HSPA12A Is aNovel Player in Nonalcoholic Steatohepatitisvia Promoting Nuclear PKM2-Mediated M1 MacrophagePolarizationQiuyue 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 themost prevalentcause of chronic liver disease worldwide. Macrophage-mediated inflammation plays a critical role in NASHpathogenesis; however, optimum therapies for macro-phage activation and NASH remain elusive. HSPA12Aencodes a novel member of the HSP70 family. Here,we report that NASH patients showed increased hepaticHSPA12A expression and serum HSPA12A contents. In-triguingly, knockout of HSPA12A (Hspa12a2/2) in miceattenuated high-fat diet (HFD)–induced hepatic steatosisand injury. HFD-induced macrophage polarization to-ward an M1 phenotype and inflammatory responsesin the liver of Hspa12a2/2 mice were also attenuated.Loss- and gain-of-function studies revealed that the denovo lipogenesis in hepatocytes was regulated by theparacrine effects of macrophage HSPA12A rather thanby hepatocyte HSPA12A. In-depth molecular analysisrevealed that HSPA12A interacted with the M2 isoformof pyruvate kinase (PKM2) in macrophages and in-creased its nuclear translocation, thereby promotingM1 polarization and secretion of proinflammatory M1cytokines; this led, ultimately, to hepatocyte steatosisvia paracrine effects. Taken together, these findingsshow that HSPA12A acts as a novel regulator of M1macrophage polarization and NASH pathogenesis byincreasing nuclear PKM2. Strategies that inhibit macro-phage HSPA12A might be a potential therapeutic inter-vention for NASH.

Nonalcoholic fatty liver disease (NAFLD) affects;22–28%of the global population (1). The spectrum of NAFLDbegins with isolated steatosis, with subsequent develop-ment of steatohepatitis (NASH) and steatofibrosis, even-tually leading to cirrhosis and hepatocellular carcinoma.Patients with NAFLD, particularly NASH, show reducedsurvival, primarily due to cardiovascular and liver-relateddiseases (2). NASH livers are more injured than livers withisolated steatosis, which more likely leads to progressivefibrosis and eventual liver-associated illness and death (3).The molecular players that modulate disease pathogenesisare starting to be identified, enabling translation of re-search findings to clinical trials (4,5). However, no opti-mum therapy is yet available, suggesting that a morecomprehensive understanding of the onset and progres-sion of NAFLD is needed.

The shift from isolated steatosis to NASH is definedhistologically as emergence of liver inflammation andhepatocellular injury. The most common theory explainingthis shift is the “double-hit hypothesis” (3,5,6). The firsthit consists mainly of lipid accumulation, whereas thesecond comprises sequential innate immune responses(6–8). Compelling evidence indicates that liver macro-phages play a key role in regulating the second hit. Macro-phages are a highly heterogeneous, plastic cell populationthat undergoes pleiotropic and coordinated responses to theimmunological environment; two common phenotypes

1Department of Anesthesiology, First Affiliated Hospital of Nanjing MedicalUniversity, Nanjing, China2Department of Geriatrics, First Affiliated Hospital of Nanjing Medical University,Nanjing, China3Department of Pathology, First Affiliated Hospital of Nanjing Medical University,Nanjing, China4Department of Surgery, East Tennessee State University, Johnson City, TN5Department of Pathophysiology, Nanjing Medical University, Nanjing, China6State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Labo-ratory of Molecular Medicine and School of Medicine, Nanjing University, Nanjing,China7Medical School of Nanjing University, Nanjing, China

Corresponding authors: Zhengnian Ding, zhengnianding@njmu.edu.cn, and Li Liu,liuli@njmu.edu.cn

Received 12 January 2018 and accepted 23 October 2018

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0035/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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PATHOPHYSIO

LOGY

are “proinflammatory” M1 and “immunoregulatory” M2macrophages (7,9,10). Polarization of macrophages towardthe M1 phenotype plays a critical role in pathogenesis ofseveral chronic inflammatory disorders, including NASH.By contrast, M2 macrophages promote resolution of in-flammation (10). Indeed, suppressing M1 polarizationalleviates diet-induced NASH (7,10–12).

The M2 isoform of pyruvate kinase (PKM2) is a rate-limiting enzyme of glycolysis. Cis-trans isomerization andconversion of PKM2 from a tetramer to a dimer or mono-mer lead to nuclear translocation and regulate expressionof target genes that play roles in the Warburg effect andcell cycle progression (13,14). Intriguingly, nuclear PKM2is a critical determinant of macrophage M1 activation andis involved in inflammatory diseases such as coronaryartery disease (13,15,16). Nuclear PKM2 increasesmacrophage M1 activation in response to lipopolysac-charides (LPS), mainly through TLR4 signaling (13,15,17,18). A recent study shows that PKM2 expression isassociated with liver mass (19); however, the role of PKM2in NASH pathogenesis is largely unknown.

HSPA12A is a novel and atypical member of the HSP70family (20). Hspa12a mRNA is expressed at high levels inthe brain of humans andmice under normal conditions butis decreased in the prefrontal cortex of patients withschizophrenia (20,21). Of particular interest, expressionof HSPA12A is associated with shortened survival ofpatients with hepatocellular carcinoma (22), suggestingpossible involvement of HSPA12A in modulating hepatichomeostasis. However, the role of HSPA12A in any dis-orders, including liver disease, is unknown.

Here, we report that NASH patients showed increasedhepatic HSPA12A expression and serum HSPA12A con-tents. Intriguingly, knockout of HSPA12A (Hspa12a2/2) inmice attenuated high-fat diet (HFD)–induced NASH path-ogenesis and M1 macrophage activation. Further molecu-lar analyses revealed that regulation of de novo lipogenesisin hepatocytes was regulated by the paracrine effects ofmacrophage HSPA12A via promotion of nuclear PKM2-mediated M1 polarization. Taken together, these findingssuggest that HSPA12A acts as a novel regulator of mac-rophage polarization and NASH pathogenesis. Strategiesdesigned to inhibit macrophage-specific expression ofHSPA12A might have therapeutic potential for patientswith NASH.

