected using immunofluo- 2/2 rescence staining. Briefly, Ppara+/+ and Ppara2/2 BMDM were stimulated BCG infection. In addition, Ppara mice responded to i.v. BCG with GW7647 or Wy14643 for 1 h and then fixed with 4% paraformal- challenge with significantly increased granuloma-like lesions in 4 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. PPAR-a is required for antimicrobial host defense against mycobacterial infection. (A and B) Ppara+/+ and Ppara2/2 BMDM were infected with M. tuberculosis [MOI = 1, 5, or 10; (A)] or BCG [MOI = 1, 5, or 10; (B)] for 3 d and then intracellular bacterial loads determined. (C) Ppara+/+ and Ppara2/2 mice (n = 8 per group) were infected intravenously with M. tuberculosis (1 3 106 CFU). Mice were sacrificed after 21 d of M. tuberculosis infection, and the bacterial loads in lung, liver, and spleen were determined by CFU assay. (D) Ppara+/+ and Ppara2/2 mice (n = 6–8 per group) were infected intravenously with BCG (1 3 107 CFU). Mice were sacrificed after 14 or 21 d of BCG infection, and the bacterial loads in the lung were determined by CFU assay. (E and F) Representative H&E stained images in lung tissue from Ppara+/+ and Ppara2/2 mice (n = 7 per group) infected with BCG or PBST (n = 3) for 21 d. (E) Representative images. Scale bar, 200 mm. (F) Quantitative analysis of histopathology scores in 10 random fields. (G and H) Neutrophil infiltration (G) and COX-2 expression (H) in lung tissues from Ppara+/+ and Ppara2/2 mice (n = 8 per group) infected with BCG or PBST (n = 3) for 21 d. Representative images. Scale bars, 50 mm(G) or 100 mm(H). (I and J) Quantitative scoring of brown color neutrophils (I) and COX-2+ cells (J) were counted from eight random fields. (K and L) Ppara+/+ and Ppara2/2 mice (n = 8 per group) were infected intravenously with M. tuberculosis (K)or BCG (L) for 21 d. qPCR analysis of Tnf and Il6 mRNA levels in lung (left) and spleen (right) tissue samples. (M) Ppara+/+ and Ppara2/2 mice (n = 8 per group) were infected intravenously with BCG for 21 d. Representative immunofluorescence images of TNF-a in lung tissues. Scale bar, 50 mm. Data shown are mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). Mtb, M. tuberculosis. The Journal of Immunology 5 lungs compared with Ppara+/+ mice (Fig. 1E, 1F). In lung granu- sponses during mycobacterial infection remain unclear. We thus lomas after BCG challenge, neutrophil infiltration and COX-2 levels compared the expression of three proinflammatory cytokines were increased in Ppara2/2 mice compared with Ppara+/+ mice (TNF-a, IL-6, and IL-1b) between Ppara+/+ and Ppara2/2 (Fig. 1G–J). We then assessed expression of the proinflammatory BMDM. Tnf, Il6,andIl1b mRNA levels were significantly in- cytokines TNF-a and IL-6 in lung tissues from Ppara+/+ and creased in Ppara2/2 BMDM after M. tuberculosis infection Ppara2/2 mice. Tnf and Il6 mRNA levels were markedly elevated (Supplemental Fig. 1A). Consistent with these findings, the reporter in the lung and spleen tissues of Ppara2/2 mice compared with the gene activities of NF-kB were markedly increased in Ppara2/2 corresponding tissues of Ppara+/+ mice (Fig. 1K, 1L). TNF-a BMDM after M. tuberculosis infection (Supplemental Fig. 1B). In production was also significantly elevated in the lung tissues of addition, the production of proinflammatory cytokines was mark- Ppara2/2 mice, compared with Ppara+/+ mice, after mycobacterial edly increased in Ppara2/2 compared with Ppara+/+ BMDM after infection (Fig. 1M). These results indicate that PPAR-a is required M. tuberculosis infection (Supplemental Fig. 1C). for antimicrobial host defense and attenuates systemic inflammation We then examined the effects of PPAR-a agonists on proin- during mycobacterial infection. flammatory cytokine production in BMDM after mycobacterial infection. As shown in Fig. 2E, PPAR-a agonists (GW7647 and PPAR-a agonists enhance antimicrobial defense, but suppress Wy14643) caused a dose-dependent inhibition of Tnf and Il6 inflammatory responses against mycobacterial infection in mRNA levels in Ppara+/+ after 6 h of infection with M. tuber- BMDM in a PPAR-a–dependent manner culosis or BCG. Similar to these findings, proinflammatory cyto- We assessed whether PPAR-a agonists enhance antimicrobial kine production in response to M. tuberculosis or BCG was dose- +/+ 2/2 responses in macrophages from Ppara and Ppara mice. dependently decreased by PPAR-a agonists (GW7647 and Downloaded from +/+ 2/2 Ppara and Ppara BMDM were infected with M. tubercu- Wy14643) in BMDM after 18 h of M. tuberculosis or BCG in- losis or BCG for 3 d with or without various PPAR-a agonists fection (Fig. 2F). However, PPAR-a agonists did not modulate (GW7647 or Wy14643), and the survival of intracellular myco- proinflammatory cytokine mRNA and protein levels in Ppara2/2 bacteria was determined by a CFU assay. PPAR-a agonists BMDM after M. tuberculosis or BCG infection (Fig. 2E, 2F). (GW7647 and Wy14643) (3, 25) exhibited significant killing ef- These data suggest that PPAR-a agonists promote innate antimi-

