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1、116STEATOHEPATITIS/METABOLIC LIVER DISEASE|Hepatology,Vol.74,No.1,2021 Myeloid-Cell Specific IL-6 Signaling Promotes MicroRNA-223-Enriched Exosome Production to Attenuate NAFLD-Associated FibrosisXin Hou,1,2*Shi Yin,2,3*,#Ruixue Ren,4,5 Siqi Liu,1 Liang Yong,1 Yuxiao Liu,6 Yu Li,6 Ming-Hua Zheng,7 G
2、eorge Kunos,8 Bin Gao,2 and Hua Wang4,5SEE EDITORIAL ON PAGE 5BaCKgRoUND aNDS aIMS:NAFLD is associated with elevation of many cytokines,particularly IL-6;however,the role of IL-6 in NAFLD remains obscure.The aim of this study was to examine how myeloid-specific IL-6 signaling af-fects NAFLD by the r
3、egulation of antifibrotic microRNA-223(miR-223)in myeloid cells.appRoaCH aND ReSUltS:Patients with NAFLD or NASH and healthy controls were recruited,and serum IL-6 and soluble IL-6 receptor (sIL-6R)were measured.Compared to controls,serum IL-6 and sIL-6R levels were elevated in NAFLD/NASH patients.I
4、L-6 levels correlated positively with the number of circulating leukocytes and monocytes.The role of IL-6 in NAFLD was investigated in Il6 knockout(KO)and Il6 receptor A(Il6ra)conditional KO mice after high-fat diet(HFD)feeding.HFD-fed Il6 KO mice had worse liver injury and fibrosis,but less inflamm
5、ation,compared to wild-type mice.Hepatocyte-specific Il6ra KO mice had more steatosis and liver injury,whereas myeloid-specific Il6ra KO mice had a lower number of hepatic infiltrating macrophages(IMs)and neutro-phils with increased cell death of these cells,but greater liver fibrosis(LF),than WT mi
6、ce.Mechanistically,the increased LF in HFD-fed,myeloid-specific Il6ra KO mice was attributable to the reduction of antifibrotic miR-223 and subsequent up-regulation of the miR-223 target gene,transcriptional activa-tor with PDZ-binding motif(TAZ),a well-known factor to promote NASH fibrosis.In vitro
7、,IL-6 treatment up-regulated exosome biogenesis-related genes and subsequently promoted macrophages to release miR-223-enriched exosomes that were able to reduce profibrotic TAZ expression in hepatocytes by exosomal transfer.Finally,serum IL-6 and miR-223 levels were elevated and correlated with eac
8、h other in NAFLD patients.CoNClUSIoNS:Myeloid-specific IL-6 signaling inhibits LF through exosomal transfer of antifibrotic miR-223 into hepatocytes,providing therapeutic targets for NAFLD therapy.(Hepatology 2021;74:116-132).NAFLD is the most prominent cause of chronic hepatic disease worldwide.(1,
9、2)It rep-resents a spectrum of disorders that ranges from simple steatosis to NASH,cirrhosis,and HCC.(3)Inflammation is generally believed to play a key role in the progression of NAFLD.(3-5)However,the exact effects of inflammation on NAFLD progression,especially on liver fibrosis(LF),remain obscur
10、e.(4,5)Abbreviations:Acc1,acetyl-coenzyme A carboxylase;ALIX,ALG-2-interacting protein X;ALT,alanine aminotransferase;AST,aspartate aminotransferase;BSA,bovine serum albumin;CLEC-4F,C-type lectin domain family 4 member F;Col1a1,collagen type I alpha 1 chain;Ctgf,connective tissue growth factor;CXCL1
11、0,C-X-C motif chemokine 10;ESCRT,endosomal sorting complexrequired for transport;gp130,glycoprotein 130;HC,healthy control;HFD,high-fat diet;IBA-1,ionized calcium-binding adaptor molecule 1;Ihh,Indian hedgehog;Il6ra,Il6 receptor A;IL-6R,IL-6 receptor;IL6raHep/,hepatocyte-specific Il6ra KO;Il6raMye/,
12、myeloid-specific Il6ra KO;IMs,infiltrating macrophages;ISH,in situ hybridization;KCs,Kupffer cells;KO,knockout;LF,liver fibrosis;Ly6g,lymphocyte antigen 6 complex locus G6D;MCP1,monocyte chemoattractant protein 1;Mip,macrophage inflammatory protein;miR-223,microRNA-223;MMPs,matrix-degrading metallop
