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• W2020004405 abstract "Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability. Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability. MicroRNAs (miRs) 3The abbreviations used are: miR, microRNA; ALD, alcoholic liver disease; KC, Kupffer cell; DMSO, dimethyl sulfoxide; qPCR, quantitative PCR. are small non-coding RNA molecules that regulate the expression of target genes involved in a wide range of biological processes (1Baltimore D. Boldin M.P. O'Connell R.M. Rao D.S. Taganov K.D. Nat. Immunol. 2008; 9: 839-845Crossref PubMed Scopus (927) Google Scholar, 2Bi Y. Liu G. Yang R. J. Cell. Physiol. 2009; 218: 467-472Crossref PubMed Scopus (146) Google Scholar). Innate immune responses and inflammation are fine-tuned by miR-125b, miR-146a, and miR-155. Upon lipopolysaccharide (LPS) stimulation, both miR-146a and miR-155 are up-regulated in monocytes and macrophages (3O'Connell R.M. Taganov K.D. Boldin M.P. Cheng G. Baltimore D. Proc Natl. Acad. Sci. 2007; 104: 1604-1609Crossref PubMed Scopus (1559) Google Scholar, 4Perry M.M. Moschos S.A. Williams A.E. Shepherd N.J. Larner-Svensson H.M. Lindsay M.A. J. Immunol. 2008; 180: 5689-5698Crossref PubMed Scopus (385) Google Scholar), whereas miR-125b is down-regulated (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar). Consistent with this, miR-155 exerts a positive regulation on the release of tumor necrosis factor α (TNFα) through enhancing its translation (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar). In contrast, miR-146a acts as a negative regulator and decreases the release of inflammatory mediators, such as interleukin (IL)-1β or IL-8 (4Perry M.M. Moschos S.A. Williams A.E. Shepherd N.J. Larner-Svensson H.M. Lindsay M.A. J. Immunol. 2008; 180: 5689-5698Crossref PubMed Scopus (385) Google Scholar), whereas miR-125b acts as a post-transcriptional repressor of TNFα (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar). In alcoholic liver disease (ALD), TNFα production by the resident liver macrophages, Kupffer cells (KCs), plays a central role in disease pathogenesis (6Thurman R.G. Am. J. Physiol. 1998; 275: G605-G611PubMed Google Scholar). Chronic alcohol exposure in vitro and in vivo increases inflammatory cell responses, particularly to LPS stimulation (7Romics Jr., L. Mandrekar P. Kodys K. Velayudham A. Drechsler Y. Dolganiuc A. Szabo G. Alcohol Clin. Exp. Res. 2005; 29: 1018-1026Crossref PubMed Scopus (23) Google Scholar, 8Mandrekar P. Bala S. Catalano D. Kodys K. Szabo G. J. Immunol. 2009; 183: 1320-1327Crossref PubMed Scopus (149) Google Scholar). Alcohol-induced sensitization of KCs to gut-derived LPS was shown to contribute to the initiation and progression of ALD (9Nagy L.E. Exp. Biol. Med. 2003; 228: 882-890Crossref PubMed Scopus (200) Google Scholar). KC-derived TNFα has been identified as an important mediator of steatosis, inflammation, and hepatocyte damage in ALD (10Iimuro Y. Gallucci R.M. Luster M.I. Kono H. Thurman R.G. Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (449) Google Scholar, 11Uesugi T. Froh M. Arteel G.E. Bradford B.U. Wheeler M.D. Gäbele E. Isayama F. Thurman R.G. J. Immunol. 2002; 168: 2963-2969Crossref PubMed Scopus (154) Google Scholar). Although the involvement of various signaling pathways such as nuclear factor-κB (NF-κB) and Erk and mRNA stability has been studied in KCs from ALD (8Mandrekar P. Bala S. Catalano D. Kodys K. Szabo G. J. Immunol. 2009; 183: 1320-1327Crossref PubMed Scopus (149) Google Scholar, 12Kishore R. McMullen M.R. Nagy L.E. J. Biol. Chem. 2001; 276: 41930-41937Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 13Kishore R. Hill J.R. McMullen M.R. Frenkel J. Nagy L.E. Am. J. Physiol. Gastrointest. Liver Physiol. 2002; 282: G6-G15Crossref PubMed Scopus (105) Google Scholar), the role of miRs is unknown in resident liver macrophages. A recent report has described the miR expression profile in a murine model of ALD (14Dolganiuc A. Petrasek J. Kodys K. Catalano D. Mandrekar P. Velayudham A. Szabo G. Alcohol Clin. Exp. Res. 2009; 33: 1704-1710Crossref PubMed Scopus (164) Google Scholar), but the functions and physiological activity of specific miRs and their cell-specific role and expression remain to be elucidated. Considering the potential role of miRs in LPS-induced TNFα production and the importance of macrophage inflammatory activation in ALD, we hypothesized that miR-155, miR-146a, and/or miR-125b could play a role in the development of alcoholic liver injury. Here we report for the first time that chronic alcohol induces miR-155 in macrophages via NF-κB and that elevated miR-155 results in increased TNFα production by increasing TNFα mRNA stability. All animals received proper care in agreement with animal protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. Eight-week-old female mice (C57BL/6) were divided into two groups (15–30 mice/group depending on the experiment). The alcohol-fed group received the Lieber-DeCarli diet (Bio-Serv, Frenchtown, NJ) with 5% (v/v) ethanol (32.4% alcohol-derived calories) for 4 weeks; pair-fed control mice received an equal amount of calories as their alcohol-fed counterparts with the alcohol-derived calories substituted with dextrin maltose. Mice were bled by submandibular venipuncture, and serum was separated from whole blood and frozen at −80 °C. For some mice, livers were fixed in formalin and were further paraffin-embedded, sectioned, and stained with hematoxylin and eosin for microscopic analysis. The rest of the mice received anesthesia with ketamine (100 mg/kg), and KCs were isolated as described previously (15Hritz I. Mandrekar P. Velayudham A. Catalano D. Dolganiuc A. Kodys K. Kurt-Jones E. Szabo G. Hepatology. 2008; 48: 1224-1231Crossref PubMed Scopus (325) Google Scholar). Briefly, the livers were perfused with saline solution for 10 min followed by in vivo digestion with liberase enzyme for 5 min and in vitro digestion for 30 min. The non-hepatocyte content was separated by Percoll gradient and centrifuged for 60 min at 800 × g. The intercushion fraction was washed and adhered to plastic in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. The non-adherent fraction was washed, and the adherent KC population was adjusted to 2 × 106/ml in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Depending on experimental conditions, cells from 5–10 mice were pooled for each experiment given the limited number of KCs available from each animal. For in vitro stimulation, cells were rested overnight, and on the next day, they were stimulated with 25 mm alcohol or 100 ng/ml LPS or both for 6 h; supernatants were collected for TNFα analysis, and total RNA was isolated from cells for miR-155 expression as indicated in Fig. 4, D–E legend. Serum alanine aminotransferase activity was determined using a kinetic method (D-Tek LLC, Bensalem, PA), serum endotoxin levels were measured using the limulus amebocyte lysate assay (Lonza, MD), and serum alcohol levels were determined using an alcohol analyzer (Analox Instruments). RAW 264.7 macrophages were purchased from the American Type Culture Collection and maintained in Dulbecco's modified medium (Invitrogen) containing 10% FBS (HyClone, South Logan, UT) at 37 °C in a 5% CO2 atmosphere. For prolonged alcohol exposure, cells exposed to 50 mm alcohol were placed in a C.B.S. Scientific incubation culture chamber with twice the alcohol concentration in the bottom of the chamber to saturate the chamber and maintain a stable alcohol concentration, as described previously (8Mandrekar P. Bala S. Catalano D. Kodys K. Szabo G. J. Immunol. 2009; 183: 1320-1327Crossref PubMed Scopus (149) Google Scholar). Actinomycin D, MG-132, Bay11-7082, and LPS (Escherichia coli strain 0111:B4) were purchased from Sigma-Aldrich. RAW 264.7 macrophages were stimulated with E. coli-derived LPS (100 ng/ml), 50 mm alcohol, or the combination of LPS and alcohol at the times indicated in Figs. 1, A and B, 2, A–D, 5, A–E, 6, A–D, and 7, A–C legends.FIGURE 2TNFα production is increased in RAW 264.7 macrophages after LPS and/or alcohol treatment and correlates with miR-155 expression. A, RAW 264.7 macrophages were stimulated with 50 mm alcohol for the indicated time points, and TNFα levels were measured in supernatants by ELISA. B and C, RAW 264.7 macrophages were stimulated with 50 mm alcohol for 48 h or LPS for 6 h or with LPS for 6 h after 48 h of alcohol pretreatment. TNFα levels were measured