fects against M. tuberculosis (Fig. 2A, 2B) and BCG (Fig. 2C, 2D) crobial defense, but negatively regulate M. tuberculosis– or BCG- http://www.jimmunol.org/ +/+ in Ppara BMDM in a dose-dependent manner. The intracel- induced production of proinﬂammatory cytokines in BMDM in a lular survival levels of M. tuberculosis and BCG were similarly PPAR-a–dependent manner. inhibited in Ppara+/+ BMDM by PPAR-a agonists 2 d postin- a fection (data not shown). In contrast, the PPAR-a agonists did not PPAR- activation enhances autophagy and expression of exert antimycobacterial effects in Ppara2/2 BMDM (Fig. 2A–D), autophagy-related genes in macrophages suggesting that the antimicrobial activities were dependent on Previous studies showed that pharmacological activation of PPAR- PPAR-a activation. a promotes hepatic autophagy through transcriptional activation PPAR-a has been reported to control inﬂammatory responses of autophagy-related genes (Atgs) (25). To determine the

(26, 27). However, the effects of PPAR-a in inﬂammatory re- autophagy-inducing capacity of PPAR-a agonists in macrophages, by guest on September 28, 2021

FIGURE 2. PPAR-a agonists activate antimicrobial responses but inhibit inﬂammatory responses against mycobacterial infection in macrophages. (A–F) Ppara1/1 and Ppara2/2 BMDM were infected with M. tuberculosis (MOI = 5) or BCG (MOI = 5) for 4 h and then stimulated with GW7647 (2, 10, or 20 mM) or Wy14643 (20, 50, or 100 mM). (A–D) Intracellular bacterial loads were determined by CFU assay after 3 d of infection. (E) Cells were lysed and subjected to qPCR analysis (for 6 h) of Tnf and Il6.(F) TNF-a and IL-6 in supernatants were determined by ELISA (for 18 h). Data shown are mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; ns, non- signiﬁcant; SC, solvent control (0.1% DMSO); Wy, Wy14643. 6 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY

Ppara+/+ and Ppara2/2 BMDM were treated with PPAR-a ago- by PPAR-a agonists in Ppara+/+ BMDM (Fig. 3E, 3F). However, nists, and autophagic activation was evaluated by assessing LC3 PPAR-a–induced LC3B expression was not significantly upregu- punctae formation by immunofluorescence analysis. As shown in lated in Ppara2/2 BMDM, when compared with that in Ppara+/+ Fig. 3A and 3B, the number of LC3 punctae was significantly BMDM (Fig. 3E, 3F). We next monitored autophagic flux in increased in Ppara+/+ BMDM after treatment with PPAR-a ag- macrophages using a mCherry-EGFP-LC3B and measured mRFP- onists (GW7647 and Wy14643), when compared with that in GFP-LC3 delivery to lysosomes in BMDM treated with the Ppara2/2 BMDM. In addition, PPAR-a agonists (GW7647 and PPAR-a agonists. As shown in Fig. 3G, the number of red punctae Wy14643) robustly upregulated the lipidated form of LC3 in was increased in Ppara+/+ BMDM, whereas only the yellow Ppara+/+ BMDM, as determined by immunoblotting of autopha- punctae were observed in Ppara2/2 BMDM, in response to gosomal membrane–associated LC3-II fractions (Fig. 3C, 3D). In PPAR-a agonists. contrast, the level of lipidated LC3 was not significantly upregu- A number of Atgs identified in yeasts and mammals are asso- lated in Ppara2/2 BMDM (Fig. 3C, 3D). Flow cytometric analysis ciated with key functions of the autophagy pathway (28). In ad- using an LC3B-specific Ab revealed an increase in LC3B ex- dition, PPAR-a activation leads to transcriptional activation of pression, which is correlated with autophagosomal formation (21), numerous Atgs by directly binding to their promoter regions (25). Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. PPAR-a activation enhances autophagy and increases autophagy-related gene expression in macrophages. (A–F) Ppara+/+ and Ppara2/2 BMDM were stimulated with GW7647 (10 mM) or Wy14643 (50 mM). (A and B) Alexa488-conjugated LC3 (green) and DAPI (blue) were detected by confocal microscopic analysis at 8 h of GW7647 or Wy14643 stimulation. (A) Representative images. Scale bar, 5 mm. (B) Statistical analysis of LC3 punctate foci per cell. (C and D) Cells were stimulated with GW7647 or Wy14643 for the time as indicated. Cell lysates were subjected to immunoblot analysis of LC3 and Actin. The densitometry values for LC3 II were normalized to Actin in each lower panel. (E and F) Flow cytometric analysis of LC3B expression at 24 h of GW7647 or Wy14643 treatment (E). (F) Statistical analysis of LC3B by flow cytometry. (G) Ppara+/+ and Ppara2/2 BMDM were transduced with a retrovirus expressing a tandem LC3 plasmid (mCherry-EGFP-LC3B) and stimulated with GW7647 or Wy14643 for 24 h. Cells were fixed, and mCherry- or EGFP-expressing LC3 was detected by confocal microscopy. Scale bar, 5 mm. (H) Ppara+/+ and Ppara2/2 BMDM were stimulated with GW7647 or Wy14643 for 6 or 18 h. Becn1, Atg7, Lc3b, Lamp1, Lamp2, and Rab7 mRNA levels were determined by qPCR analysis. Representative confocal microscopic images from three independent samples are shown, with each experiment including at least 50 cells scored from seven random fields (A and G). Data shown are mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; SC, solvent control (0.1% DMSO); Wy, Wy14643. The Journal of Immunology 7