RESEARCH DESIGN AND METHODS

Human Samples

Liver SpecimensLiver specimens were collected from patients who under-went hepatic hemangioma surgery at the First AffiliatedHospital of Nanjing Medical University. Patients providedinformed consent at the time of recruitment. Normal orNASH livers were identified using hematoxylin-eosin (H-E)staining, Oil Red O (ORO) staining, and serum alanineaminotransferase (ALT) and AST activity measurements,

according to the NASH Clinical Research Network–mod-ified Brunt methodology and other studies (3,23). Liverswith bridging fibrosis or cirrhosis were excluded usingMasson’s staining (Supplementary Fig. 1A and B andSupplementary Table 1A and B).

Blood SamplesHuman blood samples were collected from bariatric sur-gery patients with NASH diagnosed after clinic and B-typeultrasonographic assessment (Supplementary Table 2Aand B). Patients were fasted for 12 h before blood sam-pling. The study was approved by the Ethical Board of theFirst Affiliated Hospital of Nanjing Medical University(2016-SR-122 and 2016-SR-123). All human studieswere conducted according to the World Medical Associa-tion Declaration of Helsinki.

Hspa12a Knockout MiceConditional Hspa12a knockout mice were generated usingthe loxP and Cre recombinant system. The Hspa12a target-ing vector was constructed using the bacterial artificialchromosome (BAC) retrieval method. In brief, the regionof the Hspa12a gene containing exons 2–4 was retrievedfrom a 129/sv BAC clone (BACPAC Resources Center,Oakland, CA) using a retrieval vector containing twohomologous arms. Exons 2 and 3 were replaced by loxPsites flanking a PGK-neo cassette (a positive selectionmarker) (Supplementary Fig. 2A). The breeding strategy togenerate wild-type (WT) (Hspa12aflox/flox) and Hspa12a2/2

(Hspa12aflox/flox, CreTg) mice is shown in SupplementaryFig. 2B. Mice were bred at the Model Animal ResearchCenter of Nanjing University and maintained on a 12-hlight/dark cycle at 23 6 1°C with access to food and waterad libitum. All experiments conformed to the Guide for theCare and Use of Laboratory Animals published by the U.S.National Institutes of Health (8th edition, 2011). Theanimal care and experimental protocols were approvedby the Committee on Animal Care at Nanjing University.

HFD Feeding ProtocolFor the HFD experiments, mice were fed an HFD (60% ofkcal from fat, D-12492; Research Diets, New Brunswick,NJ) for 14 weeks beginning at age 5 weeks. Mice that werefed a normal chow diet and received only 6% of kcal fromfat served as controls.

Immunoblotting and Immunoprecipitation-ImmunoblottingCytosolic and nuclear proteins were prepared from liversor cells using the NucBuster protein extraction kit accord-ing to the manufacturer’s instructions (Novagen, Darm-stadt, Germany). Western blotting was performed accordingto our previous methods (24,25). To control for laneloading, the membranes were probed with anti-GAPDHor anti–a-tubulin antibodies for cytosolic proteins andanti-histone H3 or anti-lamin A/C antibodies for nuclearproteins.

362 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

For analyzing the interaction between HSPA12Aand PKM2 by immunoprecipitation-immunoblotting,Raw264.7 macrophages were overexpressed with flag-tagged HSPA12A. After challenge with LPS or vehiclefor 16 h, cells were collected for protein extraction. Ali-quots of equal volume and protein content were precip-itated with anti-flag antibodies, followed by Westernblotting with anti-PKM2 and anti-HSPA12A antibodies,as described previously (26). The antibodies used in theexperiments are listed in Supplementary Table 3.

Quantitative Real-time PCRQuantitative real-time PCR was performed as describedpreviously (27). In brief, total RNA was extracted for cDNAsynthesis using the oligo (dT) primer. After cDNA synthe-sis, the expressions of indicated genes were estimated byreal-time PCR using the SYBR Green master mix (Roche,Indianapolis, IN). The PCR results of b-actin served asinternal controls. The primers used for PCR are listed inSupplementary Table 4.

Biochemical AnalysisLevels of HSPA12A in human serum and insulin in mouseserum and secretion of HSPA12A, IL-1b, IL-6, IL-12, andTNF-a from macrophages were detected using ELISA kits(Supplementary Table 5) according to the manufacturer’sinstructions. Serum ALT and AST activity, glucose, LDLcholesterol, HDL cholesterol, total cholesterol, and tri-glyceride (TAG) were measured using a Beckman CoulterAU5800 Chemistry System analyzer (Brea, CA).

Measurement of Lipid SynthesisLipid content was evaluated using both ORO (Sigma-Aldrich, St. Louis, MO) staining and a TAG assay. OROstaining was performed on 4% paraformaldehyde-fixedfrozen liver sections or hepatocyte cultures. Quantificationwas performed using spectrophotometry at a wavelengthof 510 nm after extraction of the stained ORO in cellcultures. TAG was evaluated in liver tissues or hepatocytecultures using a TAG assay kit according to the manufac-turer’s instructions (Jiancheng Biotech, Nanjing, China).

Histological Analysis, Masson’s Staining, andImmunofluorescence StainingParaffin-embedded liver sections were stained with H-E toevaluate the histological changes and averaged hepatocyteareas. Masson’s staining was also performed to indicatefibrosis, as described previously (24). Immunofluorescencestaining of frozen liver sections or Raw264.7 macrophageswas performed as described previously (24,25) using ap-propriate antibodies (Supplementary Table 3). In brief,after incubation with the indicated primary antibodiesovernight at 4°C, Cy3- or FITC-conjugated appropriatesecondary antibodies were added to the sections to visu-alize the staining. Hoechst 33342 reagent was used tocounterstain the nuclei. The staining was observed andquantified in 7–10 randomly selected areas of each sample

using a fluorescence microscope with cellSens Dimension1.15 software (Olympus, Tokyo, Japan).

Adenovirus ConstructionThe adenoviral vector containing a three flags–taggedcoding region of mouse Hspa12a (NM_175199) was gen-erated by GeneChem (Shanghai, China). A schematic over-view of virus construction is shown in SupplementaryFig. 3.