in supernatants by ELISA, and TNFα mRNA was quantified using specific primers in real-time PCR. Data represent the mean value (S.E. indicated by error bars) of at least three independent experiments. (* indicates p ≤ 0.05 versus unstimulated cells). Statistically significant differences are shown. D, the correlation between miR-155 expression and TNFα production in RAW 264.7 macrophages under different conditions (50 mm alcohol for 6, 24, and 48 h and 100 ng/ml LPS for 6 h with or without alcohol pretreatment) is shown (R2 = 0.94, p < 0.01). Expression of miR-155 was assayed by qPCR, and data were normalized to sno202 control. TNFα levels were measured in supernatants by ELISA after collection of the medium in the same samples. Each dot represents the average of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5miR-155 regulates TNFα production. A–E, RAW 264.7 macrophages were transfected with anti-miR-control or anti-miR-155 (A and B) with pre-miR-control or pre-miR-155 (C–E), exposed to 50 mm alcohol for 48 h (A, B, D, and E), and further stimulated or not with 100 ng/ml LPS for 6 h as indicated. Culture medium was collected, and supernatants were analyzed for TNFα production by ELISA. Mean values of TNFα (S.E. indicated by error bars) from three independent experiments are shown. C, mature miR-155 expression was assayed by qPCR and normalized to sno202 control after transfection with pre-miR-control or pre-miR-155. Data from two experiments (mean ± S.E.) are shown. F, Kupffer cells from alcohol-fed mice were transfected with anti-miR-control or anti-miR-155 using an Amaxa transfection kit, medium was changed after 4–6 h of transfection and stimulated on the next day or not with 100 ng/ml LPS for 6 h, and culture supernatant was collected and analyzed for TNFα production by ELISA. Mean values of TNFα from three independent experiments are shown. Statistically significant differences are shown (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6miR-155 increases TNFα secretion by means of affecting TNFα mRNA stability. A–D, RAW 264.7 macrophages were transfected with pre-miR-control or pre-miR-155 and treated or not with alcohol for 48 h (A) or transfected with anti-miR-control or anti-miR-155, treated with LPS or alcohol plus LPS or alcohol alone as indicated (B–D), and further cultured in the presence of 5 μg/ml actinomycin D (A–D). Total RNA was isolated at the times shown, and TNFα mRNA was quantified using specific primers in real-time PCR. Data were normalized with the housekeeping gene 18 S, and half-life is indicated as the percentage of remaining TNFα at different time points showing one experiment out of three with similar results or as absolute numbers (mean ± S.E. (error bars)) from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Treatment with NF-κB inhibitors Bay 11-7082 or MG-132 prevented miR-155 increase in response to alcohol. A, RAW 264.7 macrophages were treated with alcohol for 48 h and treated or not with LPS for 30 min, and nuclear proteins were subjected to EMSA. B and C, for NF-κB inhibition, RAW 264.7 macrophages were pretreated with Bay11-7082 (0.1 μm) or MG-132 (0.25 μm) or DMSO as a negative control for 30 min and then exposed or not to alcohol (50 mm) for 48 h and further treated or not with LPS for 6 h. Expression of miR-155 was assayed by qPCR, and data were normalized to sno202 control. The -fold increase in the expression of miR-155 versus non-stimulated cells is shown. Data represent the mean value (S.E. indicated by error bars) of at least three independent experiments. D, Kupffer cells isolated from pair-fed or alcohol-fed animals (n = 8) were pooled and treated or not with 100 ng/ml LPS for 30 min, and 10 μg of whole cell lysate were subjected to EMSA to evaluate NF-κB activation. E, for NF-κB inhibition, Kupffer cells from pair-fed or alcohol-fed animals (n = 10) were pretreated with MG-132 (2.5 μm) or DMSO for 30 min and further stimulated or not with 100 ng/ml LPS for 6 h. Expression of miR-155 was assayed by qPCR, data were normalized to sno202 control, and the -fold increase in the expression of miR-155 in Kupffer cells from alcohol-fed mice versus Kupffer cells from pair-fed mice is shown. Data represent the mean value (S.E. indicated by error bars). Statistically significant differences are shown (*, p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) For inhibition of miR-155, RAW 264.7 macrophages were transfected with anti-miR-155 and anti-miR-negative control 1 (anti-miR-control), and for overexpression, pre-miR-155 and pre-miR-negative control 1 (pre-miR-control) were used to transfect the cells using siPORT NeoFx transfection agent. All these reagents were purchased from Ambion Inc. (Austin, TX). Knockdown efficiency was determined by transfecting the cells with GAPDH siRNA (Ambion), and overexpression efficiency was checked by determining mature miR-155 levels in transfected cells. Transfected cells were treated with and without 50 mm alcohol for 48 h and, where indicated, stimulated with LPS (100 ng/ml) and treated in the presence or absence of actinomycin D according to experimental requirements before the isolation of RNA (RNeasy kit, Qiagen) or supernatant collection. Amaxa mouse macrophage Nucleofector kit was used to transfect KCs according to manufacturer instructions using the Y-001 program (Lonza). Medium was changed after 4–6 h of transfection, and on the next day, KCs were stimulated with LPS as indicated in Fig. 5F legend. Total RNA was isolated using the mirVanaTM miRNA isolation kit (Ambion). The quality of RNA was routinely checked by measurement of optical density (260/280 and 260/230 ratio) and gel electrophoresis. Quantitative RT-PCR analyses for miR-125b, miR-146a, miR-155, and sno202, used as normalizing control, were performed using TaqMan miRNA assays with reagents, primers, and probes obtained from Ambion. In brief, a stem loop primer was used for reverse transcription (30 min, 16 °C; 30 min, 42 °C; 5 min 85 °C) followed by qPCR employing TaqMan probes and primers in an Eppendorf Realplex Mastercycler (Eppendorf, Westbury, NY). For TNFα and 18 S mRNA expression, RNA was cDNA transcribed with the reverse transcription system (Promega Corp., Madison, WI). Real-time quantitative polymerase chain reaction was performed using the iCycler (Bio-Rad Laboratories), as described (16Romics Jr., L. Kodys K. Dolganiuc A. Graham L. Velayudham A. Mandrekar P. Szabo G. Hepatology. 2004; 40: 376-385Crossref PubMed Scopus (80) Google Scholar). The primer sequences were as follows: 18 S, forward, 5′-GTA ACC CGT TGA ACC CCA TT-3′, and reverse, 5′-CCA TCC AAT CGG TAG TAG CG-3′; TNFα, forward, 5′-CAC CAC CAT CAA GGA CTC AA-3′, and reverse, 5′-AGG CAA CCT GAC CAC TCT CC-3′. Relative expression was calculated using the comparative threshold cycle (Ct) method. To examine NF-κB activation, RAW 264.7 macrophages were exposed to 50 mm alcohol for 48 h and treated or not with 100 ng/ml LPS for 30 min, and nuclear proteins were isolated. KCs from pair-fed or alcohol-fed mice were isolated, rested overnight, and treated or not with LPS (100 ng/ml) for 30 min, and whole cell lysate was isolated given the limited number of cells. Five μg of nuclear protein from RAW 264.7 macrophages or 10 μg of whole cell lysate from KCs were labeled with NF-κB probe as described (17Mandrekar P. Jeliazkova V. Catalano D. Szabo G. J. Immunol. 2007; 178: 7686-7693Crossref PubMed Scopus (43) Google Scholar). To inhibit NF-κB activity, RAW 264.7 macrophages were first treated with Bay11-7082 or MG-132 for 30 min prior to alcohol or LPS stimulation, and then cells were exposed or not to 50 mm alcohol for 48 h as indicated in the Fig. 7, B–C legend. For KCs, cells were rested overnight and then treated with MG-132 for 30 min prior to any in vitro stimulation as described in Fig. 7E legend. Total RNA was extracted with the mirVana miRNA isolation kit and analyzed for miR-155 expression as described earlier. The amount of TNFα in cell-free supernatants was determined by ELISA according to the manufacturer's instructions (BD Biosciences). Data are presented as mean ± S.E., and groups were compared by means of Student's t test or Mann-Whitney U test according to data distribution. Correlation was assessed by means of Spearman's rho test. p < 0.05 was regarded as significant. Prolonged alcohol exposure leads to increased inflammatory cell responses, particularly up-regulation of LPS-induced TNFα production in macrophages and KCs (8Mandrekar P. Bala S. Catalano D. Kodys K. Szabo G. J. Immunol. 