We thus examined whether PPAR-a activation increases tran- these genes (Fig. 5D), suggesting that TFEB is required for the scriptional activation of Atgs in macrophages. Application of the expression of Atgs in response to PPAR-a activation. PPAR-a agonists significantly increased Becn1, Atg7, Lc3b, Rab7, We next investigated the effect of TFEB on PPAR-a–induced lysosomal-associated membrane protein (Lamp)1, and Lamp2 autophagic activation in BMDM. As shown in Fig. 5E and 5F, mRNA levels in Ppara+/+ BMDM (Fig. 3H). In addition, PPAR-a knockdown of TFEB using lentiviral shRNA targeting TFEB activation was required for this induction of expression, because significantly decreased the LC3B expression in BMDM after the transcriptional activation of these genes was not significantly treatment with GW7647 or Wy14643. Moreover, UVRAG protein upregulated in Ppara2/2 BMDM, when compared with that in levels in BMDM decreased in response to treatment with GW7647 Ppara+/+ BMDM (Fig. 3H). Thus, these data indicate that PPAR-a or Wy14643 (data not shown). We further assessed the role of activation robustly enhances autophagosome formation and TFEB in PPAR-a–induced LAMP2 expression and puncta for- autophagic flux in BMDM. mation in BMDM. Silencing of TFEB led to significant inhibition of LAMP2 expression and puncta formation in BMDM (Fig. 5G, a PPAR- is required for phagosomal maturation in macrophages 5H). These data collectively suggest that PPAR-a activation re- during mycobacterial infection sults in the induction and nuclear translocation of TFEB, which To determine the importance of PPAR-a–induced autophagic acti- are required for expression of autolysosome-related genes, auto- vation during mycobacterial infection, we assessed the effects of phagic activation, and lysosomal biogenesis, in BMDM. PPAR-a on the colocalization of M. tuberculosis phagosomes a with autophagosomes or lysosomes in macrophages infected with PPAR- –induced TFEB expression enhances phagosomal

