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• W2083050494 abstract "Background & Aims: Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. Methods: We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and “non-obese ob/ob” mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. Results: Antibiotic treatment decreased liver infiltration with CD4+ T, CD8+ T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4+ T, CD8+ T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4+ T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in “non-obese ob/ob” mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. Conclusions: Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization. Background & Aims: Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. Methods: We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and “non-obese ob/ob” mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. Results: Antibiotic treatment decreased liver infiltration with CD4+ T, CD8+ T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4+ T, CD8+ T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4+ T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in “non-obese ob/ob” mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. Conclusions: Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization. Nonalcoholic fatty liver disease (NAFLD) is an increasingly recognized condition that may lead to end-stage liver disease. NAFLD corresponds to a wide spectrum of pathologic lesions, ranging from pure steatosis to steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Nonalcoholic steatohepatitis (NASH) is merely a stage in NAFLD: steatosis—the retention of lipids in hepatocytes—predisposes the liver to the development of more severe lesions.1Perlemuter G. Bigorgne A. Cassard-Doulcier A.M. et al.Nonalcoholic fatty liver disease: from pathogenesis to patient care.Nat Clin Pract Endocrinol Metab. 2007; 3: 458-469Crossref PubMed Scopus (83) Google Scholar However, steatosis per se may not be a severe disease because many patients do not progress to necroinflammation or fibrosis.2Angulo P. Nonalcoholic fatty liver disease.N Engl J Med. 2002; 346: 1221-1231Crossref PubMed Scopus (4170) Google Scholar Steatohepatitis may be seen as a “double-hit” lesion,3Day C.P. James O.F. Steatohepatitis: a tale of two “hits?”.Gastroenterology. 1998; 114: 842-845Abstract Full Text Full Text PDF PubMed Scopus (3483) Google Scholar with steatosis (the first hit) rendering the liver vulnerable to further injury. A second hit, such as lipid peroxidation or cytokine secretion,4Seki S. Kitada T. Sakaguchi H. Clinicopathological significance of oxidative cellular damage in non-alcoholic fatty liver diseases.Hepatol Res. 2005; 33: 132-134Crossref PubMed Scopus (74) Google Scholar, 5Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (843) Google Scholar then triggers the recruitment of inflammatory cells by the liver.6Brunt E.M. Nonalcoholic steatohepatitis: definition and pathology.Semin Liver Dis. 2001; 21: 3-16Crossref PubMed Scopus (771) Google Scholar Several lines of evidence suggest a role for endotoxin in fatty liver inflammation. Lipopolysaccharide (LPS) is a membrane component of gram-negative commensal bacteria present in the digestive tract. A role for endotoxin in the pathogenesis of NASH is suggested by the high incidence of NASH and cirrhosis following jejuno-ileal bypass surgery for obesity. This situation can induce bacterial overgrowth in the defunctionalized small intestine, resulting in LPS absorption.7Lichtman S.N. Sartor R.B. Keku J. et al.Hepatic inflammation in rats with experimental small intestinal bacterial overgrowth.Gastroenterology. 1990; 98: 414-423Abstract PubMed Google Scholar The hepatotoxicity of LPS results from the release of cytokines, such as interferon γ, which increases the sensitivity of hepatocytes to tumor necrosis factor α.8Yang S.Q. Lin H.Z. Lane M.D. et al.Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis.Proc Natl Acad Sci U S A. 1997; 94: 2557-2562Crossref PubMed Scopus (691) Google Scholar C57BL/6 ob/ob mice have a mutation that prevents leptin synthesis, resulting in a phenotype of obesity, hepatomegaly, severe steatosis, and high serum levels of aminotransferase.9Koteish A. Mae Diehl A. Animal models of steatohepatitis.Best Pract Res Clin Gastroenterol. 2002; 16: 679-690Abstract Full Text PDF PubMed Scopus (152) Google Scholar Ob/ob mice are much more sensitive to LPS than their lean littermates, rapidly developing liver inflammation after exposure to LPS.8Yang S.Q. Lin H.Z. Lane M.D. et al.Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis.Proc Natl Acad Sci U S A. 1997; 94: 2557-2562Crossref PubMed Scopus (691) Google Scholar, 10Faggioni R. Fantuzzi G. Gabay C. et al.Leptin deficiency enhances sensitivity to endotoxin-induced lethality.Am J Physiol. 1999; 276: R136-R142PubMed Google Scholar, 11Romics Jr, L. Mandrekar P. Kodys K. et al.Increased lipopolysaccharide sensitivity in alcoholic fatty livers is independent of leptin deficiency and toll-like receptor 4 (TLR4) or TLR2 mRNA expression.Alcohol Clin Exp Res. 2005; 29: 1018-1026Crossref PubMed Scopus (23) Google Scholar The mechanism underlying the role of LPS in the onset of lymphocyte homing to fatty liver remains unknown. CXC chemokine expression has been shown to be induced in endotoxemic animals.12Li X. Klintman D. Liu Q. et al.Critical role of CXC chemokines in endotoxemic liver injury in mice.J Leukoc Biol. 2004; 75: 443-452Crossref PubMed Scopus (90) Google Scholar CXCL12 (stromal cell-derived factor 1α [SDF-1α]) is expressed in a wide variety of tissues, including bile duct epithelial cells.13Coulomb-L'Hermin A. Amara A. Schiff C. et al.Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.Proc Natl Acad Sci U S A. 1999; 96: 8585-8590Crossref PubMed Scopus (93) Google Scholar CXCL13 (B cell chemoattractant chemokine, BLC/BCA-1) selectively attracts B cells and is expressed in the liver.14Legler D.F. Loetscher M. Roos R.S. et al.B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5.J Exp Med. 1998; 187: 655-660Crossref PubMed Scopus (627) Google Scholar, 15Meijer J. Zeelenberg I.S. Sipos B. et al.The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver.Cancer Res. 2006; 66: 9576-9582Crossref PubMed Scopus (80) Google Scholar Several studies have suggested that the homing of circulating lymphocytes to the liver may increase during inflammation, but no study has yet formally confirmed this hypothesis. We explored the pathogenesis of NASH further by investigating the role of digestive LPS in the onset of liver inflammation and the mechanisms of lymphocyte homing to fatty liver in ob/ob mice. We first demonstrate that digestive bacterial compounds are involved in liver inflammation in ob/ob mice. We then show that CD4+ T, CD8+ T, and B cells from ob/ob mice have an intrinsic propensity to migrate specifically to the liver, which is enhanced by LPS challenge. Moreover, we obtain similar results in a model of high-fat diet (HFD)-induced obesity. Lymphocytes from ob/ob mice are hyperresponsive to at least 2 CXC chemokines expressed in the liver. We demonstrate that, in the ob/ob model, obesity itself—rather than leptin deficiency—is directly involved in liver inflammation and lymphocyte sensitivity to chemokines. Our study provides the first evidence that liver inflammation in obesity is due to both steatosis and lymphocyte hyperresponsiveness to chemotactic agents, which are reversible with weight normalization. Five-week-old male C57BL/6 ob/ob mice and controls (lean littermates) were purchased from Janvier (Le Genest St. Isle, France). Mice were treated in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). They were fed a diet consisting of 22% protein, 4.3% lipid, and 70% carbohydrate supplied ad libitum. “Non-obese ob/ob” mice were supplied daily with the same caloric intake as lean controls, from the age of 5 weeks, and experiments were performed on 12-week-old mice. C57BL/6 mice fed a HFD were purchased at the age of 3 weeks and fed a diet consisting of 15.5% protein, 46% lipid, and 38.5% carbohydrate (Safe, Augy, France) supplied ad libitum, and experiments were performed after 18 weeks on the HFD. Ciprofloxacin (Bayer Pharma, Germany) and ornidazol (Roche, Switzerland) were given in drinking water at a daily dose of 10 μg/g and 15 μg/g body weight, respectively, for 10 days, whereas the control group received water only. Blood was collected by retroorbital vein puncture. The serum was stored at −20°C until use for alanine and aspartate aminotransferase (ALT, AST, respectively), triglyceride, glucose, total cholesterol, and high-density lipoprotein-cholesterol determinations (OLYMPUS AU400). Serum insulin was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). Insulin resistance was determined by the product: fasting serum glucose (mmol/L) × insulin (mU/L). Liver samples of 6 mice from each group were fixed in 36% formaldehyde and embedded in paraffin. Sections (5 μm) were cut and stained with hematoxylin, eosin, and saffron. Slides were read blindly. Lobular inflammation was defined according to Kleiner's score16Kleiner D.E. Brunt E.M. Van Natta M. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7206) Google Scholar: 0, no focus; 1, <2 foci; 2, 2–4 foci; 3, >4 foci per 200× field. For quantification and phenotyping of lymphocytes infiltrating the liver, lymphocytes were isolated from mouse liver by a 2-step perfusion procedure. In the first step, circulating cells were eliminated by perfusing the liver in situ through the portal vein with Ca2+- and Mg2+-free HEPES (pH 7.65) supplemented with 5 mmol/L EDTA at 37°C, at a flow rate of 4 mL/min. The liver was then perfused with 0.05% collagenase IV (Sigma–Aldrich, St. Louis, MO) buffered with 0.1 mol/L HEPES at 37°C for 5 minutes, excised, and homogenized. The suspension was filtered through a 70-μm filter, and the filtrate was centrifuged at 50g at room temperature for 2 minutes to remove liver parenchymal cells. The lymphocyte fraction was stained with monoclonal antibodies (mAbs) for flow cytometry analysis. Lean donor mice were challenged intraperitoneally (IP) with LPS (0.5 μg/g body weight) (Alexis, Switzerland) diluted in sterile saline and killed 6 hours later. A lymphocyte suspension was obtained by tearing the spleen with fine forceps in 1X phosphate-buffered saline (PBS) in sterile conditions. Lymphocytes were purified by centrifuging this suspension through a sterile Lympholyte density gradient (Cedarlane, Canada) at 800g for 20 minutes at room temperature. Lymphocytes were labeled with fluorescent cell tracers for living cells (CellTracker Orange CMTMR; Molecular Probes, Eugene, OR). A suspension of 106 cells/mL was incubated for 30 minutes at 37°C with Orange CMTMR (1:250). We transferred 6 × 106 lymphocytes intravenously (IV) to ob/ob and control recipient mice 6 hours after IP challenge with LPS (0.5 μg/g body weight). Recipient mice were killed 18 hours after lymphocyte transfer. Circulating cells were eliminated by the first perfusion step, as described previously. A fraction of the liver was then excised and weighed before digestion in a Petri dish with 0.05% collagenase IV buffered with 0.1 mol/L HEPES at 37°C for 10 minutes. The lymphocyte fraction was stained with mAbs. Lymphocyte migration was quantified by flow cytometry and expressed as the number of fluorescent lymphocytes recruited per gram of liver. For each experiment, 2 ob/ob and 2 control donor mice were challenged IP with LPS and killed 6 hours later. Lymphocytes were purified as described previously. A suspension of 106 cells/mL was incubated for 30 minutes at 37°C with either Calcein AM (1:1000) (Molecular Probes) or CellTracker Orange CMTMR (1:250), according to their source. We pooled 6 × 106 cells labeled with each cell tracer and transferred them IV into lean recipient mice 6 hours after LPS challenge. Recipient mice were killed 18 hours after lymphocyte transfer. Lymphocytes were isolated from the spleen, lymph node, and thymus by homogenizing and seeping through a 40-μm filter in 1X PBS and from the liver by a 2-step perfusion procedure. The lymphocyte fraction was stained with mAbs. Lymphocyte migration was quantified by flow cytometry, according to lymphocyte source and phenotype. Results are expressed as the number of fluorescent cells recruited to the liver per 105 transferred cells. Lymphocyte chemotaxis was evaluated with the Transwell system (Corning Costar, MA). Lymphocytes were isolated from the spleen of ob/ob or control mice as described above 6 hours after LPS challenge and stained with fluorescent mAbs. We added 1.5 × 106 cells in 150 μL RPMI supplemented with 20 mmol/L HEPES and 1% calf fetal serum (Perbio Sciences, Belgium) to the upper chamber and 600 μL of the same medium with (1 μg/mL) or without murine CXCL12, CXCL13, CCL19, and CCL21 (R&D Systems) to the lower chamber. A sample of 1.5 × 106 cells was used as input control. Cells were incubated at 37°C for 1 hour (for CXCL13 and CCL19) or 2 hours (for CXCL12 and CCL21). Results are expressed as the percentage of cells added to the upper chamber that migrated to the lower chamber for each phenotype. Lymphocytes were stained with allophycocyanin-conjugated rat anti-CD4, anti-CD8, and anti-CD19 mAbs; mouse anti-NK1.1; and PerCP-Cy5.5-conjugated hamster anti-CD3 mAbs (Pharmingen, CA). Lymphocyte subpopulations were analyzed by 4-color flow cytometry, using a FACScalibur machine (Becton Dickinson Immunocytometry Systems, NJ). Immunofluorescence was performed on frozen liver