HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis Via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization

Diabetes Volume 68, February 2019 361

HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization

Qiuyue Kong,1 Nan Li,1 Hao Cheng,1 Xiaojin Zhang,2 Xiaofei Cao,1 Tao Qi,1 Leyang Dai,1 Zhihong Zhang,3 Xuan Chen,1 Chuanfu Li,4 Yuehua Li,5 Bin Xue,6 Lei Fang,7 Li Liu,2 and Zhengnian Ding1

Diabetes 2019;68:361–376 | https://doi.org/10.2337/db18-0035

Nonalcoholic steatohepatitis (NASH) is the most prevalent Nonalcoholic fatty liver disease (NAFLD) affects ;22–28% cause of chronic liver disease worldwide. Macrophage- of the global population (1). The spectrum of NAFLD mediated inflammation plays a critical role in NASH begins with isolated steatosis, with subsequent develop- pathogenesis; however, optimum therapies for macro- ment of steatohepatitis (NASH) and steatofibrosis, even- phage activation and NASH remain elusive. HSPA12A tually leading to cirrhosis and hepatocellular carcinoma. encodes a novel member of the HSP70 family. Here, Patients with NAFLD, particularly NASH, show reduced PATHOPHYSIOLOGY we report that NASH patients showed increased hepatic survival, primarily due to cardiovascular and liver-related HSPA12A expression and serum HSPA12A contents. In- 2/2 diseases (2). NASH livers are more injured than livers with triguingly, knockout of HSPA12A (Hspa12a ) in mice isolated steatosis, which more likely leads to progressive – attenuated high-fat diet (HFD) induced hepatic steatosis fibrosis and eventual liver-associated illness and death (3). and injury. HFD-induced macrophage polarization to- The molecular players that modulate disease pathogenesis ward an M1 phenotype and inflammatory responses 2 2 are starting to be identified, enabling translation of re- in the liver of Hspa12a / mice were also attenuated. search findings to clinical trials (4,5). However, no opti- Loss- and gain-of-function studies revealed that the de mum therapy is yet available, suggesting that a more novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A rather than comprehensive understanding of the onset and progres- by hepatocyte HSPA12A. In-depth molecular analysis sion of NAFLD is needed. fi revealed that HSPA12A interacted with the M2 isoform The shift from isolated steatosis to NASH is de ned fl of pyruvate kinase (PKM2) in macrophages and in- histologically as emergence of liver in ammation and creased its nuclear translocation, thereby promoting hepatocellular injury. The most common theory explaining “ ” fi M1 polarization and secretion of proinflammatory M1 this shift is the double-hit hypothesis (3,5,6). The rst cytokines; this led, ultimately, to hepatocyte steatosis hit consists mainly of lipid accumulation, whereas the via paracrine effects. Taken together, these findings second comprises sequential innate immune responses showthatHSPA12AactsasanovelregulatorofM1 (6–8). Compelling evidence indicates that liver macro- macrophage polarization and NASH pathogenesis by phages play a key role in regulating the second hit. Macro- increasing nuclear PKM2. Strategies that inhibit macrophages are a highly heterogeneous, plastic cell population phage HSPA12A might be a potential therapeutic inter- that undergoes pleiotropic and coordinated responses to the vention for NASH. immunological environment; two common phenotypes

1Department of Anesthesiology, First Affiliated Hospital of Nanjing Medical Corresponding authors: Zhengnian Ding, [email protected], and Li Liu, University, Nanjing, China [email protected] 2 fi Department of Geriatrics, First Af liated Hospital of Nanjing Medical University, Received 12 January 2018 and accepted 23 October 2018 Nanjing, China This article contains Supplementary Data online at http://diabetes 3Department of Pathology, First Affiliated Hospital of Nanjing Medical University, .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0035/-/DC1. Nanjing, China 4Department of Surgery, East Tennessee State University, Johnson City, TN © 2018 by the American Diabetes Association. Readers may use this article as 5Department of Pathophysiology, Nanjing Medical University, Nanjing, China long as the work is properly cited, the use is educational and not for profit, and the 6State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Labo- work is not altered. More information is available at http://www.diabetesjournals. ratory of Molecular Medicine and School of Medicine, Nanjing University, Nanjing, org/content/license. China 7Medical School of Nanjing University, Nanjing, China 362 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019 are “proinflammatory” M1 and “immunoregulatory” M2 according to the NASH Clinical Research Network–mod- macrophages (7,9,10). Polarization of macrophages toward ified Brunt methodology and other studies (3,23). Livers the M1 phenotype plays a critical role in pathogenesis of with bridging fibrosis or cirrhosis were excluded using several chronic inflammatory disorders, including NASH. Masson’s staining (Supplementary Fig. 1A and B and By contrast, M2 macrophages promote resolution of in- Supplementary Table 1A and B). flammation (10). Indeed, suppressing M1 polarization alleviates diet-induced NASH (7,10–12). Blood Samples The M2 isoform of pyruvate kinase (PKM2) is a rate- Human blood samples were collected from bariatric sur- limiting enzyme of glycolysis. Cis-trans isomerization and gery patients with NASH diagnosed after clinic and B-type conversion of PKM2 from a tetramer to a dimer or mono- ultrasonographic assessment (Supplementary Table 2A mer lead to nuclear translocation and regulate expression and B). Patients were fasted for 12 h before blood sam- of target genes that play roles in the Warburg effect and pling. The study was approved by the Ethical Board of the fi cell cycle progression (13,14). Intriguingly, nuclear PKM2 First Af liated Hospital of Nanjing Medical University is a critical determinant of macrophage M1 activation and (2016-SR-122 and 2016-SR-123). All human studies is involved in inflammatory diseases such as coronary were conducted according to the World Medical Associa- artery disease (13,15,16). Nuclear PKM2 increases tion Declaration of Helsinki. macrophage M1 activation in response to lipopolysac- Hspa12a Knockout Mice charides (LPS), mainly through TLR4 signaling (13,15, Conditional Hspa12a knockout mice were generated using 17,18). A recent study shows that PKM2 expression is the loxP and Cre recombinant system. The Hspa12a target- associated with liver mass (19); however, the role of PKM2 ing vector was constructed using the bacterial artificial in NASH pathogenesis is largely unknown. chromosome (BAC) retrieval method. In brief, the region HSPA12A is a novel and atypical member of the HSP70 of the Hspa12a gene containing exons 2–4 was retrieved family (20). Hspa12a mRNA is expressed at high levels in from a 129/sv BAC clone (BACPAC Resources Center, the brain of humans and mice under normal conditions but Oakland, CA) using a retrieval vector containing two is decreased in the prefrontal cortex of patients with homologous arms. Exons 2 and 3 were replaced by loxP schizophrenia (20,21). Of particular interest, expression sites flanking a PGK-neo cassette (a positive selection of HSPA12A is associated with shortened survival of marker) (Supplementary Fig. 2A). The breeding strategy to patients with hepatocellular carcinoma (22), suggesting fl fl 2 2 generate wild-type (WT) (Hspa12a ox/ ox)andHspa12a / possible involvement of HSPA12A in modulating hepatic fl fl (Hspa12a ox/ ox, CreTg) mice is shown in Supplementary homeostasis. However, the role of HSPA12A in any dis- Fig. 2B. Mice were bred at the Model Animal Research orders, including liver disease, is unknown. Center of Nanjing University and maintained on a 12-h Here, we report that NASH patients showed increased light/dark cycle at 23 6 1°C with access to food and water hepatic HSPA12A expression and serum HSPA12A con- 2 2 ad libitum. All experiments conformed to the Guide for the tents. Intriguingly, knockout of HSPA12A (Hspa12a / )in Care and Use of Laboratory Animals published by the U.S. mice attenuated high-fat diet (HFD)–induced NASH path- National Institutes of Health (8th edition, 2011). The ogenesis and M1 macrophage activation. Further molecu- animal care and experimental protocols were approved lar analyses revealed that regulation of de novo lipogenesis by the Committee on Animal Care at Nanjing University. in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A via promotion of nuclear PKM2- mediated M1 polarization. Taken together, these findings HFD Feeding Protocol suggest that HSPA12A acts as a novel regulator of mac- For the HFD experiments, mice were fed an HFD (60% of rophage polarization and NASH pathogenesis. Strategies kcal from fat, D-12492; Research Diets, New Brunswick, designed to inhibit macrophage-specific expression of NJ) for 14 weeks beginning at age 5 weeks. Mice that were HSPA12A might have therapeutic potential for patients fed a normal chow diet and received only 6% of kcal from with NASH. fat served as controls.