M. tuberculosis–ERFP.AsshowninFig.4A,treatmentofM. maturation and antimicrobial responses but attenuates Downloaded from tuberculosis–infectedBMDMwithPPAR-a agonists resulted in inflammatory responses during mycobacterial infection increased colocalization of the autophagosomal marker LC3 with We elucidated the role of TFEB in PPAR-a–mediated phagosomal M. tuberculosis phagosomes. Immunofluorescence analyses using maturation and antimicrobial responses in BMDM during myco- an anti-LAMP-2A Ab also showed an increase in the colocalization bacterial infection. Similar to the findings described above (Fig. 4A, of M. tuberculosis and BCG phagosomes with LAMP-2A-positive 4B), PPAR-a stimulation resulted in a significant increase in +/+ lysosomes in Ppara BMDM after treatment with the PPAR-a colocalization of M. tuberculosis phagosomes with the autophago- http://www.jimmunol.org/ agonists (Fig. 4B, Supplemental Fig. 2). The PPAR-a agonists led somal marker LC3 (Fig. 6A, 6B) and lysosomal marker LAMP2 to markedly increased amounts of LC3+ autophagosomes and ly- (Fig. 6C, 6D) in BMDM transduced with nonspecific lentiviral sosomes in Ppara+/+ BMDM infected with M. tuberculosis or BCG shRNA. Silencing of TFEB by lentiviral shRNA specific to Tfeb (Fig. 4C, 4D, Supplemental Fig. 2). However, these responses were (shTFEB) markedly inhibited PPAR-a–induced colocalization of not significantly up-regulated in Ppara2/2 BMDM (Fig. 4C, 4D, M. tuberculosis phagosomes with autophagosomes and lysosomes Supplemental Fig. 2). In addition, the fusion of mycobacterial in BMDM (Fig. 6A–D). It was noted that the PPAR-a-mediated phagosomes containing M. tuberculosis with late endosomes and increases in LC3 and LAMP2 levels, representing autophagosomal lysosomes was arrested (29). Rab7 functions as a checkpoint in the and lysosomal biogenesis, respectively, in nonspecific lentiviral regulation of phagosomal maturation upon recruitment into M. shRNA–transduced/M. tuberculosis–infected BMDM were signifi- by guest on September 28, 2021 tuberculosis– or BCG-containing phagosomes (30, 31). Because cantly decreased in shTFEB-transduced BMDM (Fig. 6A, 6C). application of PPAR-a agonists led to an increase in Rab7 ex- Additionally, lentiviral knockdown of TFEB significantly suppression (Fig. 3H), we determined whether PPAR-a agonists also pressed the PPAR-a–induced antimicrobial responses against M. enhance the recruitment of Rab7 into phagosomes containing M. tuberculosis infection in BMDM (Fig. 6E, 6F). We further inves- tuberculosis. As shown in Fig. 4E and 4F treatment of BMDM with tigated the role of TFEB in the regulation of inflammatory re- PPAR-a agonists increased Rab7 recruitment into M. tuberculosis– sponses in BMDM during mycobacterial infection. As shown in containing phagosomes. Therefore, PPAR-a is required for phag- Fig. 6G and 6H, silencing of TFEB increased production of the osomal maturation and recruitment of Rab7 into M. tuberculosis proinflammatory cytokines TNF-a and IL-6 in BMDM in response phagosomes in macrophages during mycobacterial infection. to M. tuberculosis or BCG. These data suggest that TFEB is required for PPAR-a–mediated phagosomal maturation and killing of a PPAR- agonists enhance the expression and nuclear mycobacteria as well as the suppression of proinflammatory re- translocation of TFEB, which is essential for induction of sponses in BMDM during M. tuberculosis infection. autophagy-related genes and lysosomal biogenesis in macrophages PPAR-a activation is required for lipid catabolism and Because PPAR-a was found to be essential for the expression of increased mitochondrial respiratory function in macrophages multiple Atgs in BMDM (Fig. 3H), we evaluated the effects of infected with M. tuberculosis PPAR-a on the transcriptional activation and nuclear translocation The accumulation of lipid bodies is a hallmark of the foamy of TFEB. As shown in Fig. 5A–C, treatment with PPAR-a ago- macrophages generated upon virulent mycobacterial infection (32). nists led to robust expression (Fig. 5A) and rapid nuclear trans- M. tuberculosis or BCG phagosomes merge with intracellular lipid location of TFEB (Fig. 5B, 5C). Although PPAR-a stimulation bodies in foamy macrophages, a secure reservoir in which M. significantly increased translocation of cytosolic TFEB to the tuberculosis can hide and survive and from which they are re- nucleus in Ppara+/+ BMDM, this was markedly decreased in leased (32–34). Next, we investigated whether PPAR-a activation Ppara2/2 BMDM (Fig. 5B, 5C). We next examined the role of contributes to the reduction in fatty acid–rich lipid body formation TFEB in PPAR-a–mediated induction of genes involved in auto- in BMDM infected with M. tuberculosis or BCG. Bodipy 493/503 phagosomal formation and maturation into autolysosomes. As fluorescence staining revealed an increase in neutral lipid levels in expected, PPAR-a activation markedly increased the transcrip- the cytoplasm of M. tuberculosis–infected macrophages (Fig. 7A). tional activation of several genes involved in autophagosomal and Following treatment with PPAR-a agonists, the number and size autolysosomal formation (Uvrag, Lc3b, Vps11, and Vps34)in of lipid bodies were greatly reduced in M. tuberculosis–infected BMDM at 6 and 18 h. Importantly, lentiviral-mediated knockdown macrophages (Fig. 7A–C). Infection of BMDM with BCG resulted of TFEB significantly decreased the transcriptional activation of in significantly reduced formation of lipid bodies in the cytosol 8 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. PPAR-a is required for phagosomal maturation in macrophages during mycobacterial infection. (A–D) Ppara+/+ and Ppara2/2 BMDM were infected with M. tuberculosis–ERFP (MOI = 5) for 4 h and then stimulated with GW7647 (10 mM) or Wy14643 (50 mM) for 24 h. (A) M. tuberculosis– ERFP (red), Alexa-488 conjuated-LC3 (green), and DAPI (blue) were detected by confocal microscopy. Scale bar, 5 mm. (C) Statistical analysis of M. tuberculosis–ERFP and LC3 colocalization per cell containing 200 internalized mycobacteria. (B) M. tuberculosis-ERFP (red), Alexa488-conjuated LAMP2 (green), and DAPI (blue) were detected by confocal microscopy. Scale bar, 5 mm. Representative three-dimensional reconstructed Z-stack immunofluorescence images are shown in each lower panel. (D) Statistical analysis of M. tuberculosis and LAMP2 colocalization per cell containing 200 internalized mycobacteria. Representative confocal microscopic images from three independent samples are shown, with each experiment including at least 100 cells scored from seven random fields (A–D). (E and F) Ppara+/+ and Ppara2/2 BMDM were infected with M. tuberculosis–ERFP (MOI = 5, for 4 h) and then stimulated with GW7647 (10 mM) or Wy14643 (50 mM) for 24 h. (E) M. tuberculosis–ERFP (red), Alexa488-conjugated LAMP2 (green), and DAPI (blue) were detected by confocal microscopy. Scale bar, 5 mm. (F) Statistical analysis of M. tuberculosis–ERFP and Rab7 colocalization. Repre- sentative confocal microscopic images from three independent samples are shown, with each experiment including at least 50 cells scored from seven random fields. Data shown are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; SC, solvent control (0.1% DMSO); Wy, Wy14643; ns, nonsignificant. compared with M. tuberculosis infection (Supplemental Fig. 3). lysosomal acid lipase; Cd36, lipid uptake; Cpt1a and Acadl, lipid Similar to M. tuberculosis infection, PPAR-a stimulation led to a metabolism and FAO) in uninfected (Supplemental Fig. 4) or M. significant reduction in lipid body number and size in BCG- tuberculosis–infected Ppara+/+ BMDM (Fig. 7E). In addition, the infected BMDM (Supplemental Fig. 3). Importantly, the PPAR- transcriptional activation of these genes was markedly abrogated a–mediated reduction in lipid body formation was absent in in Ppara2/2 BMDM, when compared with that in Ppara+/+ Ppara2/2 BMDM during M. tuberculosis or BCG infection (Fig. BMDM (Fig. 7E). Collectively, these data indicate that PPAR-a 7A–C, Supplemental Fig. 3). We then quantified the cellular ox- activation is required for lipid catabolism, increased mitochondrial ygen consumption rate as a measure of mitochondrial respiration respiratory function, and upregulation of FAO in BMDM during and FAO in unstimulated and GW7647- or Wy14643-treated mycobacterial infection. BMDM. PPAR-a agonist treatment increased the basal and maximum oxygen consumption rates, both of which were signif- Discussion icantly decreased in Ppara2/2 BMDM (Fig. 7D). The findings of this work suggest that PPAR-a is required for In addition, PPAR-a agonist treatment upregulated the expres- innate host responses in macrophages to mycobacterial infection. sion of genes involved in lipid uptake, lipolysis, and FAO (Lipa, They also suggest that PPAR-a is a critical and master regulator of The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 5. PPAR-a-induced TFEB expression is essential for autophagy-related gene induction and lysosomal biogenesis in macrophages. (A–C) Ppara1/1 and Ppara2/2 BMDM were stimulated with GW7647 (10 mM) or Wy14643 (50 mM) for 6 (A)or18h(A–C). (A) Tfeb expression level was