Cell Culture and Treatment

CulturePrimary hepatocytes and liver macrophages (Kupffer cells)were isolated from 7- to 10-week-old mice by digestionwith 0.06% collagenase type IV, followed by centrifugationon a 25–50% Percoll gradient, as described previously (28).Primary hepatocytes were grown in DMEM supplementedwith 10% FBS and 0.01 mmol/L dexamethasone. MouseRaw264.7 macrophages and human hepatocellular carci-noma HepG2 cells were grown in DMEM supplementedwith 10% FBS. Mouse AML-12 hepatocytes were culturedin DMEM/F12 supplemented with 10% FBS, ITS (5 mg/mLinsulin, 5 mg/mL transferrin, and 5 ng/mL selenium), and40 ng/mL dexamethasone.

Overexpression of HSPA12A (Hspa12ao/e)Cells were infected for 48 h with adenovirus carryingthe Hspa12a expression sequence. Cells infected withempty adenovirus served as normal expression con-trols (Hspa12an/e).

Effect of HSPA12A on Hepatocyte SteatosisHspa12ao/e or Hspa12a2/2 hepatocytes were incubatedwith 200 mmol/L oleic acid (OA) for 24 h.

Collection of Conditioned Medium from MacrophagesHspa12ao/e or Hspa12an/e Raw264.7 macrophages weretreated for 16 h with LPS (500 ng/mL) to induce M1polarization. The medium was then collected (referred tohere as Hspa12ao/e conditioned medium [CM] or Hspa12an/e

CM).

Paracrine Effects of Macrophage HSPA12A onHepatocyte SteatosisAML-12 hepatocytes were incubated for 24 h withHspa12ao/e CM or Hspa12an/e CM in the presence of OA(200 mmol/L).

Activation of Kupffer CellsAfter they were grown for 48 h, WT and Hspa12a2/2

Kupffer cells were incubated with LPS (200 ng/mL) for16 h. Kupffer cells were harvested for immunoblottinganalysis, and the medium were collected and used as WTCM and Hspa12a2/2 CM, respectively.

Cross-linkingRAW 264.7 macrophages were treated with 500 ng/mLLPS for 16 h. Cross-linking was performed using

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500 mmol/L disuccinimidyl suberate (Thermo Fisher Sci-entific, Waltham, MA) for 30 min. Lysates were analyzedby Western blot as previously described (15).

Bone Marrow TransplantationTo examine the effects of macrophage HSPA12A on HFD-induced NASH, we transplanted the bone marrows ofHspa12a2/2 mice to WT mice according to previouslydescribed methods (29). In brief, the recipient C57BL/6WT mice (6 weeks old) were given acidified and antibioticwater (100 mg/L neomycin and 10 mg/L polymyxin) for1 week before irradiation. At the day of transplantation,recipient mice were given a lethal irradiation (9 Gy). Fourhours after irradiation, a total of 5 3 106 bone marrowcells isolated from donor WT orHspa12a2/2mice (4 weeksold) were intravenously injected into a recipient WTmousevia tail vein, referred to as Hspa12a2/2/WT and WT/WTmice, respectively. After receiving acidified and antibioticwater for 4 weeks after transplantation, the bone marrowtransplantation (BMT) mice were fed with HFD for14 weeks.

Liquid Chromatography–Tandem Mass SpectrometryAnalysisPrimary mouse adipocytes differentiated from preadipo-cytes were used in the experiments. The primary mousepreadipocytes were used as controls. The proteins thatspecifically interact with HSPA12A were pulled down byHSPA12A antibody and were separated by SDS-PAGEfollowed by Coomassie blue staining, digestion in gel withtrypsin, and analysis by liquid chromatography–tandemmass spectrometry. In brief, peptides were dissolved insolvent A (2% formic acid in 3% acetonitrile) and loadeddirectly onto a reversed-phase trap column (Chrom XPC18-CL-3m 120A; Eksigent). Peptide separation was per-formed using a reversed-phase analytical column (3 mm,120A) (3C18-CL-120; Eksigent). Eluting peptides from thecolumn were analyzed using an AB Sciex 5600+ TripleTOFsystem. Tandem mass spectrometry data were processedusing ProteinPilot Software 4.5 (AB Sciex). Tandem massspectra were searched against the UniProt mouse database(16,923 sequences) concatenated with a reverse decoydatabase. Trypsin/P was specified as the cleavage enzyme,allowing up to three missing cleavages, four modificationsper peptide, and five charges.

Statistical AnalysisData are expressed as the mean 6 SD. Groups werecompared using an unpaired, two-tailed Student t test orusing one-way or two-way ANOVA followed by Tukey posthoc test. A P value of ,0.05 was considered significant.

RESULTS

Liver Macrophages Express High Levels of HSPA12AThe expression profile of HSPA12A protein in liver andcells is unclear. Western blotting revealed low-level ex-pression of HSPA12A in mouse liver (Supplementary Fig.

4). In mouse liver, expression of HSPA12A bymacrophageswas 17.2-fold higher than that in hepatocytes (Fig. 1A).Macrophages in human livers also showed abundant ex-pression of HSPA12A, whereas expression by hepatocyteswas barely detectable (Fig. 1B and Supplementary Fig. 5).

Upregulation of HSPA12A in Human Patients Showsa Positive Correlation with NASHTo investigate the clinical significance of HSPA12A inNASH, we evaluated expression of HSPA12A in NASHpatients. Expression ofHspa12amRNA in NASH livers wassignificantly higher than that in non-NASH controls (Fig.1C). Moreover, we observed a positive correlation betweencirculating HSPA12A levels and ALT and AST activity inNASH patients (Fig. 1D and Supplementary Fig. 6). Un-expectedly, expression of HSPA12A decreased in the cy-tosolic fraction but increased in the nuclear fraction,suggesting nuclear translocation of HSPA12A in the hu-man NASH liver (Fig. 1E). Similar distribution of HSPA12Awas observed in the livers of mice fed an HFD (Fig. 1F).Therefore, NASH is associated with upregulated expres-sion of HSPA12A.

HSPA12A Deficiency Ameliorates HFD-Induced NASHBecause expression of HSPA12A was upregulated in NASHlivers, it is conceivable that HSPA12A knockout may havetherapeutic potential. Therefore, we generated HSPA12Aknockout mice (Hspa12a2/2) using the Cre-loxP recombi-nant system (Supplementary Fig. 2A and B). Successfulknockout of HSPA12A in the liver was verified by proteinanalysis (Fig. 2A). The body weight of adult Hspa12a2/2

mice, along with serum levels of glucose and lipids, wassimilar to that in WTmice with access to food and water adlibitum, as were fasting serum insulin levels (Supplemen-tary Fig. 7A–C). Liver weight, hepatocyte size, and lipidamount were similar in Hspa12a2/2 and WT mice feda normal chow diet (Fig. 2B–D). However, HFD-inducedincreases in liver weight and hepatocyte size were de-creased in Hspa12a2/2 compared with WT mice (Fig. 2Band C). Furthermore, HFD-induced steatosis was lessmarked in Hspa12a2/2 mice, as indicated by reducedTAG content, histological alteration, and ORO staining(Fig. 2C and D). Notably, HFD-induced liver injury, asindicated by elevated serum ALT and AST activities, wasabolished in Hspa12a2/2 mice (Fig. 2E). No significantfibrosis, indicated by Masson’s staining, was observed inboth genotypes after HFD feeding for 14 weeks (Supple-mentary Fig. 8). Taken together, these findings stronglysuggest that HSPA12A deficiency prevents progression ofNAFLD to NASH.

Hspa12a2/2 Mice Show Decreased Expression ofGenes Involved in De Novo Fat Synthesis Upon HFDFeedingTo gain insight into the mechanisms underlying HSPA12Adeficiency–mediated inhibition of NAFLD progression, weexamined expression of genes linked to lipid metabolism in

364 HSPA12A Regulates NASH Pathogenesis Diabetes Volume 68, February 2019

Figure 1—HSPA12A upregulation was positively correlated with human NASH. A: HSPA12A expression was examined in primaryhepatocytes andmacrophages. Primary cells were isolated frommice for immunoblotting analysis. **P, 0.01; mean6SD; n = 3mice/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 wasused to counterstain the nuclei. Note that HSPA12A, indicated by arrows in images, was preferably expressed in macrophages (right panel)comparedwith hepatocytes (left panel). Scale bar = 5mm. **P, 0.01; mean6 SD; n = 3 human subjects/group.C: IncreasedHspa12amRNAexpression 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

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the livers of HFD-fed mice. We noted a significant decreasein transcription of lipogenic transcription factors (Chrebp,Srebp-1c, Ppara, and Pparg) in Hspa12a2/2 livers comparedwith WT livers (Fig. 2F). In agreement, the expression oftarget genes, including Gck, Lpk, Acc, Fas, Scd1, Gpat,Dgat2, and Elovl6, involved in de novo fat synthesiswere reduced in Hspa12a2/2 livers compared with WTlivers (Fig. 2F). We also noticed that genes regulating lipidstorage and transportation (Cidea, Plin1, and Apob) andlipolysis (Hsl and Cgi58) were downregulated inHspa12a2/2 livers (Supplementary Fig. 9A and B). Asthe most prominent changes in expression were observedfor transcription factors and target genes involved in denovo fat synthesis, we examined expression of PPARgand SCD1 proteins. In line with the aforementionedresults, expression of both proteins was reduced in theliver of Hspa12a2/2 compared with WT controls uponHFD feeding (Supplementary Fig. 9C). The findings sug-gest that HSPA12A regulates de novo fat synthesis andother lipid metabolic signaling in the liver upon HFDfeeding.

Lipid Accumulation in Hepatocytes Is Regulated by theParacrine Effects of Macrophage HSPA12ATo investigate how HSPA12A regulates accumulation oflipids in hepatocytes, we examined the effects of loss- andgain-of-HSPA12A function on lipid deposition using threetypes of hepatocyte culture: primary mouse hepatocytes,mouse hepatocyte AML-12 cells, and human hepatocellularcarcinoma HepG2 cells. Unexpectedly, we found thatOA-induced lipid deposition in primary hepatocytes wasnot affected by either Hspa12a deficiency or overexpres-sion (Fig. 3A and B). Also, overexpression of HSPA12A didnot affect OA-induced lipid deposition in either AML-12 orHepG2 hepatocytes (Fig. 3B).

Because hepatic macrophages showed higher HSPA12Aexpression than hepatocytes (Fig. 1A and B), we designedculture experiments to evaluate the interaction betweenmacrophage HSPA12A and hepatocyte lipid deposition. Todo this, we overexpressed HSPA12A (Hspa12ao/e) inRaw264.7 macrophages by infection with the adenoviruscarrying the Hspa12a expression sequence; macrophagesinfected with empty virus served as normal expressioncontrols (Hspa12an/e). The CM collected from these mac-rophage cultures were referred to as Hspa12ao/e CM andHspa12an/e CM. Notably, Hspa12ao/e CM increased theOA-induced lipid deposition in AML-12 hepatocytes

(Fig. 3C). The regulation of primary Kupffer cell HSPA12Aon hepatocyte lipid deposition was also examined. The CMwas collected from LPS-treated primary Hspa12a2/2 andWT Kupffer cell cultures, referred to as Hspa12a2/2 CMand WT CM, respectively. AML-12 hepatocytes incubatedwith Hspa12a2/2 CM accumulated significantly less lipidthan cells treated with WT CM in the presence of OA (Fig.3D). Taken together, these data indicate that macrophageHSPA12A regulates hepatocyte steatosis through para-crine effects.

HSPA12A Deficiency Reduces Both MacrophageRecruitment and M1 Polarization in the Liver of HFD-Fed MiceThe effects of macrophage HSPA12A on hepatocyte lipiddeposition prompted us to focus on macrophage activationand inflammatory responses because they play criticalroles in NASH pathogenesis (7,10). Indeed, upon HFDfeeding, we found a significant reduction in the number ofF4/80-positive cells in the liver of Hspa12a2/2 mice com-pared with that of WT mice (Fig. 4A). Accordingly, analysisof mRNA levels revealed markedly lower expressionof proinflammatory mediators and M1 markers, includingIl-1b, Il-6, Tnfa, Nfkb, Tlr4, Inos, Mcp1, Ifng, Il-12, Cd86,and Cd68, in livers ofHspa12a2/2mice compared with WTcontrols upon HFD (Fig. 4B and C). By contrast, the liversof Hspa12a2/2 mice showed markedly higher expressionof mRNA encoding M2 markers, including Cd163, Arg1,and Cd206, than that of WT mice fed an HFD (Fig. 4C). Inagreement, protein analysis confirmed reduced expres-sion of iNOS (an M1 marker) and increased expres-sion of CD206 (an M2 marker) in livers from HFD-fedHspa12a2/2 mice (Fig. 4D). Moreover, livers fromHspa12a2/2 mice showed lower expression of TLR4,MyD88, and phosphorylated (p-) NF-kB (p65) than thosefrom WT controls upon HFD (Fig. 4D). Collectively, thesedata indicate that HSPA12A deficiency attenuates bothmacrophage recruitment and M1 polarization in the liverof HFD-fed mice.

Overexpression of HSPA12A Promotes M1Macrophage Polarization by Increasing NuclearTranslocation of PKM2

HSPA12A Is Upregulated During M1 MacrophagePolarizationTo investigate how HSPA12A regulates macrophagepolarization, we first examined whether HSPA12A

unpaired, two-tailed Student t test. D: Increased circulating HSPA12A content correlated with hepatic injury in NASH patients. Hepatic injurywas 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;mean6SD; n = 17 for the normal ALT group and n = 11 for elevated ALT group, and n = 22 for the normal AST groupand n = 6 for elevated AST group. Data were analyzed by unpaired, two-tailed Student t test. E: Nuclear translocation of HSPA12A in livers ofhuman NASH patients. Cytosolic and nuclear protein extracts were prepared from human livers with or without NASH. HSPA12A expressionwas analyzed by immunoblotting analysis. Blots for GAPDH or histone H3 served as loading controls. **P, 0.01; mean6 SD; n = 3 humanindividuals/group. Datawere analyzed by unpaired, two-tailed Student t test. F: Nuclear translocation of HSPA12A in livers of NASHmice.WTmice were fed with HFD for 14 weeks to induce NASH. Mice fed with normal chow served as controls. Cytosolic and nuclear protein extractswere prepared for immunoblotting against HSPA12A. *P , 0.05 and **P , 0.01; mean 6 SD; n = 4 mice/group. Data were analyzed byunpaired, two-tailed Student t test.

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Figure 2—Deficiency of HSPA12A attenuated HFD-induced hepatic steatosis and injury. A: HSPA12A expression was absent in livers ofHspa12a2/2 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) wasnormalized with body length (BL). **P , 0.01; mean6 SD; sample numbers are indicated. Data were analyzed by two-way ANOVA analysisfollowed by Tukey test.C: Hepatic histology. After feeding with HFD or chow for 14 weeks, livers were removed and analyzed by H-E stainingon paraffin sections. Hepatocyte areas weremeasured. Arrows indicate the lipid droplet cavitation. **P, 0.01; mean6 SD; n = 7 for HFD-WTgroup and n = 5 for other groups. Data were analyzed by two-way ANOVA analysis followed by Tukey test. Scale bar = 20 mm. D: Lipoidaccumulation. Top panel: after feeding with HFD or chow for 14 weeks, frozen liver sections were prepared for ORO staining to indicate lipiddeposition. n = 4 for HFD-WT group and n = 5 for other groups. Bottom panel: after feeding with HFD for 14 weeks, livers were collected forTAG content analysis. *P, 0.05; mean6SD; n = 4 forWT group and n = 5 forHspa12a2/2 group. Data were analyzed by unpaired, two-tailedStudent t test. Scale bar = 20 mm. E: Hepatic function. Serum samples were collected from mice fed with HFD or chow diet for 14 weeks forALT 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:

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expression is altered upon activation of M1 macrophages.Liver macrophages from NASH patients showed markedlyhigher protein expression and nuclear translocation ofHSPA12A than controls, as illustrated by immunostaining(Fig. 5A and Supplementary Fig. 10). The same pattern ofHSPA12A expression and distribution was observed inRaw264.7 macrophages after LPS treatment, as indicatedby immunoblotting and immunostaining (Fig. 5B and C).We also observed increased expression of HSPA12A inRaw264.7 macrophages exposed to OA (SupplementaryFig. 11). These findings suggest that HSPA12A in macro-phages is upregulated and nuclear translocated during anM1 response.

HSPA12A Promotes M1 Macrophage PolarizationNext, we examined the direct effects of HSPA12A on theM1 macrophage response. Overexpression of HSPA12Aincreased mRNA expression of M1 markers (Inos, Il-1b,Tnfa, Mcp1, Ccl3, and Ccl4) in LPS-treated Raw264.7macrophages (Fig. 5D). In agreement, expression ofiNOS and TNF-a protein in Hspa12ao/e macrophageswas higher than that in Hspa12an/e macrophages in thepresence of LPS (Fig. 5E). Expression of TLR4 and p-NF-kB, both critical for M1 macrophage activation, also in-creased in Hspa12ao/e macrophages (Fig. 5E). By contrast,primary Kupffer cells from Hspa12a2/2 mice showed de-creased expression of iNOS, TLR4 MyD88, and p-NF-kBprotein compared with WT Kupffer cells after LPS stim-ulation (Supplementary Fig. 12). These findings confirmthe aforementioned in vivo results showing that HSPA12Adeficiency attenuates M1 macrophage polarization in theliver of HFD-fed mice (Fig. 4B–D).