Immunoblotting and Immunoprecipitation- RESEARCH DESIGN AND METHODS Immunoblotting Human Samples Cytosolic and nuclear proteins were prepared from livers Liver Specimens or cells using the NucBuster protein extraction kit accord- Liver specimens were collected from patients who under- ing to the manufacturer’s instructions (Novagen, Darm- went hepatic hemangioma surgery at the First Afﬁliated stadt, Germany). Western blotting was performed according Hospital of Nanjing Medical University. Patients provided to our previous methods (24,25). To control for lane informed consent at the time of recruitment. Normal or loading, the membranes were probed with anti-GAPDH NASH livers were identiﬁed using hematoxylin-eosin (H-E) or anti–a-tubulin antibodies for cytosolic proteins and staining, Oil Red O (ORO) staining, and serum alanine anti-histone H3 or anti-lamin A/C antibodies for nuclear aminotransferase (ALT) and AST activity measurements, proteins. diabetes.diabetesjournals.org Kong and Associates 363

For analyzing the interaction between HSPA12A using a fluorescence microscope with cellSens Dimension andPKM2byimmunoprecipitation-immunoblotting, 1.15 software (Olympus, Tokyo, Japan). Raw264.7 macrophages were overexpressed with flag- tagged HSPA12A. After challenge with LPS or vehicle Adenovirus Construction for 16 h, cells were collected for protein extraction. Ali- The adenoviral vector containing a three flags–tagged quots of equal volume and protein content were precip- coding region of mouse Hspa12a (NM_175199) was gen- itated with anti-flag antibodies, followed by Western erated by GeneChem (Shanghai, China). A schematic over- blotting with anti-PKM2 and anti-HSPA12A antibodies, view of virus construction is shown in Supplementary as described previously (26). The antibodies used in the Fig. 3. experiments are listed in Supplementary Table 3. Cell Culture and Treatment Quantitative Real-time PCR Culture Quantitative real-time PCR was performed as described Primary hepatocytes and liver macrophages (Kupffer cells) previously (27). In brief, total RNA was extracted for cDNA were isolated from 7- to 10-week-old mice by digestion synthesis using the oligo (dT) primer. After cDNA synthe- with 0.06% collagenase type IV, followed by centrifugation sis, the expressions of indicated genes were estimated by on a 25–50% Percoll gradient, as described previously (28). real-time PCR using the SYBR Green master mix (Roche, Indianapolis, IN). The PCR results of b-actin served as Primary hepatocytes were grown in DMEM supplemented with 10% FBS and 0.01 mmol/L dexamethasone. Mouse internal controls. The primers used for PCR are listed in Raw264.7 macrophages and human hepatocellular carci- Supplementary Table 4. noma HepG2 cells were grown in DMEM supplemented Biochemical Analysis with 10% FBS. Mouse AML-12 hepatocytes were cultured in DMEM/F12 supplemented with 10% FBS, ITS (5 mg/mL Levels of HSPA12A in human serum and insulin in mouse insulin, 5 mg/mL transferrin, and 5 ng/mL selenium), and serum and secretion of HSPA12A, IL-1b, IL-6, IL-12, and 40 ng/mL dexamethasone. TNF-a from macrophages were detected using ELISA kits ’ (Supplementary Table 5) according to the manufacturer s Overexpression of HSPA12A (Hspa12ao/e) instructions. Serum ALT and AST activity, glucose, LDL Cellswereinfectedfor48hwithadenoviruscarrying cholesterol, HDL cholesterol, total cholesterol, and tri- the Hspa12a expression sequence. Cells infected with glyceride (TAG) were measured using a Beckman Coulter empty adenovirus served as normal expression con- AU5800 Chemistry System analyzer (Brea, CA). trols (Hspa12an/e).

Measurement of Lipid Synthesis Effect of HSPA12A on Hepatocyte Steatosis o/e 2/2 Lipid content was evaluated using both ORO (Sigma- Hspa12a or Hspa12a hepatocytes were incubated Aldrich, St. Louis, MO) staining and a TAG assay. ORO with 200 mmol/L oleic acid (OA) for 24 h. staining was performed on 4% paraformaldehyde-fixed Collection of Conditioned Medium from Macrophages frozen liver sections or hepatocyte cultures. Quantification Hspa12ao/e or Hspa12an/e Raw264.7 macrophages were was performed using spectrophotometry at a wavelength treated for 16 h with LPS (500 ng/mL) to induce M1 of 510 nm after extraction of the stained ORO in cell polarization. The medium was then collected (referred to cultures. TAG was evaluated in liver tissues or hepatocyte here as Hspa12ao/e conditioned medium [CM] or Hspa12an/e cultures using a TAG assay kit according to the manufac- CM). turer’s instructions (Jiancheng Biotech, Nanjing, China). Paracrine Effects of Macrophage HSPA12A on Histological Analysis, Masson’s Staining, and Hepatocyte Steatosis Immunofluorescence Staining AML-12 hepatocytes were incubated for 24 h with Paraffin-embedded liver sections were stained with H-E to Hspa12ao/e CM or Hspa12an/e CM in the presence of OA evaluate the histological changes and averaged hepatocyte (200 mmol/L). areas. Masson’s staining was also performed to indicate fibrosis, as described previously (24). Immunofluorescence Activation of Kupffer Cells 2/2 staining of frozen liver sections or Raw264.7 macrophages After they were grown for 48 h, WT and Hspa12a was performed as described previously (24,25) using ap- Kupffer cells were incubated with LPS (200 ng/mL) for propriate antibodies (Supplementary Table 3). In brief, 16 h. Kupffer cells were harvested for immunoblotting after incubation with the indicated primary antibodies analysis, and the medium were collected and used as WT 2/2 overnight at 4°C, Cy3- or FITC-conjugated appropriate CM and Hspa12a CM, respectively. secondary antibodies were added to the sections to visualize the staining. Hoechst 33342 reagent was used to Cross-linking counterstain the nuclei. The staining was observed and RAW 264.7 macrophages were treated with 500 ng/mL quantified in 7–10 randomly selected areas of each sample LPS for 16 h. Cross-linking was performed using 364 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