measured by qPCR analysis. (B) Immunofluorescence microscopic analysis of TFEB nuclear translocation. Alexa488-conjugated TFEB (green) and DAPI by guest on September 28, 2021 (blue) were detected by confocal microscopic analysis. Scale bar, 10 mm. (C) Quantitative analysis of TFEB nuclear translocation. (D–H) BMDM were transduced with shTFEB–expressing lentivirus and then stimulated with GW7647 or Wy14643 for 6 (D)or18h(D, G, and H). (D) Uvrag, Lc3b, Vps11, and Vps34 expression levels were detected by qPCR analysis. (E and F) Alexa 488-conjugated LC3B was analyzed by flow cytometry. Representative his- tograms (E) and statistical analysis of LC3B expression (F). (G and H) Alexa488-conjugated LAMP2 (green) and DAPI (blue) were detected by confocal microscopic analysis. (G) Representative of images. Scale bar, 5 mm. (H) Statistical analysis of LAMP2 punctate foci per cell. Representative confocal microscopic images from three independent samples are shown, with each experiment including at least 50 cells scored from seven random fields (B and G). Data shown are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; SC, solvent control (0.1% DMSO); Wy, Wy14643. host antimycobacterial responses through transcriptional auto- fection is in its infancy. Advances have recently been made toward phagic activation, lysosomal biogenesis, promotion of phagosomal our understanding of the transcriptional regulation of Atgs that maturation, and induction of TFEB in macrophages. We discov- function in autophagic processes and numerous physiological re- ered that TFEB mediates innate host defense against mycobacte- sponses, including innate host defense against mycobacterial infec- rial infection by enhancing Atg activation, lysosomal biogenesis, tion. As an example, recent studies have shown that activation of phagosomal maturation, and transcriptional activation of genes NR1D1 (an adopted orphan nuclear receptor subfamily 1, group D, involved in FAO. Our studies have broad implications for mo- member 1) enhances autophagy and the expression of LAMP1 and lecular regulation of the autophagy pathway and lysosomal bio- TFEB, to promote antimycobacterial effects in human macrophages genesis by which PPAR-a activation reinforces antimicrobial host (37). We reported previously that the AMPK-PGC1a pathway is defense and prevents infectious diseases. essential for transcriptional activation of Atgs, including Atg5, Autophagy is an intracellular degradative process with various Becn1,andAtg7, in macrophages via C/EBP-b signaling activation homeostatic roles, including functions in innate immune regulation (18). The findings of the current study showed that PPAR-a acti- and cell-autonomous defense against intracellular microbes including vation led to activation of autophagy and promoted the expression of M. tuberculosis (5). Atgs exert a concerted effect in autophagy, numerous Atgs, including Becn1, Atg7, Lc3b, Lamp1, Lamp2, Rab7, i.e., autophagosome initiation and elongation and lysosomal func- and Tfeb, in macrophages. We also found that PPAR-a deficiency tions (28). Various Atgs play diverse roles in innate and inflamma- led to excessive inflammatory responses in macrophages after M. tory responses, such as the eradication of intracellular pathogens, tuberculosis infection. These data in part support previous reports murine gammaherpesvirus 68 reactivation from latency, Ag pre- that pharmacological activation of PPAR-a enhances the transcrip- sentation, and innate immune signaling in a cell type–specific tion of Atgs in the mouse liver (25), and that PPAR-a activation manner (28, 35, 36). Despite these findings, research on the mo- ameliorates hepatic injury and inflammatory responses through lecular mechanisms of Atg regulation in macrophages during in- autophagic activation (27). 10 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY Downloaded from http://www.jimmunol.org/