2009; 183: 1320-1327Crossref PubMed Scopus (149) Google Scholar, 18Mandrekar P. Szabo G. J. Hepatol. 2009; 50: 1258-1266Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Recent studies demonstrated that TNFα is regulated by miR-125b (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar), miR-146a (19Taganov K.D. Boldin M.P. Chang K.J. Baltimore D. Proc Natl. Acad. Sci. 2006; 103: 12481-12486Crossref PubMed Scopus (3545) Google Scholar), and miR-155 (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar). Thus, we hypothesized that alcohol may affect TNFα production via regulation of miRs. First, we studied RAW 264.7 cells that represent a surrogate model of KCs with respect to alcohol-induced TNFα production (20Szabo G. Mandrekar P. Alcohol Clin. Exp. Res. 2009; 33: 220-232Crossref PubMed Scopus (289) Google Scholar). Alcohol treatment at a 50 mm dose, which corresponds to a 0.2 g/dl blood alcohol level found in chronic alcoholics, resulted in significant up-regulation of miR-155 (Fig. 1A). Alcohol increased miR-155 within 6–72 h with the highest induction after prolonged alcohol exposure for 72 h (Fig. 1A). We identified that the alcohol-induced increase was specific to miR-155 as there were no significant changes in miR-146a or miR-125b levels (Fig. 1A). This observation led us to further study miR-155 in alcohol-treated macrophages. The Toll-like receptor 4 (TLR4) ligand, LPS, has been implicated in the pathogenesis of ALD (15Hritz I. Mandrekar P. Velayudham A. Catalano D. Dolganiuc A. Kodys K. Kurt-Jones E. Szabo G. Hepatology. 2008; 48: 1224-1231Crossref PubMed Scopus (325) Google Scholar), and it has been proposed that alcohol sensitizes liver macrophages to LPS-induced TNFα production. Although alcohol or LPS treatment alone resulted in a significant increase in miR-155 expression in RAW macrophages, alcohol pretreatment augmented the LPS-induced increase in miR-155 levels (Fig. 1B). These data suggest that miR-155 could be involved in alcohol-induced changes in macrophage activation. Because miR-155 is involved in LPS-induced TNFα regulation (5Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. J. Immunol. 2007; 179: 5082-5089Crossref PubMed Scopus (1144) Google Scholar) and TNFα is increased in ALD (18Mandrekar P. Szabo G. J. Hepatol. 2009; 50: 1258-1266Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar), next we confirmed enhanced TNFα production in chronic alcohol-treated macrophages. Prolonged alcohol treatment alone resulted in a time-dependent induction of TNFα in RAW cells (Fig. 2A), and LPS alone also stimulated TNFα production (Fig. 2, B and C). More important, prolonged alcohol pretreatment augmented LPS-induced TNFα protein production (Fig. 2B) and mRNA expression (Fig. 2C). We identified that changes in TNFα production and miR-155 expression were parallel in macrophages. Indeed, a significant positive correlation (R2 = 0.94) existed between TNFα levels and miR-155 expression after alcohol and/or LPS stimulation (Fig. 2D). KCs, the resident macrophages in the liver, are the main source of TNFα in this organ (21Dong Z. Wei H. Sun R. Tian Z. Cell. Mol. Immunol. 2007; 4: 241-252PubMed Google Scholar), and activation of KCs plays a key role in ALD (18Mandrekar P. Szabo G. J. Hepatol. 2009; 50: 1258-1266Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Therefore, we next evaluated whether KCs in vivo would have increased miR-155 levels in ALD. Chronic alcohol feeding in mice resulted in increased serum alcohol levels (Fig. 3A) and significant liver damage indicated by increased serum alanine aminotransferase levels when compared with isocaloric diet feeding (Fig. 3B). We found increased serum endotoxin levels in alcohol-fed mice, suggesting a role for LPS (Fig. 3C). Evaluation of liver histology revealed features of ALD including steatosis and inflammatory cell infiltrates in alcohol-fed but not in pair-fed mice (Fig. 3D). Given the features of ALD in vivo, next we tested the hypothesis that miR-155 and TNFα were increased in vivo in KCs in ALD. We determined that KCs isolated from alcohol-fed mice showed increased TNFα production and TNFα mRNA expression when