500 mmol/L disuccinimidyl suberate (Thermo Fisher Sci- 4). In mouse liver, expression of HSPA12A by macrophages entific, Waltham, MA) for 30 min. Lysates were analyzed was 17.2-fold higher than that in hepatocytes (Fig. 1A). by Western blot as previously described (15). Macrophages in human livers also showed abundant expression of HSPA12A, whereas expression by hepatocytes Bone Marrow Transplantation was barely detectable (Fig. 1B and Supplementary Fig. 5). To examine the effects of macrophage HSPA12A on HFD- induced NASH, we transplanted the bone marrows of Upregulation of HSPA12A in Human Patients Shows 2 2 Hspa12a / mice to WT mice according to previously a Positive Correlation with NASH described methods (29). In brief, the recipient C57BL/6 To investigate the clinical significance of HSPA12A in WT mice (6 weeks old) were given acidified and antibiotic NASH, we evaluated expression of HSPA12A in NASH water (100 mg/L neomycin and 10 mg/L polymyxin) for patients. Expression of Hspa12a mRNA in NASH livers was 1 week before irradiation. At the day of transplantation, significantly higher than that in non-NASH controls (Fig. recipient mice were given a lethal irradiation (9 Gy). Four 1C). Moreover, we observed a positive correlation between hours after irradiation, a total of 5 3 106 bone marrow circulating HSPA12A levels and ALT and AST activity in 2 2 cells isolated from donor WT or Hspa12a / mice (4 weeks NASH patients (Fig. 1D and Supplementary Fig. 6). Un- old) were intravenously injected into a recipient WT mouse expectedly, expression of HSPA12A decreased in the cy- 2 2 via tail vein, referred to as Hspa12a / /WT and WT/WT tosolic fraction but increased in the nuclear fraction, mice, respectively. After receiving acidified and antibiotic suggesting nuclear translocation of HSPA12A in the hu- water for 4 weeks after transplantation, the bone marrow man NASH liver (Fig. 1E). Similar distribution of HSPA12A transplantation (BMT) mice were fed with HFD for was observed in the livers of mice fed an HFD (Fig. 1F). 14 weeks. Therefore, NASH is associated with upregulated expression of HSPA12A. Liquid Chromatography–Tandem Mass Spectrometry Analysis HSPA12A Deficiency Ameliorates HFD-Induced NASH Primary mouse adipocytes differentiated from preadipo- Because expression of HSPA12A was upregulated in NASH cytes were used in the experiments. The primary mouse livers, it is conceivable that HSPA12A knockout may have preadipocytes were used as controls. The proteins that therapeutic potential. Therefore, we generated HSPA12A fi 2 2 speci cally interact with HSPA12A were pulled down by knockout mice (Hspa12a / ) using the Cre-loxP recombi- HSPA12A antibody and were separated by SDS-PAGE nant system (Supplementary Fig. 2A and B). Successful followed by Coomassie blue staining, digestion in gel with knockout of HSPA12A in the liver was verified by protein – 2 2 trypsin, and analysis by liquid chromatography tandem analysis (Fig. 2A). The body weight of adult Hspa12a / mass spectrometry. In brief, peptides were dissolved in mice, along with serum levels of glucose and lipids, was solvent A (2% formic acid in 3% acetonitrile) and loaded similar to that in WT mice with access to food and water ad directly onto a reversed-phase trap column (Chrom XP libitum, as were fasting serum insulin levels (Supplemen- C18-CL-3m 120A; Eksigent). Peptide separation was per- tary Fig. 7A–C). Liver weight, hepatocyte size, and lipid 2 2 formed using a reversed-phase analytical column (3 mm, amount were similar in Hspa12a / and WT mice fed 120A) (3C18-CL-120; Eksigent). Eluting peptides from the a normal chow diet (Fig. 2B–D). However, HFD-induced column were analyzed using an AB Sciex 5600+ TripleTOF increases in liver weight and hepatocyte size were de- 2 2 system. Tandem mass spectrometry data were processed creased in Hspa12a / compared with WT mice (Fig. 2B using ProteinPilot Software 4.5 (AB Sciex). Tandem mass and C). Furthermore, HFD-induced steatosis was less 2 2 spectra were searched against the UniProt mouse database marked in Hspa12a / mice, as indicated by reduced (16,923 sequences) concatenated with a reverse decoy TAG content, histological alteration, and ORO staining fi database. Trypsin/P was speci ed as the cleavage enzyme, (Fig. 2C and D). Notably, HFD-induced liver injury, as fi allowing up to three missing cleavages, four modi cations indicated by elevated serum ALT and AST activities, was fi 2 2 per peptide, and ve charges. abolished in Hspa12a / mice (Fig. 2E). No significant fibrosis, indicated by Masson’s staining, was observed in Statistical Analysis both genotypes after HFD feeding for 14 weeks (Supple- Data are expressed as the mean 6 SD. Groups were mentary Fig. 8). Taken together, these findings strongly compared using an unpaired, two-tailed Student t test or suggest that HSPA12A deficiency prevents progression of using one-way or two-way ANOVA followed by Tukey post NAFLD to NASH. hoc test. A P value of ,0.05 was considered significant. Hspa12a2/2 Mice Show Decreased Expression of RESULTS Genes Involved in De Novo Fat Synthesis Upon HFD Liver Macrophages Express High Levels of HSPA12A Feeding The expression profile of HSPA12A protein in liver and To gain insight into the mechanisms underlying HSPA12A cells is unclear. Western blotting revealed low-level ex- deficiency–mediated inhibition of NAFLD progression, we pression of HSPA12A in mouse liver (Supplementary Fig. examined expression of genes linked to lipid metabolism in diabetes.diabetesjournals.org Kong and Associates 365

