1. Volume 143, Issue 1, Pages 188-198.e7 (July 2012)
Interleukin-22 Promotes Proliferation of Liver Stem/Progenitor Cells in Mice and Patients With Chronic Hepatitis B Virus Infection 
Dechun Feng, Xiaoni Kong, Honglei Weng, Ogyi Park, Hua Wang, Steven Dooley, M. Eric Gershwin, Bin Gao 
Gastroenterology 
Volume 143, Issue 1, Pages e7 (July 2012)
DOI: /j.gastro
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2. Figure 1
Positive correlation between IL-22+ inflammatory cells and CK19+ LPCs in patients with chronic HBV. (A) Representative immunostaining with anti–IL-22 and anti-CK19 antibodies of serial liver sections from an HBV patient. Please note that most IL-22+ inflammatory cells and CK19+ LPCs are colocalized outside the dotted line. (B) Spearman's nonparametric rank correlation coefficient between the number of IL-22+ inflammatory cells and CK19+ LPCs from 64 HBV patients.
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3. Figure 2
The IL-22TG mice have increased LPC proliferation after being fed a DDC diet compared with the WT mice. The mice were fed a DDC diet for up to 12 weeks. (A) H&E staining of liver tissues from DDC-fed mice at different time periods. (B) Representative immunohistochemical analyses with an anti–pan-CK antibody of liver tissues from mice fed a DDC diet for 2 weeks. Arrows indicate ductular reactions. (C, D) Pan-CK (green)/BrdU (red) double staining of liver tissues from mice fed a DDC diet for 2 or 12 weeks with a BrdU injection 2 h before sacrifice. (E, F) The number of pan-CK+ and BrdU+pan-CK+ double-positive cells was quantified. **P < .01 and ***P < Representative photographs from 3 independent experiments with similar results are shown.
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4. Figure 3
STAT3 contributes to the increased susceptibility of the IL-22TG mice to DDC-induced LPC proliferation. WT, IL-22TG, and IL-22TGSTAT3LKO double-mutant mice were fed a DDC diet for 2 or 12 weeks. (A) Immunohistochemical staining with an anti–pSTAT3 antibody and H&E staining. Arrows indicate pSTAT3+ LPC nuclei. (B) Pan-CK (green)/BrdU (red) double staining of liver tissues with a BrdU injection 2 h before sacrifice. (C) Quantification of pan-CK+ and BrdU+pan-CK+ areas. **P < .01 and ***P < Representative photographs from 3 independent experiments with similar results are shown.
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5. Figure 4
Injection of Ad-IL-22 stimulates LPC proliferation in the WT but not in the STAT3LKO mice. WT and STAT3LKO mice were fed a DDC diet for 4 weeks followed by injection with an Ad-vector or Ad-IL-22 virus. The mice were then maintained on a DDC diet for 2 more weeks and sacrificed. (A) A schematic diagram of the experimental design. (B) Serum IL-22 levels. (C) H&E staining. (D) Double staining with pan-CK (green)/BrdU (red) antibodies. (E) Quantification of the pan-CK+ area and BrdU+pan-CK+ double area. *P < .05 and **P < .01. Representative photographs from 3 independent experiments with similar results are shown.
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6. Figure 5
The expression of IL-22R1 and IL-10R2 on LPCs. (A) Liver nonparenchymal cells from the DDC-fed mice were isolated and analyzed by flow cytometry with fluorescein isothiocyanate (FITC)-anti-CD45, phycoerythrin (PE)–anti-EpCAM, and allophycocyanin (APC)–anti-IL-22R1 or APC–anti-IL-10R2 antibodies (Ab). Data are representative of 3 different experiments. (B) Real-time polymerase chain reaction analyses of IL-22R1 and IL-10R2 expression in primary mouse hepatocytes and purified LPCs from the DDC-treated mice. **P < .01.
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7. Figure 6
IL-22 promotes LPC proliferation in vitro in a STAT3-dependent manner. LPCs were purified from the DDC-fed WT or STAT3LKO mice and were then cultured in vitro. (A) These purified LPCs were serum starved for 4 h and were then treated with IL-22 (100 ng/mL) in vitro for 30 min. Pan-CK (green) and pSTAT3 (red) double staining were performed. (B) Serum-starved LPCs were also treated with IL-22 (100 ng/mL) in vitro, followed by incubation with BrdU for 24 h. Pan-CK (green) and BrdU (red) double staining were performed. Arrows indicate positive staining. The number of pan-CK+BrdU+ LPCs was counted and shown in the right panel. (C) Real-time polymerase chain reaction analysis of several anti-apoptotic and cell-cycle–associated genes from purified LPCs treated with IL-22 for 6 h. *P < .05; **P < .01; and ***P < Data are representative of 3 different experiments.
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8. Figure 7
IL-22 promotes the proliferation and prevents the apoptosis of BMOL cells in vitro. (A) BMOL cells were treated with IL-22 for 30 min before Western blot analysis of pSTAT3 and STAT3. (B) BMOL cells were starved overnight and then treated with IL-22 for 24 h, and BrdU was added 2 h before harvest. BrdU+ cells were detected by immunostaining. (C) BMOL cells were pretreated with IL-22 for 2 h before treatment with cycloheximide for 6 h. Cell apoptosis was then determined. (D, E) BMOL cells were treated with IL-22 for 24 h before being subjected to Western blot or reverse transcription polymerase chain reaction analysis. *P < .05; **P < .01; and ***P < .001.
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9. Supplementary Figure 1
CD3+ T cells are the major source of IL-22 in the livers from HBV patients. Liver samples from human HBV patients were stained with anti-IL-22 and anti-CD3 antibodies. Most IL-22+ cells were co-localized with CD3+ T cells. Representative photographs from 25 HBV-infected livers with similar results are shown.
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10. Supplementary Figure 2
Similar LPC proliferation in WT mice and IL-22 KO mice after DDC diet feeding. (A) Mice were fed DDC diet for various time points, serum IL-22 levels were measured. (B) H&E staining of liver tissues from 2-week DDC-fed WT and IL-22 KO mice. (C) Pan-CK (green)/BrdU (red) double staining of liver tissues from 2-weel DDC fed-WT and IL-22TG mice with BrdU injection 2 h before sacrifice. (D) Quantification of the pan-CK+ area and BrdU+pan-CK+ double positive cells. (E) Quantification of BrdU+hepatocytes in WT mice and IL-22 KO 32 and 40 h after 2/3 partial hepatectomy. Values represent means ± SD (n=6=10).
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11. Supplementary Figure 3
WT and IL-22TG mice were fed DDC diet for various time periods. (A) Serum ALT and AST were measured.
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12. Supplementary Figure 4
Increased LPCs in IL-22TG mice after CDE diet feeding. Mice were fed CDE diet for 4 weeks. (A) H&E staining of liver tissues from CDE-fed WT and IL-22TG mice. (B) Pan-CK (green)/BrdU (red) double staining of liver tissues from CDE-fed WT and IL-22TG mice with BrdU injection 2 h before sacrifice. (C) Quantification of the pan-CK+ area and Brdu+pan-CK+ double positive cells. **P < .01, ***P < .001.
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13. Supplementary Figure 5
STAT3 was deleted in LPCs of IL-22TGSTAT3LKO mice. WT,IL-22TG and IL-22TGSTAT3LKO double mutant mice were fed DDC diet for 4 weeks. Liver sections were stained with anti-STAT3 antibody and visualized by DAB. Brown staining is positive for STAT3 protein.
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14. Supplementary Figure 6
(A) LPCs were purified from DDC-fed mice by using two-step MACS purification process. Liver nonparenchymal cells were stained with FITC-anti-CD45 and PE-anti-EpCAM. CD45+ cells were depleted in the first step and EpCAM+ cells were positively selected in the second step. (B) Primary LPCs were isolated from DDC-fed mice, cultured in vitro and stained with anti-pan-CK antibody. Nuclei were stained with Propidium iodide. About 70-80% purified cells are pan-CK positive. LPCs from the DDC-fed IL-22TG mice proliferate faster in vitro compared with those from the DDC-fed WT mice, which is suppressed by the treatment with an IL22 neutralizing antibody. WT and IL-22TG mice were fed a DDC diet for 4 weeks. (C) LPCs were purified and cultured in vitro for 24h, and the supernatants were then collected for the measurement of IL-22 levels. (D) Purified LPCs were cultured in serum free medium with or with out IL-22 blocking antibody (5 μg/mL, from R&D system). BrdU (10 μM) was added to culture medium 24 h before harvest.Pan-CK (green) and BrdU (red) double staining were performed to evaluate cell proliferation. (E) Quantification of percentage of BrdU+pan-CK+ cells in total pan-CK+ cells. **P < .01, *P < .05.
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15. Supplementary Figure 7
IL-22 does not upregulate IL-20 mRNA expression in BMOL cells, and IL-20 does not upregulate expression of cyclin D, Bcl-xL, and Bcl-2 in BMOL cells.
Gastroenterology  , e7DOI: ( /j.gastro )
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