FIGURE 6. PPAR-a-induced TFEB enhances phagosomal maturation and antimicrobial responses but decreases inﬂammatory responses during M. tuberculosis infection. (A–D) BMDM were transduced with shTFEB-expressing lentivirus, infected with M. tuberculosis–ERFP (MOI = 5, for 4 h), and then stimulated with GW7647 (10 mM) or Wy14643 (50 mM) for 24 h. (A and B) M. tuberculosis-ERFP, Alexa488-conjugated LC3 and DAPI were detected by confocal microscopy. (A) Representative of images. Scale bar, 5 mm. (B) Statistical analysis of M. tuberculosis-ERFP and LC3 colocalization per cell containing 200 internalized mycobacteria. (C and D) M. tuberculosis-ERFP (red), Alexa488-conjugated LAMP2 (green) and DAPI (blue) were detected by by guest on September 28, 2021 confocal microscopy. (C) Representative of images. Scale bar, 5 mm. (D) Statistical analysis of M. tuberculosis-ERFP and LAMP2 colocalization per cell containing 200 internalized mycobacteria. (E and F) BMDM were transduced with shTFEB-expressing lentivirus, infected with M. tuberculosis (MOI = 5, for 4 h), and then stimulated with GW7647 [(E); 2, 10, or 20 mM] or Wy14643 [(F); 20, 50, or 100 mM] for 3 d. Intracellular bacterial loads were determined by CFU assay. (G and H) BMDM were transduced with shTFEB-expressing lentivirus and infected with M. tuberculosis (G) or BCG [(H); MOI = 5] for 6 or 18 h. TNF-a and IL-6 levels in culture supernatants were determined by ELISA. Representative confocal microscopic images from three independent samples are shown (A and C). Data shown are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; SC, solvent control (0.1% DMSO); Un, uninfected; Wy, Wy14643.