Figure 1—HSPA12A upregulation was positively correlated with human NASH. A: HSPA12A expression was examined in primary hepatocytes and macrophages. Primary cells were isolated from mice for immunoblotting analysis. **P , 0.01; mean 6 SD; n = 3 mice/group. Data were analyzed by unpaired, two-tailed Student t test. B: HSPA12A expression was examined in human normal livers. Immunoflu- orescence staining of HSPA12A, CD68 (macrophage), and HEP1 (hepatocyte) was performed on frozen liver sections. Hoechst 33342 was used to counterstain the nuclei. Note that HSPA12A, indicated by arrows in images, was preferably expressed in macrophages (right panel) compared with hepatocytes (left panel). Scale bar = 5 mm. **P , 0.01; mean 6 SD; n = 3 human subjects/group. C: Increased Hspa12a mRNA expression in livers of NASH patients. **P , 0.01; mean 6 SD; n = 7 for the control and n = 12 for NASH group. Data were analyzed by 366 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019 the livers of HFD-fed mice. We noted a significant decrease (Fig. 3C). The regulation of primary Kupffer cell HSPA12A in transcription of lipogenic transcription factors (Chrebp, on hepatocyte lipid deposition was also examined. The CM 2 2 2 2 Srebp-1c, Ppara, and Pparg)inHspa12a / livers compared was collected from LPS-treated primary Hspa12a / and 2 2 with WT livers (Fig. 2F). In agreement, the expression of WT Kupffer cell cultures, referred to as Hspa12a / CM target genes, including Gck, Lpk, Acc, Fas, Scd1, Gpat, and WT CM, respectively. AML-12 hepatocytes incubated 2 2 Dgat2,andElovl6, involved in de novo fat synthesis with Hspa12a / CM accumulated significantly less lipid 2 2 were reduced in Hspa12a / livers compared with WT than cells treated with WT CM in the presence of OA (Fig. livers (Fig. 2F). We also noticed that genes regulating lipid 3D). Taken together, these data indicate that macrophage storage and transportation (Cidea, Plin1, and Apob) and HSPA12A regulates hepatocyte steatosis through para- lipolysis (Hsl and Cgi58) were downregulated in crine effects. 2 2 Hspa12a / livers (Supplementary Fig. 9A and B). As the most prominent changes in expression were observed HSPA12A Deficiency Reduces Both Macrophage for transcription factors and target genes involved in de Recruitment and M1 Polarization in the Liver of HFD- novo fat synthesis, we examined expression of PPARg Fed Mice and SCD1 proteins. In line with the aforementioned The effects of macrophage HSPA12A on hepatocyte lipid results, expression of both proteins was reduced in the deposition prompted us to focus on macrophage activation 2 2 liver of Hspa12a / compared with WT controls upon and inflammatory responses because they play critical HFD feeding (Supplementary Fig. 9C). The findings sug- roles in NASH pathogenesis (7,10). Indeed, upon HFD gest that HSPA12A regulates de novo fat synthesis and feeding, we found a significant reduction in the number of 2/2 other lipid metabolic signaling in the liver upon HFD F4/80-positive cells in the liver of Hspa12a mice com- feeding. pared with that of WT mice (Fig. 4A). Accordingly, analysis of mRNA levels revealed markedly lower expression Lipid Accumulation in Hepatocytes Is Regulated by the of proinflammatory mediators and M1 markers, including Paracrine Effects of Macrophage HSPA12A Il-1b, Il-6, Tnfa, Nfkb, Tlr4, Inos, Mcp1, Ifng, Il-12, Cd86, 2 2 To investigate how HSPA12A regulates accumulation of and Cd68, in livers of Hspa12a / mice compared with WT lipids in hepatocytes, we examined the effects of loss- and controls upon HFD (Fig. 4B and C). By contrast, the livers 2 2 gain-of-HSPA12A function on lipid deposition using three of Hspa12a / mice showed markedly higher expression types of hepatocyte culture: primary mouse hepatocytes, of mRNA encoding M2 markers, including Cd163, Arg1, mouse hepatocyte AML-12 cells, and human hepatocellular and Cd206, than that of WT mice fed an HFD (Fig. 4C). In carcinoma HepG2 cells. Unexpectedly, we found that agreement, protein analysis confirmed reduced expres- OA-induced lipid deposition in primary hepatocytes was sion of iNOS (an M1 marker) and increased expres- not affected by either Hspa12a deficiency or overexpression of CD206 (an M2 marker) in livers from HFD-fed 2 2 sion (Fig. 3A and B). Also, overexpression of HSPA12A did Hspa12a / mice (Fig. 4D). Moreover, livers from 2 2 not affect OA-induced lipid deposition in either AML-12 or Hspa12a / mice showed lower expression of TLR4, HepG2 hepatocytes (Fig. 3B). MyD88, and phosphorylated (p-) NF-kB (p65) than those Because hepatic macrophages showed higher HSPA12A from WT controls upon HFD (Fig. 4D). Collectively, these expression than hepatocytes (Fig. 1A and B), we designed data indicate that HSPA12A deficiency attenuates both culture experiments to evaluate the interaction between macrophage recruitment and M1 polarization in the liver macrophage HSPA12A and hepatocyte lipid deposition. To of HFD-fed mice. do this, we overexpressed HSPA12A (Hspa12ao/e)in Raw264.7 macrophages by infection with the adenovirus Overexpression of HSPA12A Promotes M1 carrying the Hspa12a expression sequence; macrophages Macrophage Polarization by Increasing Nuclear infected with empty virus served as normal expression Translocation of PKM2 controls (Hspa12an/e). The CM collected from these mac- HSPA12A Is Upregulated During M1 Macrophage rophage cultures were referred to as Hspa12ao/e CM and Polarization Hspa12an/e CM. Notably, Hspa12ao/e CM increased the To investigate how HSPA12A regulates macrophage OA-induced lipid deposition in AML-12 hepatocytes polarization, we first examined whether HSPA12A unpaired, two-tailed Student t test. D: Increased circulating HSPA12A content correlated with hepatic injury in NASH patients. Hepatic injury was reflected by abnormal elevated activities of ALT (males .50 and females .40 units/L) and AST (males .40 and females .35 units/L). *P , 0.05 and **P , 0.01; mean 6 SD; n = 17 for the normal ALT group and n = 11 for elevated ALT group, and n = 22 for the normal AST group and n = 6 for elevated AST group. Data were analyzed by unpaired, two-tailed Student t test. E: Nuclear translocation of HSPA12A in livers of human NASH patients. Cytosolic and nuclear protein extracts were prepared from human livers with or without NASH. HSPA12A expression was analyzed by immunoblotting analysis. Blots for GAPDH or histone H3 served as loading controls. **P , 0.01; mean 6 SD; n = 3 human individuals/group. Data were analyzed by unpaired, two-tailed Student t test. F: Nuclear translocation of HSPA12A in livers of NASH mice. WT mice were fed with HFD for 14 weeks to induce NASH. Mice fed with normal chow served as controls. Cytosolic and nuclear protein extracts were prepared for immunoblotting against HSPA12A. *P , 0.05 and **P , 0.01; mean 6 SD; n = 4 mice/group. Data were analyzed by unpaired, two-tailed Student t test. diabetes.diabetesjournals.org Kong and Associates 367