compared with pair-fed controls even without ex vivo stimulation (Fig. 4, A and B). LPS stimulation in KCs from alcohol-fed mice resulted in significantly higher TNFα production at both protein and mRNA levels when compared with KCs from LPS-stimulated pair-fed mice as well as from LPS-naive KCs of alcohol fed mice (Fig. 4, A and B). In addition, we found significantly higher expression of miR-155 in KCs of mice with alcohol feeding when compared with mice with isocaloric control diet (Fig. 4C). Interestingly, the level of miR-125b but not miR-146a was also increased after alcohol feeding in KCs (Fig. 4C). In vitro LPS stimulation amplified alcohol-induced miR-155 expression in KCs isolated from alcohol-fed mice when compared with pair-fed mice (Fig. 4D). In contrast, in vitro alcohol exposure showed no significant effect on miR-155 level in KCs from alcohol-fed mice with or without in vitro LPS stimulation (Fig. 4D). A similar pattern was seen on TNFα level because in vitro alcohol exposure showed no significant effect on TNFα production in KCs from alcohol-fed mice treated or not with LPS in vitro (Fig. 4E). These data suggested that miR-155 up-regulation occurs in vivo in KCs after chronic alcohol intake in the liver. The correlation between increased miR-155 expression and TNFα levels after alcohol treatment prompted us to evaluate whether a causative relationship existed between the alcohol-induced increases in miR-155 and TNFα levels. First, transfection with a miR-155-specific inhibitor reduced alcohol-induced TNFα production (Fig. 5A). More important, inhibition of miR-155 not only reduced LPS-induced TNFα production but also decreased TNFα production in response to alcohol plus LPS (Fig. 5B). These results revealed that up-regulation of TNFα could be prevented by the anti-miR-155 but not by the anti-miR-control in RAW 264.7 macrophages treated with alcohol, LPS, or their combination (Fig. 5, A and B). Next, we sought to evaluate whether miR-155 overexpression could augment LPS-induced TNFα production because this approach could be expected to mimic up-regulation of TNFα in alcohol-treated cells. Overexpression of miR-155 by transfection of pre-miR-155, but not the pre-miR-control, resulted in increased mature miR-155 levels as measured by real-time PCR for mature miR-155 expression (Fig. 5C). Overexpression of miR-155 increased TNFα production in alcohol-treated cells when compared with cells transfected with pre-miR-control (Fig. 5D). Likewise, increased TNFα production after LPS stimulation was found in cells transfected with pre-miR-155 in both alcohol-naive and alcohol-pretreated cells (Fig. 5E), suggesting that miR-155 could augment the effect of LPS on TNFα production. However, we found no significant increase in TNFα production in cells transfected with pre-mir-155 and treated with alcohol plus LPS when compared with LPS alone-stimulated cells. These data suggest that cells may reach a threshold in miR-155 effect on TNFα after LPS stimulation, where the miR-155 effect is saturated and cannot result in further increase in TNFα production (Fig. 5E). Next, we transfected KCs from alcohol-fed mice with anti-miR-155 or anti-miR-control using the Amaxa mouse macrophage Nucleofector kit. Inhibition of miR-155 resulted in decreased TNFα production when compared with cells transfected with anti-miR-control (Fig. 5F). These results suggest that miR-155 regulates TNFα production in KCs in response to alcohol. Previous reports indicated that prolonged alcohol treatment increases LPS-induced TNFα mRNA stability both in RAW 264.7 macrophages and in primary rat KCs (22Nagy L.E. Alcohol. 2004; 33: 229-233Crossref PubMed Scopus (27) Google Scholar). Thus, we evaluated the hypothesis that miR-155 could affect TNFα production in part by regulating mRNA stability in alcohol-treated cells. First, we found that alcohol treatment alone increased TNF" @default.
• W2020004405 created "2016-06-24" @default.
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• W2020004405 date "2011-01-01" @default.
• W2020004405 modified "2023-09-30" @default.
• W2020004405 title "Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease" @default.
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