sections for CXCL12, CXCL13, and CD31 (endothelial cells). Immunohistochemistry was performed on paraffin-embedded sections for CK19 (bile duct epithelial cells) and CXCL12 (see Supplementary Material online at www.gastrojournal.org for technical details). Data are expressed as means ± SEM. Quantitative data were compared using nonparametric (Mann–Whitney) tests and Kruskall–Wallis variance analysis. Multiple comparisons were performed, using Fisher PLSD test. A paired, 2-group Wilcoxon signed-rank test was used to compare the contributions of lymphocytes from obese and control mice to homing to the liver. P values less than .05 were considered significant. We investigated the possible role of digestive bacterial compounds in the occurrence of inflammation in fatty liver by administering oral antibiotics against aerobic and anaerobic bacteria to ob/ob and control mice. Antibiotic treatment did not modify body weight (Table 1). Basal liver weight, expressed as a percentage of total body weight, was higher in ob/ob than in control mice. Liver weight significantly decreased in ob/ob mice after a 10-day course of antibiotic treatment (from 6.7% ± 0.8% to 5.8% ± 0.8%, respectively, P < .05) (Table 1). We assessed the impact of this treatment on serum aminotransferase levels. Antibiotic treatment strongly decreased ALT and AST levels in ob/ob mice but had no significant effect in control mice (Table 1). We then assessed the impact of digestive decontamination on liver inflammation. Fewer foci were observed in antibiotic-treated ob/ob mice (Kleiner’s score, 1.1 ± 0.7) than in untreated ob/ob mice (Kleiner’s score, 1.9 ± 0.5) (P < .001) (Table 1, Figure 1A and B). The number of CD4+ T (CD3+CD4+), CD8+ T (CD3+CD8+), natural killer (NK)T (CD3+NK1.1+), B (CD3−CD19+), and NK (CD3−NK1.1+) cells in the liver of ob/ob mice was significantly decreased by antibiotic treatment (Figure 1C). Overall, liver weight, biochemistry, pathology, and flow cytometry results demonstrated that reducing the level of endogenous digestive bacterial products decreased liver inflammation.Table 1Decreased Liver Inflammation in ob/ob Mice After Antibiotic TreatmentControl miceOb/ob miceUntreatedAntibioticsUntreatedAntibioticsBody weight (g)27.6 ± 0.627.8 ± 0.548.7 ± 1.347.3 ± 0.7Liver weight (%)4.8 ± 0.55.3 ± 0.66.7 ± 0.85.8 ± 0.8aP < .05,ALT (IU/L)58 ± 1266 ± 16419 ± 42158 ± 23bP < .001,AST (IU/L)175 ± 34148 ± 21400 ± 30224 ± 29cP < .01).Inflammation score (Kleiner's score)0.5 ± 0.50.1 ± 0.41.9 ± 0.51.1 ± 0.7bP < .001,NOTE. Table 1 shows means ± SEM of 6 mice per group. Statistically significant differences between antibiotic-treated and untreated mice are indicated (nonparametric variance analysis [Mann–Whitney test],a P < .05,b P < .001,c P < .01). Open table in a new tab NOTE. Table 1 shows means ± SEM of 6 mice per group. Statistically significant differences between antibiotic-treated and untreated mice are indicated (nonparametric variance analysis [Mann–Whitney test], The low-grade liver inflammation induced by digestive bacterial products was amplified by challenging mice with LPS to study lymphocyte homing to fatty liver. We developed an adoptive transfer system for evaluating the contribution of liver steatosis to lymphocyte recruitment. Lymphocytes were isolated from LPS-challenged lean donor mice, labeled with a fluorescent cell tracer, and transferred to ob/ob or control LPS-challenged recipient mice (Figure 2A). The level of recruitment for CD4+ T, CD8+ T, NKT, B, and NK fluorescent cells to fatty liver, normalized for liver weight, was higher than the level of recruitment to control liver (Figure 3). Fatty livers therefore have a higher capacity to recruit B and T cells after LPS challenge than control livers.Figure 3The contribution of steatosis to lymphocyte homing. Recruitment of (A) CD4+ T cells, (B) CD8+ T cells, (C) NKT cells, (D) B cells, and (E) NK cells. Results are expressed as the number of fluorescent lymphocytes recruited per gram of liver. Graphs show means ± SEM of 3 experiments. Statistically significant differences (P < .05) between ob/ob and control livers are indicated by asterisks (nonparametric variance analysis [Mann–Whitney test]).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then investigated whether inflammatory cells isolated from ob/ob mice displayed an intrinsic capacity to migrate to the liver. We first isolated and phenotyped lymphocytes from the spleens of ob/ob and control mice. There was no significant difference in the number of T and B cells between ob/ob and control mice. LPS challenge did not affect the composition of lymphocyte subsets (data not shown). Lymphocytes isolated from the spleens of LPS-challenged ob/ob and control donors were used for adoptive transfer. They were labeled to identify their source (ie, ob/ob or control) and transferred to recipient mice (Figure 2B). We found that the level of CD4+ T- and CD8+ T-cell migration to the liver was higher for ob/ob than for control lymphocytes: 46.3 ± 9.2 vs 30.7 ± 7.9 (P < .05) and 44.0 ± 10.5 vs 25.7 ± 7.0 cells recruited to the liver for 105 transferred cells (P < .01), respectively. The level of B-cell migration into the liver was also higher for ob/ob than for control B cells: 17.1 ± 2.6 vs 10.1 ± 2.0 cells, respectively, recruited to the liver for 105 transferred cells (P < .001) (Figure 4, left column). No significant difference was observed for NK cells (data not shown). Lymphocyte migration into the spleen, thymus, and lymph nodes did not differ significantly between ob/ob and control cells (data not shown). Thus, the higher level of ob/ob CD4+ T-, CD8+ T-, and B-cell migration was specific to the liver. We validated the results obtained for ob/ob mice in another model of obesity by feeding C57BL/6 mice a HFD. These mice had a higher body weight than C57BL/6 control mice (42.8 ± 1.0 vs 30.8 ± 0.7 g, respectively; P < .05). Serum glucose, triglyceride, total cholesterol, high-density lipoprotein-cholesterol, and insulin × glucose product were significantly higher in HFD mice than in controls (see Supplementary Table online at www.gastrojournal.org). HFD mice showed steatosis and mild lobular inflammation (Kleiner's score: 1.4 ± 0.2 vs 0.75 ± 0.16, respectively; P < .05; see Supplementary Figure 1 online at www.gastrojournal.org). Using the adoptive transfer procedure described above, we showed that the level of CD4+ T-, CD8+ T-, and B-cell migration to the liver was significantly higher for HFD lymphocytes than for controls (see Supplementary Figure 2 online at www.gastrojournal.org). Thus, obesity, whether genetic or diet induced, leads to an increase in the homing of circulating lymphocytes to the liver. We investigated whether the activation of ob/ob lymphocytes by LPS challenge contributed to increases in lymphocyte migration to the liver by transferring lymphocytes isolated from unchallenged donors. The level of ob/ob lymphocyte migration was significantly lower than that for LPS-challenged donors for CD4+ T (Figure 4A and 4D, solid columns), CD8+ T (Figure 4B and 4E, solid columns), and B cells (Figure 4C and 4F, solid columns). Significantly lower levels of control lymphocyte migration were also observed in the absence of LPS challenge to donors (Figure 4, open columns). Recruitment to lymphoid organs was also decreased by the absence of LPS challenge of donors (data not shown). Thus, LPS enhances lymphocyte migration to several organs, including the liver. However, despite the low level of lymphocyte migration in the absence of LPS challenge, ob/ob lymphocytes still displayed higher levels of migration to the liver than control cells. This was found for CD4+ T, CD8+ T, and B cells (Figure 4, right columns). Migration was similar for ob/ob and control lymphocytes in the spleen, thymus, and lymph nodes in the absence of LPS challenge (data not shown). Our results show that ob/ob CD4+ T, CD8+ T, and B cells display higher levels of migration to the liver than control cells, whether or not they are exposed to LPS. We next explored the mechanisms underlying the preferential homing of ob/ob lymphocytes to the liver. In Transwell experiments (Figure 5A), CXCL12 induced the chemotaxis of both B and T cells, and CXCL13 had only a mild chemotactic effect on T cells but induced B-cell chemotaxis. CCL19 and CCL21 preferentially attracted CD4+ T and CD8+ T cells, but no difference was observed for the migration of ob/ob and control lymphocytes. In contrast, ob/ob CD4+ T cells responded more strongly to CXCL12 than controls, and ob/ob B cells responded more strongly to CXCL13 than controls. Thus, ob/ob lymphocytes were more sensitive to both CXCL12 and CXCL13 than control cells, with statistically significant differences observed for the CXCL12/CD4+ T-cell and CXCL13/B-cell combinations. We analyzed by immunochemistry and immunofluorescence the source of chemokines in the liver. CXCL12 and CXCL13 were colocalized (Figure 5B) both in control and fatty livers. The chemokine-producing cells were CD31 negative (Figure 5B) and had the same