The roles of several nuclear receptors and their mechanisms in (43). Our data are important in suggesting a novel function for the regulation of antimycobacterial immunity have been investi- PPAR-a in the promotion of antimicrobial responses through gated. Earlier studies revealed an essential role for vitamin D– autophagic activation and induction of TFEB expression. How- receptor signaling in the activation of antimicrobial responses in ever, PPAR-g induced by BCG infection was involved in lipid human macrophages via induction of antimicrobial peptide cath- body formation and PG E2 production in macrophages, thereby elicidin LL-37, production of IL-1b, and enhancement of auto- potentiating mycobacterial pathogenesis (33). In addition, the host phagy (38, 39). A recent study revealed that biallelic retinoic lipid-sensing nuclear receptors PPAR-g and testicular receptor 4 acid–related orphan receptor g loss-of-function mutations in seven contributed to generation of foamy macrophages, induction of the individuals lacking functional RAR-related orphan receptor g and anti-inflammatory factor IL-10, and enhancement of mycobacte- RAR-related orphan receptor g T isoforms had a defect in IL-17A/ rial survival within macrophages (44). Thus, PPAR-g and PPAR-a F–producing T cells and impaired IFN-g responses to mycobac- may play opposite roles in antimicrobial host defense against terial infection (40). In addition, a macrophage transcriptomic mycobacterial infection. profiling study showed that M. tuberculosis infection enhanced the It was of interest that PPAR-a agonists play an essential role in levels of aryl hydrocarbon receptor (AHR) and its heterodimeric anti-mycobacterial effects despite their involvement in the inhi- partners AHR nuclear translocator and RELB, and stimulated the bition of proinflammatory cytokines such as TNF-a and IL-1b, expression of AHR target genes, including IL-1b (41). Another which are critically involved in host protective immunity during recent study reported that human xenobiotic nuclear receptor mycobacterial infection (45, 46). Indeed, proinflammatory re- pregnane X receptor is essential for prolonging mycobacterial sponses are regarded as a double-edged sword between protective survival, which is mediated by induction of foamy macrophage immunity and detrimental inflammatory pathogenesis during formation and inhibition of phagolysosomal fusion (42). Impor- mycobacterial infection. Higher IL-1b expression correlated with tantly, each PPAR family member plays a distinct role in various an increased accumulation of neutrophils in the lung, suggesting a physiological functions in a context-dependent manner, driving cytokine for granulocytic inflammation and TB disease pro- i.e., depending on ligand affinities, tissue, cell type, and so on gression (47). In addition, defective recruitment of anti-inflammatory The Journal of Immunology 11 Downloaded from

FIGURE 7. PPAR-a is required for lipid catabolism and mitochondrial respiratory function in macrophages during mycobacterial infection. (A–C) +/+ 2/2 Ppara and Ppara BMDM were infected with M. tuberculosis–ERFP (MOI = 5, for 4 h) then stimulated with GW7647 (10 mM) or Wy14643 (50 mM) http://www.jimmunol.org/ for 24 h. M. tuberculosis–ERFP (red) and Bodipy 493/503 (green) were detected by confocal microscopy. (A) Representative images. Scale bar, 5 mm. (B) Representative three-dimensional reconstructed Z-stack immunofluorescence images of lipid droplets and M. tuberculosis–ERFP. Scale bar, 5 mm. (C) Statistical analysis of lipid droplets. (D) Ppara+/+ and Ppara2/2 BMDM were stimulated with GW7647 or Wy14643 for 18 h. Real-time measurement of the oxygen consumption rate by sequential treatment with oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone. (E) Ppara+/+ and Ppara2/2 BMDM were infected with M. tuberculosis (MOI = 5, for 4 h) and stimulated with GW7647 or Wy14643 for 24 h. Lipa, Cd36, Cpt1a, and Acadl mRNA levels were determined by quantitative PCR analysis. Representative confocal microscopic images from four independent samples are shown, with each experiment including at least 50 cells scored from seven random fields (A and B). Data shown are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, compared with the control condition (two-tailed Student t test). GW, GW7647; SC, solvent control (0.1% DMSO); Un, uninfected; Wy, Wy14643. by guest on September 28, 2021 dendritic cells and regulatory T cells resulted in the enhanced mediator of the anti-mycobacterial function and anti-inflammatory susceptibility to Mtb infection and excessive lung inflammation responses of PPAR-a. (48). A selective inhibitor for soluble TNF receptors was benefi- Virulent M. tuberculosis infection modulates host lipid ho- cial for the treatment of mycobacterial infections through neu- meostasis and induces macrophage differentiation into foamy tralization of excessive TNF (49). Extensive studies have reported phenotypes that constitute a nutrient-rich niche for mycobacterial the roles of PPAR-a agonists in the modulation of innate and persistence and replication within tuberculous granuloma lesions adaptive immune responses (50–52). These data thus indicate that (32, 34). In addition, lipid-laden mycobacteria exhibit a drug- PPAR-a agonists may protect hosts against excessive inflamma- resistance and dormancy-like phenotype, characteristics associ- tory responses during mycobacterial infection to promote a bal- ated with latent infection (57). A recent study reported that M. anced inflammatory response. tuberculosis–induced miR-33/33* inhibited FAO and enhanced TFEB acts as a key coordinator of lysosomal function and lipid-body formation in macrophages (23). Importantly, we found biogenesis, cellular clearance, and autophagic processes by binding that PPAR-a activation promoted lipid catabolism and upregulated to the promoters of numerous genes involved in the Co-ordinated the expression of genes involved in FAO in M. tuberculosis– Lysosomal Expression and Regulation gene network (14, 15, 53). infected macrophages. Moreover, silencing of TFEB downregu- Metabolic signals such as mTORC1 inhibition drive dephos- lated the expression of genes associated with FAO in these phorylation and nuclear translocation of TFEB, thus promoting macrophages (data not shown). Previous studies also reported that the activation of genes involved in lysosomal function and auto- TFEB plays a crucial role in the link between lipophagy and FAO phagy (14, 53). PPAR-a agonists enhance the transcriptional ac- and in the transcriptional regulation of genes involved in several tivation of TFEB via direct binding to and regulation of its steps of lipid degradation (16). Moreover, silencing of miR-33/33* promoter regions in brain cells and fly models (54, 55). In addi- decreased lipid accumulation and induced lipolysis, which were tion, TFEB was activated in murine macrophages by Staphylo- correlated with increased xenophagy and antimicrobial responses coccus aureus infection and contributed to transcriptional against M. tuberculosis in macrophages (23). Thus, these data induction of inflammatory cytokines and chemokines (56). Our suggest that PPAR-a activation induces lipid catabolism to en- data revealed a novel function of TFEB in the suppression of hance destruction of the foamy refuges of mycobacteria in host mycobacterial survival within macrophages through enhancement cells. of phagosomal acidification. We found that TFEB plays a role in In summary, the findings of this study demonstrate that PPAR-a the expression of Atgs and lysosomal biogenesis, but inhibits in- activation results in the activation of autophagy through direct and flammatory responses, in macrophages during M. tuberculosis or indirect induction of Atgs via TFEB, which is essential for lyso- BCG infection. Thus, our findings suggest that TFEB is a key somal biogenesis and enhancement of phagosomal maturation to 12 PPAR-a AGONISTS ACTIVATE ANTIBACTERIAL AUTOPHAGY promote host defense against M. tuberculosis infection. In addi- is required for antimicrobial host defense through activation of autophagy. 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Dis. 199: 1053–1063. 16: e47–e63. http://www.jimmunol.org/ by guest on September 28, 2021 1 Supplemental Figure Legends