13、roteinase enzymes;MPO,myeloperoxidase;MVBs,multivesicular bodies;NLRP3,NOD-,LRR-,and pyrin domain-containing protein 3;nsmase2,neutral sphingomyelinase 2;NTA,nanoparticle tracking analysis;PA,palmitic acid;Pdgf,platelet-derived growth factor;PPAR,peroxisome proliferator-activated receptor alpha;-sma
14、,alpha-smooth muscle actin;Stam1,signal-transducing adapter molecule 1;STAT3,signal transducer and activator of transcription 3;TAZ,transcriptional activator with PDZ-binding motif;Timp,tissue inhibitor of metalloproteinase;Tsg101,tumor susceptibility gene 101;WT,wild type.Received June 9,2020;accep
15、ted October 29,2020.Additional Supporting Information may be found at AL.117IL-6 is a pleiotropic cytokine with complex roles in inflammation,playing important roles in controlling the pathogenesis of all types of diseases,includ-ing NAFLD.(6-11)Elevated serum,hepatic,and adi-pose IL-6 levels in pat
16、ients with NASH have been implicated in insulin resistance,steatosis,and liver injury.(6-12)IL-6 levels are also elevated in mice fed a high-fat diet(HFD),and genetic deletion of Il6 enhanced HFD-induced steatosis and liver injury.(13)In contrast,deletion of Il6 gene-attenuated liver injury and infl
17、ammation in another model of NASH that was induced by feeding the methionine and choline-deficient diet.(14)These contradictory data on the role of IL-6 in NAFLD were probably attributable to the different models used and widely expressed membrane-bound IL-6 receptor (mIL-6R)and its signaling chain,
18、glycoprotein 130(gp130),as well as the existence of soluble IL-6R(sIL-6R)and soluble gp130.(15)Gp130 is ubiquitously expressed,whereas IL-6 receptor (IL-6R)expression is restricted largely to hepatocytes,megakaryocytes,and subsets of leukocytes.(15)Myeloid cells,including macrophages and neutrophils
19、,express IL-6R and are key media-tors of obesity-associated inflammation.(15)Although macrophages have been implicated in the proinflam-matory phenotype in the early phase of NASH,they may adopt a restorative phenotype in the chronic res-olution phases of liver disease to limit LF.(16-18)The role of
20、 myeloid-specific IL-6 signaling in the control of NAFLD progression remains unknown.IL-6 activates classic signaling through the binding of mIL-6R,resulting in gp130 dimerization and sub-sequent activation of Janus kinases and signal transducer and activator of transcription 3(STAT3).Activated STAT
21、3 leads to IL-6-dependent gene expression and cellular responses,such as acute phase response,prolif-eration,migration,or metabolic changes.(15)IL-6 can also signal through the soluble IL-6 receptor(sIL-6R)and gp130,which is known as trans-signaling and allows cells that do not express IL-6R to resp
22、ond to IL-6.(19)IL-6-mediated trans-signaling has been shown to play a role in a variety of liver functions,such as liver regeneration.(20)sIL-6R was previously consid-ered as a systemic buffer system by promoting IL-6*These authors contributed equally to this work.Supported,in part,by the National
23、Nature and Science Foundation of China grants(81770588 to H.W.)and by the intramural program of the NIAAA,NIH,USA(to B.G.).Dr.Xin Hou was supported by the First Affiliated Hospital of Anhui Medical University Training Foundation when she performed experiments of mouse models at the NIAAA during 2018
24、-2019.Shi Yin was a participant in the NIH Graduate Partnerships Program and a graduate student in the Anhui Medical University when she worked on this project during 2008-2011,and was affiliated with the Anhui Medical University and the NIH Graduate Partnerships Program.#Dr.Yin Shis current address
25、:Division of Life Sciences and Medicine,Department of Geriatrics,The First Affiliated Hospital,University of Science and Technology of China,Hefei,China.2020 by the American Association for the Study of Liver Diseases.View this article online at .DOI 10.1002/hep.31658Potential conflict of interest:N