HSPA12A Increases Monomeric and Dimeric PKM2Levels and Nuclear Translocation of PKM2 inMacrophagesNuclear PKM2 promotes M1 macrophage polarization(15). Because mass spectrometry analysis revealed an in-teraction between PKM and HSPA12A in differentiatedadipocytes (Supplementary Fig. 13A and B), we examinedwhether PKM2 mediates HSPA12A-induced M1 macro-phage polarization. PKM2 expression and nuclear trans-location was increased in Raw264.7 macrophages inresponse to LPS stimulation (Supplementary Fig. 14). Livermacrophages of NASH patients and NASH mice demon-strated marked increases in PKM2 expression and nucleartranslocation (Fig. 6A and B and Supplementary Fig. 15A–C). However, HSPA12A deficiency decreased Pkm2 mRNAexpression in liver and PKM2 nuclear translocation in livermacrophages of HFD-fed mice (Fig. 6C and D and Sup-plementary Fig. 16). By contrast, HSPA12A overexpressionincreased PKM2 expression and nuclear translocation in

Raw264.7 macrophages (Fig. 7A and Supplementary Fig.17). In line with this, Hspa12ao/e Raw264.7 macrophagesexpressed higher levels of monomeric and dimeric PKM2than Hspa12an/e control cells in the presence of LPS(Supplementary Fig. 18). Collectively, these data suggestthat HSPA12A regulates PKM2 expression and nucleartranslocation during M1 macrophage polarization.

Nuclear PKM2 Mediates the Effects of HSPA12Aon M1 Macrophage ActivationTo determine the role of PKM2 during HSPA12A-mediatedM1 macrophage polarization in response to LPS,Hspa12ao/e Raw264.7 macrophages were treated witha small molecule, DASA-58. DASA-58 is a specific enzymeactivator of PKM2, which can prevent PKM2 nucleartranslocation (15,30,31). Notably, DASA-58 reversed theHSPA12A overexpression-induced nuclear translocationof PKM2 and expression of M1 markers (iNOS, MCP-1,and TNF-a) in LPS-treated macrophages (Fig. 7A and B).Taken together, these findings suggest that nuclearPKM2 mediates the HSPA12A-induced M1 macrophagepolarization.

Nuclear PKM2 Mediates the Paracrine Effects ofMacrophage HSPA12A on Hepatocyte SteatosisNext, we asked whether nuclear PKM2 mediates the para-crine effects of macrophage HSPA12A on hepatocytesteatosis. We found that overexpression of HSPA12Aled to increased secretion of IL-1b, IL-6, TNF-a, andIL-12 by LPS-treated Raw264.7 macrophages; however,this increase was reversed by DASA-58 (Fig. 8A andSupplementary Fig. 19). ELISA assay detected no differ-ence in HSPA12A levels in medium from Hspa12ao/e andHspa12an/e Raw264.7 macrophages (Supplementary Fig.20).

CM collected from DASA-58–treated Hspa12ao/e mac-rophage cultures was termed DASA-58 Hspa12ao/e CM.Lipid deposition in AML-12 hepatocytes treated withDASA-58 Hspa12ao/e CM was significantly lower thanthat in AML-12 hepatocytes treated with Hspa12ao/e CM(Fig. 8B). In agreement, we noted a marked reduction inthe expression of genes associated with de novo fatsynthesis and lipolysis in AML-12 hepatocytes treatedwith DASA-58 Hspa12ao/e CM compared with AML-12hepatocytes treated with Hspa12ao/e CM (Fig. 8C).

HSPA12A Forms Complex with PKM2Simultaneous nuclear translocation of HSPA12A andPKM2 during M1 macrophage activation motivated usto investigate whether there is an interaction betweenthem. Flag-tagged HSPA12A was immunoprecipitatedfrom 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; mean6 SD; n = 6 for WT group and n = 5 forHspa12a2/2 group. Data were analyzed by unpaired, two-tailed Student t test.

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LPS or vehicle for 16 h. Precipitates were immunoblottedwith antibodies specific for PKM2 and HSPA12A. PKM2protein was recovered from the flag-tagged HSPA12Aimmunocomplexes, with more PKM2 recovered after

LPS treatment (Fig. 8D). These data suggest that HSPA12Aformed a complex with PKM2, and the formation of theHSPA12A-PKM2 complex was increased during macro-phage polarization to an M1 phenotype.

Figure 3—Hepatocyte steatosis was regulated by paracrine effects of macrophage HSPA12A. A: No effects of HSPA12A deficiency onhepatocyte steatosis. Primary hepatocytes isolated fromWT andHspa12a2/2micewere exposed to OA for 24 h. Vehicle-treated cells servedas controls. TAG contents were examined. *P, 0.05 and **P, 0.01; mean6SD. Sample numbers are indicated. Datawere analyzed by two-way ANOVA analysis followed by Tukey test. N.S., not significant; prot, protein. B: No effects of HSPA12A overexpression on hepatocytesteatosis. Three types of hepatocytes with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with OA orvehicle (control) for 24 h. Lipid accumulation was examined byORO staining. **P, 0.01; mean6SD. Scale bar = 50mm. Sample numbers areindicated in the figures. Data were analyzed by two-way ANOVA analysis followed by Tukey test. C: Paracrine effects of Raw264.7macrophage 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; mean6 SD. Scale bar = 50 mm. Sample numbersare indicated. Data were analyzed by unpaired, two-tailed Student t test.D: Paracrine effects of Primary Kupffer cell HSPA12A on hepatocytesteatosis. Primary Kupffer cells isolated from WT and Hspa12a2/2 mice were challenged with LPS (200 ng/mL) for 16 h. The CMs werecollected 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.

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Macrophage HSPA12A Regulates HFD-InducedHepatic Steatosis in Mice

To examine whether macrophage HSPA12A affectsHFD-induced hepatic steatosis, we transplanted bone

marrow from Hspa12a2/2 mice into recipient WT mice(Hspa12a2/2/WT). Recipient WT mice transplanted withWT bone marrow served as controls (WT/WT). Aftera lethal irradiation, all the mice (15/15) without BMT

Figure 4—Deficiency of HSPA12A reducedM1macrophage polarization and inflammatory responses in mouse NASH livers.A: Macrophagerecruitment. Liver tissues were collected frommice fed with HFD or chow diet for 14 weeks. Frozen sections were immunostained with F4/80to visualize macrophages. Hoechst 33342 was used to counterstain nuclei. Macrophage numbers were expressed as a percentage of totalliver cells. Scale bar = 50 mm. **P, 0.01; mean6 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 for14weeks. Real-timePCRwas performed to evaluate the indicated gene expression. *P, 0.05 and **P, 0.01; mean6SD; n = 6 forWT groupand n = 5 for Hspa12a2/2 group. Data were analyzed by unpaired, two-tailed Student t test. C: mRNA levels of M1 and M2 markers. Liverswere collected frommice fed with HFD for 14 weeks. Real-time PCRwas performed to evaluate the indicated gene expression. *P, 0.05 and**P, 0.01; mean6SD; n = 6 forWT group and and n = 5 forHspa12a2/2 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-tailedStudent t test.

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died within 15 days; however, none of the BMT mice died(Supplementary Fig. 21A). HSPA12A expression in whiteblood cells from Hspa12a2/2/WT mice was hardly detect-able compared with that in WT/WT controls (Supplemen-tary Fig. 21B and C). After an HFD for 14 weeks, liver

weight, hepatocyte area, and lipid accumulation (as in-dicated by ORO staining and TAG content) inHspa12a2/2/WT mice were reduced compared withWT/WT mice (Supplementary Fig. 21D–F). In addition,expression of iNOS, TNF-a, TLR4, and MyD88 protein,

Figure 5—HSPA12A promoted M1 macrophage polarization. A: Human NASH livers demonstrated an increase of HSPA12A nucleartranslocation in macrophages. Immunostaining against HSPA12A and CD68 (macrophage) was performed on frozen sections of humanlivers. 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. Cellswere then subjected to the analysis of HSPA12A expression in cytosolic and nuclear fractions by immunoblotting (B) and immunofluo-rescence staining (C). Scale bar = 2mm. *P, 0.05; **P, 0.01; mean6SD; n = 3/group. Data were analyzed by unpaired, two-tailed Student ttest. D: HSPA12A increased mRNA expression of M1 markers in LPS-treated macrophages. Raw264.7 macrophages with HSPA12Aoverexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cells were then collected for analyzing theindicated 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-tailedStudent t test. E: HSPA12A increased protein expression of M1 markers and the TLR4/NF-kB pathway in LPS-treated macrophages.Raw264.7macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cellswere then collected for analyzing the indicated proteins by immunoblotting. *P , 0.05 and **P , 0.01; mean 6 SD; n = 3/group. Data wereanalyzed by unpaired, two-tailed Student t test.

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along with NF-kB phosphorylation, in the livers ofHspa12a2/2/WT mice was lower than in WT/WT micefed an HFD (Supplementary Fig. 22).

DISCUSSION

Here, we identified macrophage HSPA12A as a novelregulator of hepatic inflammation and NASH pathogene-sis. Thus, strategies aimed at inhibiting macrophage-specific HSPA12A may have potential as therapeuticinterventions for NASH patients.

HSPA12A was first cloned from atherosclerotic lesions,and its cerebral expression is decreased in patients with

schizophrenia (20,21,32). Recently, a study identifieda correlation between increased HSPA12A expressionand shortened survival of patients with hepatocellularcarcinoma (22). However, there is no evidence thatHSPA12A is the direct cause of any pathophysiologicalevent. Here, we used human samples, mouse models, andcell culture models to show the following: 1) NASHpatients display increased hepatic expression of HSPA12Aand higher levels of circulating HSPA12A, which are pos-itively associated with hepatic steatosis and injury, re-spectively; 2) HSPA12A is expressed preferably in livermacrophages rather than hepatocytes; 3) HSPA12A

Figure 6—Increased nuclear PKM2 in macrophages of NASH livers and the inhibitory effect of HSPA12A deficiency. A: Human NASH liversdemonstrated increased PKM2 nuclear translocation in macrophages. Immunostaining against PKM2 and CD68 (macrophage) wasperformed 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 for14 weeks. Hoechst 33342 was used to counterstain nuclei. Scale bar = 20 mm. **P, 0.01; mean6 SD; n = 4mice/group.C andD: HSPA12Adeficiency suppressed PKM2 nuclear translocation in macrophages of NASH livers. Livers were collected from mice fed with HFD for14 weeks. PKM2 expression and nuclear translocation in macrophages were examined by immunoblotting against PKM2 in cytosolic andnuclear 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.

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deficiency either globally or in macrophages attenuatesHFD-induced hepatic steatosis, liver injury, and inflam-matory M1 macrophage responses in mice; and 4) hepa-tocyte steatosis is regulated by the paracrine effects ofmacrophage HSPA12A, which are mediated, at least inpart, by nuclear PKM2-dependent modulation of M1macrophage polarization. Taken together, these resultsprovide evidence that the paracrine effects of macrophageHSPA12A regulate hepatocyte steatosis and injury.

Activation of liver macrophages, and subsequent secre-tion of proinflammatorymediators, is a key event for onset

and progression of NAFLD (9,10). Hepatic macrophagescomprise resident (Kupffer cells) and recruited macro-phages, all of which express macrophage markers suchas CD68, F4/80, and CD11b. Macrophages are activated byLPS or free fatty acids, which induce an M1 phenotype andexpression of cytokines, chemokines, and signaling mole-cules through TLR4 signaling (7,33,34). Subsequently,chemokines attract blood-derived monocytes/macroph-ages to the liver; these cells then release cytokines thatdrive NASH progression, which is characterized by in-creased hepatocellular lipid accumulation and damage

Figure 7—Inhibition of PKM2 nuclear translocation reversed the HSPA12A-induced promotion of M1 macrophage polarization. DASA-58reversed 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. Cytosolicand nuclear protein fractions were immunoblotted with the indicated antibodies (A). Levels ofMcp-1 and TnfamRNAwere also analyzed (B).*P, 0.05 and **P, 0.01; mean6 SD; n = 3/group (A) and n = 6/group (B). Data were analyzed by one-way ANOVA followed by Tukey test.

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Figure 8—Nuclear PKM2 mediated the paracrine effect of macrophage HSPA12A on hepatocyte steatosis. A: DASA-58 reversed theHSPA12A-promoted secretion of cytokines from macrophages. Raw264.7 macrophages were challenged with LPS (500 ng/mL) for 16 h inthe 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 ANOVAfollowed by Tukey test. B and C: DASA-58 blocked the paracrine effects of macrophage HSPA12A on hepatocyte steatosis. CM wascollected from the LPS-treated Raw264.7 macrophages in the presence or absence of DASA-58 for 16 h. The CM was then applied to theOA-treated AML-12 hepatocytes for 24 h. Hepatocytes were collected for lipid analysis by ORO staining (B) (scale bar = 50 mm) and theindicated 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 wereanalyzed by one-wayANOVA followed by Tukey test.D: Interaction betweenHSPA12A and PKM2 inmacrophages. RAW264.7macrophagesoverexpressing 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 withoutimmunoprecipitation (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 isupregulated by LPS or other stimuli. By directly interacting with PKM2, HSPA12A promotes PKM2 translocation to nuclei, thereby promotingM1macrophage polarization and secretion of proinflammatory M1 cytokines, and ultimately leads to hepatocyte steatosis through paracrineeffects. Both the M1-polarized macrophage-mediated inflammation and hepatocyte steatosis are essential for the pathogenesis of NASH.FFA, free fatty acid.

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(6,34). Indeed, diet-induced NASH is alleviated by de-pleting Kupffer cells and knockout of IL-1b and TLR4(11,35,36). Thus, limiting polarization of macrophagestoward an M1 phenotype is an attractive strategy forpreventing progression of NAFLD (9,10). Here, we ob-served that deficiency of HSPA12A alleviated HFD-evoked onset and progression of NAFLD, which wereconcomitant with reduced macrophage recruitment andM1 polarization; HSPA12A deficiency also reducedexpression of chemokines and proinflammatory cyto-kines in HFD-fed mice. By contrast, overexpression ofHSPA12A increased M1 macrophage polarization in re-sponse to LPS, as indicated by increased expression ofiNOS, IL-1b, TNF-a, CCL3, CCL4, and TLR4, and bysecretion of IL-1b, IL-6, IL-12, and TNF-a. More impor-tantly, CM from HSPA12A-overexpressing macrophagesled to a marked increase in lipid deposition in hepato-cytes. However, we observed no change in HSPA12Asecretion by macrophages after LPS treatment, suggest-ing that elevated serum levels of HSPA12A in NASHpatients may be due to secretion by other cell types.Considering that hepatocellular HSPA12A had no effecton its own lipid deposition, our results suggest thathepatocyte steatosis is regulated by the paracrine effectsof macrophage HSPA12A. We detected only a modestincrease in secretion of TNF-a, IL-1b, IL-6, and IL-12by HSPA12A-overexpressing macrophages, suggestingthat the relationship between macrophage HSPA12Aand hepatocyte lipid deposition is regulated by the syn-ergistic actions of a group of secreted cytokines and/orother as yet undetermined factors.

Next, we sought answers to the following two ques-tions: does M1 polarization mediate the paracrine effectsof macrophage HSPA12A on hepatocyte steatosis, and howdoes HSPA12Amodulate macrophage polarization? Recentstudies show that PKM2 is a critical determinant ofmacrophage activation and subsequent inflammatoryresponses. The PKM2 tetramer acts as a powerful glyco-lytic enzyme that regulates glycolysis in the cytosol. How-ever, PKM monomers or dimers are enzymatically inactiveand can translocate to the nucleus, where they act ascofactors to activate expression of IL-1b et al., ultimatelyleading to activation of M1 macrophages (15–17). Induc-tion of PKM2 expression by LPS is mediated by thetranscription factors NF-kB and PPARg (14,15,19). In-terestingly, we observed that PKM2 expression in the liverof NASH patients was upregulated and that this upregu-lation was more prominent in liver macrophages showingnotable levels of nuclear translocation. The livers of HFD-fed mice also showed more nuclear translocation of PKM2,which was attenuated by HSPA12A deficiency, suggestinga regulation of HSPA12A in PKM2 nuclear translocation.Importantly, preventing nuclear translocation of PKM2 inmacrophages reversed HSPA12A-induced M1 polarizationand blocked the paracrine effects of macrophage HSPA12Aon hepatocellular steatosis. Immunoprecipitation-Westernblot analyses revealed that HSPA12A forms a complex with

PKM2 in macrophages. Collectively, these results indicatethat HSPA12A interacts with PKM2 and increases itsnuclear translocation, thereby inducing M1 macrophagepolarization and secretion of proinflammatory M1 cyto-kines; ultimately, this leads to fat accumulation in hep-atocytes via paracrine effects (Fig. 8E). However, it isunknown how HSPA12A regulates PKM2 expressionand how HSPA12A interacts with PKM2. Further studiesneed to address these issues.

In conclusion, we show that deficiency of HSPA12A hasa beneficial effect on hepatic steatosis and injury in micewith HFD-induced NASH. The underlying mechanisminvolves paracrine interactions between macrophagesand hepatocytes. The data suggest that inhibitors ofmacrophage-specific HSPA12A may improve treatmentand management of nonalcoholic liver disease.

Acknowledgments. The authors thank the Translational Medicine CoreFacilities, Medical School of Nanjing University, for generous help in massspectrum analysis.Funding. This work was supported by the National Natural Science Foundationof China (81870234, 81770854, 81571378, 81571290, 81370260, and81371450), Jiangsu Province’s Outstanding Medical Academic Leaders program(15), the Priority Academic Program Development of Jiangsu Higher EducationInstitutions, the Collaborative Innovation Center for Cardiovascular Disease Trans-lational Medicine, and Jiangsu Provincial Key Discipline of Medicine(ZDXKA2016003).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Q.K., N.L., H.C., L.D., B.X., and L.F. performed allanimal 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 NASHfeatures in humans and mice. C.L. developed the study concept and experimentaldesign. Y.L. interpreted the data and wrote the manuscript. L.L. and Z.D.developed the study concept and experimental design, interpreted the data,and wrote the manuscript. Z.D. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

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