Volume 134, Issue 5, Pages 1459-1469.e2 (May 2008) Obesity-Induced Lymphocyte Hyperresponsiveness to Chemokines: A New Mechanism of Fatty Liver Inflammation in Obese Mice  Amélie E. Bigorgne, Laurence Bouchet–Delbos, Sylvie Naveau, Ibrahim Dagher, Sophie Prévot, Ingrid Durand–Gasselin, Jacques Couderc, Philippe Valet, Dominique Emilie, Gabriel Perlemuter  Gastroenterology  Volume 134, Issue 5, Pages 1459-1469.e2 (May 2008) DOI: 10.1053/j.gastro.2008.02.055 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Oral antibiotic treatment decreases liver inflammation in ob/ob mice. Oral ciprofloxacin and ornidazol treatment. Liver pathology of (A) untreated and (B) antibiotic-treated ob/ob mice (original magnification, ×40 and ×100, respectively). Solid arrows point to inflammatory cell foci, which are enlarged in the lower panels. Representative figures of 6 mice per group. (C) Flow cytometry analysis of lymphocytes infiltrating the liver. Graphs show means ± SEM of 5 mice per group. Statistically significant differences (P < .05) between antibiotic-treated and untreated ob/ob mice are indicated by an asterisk (nonparametric variance analysis [Mann–Whitney test]). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Schematic representation of the adoptive transfer procedure. (A) Study of the contribution of steatosis to lymphocyte homing. Lymphocytes, isolated from the spleen of LPS-challenged lean donor mice, were labeled with a fluorescent cell tracer and transferred to LPS-challenged ob/ob or control mice. Liver lymphocytes of recipient mice were harvested, and recruitment was analyzed by flow cytometry. (B) Study of the contribution of lymphocytes to homing to the liver. Lymphocytes, isolated from the spleen of LPS-challenged ob/ob or control donor mice, were labeled with fluorescent cell tracers, pooled, and transferred to LPS-challenged lean mice. Liver lymphocytes of recipient mice were harvested, and recruitment was analyzed by flow cytometry. Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 The 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]). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 The contribution of lymphocytes to homing to the liver. Lymphocytes were isolated from ob/ob and control mice, challenged (left column) or not (right column) with LPS, and used for adoptive transfer experiments. Migration into the liver of (A and D) CD4+ T cells, (B and E) CD8+ T cells, and (C and F) B cells. Results are expressed as the number of fluorescent lymphocytes recruited to the liver per 105 transferred lymphocytes. Graphs show means ± SEM of 6 experiments. Statistically significant differences between ob/ob and control cells are indicated (paired 2-group Wilcoxon signed-rank test, *P < .05, **P < .01, ***P < .001). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 In vitro response to chemokines and liver expression of CXCL12 and CXCL13. (A) Lymphocytes of LPS-challenged ob/ob or control mice were isolated from the spleen. The number of ob/ob CD4+ T, CD8+ T, and B cells migrating in response to CXCL12, CXCL13, CCL19, and CCL21 was compared with that of control lymphocytes. Results are expressed as the percentage of cells that migrated in response to chemokines. Graphs show means ± SEM of 5 experiments. Statistically significant differences (P < .05) between ob/ob and control cells are indicated by asterisks (nonparametric variance analysis [Mann–Whitney test]). (B) Double immunofluorescent detection of CXCL12/CXCL13, CD31 (endothelial cells)/CXCL12, and CD31/CXCL13 in control and ob/ob liver (original magnification, ×20). (C) Immunochemistry of CK19 (bile duct epithelial cells) and CXCL12 in control and ob/ob liver (original magnification, ×100). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Decrease in liver weight, aminotransferase level, and inflammation in “non-obese ob/ob” mice. (A) Body weight. (B) Liver weight, expressed as a percentage of body weight. (C) Serum ALT and (D) serum AST levels. (E) Liver inflammation (Kleiner's score). (F) Quantification of liver lymphocytes by flow cytometry, expressed as the total number of liver lymphocytes. Graphs show means ± SEM of 8 mice per group. Statistically significant differences are indicated by asterisks (nonparametric variance analysis [Mann–Whitney test]). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Normalization of B- and T-cell responses to CXCL12 and CXCL13 and decreased lymphocyte migration to the liver in “non-obese ob/ob” mice. (A) CXCL12-induced chemotaxis of CD4+ T cells and (B) CXCL13-induced chemotaxis of B cells. Results are expressed as the percentage of cells that migrated in response to chemokines. Graphs show means ± SEM of 3 experiments. Statistically significant (P < .05) and nonsignificant (ns) results are indicated (Kruskall–Wallis and Fisher PLSD tests). (C–E) Adoptive transfer of lymphocytes from ob/ob and “non-obese ob/ob” donors. Results are expressed as the number of fluorescent lymphocytes recruited to the liver per 105 transferred lymphocytes. Migration into the liver of (C) CD4+ T cells, (D) CD8+ T cells, and (E) B cells. Graphs show means ± SEM of 9 experiments. Statistically significant differences are indicated (paired 2-group Wilcoxon signed-rank test, *P < .05, **P < .01). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 Liver histology of mice fed a high-fat diet. Liver histology of: (A) Control and (B) high-fat diet mice (original magnification 40×). Black arrow points to inflammatory cell foci. Representative figures of 8 mice per group. Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 High-fat diet-induced obesity increases lymphocyte migration to the liver. Lymphocytes were isolated from high-fat diet (HFD) or control LPS-challenged donor mice, labeled, pooled, and transferred to LPS-challenged lean recipient mice. After migration, liver lymphocytes were harvested. Results are expressed as the number of fluorescent lymphocytes recruited into the liver per 105 transferred lymphocytes. Migration into the liver of: (A) CD4+T, (B) CD8+T, and (C) B cells. Graphs show means ± SEM of 5 experiments. Statistically significant differences between control and HFD cells are indicated (paired 2-group Wilcoxon signed rank test, *P < .05). Gastroenterology 2008 134, 1459-1469.e2DOI: (10.1053/j.gastro.2008.02.055) Copyright © 2008 AGA Institute Terms and Conditions