Figure 2—Deficiency of HSPA12A attenuated HFD-induced hepatic steatosis and injury. A: HSPA12A expression was absent in livers of Hspa12a2/2 mice that were analyzed by immunoblotting. n = 10 mice/group. B: HFD-induced hepatomegaly was attenuated in Hspa12a2/2 mice. Mice aged 5 weeks were fed with HFD or normal chow diet for 14 weeks. Livers were removed and weighed. The liver weight (LW) was normalized with body length (BL). **P , 0.01; mean 6 SD; sample numbers are indicated. Data were analyzed by two-way ANOVA analysis followed by Tukey test. C: Hepatic histology. After feeding with HFD or chow for 14 weeks, livers were removed and analyzed by H-E staining on paraffin sections. Hepatocyte areas were measured. Arrows indicate the lipid droplet cavitation. **P , 0.01; mean 6 SD; n = 7 for HFD-WT group and n = 5 for other groups. Data were analyzed by two-way ANOVA analysis followed by Tukey test. Scale bar = 20 mm. D: Lipoid accumulation. Top panel: after feeding with HFD or chow for 14 weeks, frozen liver sections were prepared for ORO staining to indicate lipid deposition. n = 4 for HFD-WT group and n = 5 for other groups. Bottom panel: after feeding with HFD for 14 weeks, livers were collected for TAG content analysis. *P , 0.05; mean 6 SD; n = 4 for WT group and n = 5 for Hspa12a2/2 group. Data were analyzed by unpaired, two-tailed Student t test. Scale bar = 20 mm. E: Hepatic function. Serum samples were collected from mice fed with HFD or chow diet for 14 weeks for ALT and AST evaluation. **P , 0.01; mean 6 SD; n = 9/group. Data were analyzed by two-way ANOVA analysis followed by Tukey test. F: 368 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019 expression is altered upon activation of M1 macrophages. Raw264.7 macrophages (Fig. 7A and Supplementary Fig. Liver macrophages from NASH patients showed markedly 17). In line with this, Hspa12ao/e Raw264.7 macrophages higher protein expression and nuclear translocation of expressed higher levels of monomeric and dimeric PKM2 HSPA12A than controls, as illustrated by immunostaining than Hspa12an/e control cells in the presence of LPS (Fig. 5A and Supplementary Fig. 10). The same pattern of (Supplementary Fig. 18). Collectively, these data suggest HSPA12A expression and distribution was observed in that HSPA12A regulates PKM2 expression and nuclear Raw264.7 macrophages after LPS treatment, as indicated translocation during M1 macrophage polarization. by immunoblotting and immunostaining (Fig. 5B and C). We also observed increased expression of HSPA12A in Nuclear PKM2 Mediates the Effects of HSPA12A on M1 Macrophage Activation Raw264.7 macrophages exposed to OA (Supplementary To determine the role of PKM2 during HSPA12A-mediated Fig. 11). These findings suggest that HSPA12A in macro- M1 macrophage polarization in response to LPS, phages is upregulated and nuclear translocated during an Hspa12ao/e Raw264.7 macrophages were treated with M1 response. a small molecule, DASA-58. DASA-58 is a specific enzyme HSPA12A Promotes M1 Macrophage Polarization activator of PKM2, which can prevent PKM2 nuclear Next, we examined the direct effects of HSPA12A on the translocation (15,30,31). Notably, DASA-58 reversed the M1 macrophage response. Overexpression of HSPA12A HSPA12A overexpression-induced nuclear translocation increased mRNA expression of M1 markers (Inos, Il-1b, of PKM2 and expression of M1 markers (iNOS, MCP-1, Tnfa, Mcp1, Ccl3, and Ccl4) in LPS-treated Raw264.7 and TNF-a) in LPS-treated macrophages (Fig. 7A and B). macrophages (Fig. 5D). In agreement, expression of Taken together, these findings suggest that nuclear iNOS and TNF-a protein in Hspa12ao/e macrophages PKM2 mediates the HSPA12A-induced M1 macrophage was higher than that in Hspa12an/e macrophages in the polarization. presence of LPS (Fig. 5E). Expression of TLR4 and p-NF- kB, both critical for M1 macrophage activation, also in- Nuclear PKM2 Mediates the Paracrine Effects of creased in Hspa12ao/e macrophages (Fig. 5E). By contrast, Macrophage HSPA12A on Hepatocyte Steatosis 2 2 primary Kupffer cells from Hspa12a / mice showed de- Next, we asked whether nuclear PKM2 mediates the para- creased expression of iNOS, TLR4 MyD88, and p-NF-kB crine effects of macrophage HSPA12A on hepatocyte protein compared with WT Kupffer cells after LPS stim- steatosis. We found that overexpression of HSPA12A ulation (Supplementary Fig. 12). These findings confirm led to increased secretion of IL-1b, IL-6, TNF-a, and the aforementioned in vivo results showing that HSPA12A IL-12 by LPS-treated Raw264.7 macrophages; however, deficiency attenuates M1 macrophage polarization in the this increase was reversed by DASA-58 (Fig. 8A and liver of HFD-fed mice (Fig. 4B–D). Supplementary Fig. 19). ELISA assay detected no differ- ence in HSPA12A levels in medium from Hspa12ao/e and HSPA12A Increases Monomeric and Dimeric PKM2 Hspa12an/e Raw264.7 macrophages (Supplementary Fig. Levels and Nuclear Translocation of PKM2 in 20). Macrophages CM collected from DASA-58–treated Hspa12ao/e mac- Nuclear PKM2 promotes M1 macrophage polarization rophage cultures was termed DASA-58 Hspa12ao/e CM. (15). Because mass spectrometry analysis revealed an in- Lipid deposition in AML-12 hepatocytes treated with teraction between PKM and HSPA12A in differentiated DASA-58 Hspa12ao/e CM was significantly lower than adipocytes (Supplementary Fig. 13A and B), we examined that in AML-12 hepatocytes treated with Hspa12ao/e CM whether PKM2 mediates HSPA12A-induced M1 macro- (Fig. 8B). In agreement, we noted a marked reduction in phage polarization. PKM2 expression and nuclear trans- the expression of genes associated with de novo fat location was increased in Raw264.7 macrophages in synthesis and lipolysis in AML-12 hepatocytes treated response to LPS stimulation (Supplementary Fig. 14). Liver with DASA-58 Hspa12ao/e CM compared with AML-12 macrophages of NASH patients and NASH mice demon- hepatocytes treated with Hspa12ao/e CM (Fig. 8C). strated marked increases in PKM2 expression and nuclear translocation (Fig. 6A and B and Supplementary Fig. 15A– HSPA12A Forms Complex with PKM2 C). However, HSPA12A deficiency decreased Pkm2 mRNA Simultaneous nuclear translocation of HSPA12A and expression in liver and PKM2 nuclear translocation in liver PKM2 during M1 macrophage activation motivated us macrophages of HFD-fed mice (Fig. 6C and D and Sup- to investigate whether there is an interaction between plementary Fig. 16). By contrast, HSPA12A overexpression them. Flag-tagged HSPA12A was immunoprecipitated increased PKM2 expression and nuclear translocation in from Hspa12ao/e Raw264.7 macrophages treated with

mRNA levels. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated genes’ expression. *P , 0.05 and **P , 0.01; mean 6 SD; n = 6 for WT group and n = 5 for Hspa12a2/2 group. Data were analyzed by unpaired, two- tailed Student t test. diabetes.diabetesjournals.org Kong and Associates 369

Figure 3—Hepatocyte steatosis was regulated by paracrine effects of macrophage HSPA12A. A: No effects of HSPA12A deficiency on hepatocyte steatosis. Primary hepatocytes isolated from WT and Hspa12a2/2 mice were exposed to OA for 24 h. Vehicle-treated cells served as controls. TAG contents were examined. *P , 0.05 and **P , 0.01; mean 6 SD. Sample numbers are indicated. Data were analyzed by two- way ANOVA analysis followed by Tukey test. N.S., not significant; prot, protein. B: No effects of HSPA12A overexpression on hepatocyte steatosis. Three types of hepatocytes with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with OA or vehicle (control) for 24 h. Lipid accumulation was examined by ORO staining. **P , 0.01; mean 6 SD. Scale bar = 50 mm. Sample numbers are indicated in the figures. Data were analyzed by two-way ANOVA analysis followed by Tukey test. C: Paracrine effects of Raw264.7 macrophage HSPA12A on hepatocyte steatosis. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were challenged with LPS for 16 h. The CM of macrophage cultures was collected and applied to AML-12 hepatocytes for 24 h. Lipid deposition in AML-12 hepatocytes was then evaluated by ORO staining. **P , 0.01; mean 6 SD. Scale bar = 50 mm. Sample numbers are indicated. Data were analyzed by unpaired, two-tailed Student t test. D: Paracrine effects of Primary Kupffer cell HSPA12A on hepatocyte steatosis. Primary Kupffer cells isolated from WT and Hspa12a2/2 mice were challenged with LPS (200 ng/mL) for 16 h. The CMs were collected and applied to AML-12 hepatocytes for 24 h. Lipid deposition in AML-12 hepatocytes was then evaluated by ORO staining. **P , 0.01; mean 6 SD. Scale bar = 50 mm. n = 8/group. Data were analyzed by unpaired, two-tailed Student t test.

