1. Volume 132, Issue 1, Pages 358-371 (January 2007)
IL-28A Is a Key Regulator of T-Cell–Mediated Liver Injury via the T-Box Transcription Factor T-Bet 
Juergen Siebler, Stefan Wirtz, Benno Weigmann, Imke Atreya, Edgar Schmitt, Andreas Kreft, Peter R. Galle, Markus F. Neurath 
Gastroenterology 
Volume 132, Issue 1, Pages (January 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Expression of interleukin (IL)-28R on primary T cells and effects of IL-28A on T-helper cytokine production. (A) Primary murine CD4+ T cells and hepatocytes express both chains of the IL-28 receptor. Reverse-transcription polymerase chain reaction of total RNA isolated from unstimulated primary murine CD4+ T cells by using primers for IL-28Rα and IL-10R2 (upper panels). Primary CD4+ T cells expressed mRNA for both chains of the IL-28R. Murine hepatocytes were used as positive control (lower panels). One representative experiment is shown. (B) IL-28A induces IFN-gamma production by primary CD4+ T cells via T-bet. Primary splenic CD4+ T cells were isolated from C57Bl/6 wild-type or T-bet–deficient mice by using immunomagnetic beads. T cells were stimulated with anti-CD3 antibodies (5 μg/mL) for 2 days in the presence or absence of recombinant IL-28A (20 to 2000 ng/mL), followed by analysis of culture supernatants by specific ELISA. There was no IL-4 detectable in culture media of wild-type cells, and also, no IFN-gamma was measurable in supernatants of T-bet−/− T cells. One representative of three experiments is shown. (C) IL-28A augments IFN-gamma production by primary CD4+ T cells stimulated with IL-2, IL-12, or anti-CD28 antibodies. Primary splenic CD4+ T cells were isolated from C57Bl/6 wild-type (upper panel) or T-bet–deficient (lower panel) mice by using immunomagnetic beads and cultured in complete medium. Cells were then either left unstimulated or stimulated with anti-CD3 antibodies (5 μg/mL), anti-CD3 antibodies plus IL-2, anti-CD3 antibodies plus IL-12 (1 ng/mL), or anti-CD3 plus anti-CD28 (1 μg/mL) antibodies, as indicated. Cells were cultured in the presence (+) or absence (−) of recombinant IL-28A (200 ng/mL). Concentration of IFN-gamma and IL-4 in cell culture supernatants was determined after 3 days by specific ELISA. One representative of three experiments is shown. (D) Increased synthesis of IFN-gamma by Th1 cells and augmented production of IL-4 by Th2 cells following stimulation with IL-28A. Primary naïve CD4+ CD62+ T cells were differentiated into TH1 and TH2 cells, as specified in Materials and Methods. Cells were cultured in the presence or absence of 200 ng/mL of recombinant murine IL-28A, as indicated. Production of IFN-gamma and IL-4 in culture media was analyzed by specific ELISA. One of three independent experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Cloning of murine interleukin (IL)-28A from stimulated spleen CD4+ T cells and analysis of its bioactivity in vivo. (A) Total RNA from unstimulated (left lane) or CD3- or CD28-stimulated (right lane) CD4+ T cells isolated from C57Bl/6 mice was subjected to RT-PCR by using specific primers resulting in a fragment showing complete sequence homology with murine IL-28A. (B) The cloned IL-28A cDNA encodes for a biologically active protein. An IL-28A expression plasmid or an empty control vector was transiently transfected in 293T cells. After 36 hours, cell culture supernatants were taken and intraperitoneally injected in A2G mice. Injection of murine IFN-α (500 IU) into A2G mice served as positive control for Mx1 induction. Mx1 protein expression in liver lysates of A2G mice was detected by Western blot analysis and strongly induced in the IL-28A and IFN-α groups, as compared with in the control group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Generation of interleukin (IL)-28A–transgenic mice. (A) Transgenic construct: the IL-28A cDNA was cloned blunt end at the SmaI restriction site downstream of the CD2 promoter–enhancer yielding the CD2-IL-28A–expressing construct. (B) PCR analysis of transgenic integration in genomic DNA extracted from ear biopsies of founder animals by using upstream primers for CD2 and downstream primers for IL-28A. Two independent founder lines (lines 2 and 7), denoted tg1.1 and tg1.2, were identified. (C) RT-PCR of spleen cell poly(A)+RNA for IL-28A in wild-type and transgenic mouse lines (tg1.1 and tg1.2), by using transgene-specific primers. (D) Real-time PCR analysis of IL-28R expression in purified T cells from wild-type (wt) and transgenic (tg1.1 and tg1.2) mice. Splenic T cells were subjected to real-time PCR by using IL-28R–specific primers. CD4+ T cells from transgenic mice revealed unchanged IL-28R-α and IL-10R2 receptor levels as compared with cells from wild-type mice. The receptor expression levels in wild-type mice were defined as 1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Increased T-cell proliferation and cytokine production in interleukin (IL)-28A–transgenic mice. (A) Distribution of T-cell subsets in transgenic mice. Splenic T cells were isolated and the percentage of CD4+, CD8+, CD4+CD25+, and NK1.1 T cells was quantified in both transgenic mice and wild-type animals by FACS analysis using specific antibodies. Mean values ± SEM are reported. The percentage of cells in wild-type mice was set as 100%. No significant differences in T-cell subsets were noted between wild-type and transgenic mice. (B) Spleen CD4+ T cells were isolated from transgenic and wild-type mice and stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours. Proliferation was measured by 3H-thymidine uptake assay. One representative of three experiments is shown. (C) Analysis of cytokine production in transgenic IL-28A tg1.1 and tg1.2 mice