3 S1. PPAR-α is required for inhibition of M. tuberculosis-mediated inflammatory responses

4 in macrophages. (A and C) Ppara+/+ and Ppara-/- BMDM were infected with M. tuberculosis

5 (MOI = 5) for the indicated times. The cells were subjected to semi-quantitative RT-PCR

6 analysis for (A) or ELISA (C) of TNF-α, IL-6, and IL-1β. (B) Ppara+/+ and Ppara-/- BMDM

7 were transduced with adenovirus carrying a NF-κB p65 luciferase reporter construct for 36 h.

8 The cells were infected with M. tuberculosis for 6 h and assayed for luciferase activity. Data

9 shown are means ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001,

10 compared with the control condition (two-tailed Student’s t-test).

15 S2. PPAR-α deficiency affects phagosomal acidification against BCG in macrophages. (A

16 and B) Ppara+/+ and Ppara-/- BMDM were infected with BCG-ERFP (MOI = 5, for 4 h) and

17 then stimulated with GW7647 (10 μM) or Wy14643 (50 μM) for 24 h. (A) BCG-ERFP (red),

18 Alexa488-conjugated LAMP2 (green) and DAPI (blue) were detected by confocal

19 microscopy. Scale bar, 5 µm. (B) Statistical analysis of BCG-ERFP and LAMP2

20 colocalization. Representative confocal microscopic images from three independent samples

21 are shown, with each experiment including at least 100 cells scored from seven random fields.

22 **P<0.01, compared with the control condition (two-tailed Student’s t-test). SC, solvent

23 control (0.1% DMSO); GW, GW7647; Wy, Wy14643.

26 S3. PPAR-α activation leads to increased lipid catabolism in macrophages during BCG

27 infection. (A and B) Ppara+/+ and Ppara-/- BMDM were infected with BCG-ERFP (MOI =

28 10, for 4 h; a) and then stimulated with GW7647 (10 μM) or Wy14643 (50 μM) for 24 h. (A)

29 Bodipy 493/503 (green) and DAPI (blue) were detected by confocal microscopic analysis.

30 Representative immunofluorescence images of lipid droplets and BCG-ERFP. Scale bar, 5

31 μm. (B) Statistical analysis of lipid droplets normalized mean intensity. Representative

32 confocal microscopic images from three independent samples are shown, with each

33 experiment including at least 100 cells scored from seven random fields. **P<0.01,

34 ***P<0.001, compared with the control condition (two-tailed Student’s t-test). SC, solvent

35 control (0.1% DMSO); GW, GW7647; Wy, Wy14643.

38 S4. PPAR-α deficiency decreases the lipid/fatty acid metabolic process genes in BMDMs.

39 Ppara+/+ and Ppara-/- BMDM were stimulated with GW7647 (10 μM) or Wy14643 (50 μM)

40 for 6 or 18 h. Lipa, Cd36, Cpt1a, and Acadl mRNA levels were determined by quantitative

41 PCR analysis. Data are representative of at least three independent experiments with similar

42 results. *P<0.05, **P<0.01, ***P<0.001, compared with the control condition (two-tailed

43 Student’s t-test). GW, GW7647; Wy, Wy14643.