LPS or vehicle for 16 h. Precipitates were immunoblotted LPS treatment (Fig. 8D). These data suggest that HSPA12A with antibodies speciﬁc for PKM2 and HSPA12A. PKM2 formed a complex with PKM2, and the formation of the protein was recovered from the ﬂag-tagged HSPA12A HSPA12A-PKM2 complex was increased during macro- immunocomplexes, with more PKM2 recovered after phage polarization to an M1 phenotype. 370 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

Figure 4—Deficiency of HSPA12A reduced M1 macrophage polarization and inflammatory responses in mouse NASH livers. A: Macrophage recruitment. Liver tissues were collected from mice fed with HFD or chow diet for 14 weeks. Frozen sections were immunostained with F4/80 to visualize macrophages. Hoechst 33342 was used to counterstain nuclei. Macrophage numbers were expressed as a percentage of total liver cells. Scale bar = 50 mm. **P , 0.01; mean 6 SD; n = 5 for HFD-WT group and n = 4 for all the other groups. Data were analyzed by two- way ANOVA analysis followed by Tukey test. B: mRNA levels of inflammatory mediators. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated gene expression. *P , 0.05 and **P , 0.01; mean 6 SD; n = 6 for WT group and n = 5 for Hspa12a2/2 group. Data were analyzed by unpaired, two-tailed Student t test. C: mRNA levels of M1 and M2 markers. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated gene expression. *P , 0.05 and **P , 0.01; mean 6 SD; n = 6 for WT group and and n = 5 for Hspa12a2/2 group. Data were analyzed by unpaired, two-tailed Student t test. D: Protein expression. Livers were collected from mice fed with HFD for 14 weeks. Immunoblotting for the indicated proteins was performed. **P , 0.01; mean 6 SD; n = 4 for CD206 and p-NF-kB groups and n = 5 for the other groups. Data were analyzed by unpaired, two-tailed Student t test.

2 2 Macrophage HSPA12A Regulates HFD-Induced marrow from Hspa12a / mice into recipient WT mice 2 2 Hepatic Steatosis in Mice (Hspa12a / /WT). Recipient WT mice transplanted with To examine whether macrophage HSPA12A affects WT bone marrow served as controls (WT/WT). After HFD-induced hepatic steatosis, we transplanted bone a lethal irradiation, all the mice (15/15) without BMT diabetes.diabetesjournals.org Kong and Associates 371

Figure 5—HSPA12A promoted M1 macrophage polarization. A: Human NASH livers demonstrated an increase of HSPA12A nuclear translocation in macrophages. Immunostaining against HSPA12A and CD68 (macrophage) was performed on frozen sections of human livers. Hoechst 33342 was used to counterstain nuclei. Scale bar = 20 mm. **P , 0.01; mean 6 SD; n = 3 human subjects/group. B and C: HSPA12A nuclear translocation was increased in macrophages by LPS. Raw264.7 macrophages were stimulated with LPS for 16 h. Cells were then subjected to the analysis of HSPA12A expression in cytosolic and nuclear fractions by immunoblotting (B) and immunoﬂuo- rescence staining (C). Scale bar = 2 mm. *P , 0.05; **P , 0.01; mean 6 SD; n = 3/group. Data were analyzed by unpaired, two-tailed Student t test. D: HSPA12A increased mRNA expression of M1 markers in LPS-treated macrophages. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cells were then collected for analyzing the indicated M1 markers by real-time PCR. *P , 0.05 and **P , 0.01; mean 6 SD; n = 6/group. Data were analyzed by unpaired, two-tailed Student t test. E: HSPA12A increased protein expression of M1 markers and the TLR4/NF-kB pathway in LPS-treated macrophages. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cells were then collected for analyzing the indicated proteins by immunoblotting. *P , 0.05 and **P , 0.01; mean 6 SD; n = 3/group. Data were analyzed by unpaired, two-tailed Student t test. died within 15 days; however, none of the BMT mice died weight, hepatocyte area, and lipid accumulation (as in- (Supplementary Fig. 21A). HSPA12A expression in white dicated by ORO staining and TAG content) in 2 2 2 2 blood cells from Hspa12a / /WT mice was hardly detect- Hspa12a / /WT mice were reduced compared with able compared with that in WT/WT controls (Supplemen- WT/WT mice (Supplementary Fig. 21D–F). In addition, tary Fig. 21B and C). After an HFD for 14 weeks, liver expression of iNOS, TNF-a, TLR4, and MyD88 protein, 372 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

Figure 6—Increased nuclear PKM2 in macrophages of NASH livers and the inhibitory effect of HSPA12A deficiency. A: Human NASH livers demonstrated increased PKM2 nuclear translocation in macrophages. Immunostaining against PKM2 and CD68 (macrophage) was performed on frozen sections of human livers. Hoechst 33342 was used to counterstain nuclei. Scale bar = 50 mm. **P , 0.01; mean 6 SD; n = 3 human subjects/group. B: Mouse NASH livers demonstrated increased PKM2 nuclear translocation in macrophages. Immunostaining against PKM2 and F4/80 (macrophage) was performed on frozen liver sections of WT mice fed with HFD or chow diet for 14 weeks. Hoechst 33342 was used to counterstain nuclei. Scale bar = 20 mm. **P , 0.01; mean 6 SD; n = 4 mice/group. C and D: HSPA12A deficiency suppressed PKM2 nuclear translocation in macrophages of NASH livers. Livers were collected from mice fed with HFD for 14 weeks. PKM2 expression and nuclear translocation in macrophages were examined by immunoblotting against PKM2 in cytosolic and nuclear protein extracts (C) and immunostaining against PKM2 and F4/80 on frozen sections (D). Scale bar = 20 mm. *P , 0.05; **P , 0.01; mean 6 SD; n = 6/group (C) and n = 4/group (D). Data were analyzed by unpaired, two-tailed Student t test. along with NF-kB phosphorylation, in the livers of schizophrenia (20,21,32). Recently, a study identified 2 2 Hspa12a / /WT mice was lower than in WT/WT mice a correlation between increased HSPA12A expression fed an HFD (Supplementary Fig. 22). and shortened survival of patients with hepatocellular carcinoma (22). However, there is no evidence that DISCUSSION HSPA12A is the direct cause of any pathophysiological Here, we identified macrophage HSPA12A as a novel event. Here, we used human samples, mouse models, and regulator of hepatic inflammation and NASH pathogene- cell culture models to show the following: 1) NASH sis. Thus, strategies aimed at inhibiting macrophage- patients display increased hepatic expression of HSPA12A specific HSPA12A may have potential as therapeutic and higher levels of circulating HSPA12A, which are pos- interventions for NASH patients. itively associated with hepatic steatosis and injury, re- HSPA12A was first cloned from atherosclerotic lesions, spectively; 2) HSPA12A is expressed preferably in liver and its cerebral expression is decreased in patients with macrophages rather than hepatocytes; 3)HSPA12A diabetes.diabetesjournals.org Kong and Associates 373

Figure 7—Inhibition of PKM2 nuclear translocation reversed the HSPA12A-induced promotion of M1 macrophage polarization. DASA-58 reversed the HSPA12A-induced promotion of M1 macrophage polarization. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with DASA-58 or vehicle control in the presence of LPS for 16 h. Cytosolic and nuclear protein fractions were immunoblotted with the indicated antibodies (A). Levels of Mcp-1 and Tnfa mRNA were also analyzed (B). *P , 0.05 and **P , 0.01; mean 6 SD; n = 3/group (A) and n = 6/group (B). Data were analyzed by one-way ANOVA followed by Tukey test. deficiency either globally or in macrophages attenuates and progression of NAFLD (9,10). Hepatic macrophages HFD-induced hepatic steatosis, liver injury, and inflam- comprise resident (Kupffer cells) and recruited macro- matory M1 macrophage responses in mice; and 4) hepa- phages, all of which express macrophage markers such tocyte steatosis is regulated by the paracrine effects of as CD68, F4/80, and CD11b. Macrophages are activated by macrophage HSPA12A, which are mediated, at least in LPS or free fatty acids, which induce an M1 phenotype and part, by nuclear PKM2-dependent modulation of M1 expression of cytokines, chemokines, and signaling mole- macrophage polarization. Taken together, these results cules through TLR4 signaling (7,33,34). Subsequently, provide evidence that the paracrine effects of macrophage chemokines attract blood-derived monocytes/macroph- HSPA12A regulate hepatocyte steatosis and injury. ages to the liver; these cells then release cytokines that Activation of liver macrophages, and subsequent secre- drive NASH progression, which is characterized by in- tion of proinflammatory mediators, is a key event for onset creased hepatocellular lipid accumulation and damage 374 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

Figure 8—Nuclear PKM2 mediated the paracrine effect of macrophage HSPA12A on hepatocyte steatosis. A: DASA-58 reversed the HSPA12A-promoted secretion of cytokines from macrophages. Raw264.7 macrophages were challenged with LPS (500 ng/mL) for 16 h in the presence or absence of DASA-58 (50 mmol/L). Culture medium was collected to analyze IL-1b and IL-6 contents by ELISA assay. *P , 0.05 and **P , 0.01; mean 6 SD; n = 4/group for IL-6 analysis and n = 5/group for IL-1b analysis. Data were analyzed by one-way ANOVA followed by Tukey test. B and C: DASA-58 blocked the paracrine effects of macrophage HSPA12A on hepatocyte steatosis. CM was collected from the LPS-treated Raw264.7 macrophages in the presence or absence of DASA-58 for 16 h. The CM was then applied to the OA-treated AML-12 hepatocytes for 24 h. Hepatocytes were collected for lipid analysis by ORO staining (B) (scale bar = 50 mm) and the indicated gene expression by real-time PCR (C). *P , 0.05 and **P , 0.01; mean 6 SD; n = 8/group (B) and n = 4/group (C). Data were analyzed by one-way ANOVA followed by Tukey test. D: Interaction between HSPA12A and PKM2 in macrophages. RAW264.7 macrophages overexpressing the flag-tagged HSPA12A were challenged with or without LPS for 16 h. Cellular protein extracts were immunoprecipitated (IP) with primary antibody for flag. The immunoprecipitates were blotted (IB) with PKM2 and HSPA12A. Protein extracts without immunoprecipitation (input) served as positive controls, and immunoprecipitates from IgG incubation served as negative controls. E: Schematic represents the regulation of HSPA12A on M1 macrophage polarization and NASH pathogenesis. Macrophage HSPA12A is upregulated by LPS or other stimuli. By directly interacting with PKM2, HSPA12A promotes PKM2 translocation to nuclei, thereby promoting M1 macrophage polarization and secretion of proinflammatory M1 cytokines, and ultimately leads to hepatocyte steatosis through paracrine effects. Both the M1-polarized macrophage-mediated inflammation and hepatocyte steatosis are essential for the pathogenesis of NASH. FFA, free fatty acid. diabetes.diabetesjournals.org Kong and Associates 375

(6,34). Indeed, diet-induced NASH is alleviated by de- PKM2 in macrophages. Collectively, these results indicate pleting Kupffer cells and knockout of IL-1b and TLR4 that HSPA12A interacts with PKM2 and increases its (11,35,36). Thus, limiting polarization of macrophages nuclear translocation, thereby inducing M1 macrophage toward an M1 phenotype is an attractive strategy for polarization and secretion of proinflammatory M1 cyto- preventing progression of NAFLD (9,10). Here, we ob- kines; ultimately, this leads to fat accumulation in hep- served that deficiency of HSPA12A alleviated HFD- atocytes via paracrine effects (Fig. 8E). However, it is evoked onset and progression of NAFLD, which were unknown how HSPA12A regulates PKM2 expression concomitant with reduced macrophage recruitment and and how HSPA12A interacts with PKM2. Further studies M1 polarization; HSPA12A deficiency also reduced need to address these issues. expression of chemokines and proinflammatory cyto- In conclusion, we show that deficiency of HSPA12A has kines in HFD-fed mice. By contrast, overexpression of a beneficial effect on hepatic steatosis and injury in mice HSPA12A increased M1 macrophage polarization in re- with HFD-induced NASH. The underlying mechanism sponse to LPS, as indicated by increased expression of involves paracrine interactions between macrophages iNOS, IL-1b, TNF-a, CCL3, CCL4, and TLR4, and by and hepatocytes. The data suggest that inhibitors of secretion of IL-1b, IL-6, IL-12, and TNF-a. More impor- macrophage-specific HSPA12A may improve treatment tantly, CM from HSPA12A-overexpressing macrophages and management of nonalcoholic liver disease. led to a marked increase in lipid deposition in hepatocytes. However, we observed no change in HSPA12A secretion by macrophages after LPS treatment, suggest- Acknowledgments. The authors thank the Translational Medicine Core ing that elevated serum levels of HSPA12A in NASH Facilities, Medical School of Nanjing University, for generous help in mass spectrum analysis. patients may be due to secretion by other cell types. Funding. This work was supported by the National Natural Science Foundation Considering that hepatocellular HSPA12A had no effect of China (81870234, 81770854, 81571378, 81571290, 81370260, and on its own lipid deposition, our results suggest that 81371450), Jiangsu Province’s Outstanding Medical Academic Leaders program hepatocyte steatosis is regulated by the paracrine effects (15), the Priority Academic Program Development of Jiangsu Higher Education of macrophage HSPA12A. We detected only a modest Institutions, the Collaborative Innovation Center for Cardiovascular Disease Trans- increase in secretion of TNF-a, IL-1b, IL-6, and IL-12 lational Medicine, and Jiangsu Provincial Key Discipline of Medicine by HSPA12A-overexpressing macrophages, suggesting (ZDXKA2016003). that the relationship between macrophage HSPA12A Duality of Interest. No potential conflicts of interest relevant to this article and hepatocyte lipid deposition is regulated by the syn- were reported. ergistic actions of a group of secreted cytokines and/or Author Contributions. Q.K., N.L., H.C., L.D., B.X., and L.F. performed all other as yet undetermined factors. animal study procedures and most of the in vitro experiments. X.Z., X.Ca., T.Q., and X.Ch. collected and analyzed human samples. Z.Z. scored livers for NASH Next, we sought answers to the following two ques- features in humans and mice. C.L. developed the study concept and experimental tions: does M1 polarization mediate the paracrine effects design. Y.L. interpreted the data and wrote the manuscript. L.L. and Z.D. of macrophage HSPA12A on hepatocyte steatosis, and how developed the study concept and experimental design, interpreted the data, does HSPA12A modulate macrophage polarization? Recent and wrote the manuscript. Z.D. is the guarantor of this work and, as such, had full studies show that PKM2 is a critical determinant of access to all the data in the study and takes responsibility for the integrity of the macrophage activation and subsequent inflammatory data and the accuracy of the data analysis. responses. The PKM2 tetramer acts as a powerful glyco- References lytic enzyme that regulates glycolysis in the cytosol. How- 1. Patouraux S, Rousseau D, Bonnafous S, et al. CD44 is a key player in non- ever, PKM monomers or dimers are enzymatically inactive alcoholic steatohepatitis. J Hepatol 2017;67:328–338 and can translocate to the nucleus, where they act as 2. Rinella ME. Nonalcoholic fatty liver disease: a systematic review. JAMA 2015; cofactors to activate expression of IL-1b et al., ultimately 313:2263–2273 – leading to activation of M1 macrophages (15 17). Induc- 3. Diehl AM, Day C. Cause, pathogenesis, and treatment of nonalcoholic tion of PKM2 expression by LPS is mediated by the steatohepatitis. N Engl J Med 2017;377:2063–2072 transcription factors NF-kB and PPARg (14,15,19). In- 4. Satapathy SK, Sanyal AJ. Novel treatment modalities for nonalcoholic terestingly, we observed that PKM2 expression in the liver steatohepatitis. Trends Endocrinol Metab 2010;21:668–675 of NASH patients was upregulated and that this upregu- 5. Srivastava J, Robertson CL, Ebeid K, et al. A novel role of astrocyte elevated lation was more prominent in liver macrophages showing gene-1 (AEG-1) in regulating nonalcoholic steatohepatitis (NASH). Hepatology notable levels of nuclear translocation. The livers of HFD- 2017;66:466–480 fl fed mice also showed more nuclear translocation of PKM2, 6. Ma KL, Ruan XZ, Powis SH, Chen Y, Moorhead JF, Varghese Z. In ammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apoli- which was attenuated by HSPA12A deficiency, suggesting poprotein E knockout mice. Hepatology 2008;48:770–781 a regulation of HSPA12A in PKM2 nuclear translocation. 7. Louvet A, Teixeira-Clerc F, Chobert MN, et al. Cannabinoid CB2 receptors Importantly, preventing nuclear translocation of PKM2 in protect against alcoholic liver disease by regulating Kupffer cell polarization in macrophages reversed HSPA12A-induced M1 polarization mice. Hepatology 2011;54:1217–1226 and blocked the paracrine effects of macrophage HSPA12A 8. Wenfeng Z, Yakun W, Di M, Jianping G, Chuanxin W, Chun H. Kupffer cells: on hepatocellular steatosis. Immunoprecipitation-Western increasingly significant role in nonalcoholic fatty liver disease. Ann Hepatol 2014; blot analyses revealed that HSPA12A forms a complex with 13:489–495 376 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