and wild-type mice was performed from cell culture medium of anti-CD3– and anti-CD28–stimulated CD4+ T cells using flow cytometry analysis. One representative experiment out of three is shown. (D) Analysis of IL-4 and IFN-gamma production by NK1.1 T cells in wild-type and transgenic mice. Splenic NKT cells were isolated from wild-type and transgenic mice using NK1.1 magnetic beads and stimulated with anti-CD3 plus anti-CD28 for 24 hours. The concentration of IL-4 and IFN-gamma in culture supernatants was determined by specific ELISA. One representative experiment is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Over expression of interleukin (IL)-28A augments Con A–induced liver pathology. (A) Injection of Con A (25 mg/kg) in wild-type mice and both IL-28A–transgenic lines resulted in an up-regulation of IL-28A expression, as shown by RT-PCR of hepatic mRNA 3 hours after Con A administration. Beta-actin levels served as positive control. (B) Quantification of hepatic IL-28A expression. By using specific primers for real-time PCR, we analyzed the expression of IL-28A in wild-type mice and both transgenic mouse lines before and 3 hours after application of Con A or PBS. Expression of IL-28A was about 10-fold up-regulated in PBS-treated transgenic mice as compared with wild-type mice and could be further induced upon administration of Con A. (C) Eight hours after injection of Con A, transgenic and wild-type mice were killed, and blood was taken and livers were removed. Serum AST and ALT values from indicated groups of mice are shown as mean values ± SD from three independent experiments with 9 mice per group. Statistically significant differences between wild-type and both transgenic mouse lines are indicated (*P < .05). (D) Histopathology of the livers from Con A–treated wild-type and transgenic mice after staining with hematoxylin-eosin. Representative sections from each group are shown. Mice given PBS served as controls. (E) Increased sensitivity of IL-28 transgenic mice to low doses of Con A. IL-28A tg1.1 mice and wild-type mice were challenged with 4 mg/kg of Con A. After 8 hours, levels of AST and ALT were determined. One of three experiments is shown. (F) Expression of hepatic mRNA for IFN-gamma and IL-4 quantified by real-time PCR 4 hours after injection of Con A (25 mg/kg). Expression levels in transgenic mice are shown as x-fold induction as compared with control wild-type mice. There was an induction of both IL-4 and IFN-gamma levels in both transgenic lines as compared with wild-type mice. (G) STAT protein levels and phospho-STAT protein levels in liver lysates from wild-type (wt) and transgenic (IL-28tg) mice were analyzed by Western blots and immunoprecipitation using specific antibodies. In addition, levels of T-bet were determined in liver lysates from wild-type and transgenic mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Suppression of augmented Con A–mediated liver injury in transgenic mice by neutralizing anti–IFN-gamma but not anti–interleukin(IL)-4 antibodies. (A) 2 hours before application of Con A, IL-28A–transgenic mice were treated i.p. with neutralizing anti–IL-4 or anti–IFN-gamma antibodies or anti–IL-4 plus anti–IFN-gamma antibodies, respectively. Serum AST and ALT values are shown from at least three mice per group. (B) Histologic analysis of representative liver sections from all four groups of transgenic tg1.1. mice after staining with hematoxylin eosin. Neutralizing antibodies to IFN-gamma suppressed augmented liver injury in IL-28A–transgenic mice. (C) Transgenic tg1.1 and tg1.2 mice were given a lower dose of Con A (10 mg/kg) followed by administration of neutralizing antibodies against IFN-gamma, IL-4 or IFN-gamma plus IL-4. (D) Effects of anti–IL-4 antibody therapy on STAT-6 phosphorylation. Wild-type mice were injected with 1 mg anti–IL-4 antibody. After 12 hours, mice were killed and protein expression of STAT-6 and phospho-STAT6 was analyzed by Western blotting. Expression of beta-actin served as control. (E) T-bet is a key regulator of Con A–induced liver injury in IL-28A–transgenic mice. Tg1.1 mice were crossbred with T-bet−/− mice. IL-28A–transgenic, T-bet+/− mice were challenged with Con A (25 mg/kg). Eight hours later, liver injury was analyzed by determining levels of AST and ALT. Mean values ± SEM are shown. Statistically significant differences are indicated. (F) Western blot analysis of hepatic T-bet expression in four IL-28tg/T-bet+/− mice and four wild-type control mice. The former mice showed lower hepatic T-bet levels as compared with the latter control mice. Beta-actin expression is shown as control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
(A) Antisense oligonucleotides targeting the translation start site of IL-28A attenuate Con A–mediated liver injury in wild-type mice. Antisense oligonucleotides (AS) against the translation start site of IL-28A were intravenously injected in wild-type mice at 12 hours and 30 minutes (100 μg per treatment) before administration of Con A. Mice given nonsense oligonucleotides (NS) or phosphate-buffered saline (PBS) at the same time points served as controls. Eight hours after Con A administration, AST and ALT values were measured in all three groups of mice. Mean values ± SEM are shown. Statistically significant differences are indicated. (B) Hepatic expression of IL-28A mRNA was determined by real-time PCR after administration of Con A in mice given antisense oligonucleotides, nonsense oligonucleotides, or PBS. Administration of PBS and nonsense oligonucleotides served as controls. Amount of IL-28A expression was normalized to levels from mice treated with PBS-Con A.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions