Volume 137, Issue 6, Pages 2125-2135.e2 (December 2009) Myeloid STAT3 Inhibits T Cell-Mediated Hepatitis by Regulating T Helper 1 Cytokine and Interleukin-17 Production  Fouad Lafdil, Hua Wang, Ogyi Park, Weici Zhang, Yuki Moritoki, Shi Yin, Xin Yuan Fu, M. Eric Gershwin, Zhe–Xiong Lian, Bin Gao  Gastroenterology  Volume 137, Issue 6, Pages 2125-2135.e2 (December 2009) DOI: 10.1053/j.gastro.2009.08.004 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Activation of STAT3 and STAT1 in myeloid and T cells in Con A-induced hepatitis. C57BL/6 mice were injected with vehicle (saline) or Con A for various time points. (A) Phospho-STAT3 immunostaining on liver tissues from control and Con A-treated mice. Red and blue arrows depict pSTAT3 in hepatocytes and small cells in the sinusoids, respectively. (B) Western blot analyses of the spleen tissues. (C) Flow cytometry analyses of CD11b+ myeloid cells and CD3+ T cells from the spleen of mice treated with Con A for 2 hours with pSTAT3 or pSTAT1 antibodies. (D) Absolute number of CD11b+ and CD3+ cells stained with pSTAT3 or pSTAT1. *P < .05 and **P < .01. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Myeloid STAT3 depletion exacerbates Con A-induced hepatitis and promotes preferentially innate inflammatory Th1 (IFN-γ) cytokines and Th17 cytokines to a lesser extent without affecting Th2 (IL-4) cytokine. (A) Serum ALT levels. (B) H&E staining of liver sections 12 hours post-Con A injection. Arrows indicate necrotic areas. (C) Western blot analyses of liver protein extracts from mice 3 hours post-Con A injection. (D) Serum proinflammatory cytokines. (E) Relative induction of cytokines 3 hours post-Con A injection. The values from wild-type mice were set as 1. (F) Myeloperoxidase immunostaining of liver tissues 12 hours post-Con A injection from panel D. *P < .05, **P < .01, and ***P < .005 (n = 5–8), in comparison with the corresponding WT groups. ND, not detected. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Myeloid-cell specific STAT3 deficiency promotes T-cell STAT3 activation and IL-17 response. (A) STAT3 activation (pSTAT3+ cells) in hepatic CD11b+ and CD3+ T cells 2 hours post-Con A treatment analyzed by flow cytometry. (B and C) Real-time PCR analyses of mRNAs from spleen CD4+ T cells (B) and liver tissues (C) of mice treated with Con A. (D and E) Serum levels of IL-17 and IL-22. *P < .05, **P < .01, and ***P < .005 (n = 4–8), in comparison with the corresponding WT groups. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Deletion of STAT3 in T cells reduces IL-17 production and liver injury in Con A-induced hepatitis. (A) Phosphorylated STAT3 detection in CD3+ T cells in liver mononuclear cells (MNCs) and splenocytes from mice treated with Con A for 2 hours by flow cytometry. (B) Serum inflammatory cytokines. (C) Serum ALT levels. (D) Liver tissues stained by H&E 12 hours post-Con A injection. Arrows indicate necrotic areas. (E) Myeloperoxidase immunostaining of liver tissues 12 hours post-Con A injection. *P < .05 and **P < .01 (n = 6–12). The number of mice used in all the groups in panel B was the same. ND, not detected. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 IFN-γ plays an essential role, whereas IL-17 plays a minor but significant role in T cell-mediated liver injury: IL-17 stimulates Kupffer cells to produce cytokines but prevents IFN-γ-induced hepatocyte apoptosis. (A) Serum ALT levels 12 hours post-Con A injection. (B) H&E staining of liver sections 12 hours post-Con A injection. (C) Serum inflammatory cytokines. (D) Western blot analyses of phospho-P65 and phospho-STAT3 in IL-17-treated liver macrophages. (E) Inflammatory cytokines from IL-17-treated liver macrophage culture medium 24 hours later. (F) Isolated hepatocytes were cultured for 3 days without or with IFN-γ (10 ng/mL), IL-17 (10 ng/mL), or both cytokines. Lactate dehydrogenase (LDH) activity and caspase 3 were measured. *P < .05, **P < .01, ***P < .001. ND, not detected. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Deletion of IL-17 in STAT3Mye−/− mice does not reduce Con A-induced hepatitis. (A) Serum ALT levels 12 hours post-Con A injection. (B) H&E staining of liver sections 12 hours post-Con A injection. (C) Serum inflammatory cytokines 12 hours post-Con A injection. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Deletion of STAT1 in STAT3Mye−/− mice ameliorates liver injury and abolishes innate immune and Th1 but not Th2/Th17 cytokine production during Con A-induced hepatitis. (A) Activation of pSTAT1 in WT and STAT3Mye−/− splenocytes 2 hours post-Con A injection analyzed by Western blotting. (B and C) Serum levels of cytokines. (D) Serum ALT levels. *P < .05, **P < .01, and ***P < .005 (n = 3–6). ND, not detected. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 A model depicting the hepatoprotection of myeloid cell STAT3 in T-cell hepatitis. During T-cell hepatitis, myeloid cell STAT3 inhibits STAT1 signaling in these cells, followed by preventing IL-12/IL-27 production and subsequently inhibiting IL12/IL-27 stimulation of IFN-γ production by Th1 cells. Myeloid cell STAT3 inhibits STAT1 and NF-κB activation, followed by reducing production of inflammatory cytokines (IL-6 and TNF-α) and subsequently inhibiting IL-6 stimulation of IL-17 production by Th17 cells. IFN-γ plays an essential role in T-cell hepatitis via induction of inflammation and hepatocyte death, whereas IL-17 only weakly stimulates liver inflammation but prevents hepatocyte death, thereby playing a double-edged sword role in T-cell hepatitis. Myeloid cell STAT3 also inhibits IL-22 production via unknown mechanisms. Gastroenterology 2009 137, 2125-2135.e2DOI: (10.1053/j.gastro.2009.08.004) Copyright © 2009 AGA Institute Terms and Conditions