morphology than CK19-positive cells, demonstrating their bile duct epithelial origin (Figure 5C). We generated “non-obese ob/ob” mice to distinguish between the contributions of obesity itself and leptin deficiency in the lymphocyte response to CXCL12 and CXCL13. These mice recovered mobility, with no sign of suffering or lethality, and had a significantly lower body weight than ob/ob mice (29.22 ± 0.41 vs 56.07 ± 0.94 g, respectively; P < .01), similar to that of lean control mice (Figure 6A). Serum glucose, triglyceride, total cholesterol, high-density lipoprotein-cholesterol, and insulin × glucose product were lower in these mice, as compared with ob/ob mice (see Supplementary Table online at www.gastrojournal.org). Liver weight, serum aminotransferase, and the number of inflammatory foci returned to control levels (Figure 6B–E). The quantification of total liver lymphocytes also showed significantly lower levels of liver inflammation in “non-obese ob/ob” mice (Figure 6F). Overall, these results demonstrate that liver inflammation in ob/ob mice is related to obesity itself rather than to leptin deficiency. Because “non-obese ob/ob” mice are leptin deficient, comparisons of this model with “conventional ob/ob” mice can be used for studies of the role of obesity itself in the response of lymphocytes to CXCL12 and CXCL13. CD4+ T and B cells from “non-obese ob/ob” mice migrated significantly less in response to CXCL12 and CXCL13 than did the equivalent cells from ob/ob mice (Figure 7A and 7B). This migration level was in a similar range to that for lean controls. Our results demonstrate that the restoration of normal weight in ob/ob mice abolishes lymphocyte hyperresponsiveness to chemokines. We then investigated whether a decrease in weight in ob/ob mice prevented lymphocyte migration to the liver. Ob/ob and “non-obese ob/ob” lymphocytes were used for adoptive transfer. The level of CD4+ T-, CD8+ T-, and B-cell migration to the liver was lower for “non-obese ob/ob” lymphocytes than for ob/ob lymphocytes (Figure 7C–E). In conclusion, weight normalization abolishes both obesity-induced in vitro hypersensitivity to CXCL12 and CXCL13 and in vivo migration to the liver. This report demonstrates a new pathophysiologic mechanism of inflammation in fatty liver. We show that liver inflammation in ob/ob mice results not only from the higher potency of the fatty liver to attract circulating lymphocytes but also from the intrinsic tendency of ob/ob lymphocytes to migrate specifically to the liver. We demonstrate that LPS increases lymphocyte migration to the liver by directly acting on lymphocytes. T and B cells from ob/ob mice are hyperresponsive to CXCL12 and CXCL13 and are prone to migrate to the liver. These properties are directly due to obesity and not to leptin deficiency and are reversible with weight normalization. Overall, our study provides the first evidence that obesity leads to a lymphocyte dysfunction characterized by abnormal sensitization to chemotactic agents. The liver, which is closely linked to the gastrointestinal tract, must act as a bivalent immune organ, developing an inflammatory response against pathogens and tolerance to nonpathogen digestive bacterial compounds.17Bowen D.G. McCaughan G.W. Bertolino P. Intrahepatic immunity: a tale of two sites?.Trends Immunol. 2005; 26: 512-517Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 18Crispe I.N. Giannandrea M. Klein I. et al.Cellular and molecular mechanisms of liver tolerance.Immunol Rev. 2006; 213: 101-118Crossref PubMed Scopus (195) Google Scholar, 19Li Z. Diehl A.M. Innate immunity in the liver.Curr Opin Gastroenterol. 2003; 19: 565-571Crossref PubMed Scopus (96) Google Scholar We show that liver inflammation decreased after antibiotic treatment in ob/ob mice. Gut-derived endotoxins may trigger inflammation in the fatty liver, both in murine models of NASH and in humans.20Brun P. Castagliuolo I. Leo V.D. et al.Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G518-G525Crossref PubMed Scopus (671) Google Scholar, 21Wigg A.J. Roberts-Thomson I.C. Dymock R.B. et al.The role of small intestinal bacterial ov" @default.
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• W2083050494 title "Obesity-Induced Lymphocyte Hyperresponsiveness to Chemokines: A New Mechanism of Fatty Liver Inflammation in Obese Mice" @default.
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• W2083050494 doi "https://doi.org/10.1053/j.gastro.2008.02.055" @default.
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