26、othing to report.aRtICle INFoRMatIoN:From the 1 Anhui Provincial Laboratory of Microbiology and Parasitology,School of Basic Medical Sciences,Anhui Medical University,Hefei,China;2 Laboratory of Liver Diseases,National Institute on Alcohol Abuse and Alcoholism,National Institutes of Health,Bethesda,
27、MD;3 Department of Geriatrics,Affiliated Provincial Hospital of Anhui Medical University,Hefei,China;4 Department of Oncology,the First Affiliated Hospital of Anhui Medical University,Hefei,China;5 Inflammation and Immune Mediated Diseases Laboratory of Anhui Province,Hefei,China;6 Key Laboratory of
28、 Nutrition,Metabolism and Food Safety,Shanghai Institute of Nutrition and Health,Shanghai Institutes for Biological Sciences,Shanghai,China;7 NAFLD Research Center,Department of Hepatology,The First Affiliated Hospital of Wenzhou Medical University,Wenzhou,China;8 Laboratory of Physiologic Studies,N
29、ational Institute on Alcohol Abuse and Alcoholism,National Institutes of Health,Bethesda,MD.aDDReSS CoRReSpoNDeNCe aND RepRINt ReQUeStS to:Hua Wang,M.D.,Ph.D.Department of Oncologythe First Affiliated Hospital of Anhui Medical University 81 Meishan Road Hefei,230032,China E-mail: Tel.:+86-551-651610
30、56;Mobile:+86-135-0569089 Hepatology,July 2021HOU,YIN,ET AL.118trans-signaling through IL-6/sIL-6R.(19)However,a recent study suggests that IL-6/sIL-6R complex is formed at a much lower frequency than previously anticipated,suggesting that classic IL-6 signaling through mIL-6R is active even under c
31、onditions where sIL-6R is in a high molar excess over IL-6.(21)We have previously demonstrated that Il6 knock-out(KO)mice are more susceptible to liver injury and steatosis after 3-month HFD feeding.(13)However,LF and hepatocarcinogenesis(HCC)were not examined.In the current study,we subjected Il6 K
32、O and wild-type(WT)mice to long-term(1year)HFD feeding and examined liver injury and cancer in these mice.The role of cell-specific IL-6 signaling in NAFLD was further examined in two lines of Il6 receptor a(Il6ra)conditional KO mice,including hepatocyte-specific(Il6raHep/)and myeloid-specific(Il6ra
33、Mye/)KO mice.Of interest,Il6raMye/mice were more suscepti-ble to LF than WT mice after 3-month HFD feeding,despite less hepatic neutrophil and macrophage infil-tration in the KOs.We have identified a mechanism by which myeloid IL-6 signaling ameliorates HFD-induced LF:IL-6 promoted macrophages to re
34、lease miR-223-enriched exosomes that may get transferred into hepatocytes where they suppress the expres-sion of several miR-223 target genes,including tran-scriptional activator with PDZ-binding motif(Taz),NOD-,LRR-,and pyrin domain-containing protein 3(Nlrp3),and C-X-C motif chemokine 10(Cxcl10),w
35、hich are known to promote NASH fibrosis.(22-26)Extracellular vesicles(EVs)are small membrane-bound vesicles released to the extracellular space by cells under both physiological and pathological con-ditions.(27)Based on their cellular biogenesis and sizes,EVs can be divided into three groups:exosome
36、s(40-150nm diameter in size),microvesicles/micro-particles(50-1,000 nm in diameter),and apoptotic bodies(500nm in diameter).(28)Exosomes carry pro-teins,microRNAs(miRNAs),DNAs,and glycolipids,which upon delivery can affect the cellular functions of their recipient cells.(27)Recent studies have shown
37、 that lipotoxic hepatocytes produce exosomes,which can activate macrophages by the delivery of various contents,including mitochondrial DNA and TNF-related apoptosis-inducing ligand.(29,30)Hepatocyte-derived exosomes can also promote fibrosis resolution by inhibiting macrophage activation and cytoki
