Volume 136, Issue 3, Pages 1037-1047 (March 2009) B Cells Suppress the Inflammatory Response in a Mouse Model of Primary Biliary Cirrhosis  Yuki Moritoki, Weici Zhang, Koichi Tsuneyama, Katsunori Yoshida, Kanji Wakabayashi, Guo–Xiang Yang, Christopher Bowlus, William M. Ridgway, Yoshiyuki Ueno, Aftab A. Ansari, Ross L. Coppel, Ian R. Mackay, Richard A. Flavell, M. Eric Gershwin, Zhe–Xiong Lian  Gastroenterology  Volume 136, Issue 3, Pages 1037-1047 (March 2009) DOI: 10.1053/j.gastro.2008.11.035 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Prominent PBC-like liver pathology is noted in B cell–deficient dnTGF-βRII mice. (A) Liver sections at 5 months of age demonstrate greater levels of cellular infiltrates surrounding bile ducts in Igμ–/–dnTGF-βRII mice than in dnTGF-βRII mice. (B) The liver disease score was greater in Igμ–/–dnTGF-βRII (n = 15) than in dnTGF-βRII mice (n = 13). (C) Portal inflammation was more striking in Igμ–/–dnTGF-βRII mice compared with dnTGF-βRII mice. Each section was individually analyzed and separated into those with and without portal inflammation. Thence, the percentage was calculated from the total number of sections for each group. (D) Bile duct damage was evaluated in 5 randomly selected portal areas per sample; the frequency of bile duct damage was significantly higher in Igμ–/–dnTGF-βRII mice. (E) Loss of interlobular bile ducts (arrowhead) was occasionally found in the portal tracts of Igμ–/–dnTGF-βRII mice. (H&E stain. Blue and black scale bars indicate 400 μm and 100 μm, respectively, in A and E. *P < .05; **P < .01; ***P < .001.) Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Flow cytometric and immunohistochemical profiles of liver MNC in Igμ–/–dnTGF-βRII and dnTGF-βRII mice. (A and B) The frequency of activated (CD44high) CD4+ and CD8+ T cells (A) and the surface expression of CD44 (B) were greater in the liver of Igμ–/–dnTGF-βRII mice (n = 5) compared with dnTGF-βRII mice (n = 5). MFI, mean fluorescent intensity. (C) Frequency of Foxp3+ regulatory T cells (Treg) in hepatic CD4+ T cells was significantly lower in Igμ–/–dnTGF-βRII mice. However the absolute number or total frequency of CD4+Foxp3+ Treg in Igμ–/–dnTGF-βRII mice did not demonstrate a significant decrease in this experiment. (D) Staining for granulocytes (Gr-1+) in portal areas demonstrated a greater density of cell aggregates in Igμ–/–dnTGF-βRII than in dnTGFβRII mice, with Gr-1+ cells representing more than half of the inflammatory cells in portal tracts of Igμ–/–dnTGF-βRII mice; also, Gr-1+ cells were frequent in sinusoids of livers in Igμ–/–dnTGF-βRII mice but were scarce in portal areas and sinusoids of dnTGF-βRII mice. CD4+ T cells in portal areas were present in dnTGF-βRII mice but infrequent in Igμ–/–dnTGF-βRII mice. CD8+ T cells were present around portal areas and sinusoids of liver of dnTGF-βRII mice but were concentrated in portal areas of Igμ–/–dnTGF-βRII mice. (Scale bar, 100 μm. *P < .05.) Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Levels of inflammatory serum cytokines in Igμ–/–dnTGF-βRII mice. (A–D) Mean serum cytokine levels in mice at 5 months of age, using a cytometric bead array kit. TNF-α (A) and IL-6 (D) were greatly increased in Igμ–/–dnTGF-βRII mice (n = 13) compared with dnTGF-βRII mice (n = 12), whereas there was no significant difference in the levels of IFN-γ (B) and MCP-1 (C). Levels of IL-12p70 and IL-10 were undetectable in the sera samples from either strain (data not shown). Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Colitis is exacerbated in B cell–deficient dnTGF-βRII mice. (A) Sections of colon at 5 months of age in Igμ–/–dnTGF-βRII and dnTGFβRII mice demonstrated that Igμ–/–dnTGF-βRII mice have severe inflammation in all layers and numerous crypt abscesses (arrows) in contrast to mild inflammation in dnTGF-βRII mice. (B) Disease stages of the colon were greater for Igμ–/–dnTGF-βRII mice (n = 15) than for dnTGF-βRII mice (n = 13). (C) Immunohistochemical profiles of colon MNC in Igμ–/–dnTGF-βRII show Gr-1+ cells aggregated in damaged crypts, with a crypt abscess (arrowhead) in the colon of Igμ–/–dnTGF-βRII mouse. Gr-1+ cells (arrows) also infiltrated frequently into submucosal area to muscular layers to a moderate degree, whereas Gr-1+ cells were scattered in crypt abscesses (arrowhead) in colons of dnTGF-βRII mice. CD4+ cells were frequently observed in mucosal layers of both Igμ–/–dnTGF-βRII and dnTGF-βRII mice to similar degrees. CD8+ cells were scattered to a very mild degree in mucosal layers of colons in both of these strains, while small aggregations of CD8+ cells were frequently observed in the mucosal layer of dnTGF-βRII mice. (Blue and black scale bars indicate 400 μm and 100 μm, respectively, in A and C. ***P < .001.) Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 PBC-like liver pathology transferred by dnTGF-βRII CD8+ T cells is ameliorated by cotransfer of peritoneal cavity (PC) B cells. (A) Liver histology was examined at 8 weeks after splenic CD8+ T-cell transfer in the presence or absence of CD19+ B cells from PC or spleen (Sp) of dnTGF-βRII mice into Rag-1–/– mice. Degrees of cellular infiltrates were less around bile ducts in mice in which CD8+ T cells + PC-B cells were transferred than in mice given CD8+ T cells alone or CD8+ T cells + Sp-B cells. (B) The liver disease score was lower in mice in which CD8+ T cells + PC-B cells were transferred (n = 7) than in those given CD8+ T cells alone or CD8+ T cells + Sp-B cells (n = 6 and 7, respectively). (C) Portal predominance of liver inflammation was not significantly different between the 3 groups. (D) Bile duct damage evaluated in 5 random portal areas in each sample showed frequency of bile duct damage to be significantly lower in mice given CD8+ T cells + PC-B cells compared with that of mice given CD8+ T cells + Sp-B cells, but not that of mice given CD8+ T cells alone. (H&E stain. Blue and black scale bars indicate 400 μm and 100 μm, respectively, in A. *P < .05.) Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Number of hepatic cellular infiltrates was reduced in CD8+ T-cell + peritoneal cavity (PC) B-cell transfer. (A) Absolute number of total hepatic mononuclear cells (MNC) was examined at 8 weeks after splenic CD8+ T-cell transfer in the presence or absence of CD19+ B cells from PC or spleen (Sp) of dnTGF-βRII mice into Rag-1–/– mice. A smaller number of total liver MNC was demonstrated in mice in which CD8+ T cells + PC-B cells were transferred (n = 7) than in those given CD8+ T cells alone or CD8+ T cells + Sp-B cells (n = 6 and 7, respectively). (B) Number of B cell–excluded MNC was also lower in mice in which CD8+ T cells + PC-B cells were transferred. (C) Number of hepatic CD8+ T cells was significantly diminished in mice in which CD8+ T cells + PC-B cells were transferred. (*P < .05; **P < .01.) Gastroenterology 2009 136, 1037-1047DOI: (10.1053/j.gastro.2008.11.035) Copyright © 2009 AGA Institute Terms and Conditions