9. Smith K. Liver disease: Kupffer cells regulate the progression of ALD and 23. Kleiner DE, Brunt EM, Van Natta M, et al.; Nonalcoholic Steatohepatitis NAFLD. Nat Rev Gastroenterol Hepatol 2013;10:503 Clinical Research Network. Design and validation of a histological scoring system 10. Wan J, Benkdane M, Teixeira-Clerc F, et al. M2 Kupffer cells promote M1 for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–1321 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic 24. Li J, Zhang Y, Li C, et al. HSPA12B attenuates cardiac dysfunction and fatty liver disease. Hepatology 2014;59:130–142 remodelling after myocardial infarction through an eNOS-dependent mechanism. 11. Tosello-Trampont AC, Landes SG, Nguyen V, Novobrantseva TI, Hahn YS. Cardiovasc Res 2013;99:674–684 Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced 25. Zhou H, Qian J, Li C, et al. Attenuation of cardiac dysfunction by HSPA12B in mouse model through tumor necrosis factor-a production. J Biol Chem 2012;287: endotoxin-induced sepsis in mice through a PI3K-dependent mechanism. Car- 40161–40172 diovasc Res 2011;89:109–118 12. Zhang YY, Li C, Yao GF, et al. Deletion of macrophage mineralocorticoid 26. Inagaki Y, Nemoto T, Kushida M, et al. Interferon alfa down-regulates receptor protects hepatic steatosis and insulin resistance through ERa/HGF/Met collagen gene transcription and suppresses experimental hepatic fibrosis in mice. pathway. Diabetes 2017;66:1535–1547 Hepatology 2003;38:890–899 13. Yang L, Xie M, Yang M, et al. PKM2 regulates the Warburg effect and 27. Kong Q, Dai L, Wang Y, et al. HSPA12B attenuated acute myocardial is- promotes HMGB1 release in sepsis. Nat Commun 2014;5:4436 chemia/reperfusion injury via maintaining endothelial integrity in a PI3- 14. Yang W, Lu Z. Nuclear PKM2 regulates the Warburg effect. Cell Cycle 2013; K/Akt/mTOR-dependent mechanism. Sci Rep 2016;6:33636 12:3154–3158 28. Na TY, Han YH, Ka NL, et al. 22-S-hydroxycholesterol protects against 15. Palsson-McDermott EM, Curtis AM, Goel G, et al. Pyruvate kinase M2 ethanol-induced liver injury by blocking the auto/paracrine activation of MCP-1 regulates Hif-1a activity and IL-1b induction and is a critical determinant of the mediated by LXRa. J Pathol 2015;235:710–720 warburg effect in LPS-activated macrophages [published correction appears in 29. Ye D, Yang K, Zang S, et al. Lipocalin-2 mediates non-alcoholic steato- Cell Metab 2015;21:347]. Cell Metab 2015;21:65–80 hepatitis by promoting neutrophil-macrophage crosstalk via the induction of 16. Shirai T, Nazarewicz RR, Wallis BB, et al. The glycolytic enzyme PKM2 CXCR2. J Hepatol 2016;65:988–997 bridges metabolic and inflammatory dysfunction in coronary artery disease. J Exp 30. Corcoran SE, O’Neill LA. HIF1a and metabolic reprogramming in in- Med 2016;213:337–354 flammation. J Clin Invest 2016;126:3699–3707 17. Xie M, Yu Y, Kang R, et al. PKM2-dependent glycolysis promotes NLRP3 and 31. Giannoni E, Taddei ML, Morandi A, et al. Targeting stromal-induced pyruvate AIM2 inflammasome activation. Nat Commun 2016;7:13280 kinase M2 nuclear translocation impairs oxphos and prostate cancer metastatic 18. Weinlich R, Bortoluci KR, Chehab CF, et al. TLR4/MYD88-dependent, LPS- spread. Oncotarget 2015;6:24061–24074 induced synthesis of PGE2 by macrophages or dendritic cells prevents anti-CD3- 32. Friedman LK, Mancuso J, Patel A, et al. Transcriptome profiling of hippo- mediated CD95L upregulation in T cells. Cell Death Differ 2008;15:1901–1909 campal CA1 after early-life seizure-induced preconditioning may elucidate new 19. Panasyuk G, Espeillac C, Chauvin C, et al. PPARg contributes to PKM2 and genetic therapies for epilepsy. Eur J Neurosci 2013;38:2139–2152 HK2 expression in fatty liver. Nat Commun 2012;3:672 33. Baffy G. Kupffer cells in non-alcoholic fatty liver disease: the emerging view. J 20. Han Z, Truong QA, Park S, Breslow JL. Two Hsp70 family members ex- Hepatol 2009;51:212–223 pressed in atherosclerotic lesions. Proc Natl Acad Sci U S A 2003;100:1256–1261 34. Koyama Y, Brenner DA. Liver inflammation and fibrosis. J Clin Invest 2017; 21. Pongrac JL, Middleton FA, Peng L, Lewis DA, Levitt P, Mirnics K. Heat shock 127:55–64 protein 12A shows reduced expression in the prefrontal cortex of subjects with 35. Kamari Y, Shaish A, Vax E, et al. Lack of interleukin-1a or interleukin-1b schizophrenia. Biol Psychiatry 2004;56:943–950 inhibits transformation of steatosis to steatohepatitis and liver fibrosis in hy- 22. Yang Z, Zhuang L, Szatmary P, et al. Upregulation of heat shock proteins percholesterolemic mice. J Hepatol 2011;55:1086–1094 (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) in tumour tissues is associated 36. Csak T, Velayudham A, Hritz I, et al. Deficiency in myeloid differentiation with poor outcomes from HBV-related early-stage hepatocellular carcinoma. Int J factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis Med Sci 2015;12:256–263 and fibrosis in mice. Am J Physiol Gastrointest Liver Physiol 2011;300:G433–G441