38、ne secretion,suppressing the activation of hepatic stellate cells,and inducing extracellular matrix degradation and remodeling.(31)In addition,neutrophils and mac-rophages can also release exosomes that subsequently alter liver functions.For example,miR-223 is one of the most abundant miRNAs express
39、ed in neutrophils and macrophages,playing a key role in controlling inflammation in various types of diseases,includ-ing liver diseases.(32)After activation,neutrophils can deliver miR-223 by exosomes into macrophages(33,34)or hepatocytes,(35)thereby inhibiting NASH and LF.Macrophages can also deliv
40、er miR-223 by exosomes into a variety of cells,including hepatocytes,where they subsequently affect the functions of these cells.(36,37)In the current article,we demonstrate that IL-6 can induce macrophages and also neutrophils,albeit to a lesser extent,to release miR-223-enriched exosomes,which are
41、 then transferred into hepatocytes to suppress LF.Mechanistically,IL-6 promotes exosome biogene-sis by up-regulating exosome biogenesis-related genes without affecting pre-miRNA-223 expression.Materials and MethodsSeRUM SaMpleS FRoM NaFlD patIeNtSA total of 40 patients with biopsy-proven NAFLD in th
42、is study were recruited from a well-characterized PERSONS(Prospective Epidemic Research Specifically Of NAFLD)cohort 1 from December 2016 to July 2019(Supporting Table S1).(38)Forty age-and sex-matched healthy persons were also enrolled as healthy controls(HCs).Cohort 2 patients include 50 NASH pati
43、ents and 30 HCs(Supporting Table S2).The research protocol was approved by the ethics committee of the First Affiliated Hospital of Wenzhou Medical University and Anhui Medical University.Written informed consent was obtained from each subject before participation in the study.MICe aND HFD FeeDINg M
44、oUSe MoDelWT and Il6 global KO mice on a C57BL/6J back-ground(Jackson Laboratory,Bar Harbor,ME)at age 8-10 weeks were used.Hepatocyte-specific Il6ra KO(Il6raHep/)and myeloid-specific Il6ra KO(Il6raMye/)were generated by a few steps,including crossing Il6raflox/flox mice with Albumin Cre and Lysosome
45、 Cre mice Hepatology,Vol.74,No.1,2021HOU,YIN,ET AL.119(Jackson Laboratory),respectively.Littermate Il6raflox/flox mice were used as WT controls.Mice were fed the HFD(60%kcal%fat;Research Diet,New Brunswick,NJ)or control diet(10%kcal%fat;Research Diet)for 3months or 1year.All mouse experiments were a
46、pproved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.MiR-223 IN SITU HyBRIDIZatIoNDetection of miR-223 in situ hybridization(ISH)was performed as described.(35)Paraffin liver sections were hybridized with FAM-labeled miR-223 probe(Qiagen,Valencia,CA).U6 was
47、 used as a positive control,which suggested that the RNA was stable in the section(the image is not shown).Sheep anti-FAM-peroxidase(Abcam,Cambridge,MA)was used after slide blocking.A tyramine signal amplification kit(PerkinElmer,Waltham,MA)was performed to amplify the signal.Meanwhile,the slides we
48、re stained with albumin(catalog NBP1-32458;Novus Biologicals,Centennial,CO)or F4/80 antibody(catalog 70076;Cell Signaling Technology,Danvers,MA),as well as DAPI.Images were obtained by using an LSM 710 confocal microscope(Zeiss,Thornwood,NY).StatIStICal aNalySISData are expressed as the means SEM an
49、d were analyzed using GraphPad Prism software(GraphPad Software Inc.,La Jolla,CA).To compare values obtained from two groups,the Student t test was performed.To compare data from multiple groups,one-way ANOVA followed by Tukeys post hoc test was performed.Correlations between variables were assessed
50、 by linear regression analysis.Linear correc-tion index R square and P values were calculated.P values of0.05 were considered significant.Many other methods are described in the Supporting Information.ResultseleVatIoN oF SeRUM Il-6 aND sIl-6R leVelS IN NaFlD aND NaSH patIeNtSTo investigate the role