The IL-6–gp130–STAT3 pathway in hepatocytes triggers liver protection in T cell–mediated liver injury (original) (raw)
IL-6–gp130–dependent signaling in hepatocytes confers protection in the Con A model. The cytokine IL-6 triggers liver protection in the Con A model of T cell–mediated hepatitis. IL-6 controls gene transcription in various cell types that might be involved in protective mechanisms, e.g., hepatocytes and immune-activated cells. Hepatocytes are the predominant hepatic cells; therefore, we evaluated whether hepatocytes mediate IL-6–dependent protection in the Con A model. We generated mice with a hepatocyte-specific deletion of the IL-6 signal–transducing gp130 receptor (25) and analyzed the effect of IL-6 treatment in this experimental hepatitis model. Hepatocyte-specific gp130-deficient mice were generated by crossbreeding alfpCre and gp130loxP mice, in which LoxP sites flank exon 16, encoding the transmembrane domain of gp130 (26). Expression of Cre recombinase is controlled by a hepatocyte-specific albumin promoter, and results in a more than 90% deletion of exon 16 in adult liver (25). Since nonparenchymal cells show no gp130 deletion, the overall extent of hepatic gp130 inactivation is likely to reflect complete gp130 deletion in hepatocytes.
Gp130 wild-type (gp130LoxP/LoxP) animals were treated with IL-6 or vehicle before Con A injection. In order to monitor liver cell damage, AST levels were determined before and at various time points after treatment. IL-6 pretreatment completely blocked liver cell damage. No increase in transaminases was evident (Figure 1A). In alfpCre gp130LoxP/LoxP mice, IL-6 failed to have a protective effect, which indicates that IL-6–dependent liver protection is dependent on gp130-mediated signaling in hepatocytes (Figure 1B). Additionally, all alfpCre gp130LoxP/LoxP mice died within the first 20 hours after Con A injection. In contrast, all NaCL- and IL-6–treated wild-type animals survived during this phase, indicating a higher sensitivity of alfpCre gp130LoxP/LoxP mice toward Con A.
gp130 in hepatocytes is essential for IL-6–dependent protection in Con A–induced hepatitis. Con A hepatitis was induced by intravenous injection of 32.5 mg/kg Con A in wild-type (gp130LoxP/LoxP) and hepatocyte-specific gp130-null (alfpCre gp130LoxP/LoxP) mice. Mice were pretreated for 3 hours with NaCl (solid line) or IL-6 (200 μg/kg) (dotted line) before i.v. injection of Con A. Liver injury was quantified by detection of AST. (A) Time course of AST serum levels during Con A–induced hepatitis in wild-type mice pretreated with NaCl (solid line) or IL-6 (dotted line). #P < 0.01 and *P < 0.05 vs. corresponding IL-6–pretreated wild-type control group at the same time point. (B) Time course of AST serum levels during Con A–induced hepatitis in alfpCre gp130LoxP/LoxP mice pretreated with NaCl (solid line) or IL-6 (dotted line).
Lack of gp130 expression in hepatocytes has no impact on liver-damaging cytokine secretion after Con A injection. During Con A–induced hepatitis, immune cells are activated, and as a consequence, cytokines that promote liver cell damage are released. TNF-α and IFN-γ are essential in triggering Con A–induced liver injury. Additionally, IFN-γ and TNF-α are markers of immune-cell activation in this model. They are expressed by activated macrophages or CD4+ and CD8+ cells (1, 3, 27, 28). Therefore, we were interested in studying whether lack of gp130 expression in hepatocytes and IL-6 pretreatment have an impact on cytokine secretion.
IL-6 treatment of gp130LoxP/LoxP mice resulted in a slight reduction of TNF-α levels compared with those in animals treated with the carrier solution (Figure 2A). The same observation was evident in alfpCre gp130LoxP/LoxP animals. Slightly reduced TNF-α levels were observed in the IL-6–treated group (Figure 2B). IL-6 pretreatment had no impact on IFN-γ secretion in wild-type and alfpCre gp130LoxP/LoxP animals (Figure 2, C and D).
Cytokine secretion during Con A–induced hepatitis. Cytokine levels in the serum after Con A injection in wild-type (gp130LoxP/LoxP) and alfpCre gp130LoxP/LoxP mice. Three hours before Con A administration, animals were treated with NaCl (solid line) or IL-6 (200 μg/kg) (dotted line) by i.p. injection. (A and B) TNF-α levels of wild-type and alfpCre gp130LoxP/LoxP mice. #P < 0.01 vs. corresponding IL-6–pretreated wild-type control group at the same time point. (C and D) IFN-γ levels of wild-type and alfpCre gp130LoxP/LoxP mice. (E and F) IL-6 concentrations of wild-type and alfpCre gp130LoxP/LoxP mice. §P < 0.001 and #P < 0.01 vs. corresponding IL-6–pretreated wild-type or alfpCre gp130LoxP/LoxP mice at the same time point.
The endogenous IL-6 levels during Con A–induced hepatitis are significantly elevated in alfpCre gp130LoxP/LoxP mice. In wild-type mice, a significant increase in IL-6 serum concentration was observed during the first hours of Con A–induced hepatitis (Figure 2E). The IL-6 serum levels returned to basal levels 24 hours after Con A injection. IL-6 pretreatment reduced IL-6 serum levels at later time points (Figure 2E). A different IL-6 kinetic was evident in alfpCre gp130LoxP/LoxP animals. In both groups, a marked increase in IL-6 serum levels was obvious during the first hours after Con A injection. Additionally, IL-6 remained elevated during the first 24 hours in both groups (Figure 2F).
The IL-6 –gp130–dependent activation of STAT3 in wild-type mice is suppressed in alfpCre gp130LoxP/LoxP animals. To distinguish between the intracellular events that occur in wild-type and alfpCre gp130LoxP/LoxP animals during Con A–induced liver cell damage, we analyzed hepatic STAT1 and STAT3 activation during disease progression. Earlier studies indicated that STAT1 plays a harmful role during activation of CD4+ and NKT cells (29). Therefore, we studied nuclear translocation of phosphorylated STAT1 and STAT3 in the liver.
IL-6 pretreatment had no impact on hepatic STAT1 activation in wild-type or in alfpCre gp130LoxP/LoxP mice (Figure 3A; 0 hours, Con A) (25). Con A injection resulted in STAT1 activation in wild-type and alfpCre gp130LoxP/LoxP mice, but no remarkable differences were found between the 2 groups (Figure 3A). IL-6 pretreatment resulted in a slight increase of STAT1 phosphorylation in wild-type mice 2 hours and 4 hours after Con A injection (Figure 3A). In alfpCre gp130LoxP/LoxP animals, IL-6 injection resulted in a stronger and earlier STAT1 phosphorylation than in wild-type mice. Eight hours after Con A injection, STAT1 activation returned to basal levels (Figure 3A).
Changes in liver nuclear phosphorylation of STAT1 and STAT3 during Con A–induced hepatitis. (A) Phosphorylation of STAT1 in wild-type and alfpCre gp130LoxP/LoxP mice. Western blot analysis of STAT1 and STAT3 activation, as detected by phosphorylation of STAT1 and STAT3, in liver nuclear protein extracts of wild-type and alfpCre gp130LoxP/LoxP mice. Animals were pretreated 3 hours before Con A administration with NaCl or IL-6 (200 μg/kg) by i.p. injection as indicated. Representative results of 3 independent experiments are shown. (B) Phosphorylation of STAT3 in wild-type and alfpCre gp130LoxP/LoxP mice. (C) Double immunofluorescence of liver sections for P-STAT3 and CD11b expression. CD11b detects activated macrophages, NK cells, and monocytes. Mice were killed 2 hours after Con A injection. DAPI counterstaining was used to detect nuclei. (D) Double immunofluorescence of liver sections for P-STAT3 and CD11b expression. CD11b detects activated macrophages, NK cells, and monocytes. Mice were killed 4 hours after Con A injection. Representative areas are shown. Slides of 3 animals per group were analyzed. Magnification, ×400.
Activation of STAT3 during Con A–induced hepatitis can be divided into 3 phases (Figure 3B). The prephase, before Con A injection (0 hours), the immediate phase, 2 hours after injection, and 2 later phases, 4 and 8 hours after initiation of the disease.
To differentiate between STAT3 activation in hepatocytes and other liver cells we performed coimmunfluorescence stainings of CD11b and P-STAT3. In the prephase, STAT3α phosphorylation was exclusively detectable in hepatocytes of IL-6–pretreated wild-type animals, but not in IL-6–injected alfpCre gp130LoxP/LoxP animals (Figure 3B; 0 hours, Con A).
Two hours after Con A injection, IL-6 pretreatment resulted in stronger STAT3α activation in wild-type and alfpCre gp130LoxP/LoxP animals compared to that in the NaCl-treated groups (Figure 3B). During this phase, STAT3 was predominately expressed in activated monocytes, NK cells, and macrophages (Figure 3C; CD11b+ cells). IL-6 pretreatment enhanced the number of STAT3-positive cells (Figure 3C).
At the later phase, 4 hours after Con A injection, strong P-STAT3α and P-STAT3β signals were found exclusively in hepatocytes of NaCl-treated wild-type mice. This strong STAT3 activation was found in neither of the other groups (Figure 3, B and D). Increased STAT3 activation 4 hours after Con A injection depended on gp130 expression in hepatocytes. IL-6 pretreatment reduced STAT3 activation in hepatocytes in wild-type animals (Figure 3D). In hepatocyte-specific gp130–/– mice, only a weak STAT3 activation in nonparenchymal liver cells (CD11b+ cells) was detectable. This activation was independent of IL-6 pretreatment (Figure 3D).
Generation and molecular characterization of hepatocyte-specific gp130 ΔSTAT/LoxP and gp130Y757F/LoxP mice. Since gp130 expression in hepatocytes is essential for IL-6–induced protection in Con A–induced hepatitis, we aimed to identify the intracellular signaling pathways that are responsible for mediating protection. For this purpose, we generated mice with hepatocyte-specific deletions of either gp130-STAT1/3 or gp130-SHP2-RAS-MAPK signaling. The activation of the 2 distinct signaling cascades in response to ligand-mediated gp130 dimerization is mediated by phosphorylation of specific tyrosines (Y) in the cytoplasmatic domain of gp130. Y757 mediates the engagement of the cytoplasmic Src-homology tyrosine phosphatase (SHP2) and the activation of the RAS-ERK pathway (30). The 4-membrane distal phosphotyrosines are binding sites for the SH2 domains of the latent transcription factors STAT1 and STAT3 (31).
The alfpCre gp130ΔSTAT/LoxP and alfpCre gp130Y757F/LoxP animals were obtained by crossing alfpCre gp130LoxP/LoxP with either gp130ΔSTAT/ΔSTAT or gp130Y757F/YY757F knockin mice (32). Gp130ΔSTAT/ΔSTAT mice express a truncated gp130 protein that lacks the part of the cytoplasmic domain required for STAT1 and STAT3 binding and activation. Gp130Y757F/Y757F animals express a gp130 protein with a phenylalanine substitution of the Y757 residue which, in its phosphorylated form, is essential for SHP2 recruitment and subsequent activation of the RAS-MAPK cascade. In the resulting alfpCre gp130ΔSTAT/LoxP and alfpCre gp130Y757F/LoxP animals, the LoxP-flanked gp130 is deleted selectively in hepatocytes as expression of Cre recombinase is controlled by a hepatocyte-specific promoter (25). This approach therefore enables the selective expression of mutant gp130 protein in hepatocytes. Each mouse was genotyped by PCR analysis for the genomic existence of Cre, gp130LoxP, and the respective mutant gp130 allele.
Functional characterization of the alfpCre gp130ΔSTAT/LoxP and alfpCre gp130Y757F/LoxP animals was performed by IL-6 stimulation in primary hepatocytes isolated from the 4 strains. IL-6–induced activation of gp130-dependent pathways was studied by the monitoring of P-STAT1/3 and P-ERK2 expression. Treatment of wild-type primary hepatocytes resulted in phosphorylation of STAT3 and ERK2 (Figure 4B). Neither protein was phosphorylated (and hence activated) in hepatocytes derived from alfpCre gp130LoxP/LoxP mice (Figure 4C). However, in hepatocytes isolated from IL-6–stimulated alfpCre gp130Y757F/LoxP or alfpCre gp130ΔSTAT/LoxP mice, we observed selective activation of P-STAT3 or P-ERK2, respectively (Figure 4, D and E). P-STAT1 activation was neither induced in IL-6–stimulated primary hepatocytes isolated from wild-type, alfpCre gp130LoxP/LoxP, alfpCre gp130Y757F/LoxP, nor alfpCre gp130ΔSTAT/LoxP animals (data not shown).
Generation and functional characterization of hepatocyte-specific gp130ΔSTAT/LoxP and gp130Y757F/LoxP mice. (A) alfpCre gp130LoxP/LoxP mice were generated by breeding alfpCre mice with animals expressing LoxP-flanked gp130 alleles (25). Hepatocyte-specific gp130ΔSTAT/LoxP animals (alfpCre gp130ΔSTAT/LoxP) were generated by crossing alfpCre gp130LoxP/LoxP with gp130ΔSTAT/ΔSTAT mice, which express a truncated gp130 knockin allele encoding a truncated gp130 protein that lacks the domains mediating STAT1 and STAT3 activation (26). Hepatocyte-specific gp130Y757F/LoxP animals (alfpCre gp130Y757F/LoxP) were bred by crossing alfpCre gp130LoxP/LoxP with gp130Y757F/Y757F mice, which express a gp130 allele encoding a phenylalanine substitution of the Y757 residue (32), thereby rendering gp130 incapable of recruiting SHP2 and activating the RAS-MAPK pathway. Animals were genotyped by PCR analysis for alfpCre, gp130LoxP, and gp130Y757F alleles. (B and E) The functional characterization of hepatocyte-specific gp130 mutant mice. Gp130 downstream-signaling pathways were analyzed by monitoring of phosphorylated STAT3 and phosphorylated ERK2 (p42) expression in whole cell extracts of primary hepatocytes isolated from wild-type (B), alfpCre gp130LoxP/LoxP (C), alfpCre gp130Y757F/LoxP (D), or alfp Cre gp130ΔSTAT/LoxP mice (E). Activation of phosphorylated STAT1 was not detected in any of the 4 mouse strains.
Activation of the IL-6–gp130–STAT pathway in hepatocytes confers protection during Con A–induced liver damage. We used alfpCre gp130Y757F/LoxP and alfpCre gp130ΔSTAT/LoxP animals to identify the gp130-dependent pathways in hepatocytes essential for mediating IL-6–dependent protection during Con A–induced hepatitis. IL-6 pretreatment of alfpCre gp130Y757F/LoxP mice resulted in complete prevention of liver cell damage (Figure 5A). In contrast, alfpCre gp130ΔSTAT/LoxP animals showed enhanced sensitivity toward Con A when compared to wild-type mice (Figure 5B). Independently of IL-6 pretreatment, all alfpCre gp130ΔSTAT/LoxP mice died within 8 hours after Con A injection in contrast to a 100% survival rate for all analyzed alfpCre gp130Y757F/LoxP animals.
gp130-STAT3 signaling in hepatocytes is responsible for IL-6–induced liver protection. Con A–induced hepatitis was induced by intravenous injection of 32.5 mg/kg Con A in hepatocyte-specific gp130ΔSTAT (alfpCre gp130ΔSTAT/LoxP) and hepatocyte-specific gp130Y757F/LoxP mice (alfpCre gp130Y757F/LoxP). Mice were pretreated 3 hours before Con A injection with NaCl (solid line) or IL-6 (200 ng/g) (dotted line) by i.p. injection. (A and B) Liver damage was quantified by detection of ASTs. (A) AST levels of alfpCre gp130Y757F/LoxPmice in the time course of Con A–induced hepatitis, pretreated with NaCl (solid line) or IL-6 (dotted line). #P < 0.01 and *P < 0.05 vs. corresponding IL-6–pretreated control group at the same time point. (B) AST levels of alfpCre gp130ΔSTAT/LoxP in the time course of Con A–induced hepatitis, pretreated with NaCl (solid line) or IL-6 (dotted line). (C and D) TNF-α, IFN-γ, and IL-6 secretion in the serum during Con A–induced hepatitis in alfpCre gp130Y757F/LoxP (C) and alfpCre gp130ΔSTAT/LoxP mice (D) was determined by ELISA. §P < 0.001 vs. corresponding IL-6–pretreated alfpCre gp130Y757F/LoxP mice at the same time point.
To analyze possible modifications of immune-cell activation in these animals, serum TNF-α, IFN-γ, and IL-6 levels were determined before and after Con A injection (Figure 5, C and D). IL-6 pretreatment in alfpCre gp130Y757F/LoxP or alfpCre gp130ΔSTAT/LoxP animals had no significant impact on TNF-α serum levels (Figure 5D). In both mouse strains, IL-6 treatment resulted in slightly elevated IFN-γ serum levels (Figure 5, B and C). Additionally, IL-6 serum levels did not differ between the vehicle-treated alfpCre gp130Y757F/LoxP and alfpCre gp130ΔSTAT/LoxP animals. In both mouse strains, we detected maximal IL-6 concentrations 8 hours after Con A injection (Figure 5D). In contrast, pretreatment with recombinant IL-6 resulted in a reduction of endogenous IL-6 serum concentrations during Con A–induced hepatitis in alfpCre gp130Y757F/LoxP animals (Figure 5C). No reduction of IL-6 serum levels was observed in alfpCre gp130ΔSTAT/LoxP mice (Figure 5D).
Gene expression profiles of IL-6–gp130 pathways in hepatocytes. The analysis of hepatocyte-specific gp130 mutations during Con A–induced hepatitis demonstrated that IL-6–gp130–STAT3–dependent signals appear to confer protection. To identify potential target genes involved in mediating protection, hepatic gene expression profiles were determined in IL-6–treated wild-type, alfpCre gp130LoxP/LoxP, alfpCre gp130Y757F/LoxP, and alfpCre gp130ΔSTAT/LoxP mice. Figure 6 depicts a selection of genes that are significantly regulated by gp130-dependent pathways in hepatocytes in vivo. These can be divided into 2 groups: genes involved in controlling gp130-dependent signaling in the cell, e.g., stat-1, stat-3, socs-1 (suppressor of cytokine signaling-1) and socs-3; and genes regulating immune-mediated mechanisms, e.g., serum amyloid A2 (saa2) and keratinocyte growth factor (kc). As the gp130-STAT3–dependent pathway confers protection in the Con A model, we hypothesized that indirect immune-mediated mechanisms were essential for protection against liver damage in the Con A model.
IL-6–induced gene expression profile of wild-type, alfpCre gp130LoxP/LoxP, alfpCre gp130Y757F/LoxP, and alfpCre gp130ΔSTAT/LoxP mice. Affimetrix GeneChip analysis was used to detect gene expression of IL-6–stimulated gp130LoxP/LoxP, alfpCre gp130LoxP/LoxP, alfpCre gp130ΔSTAT/LoxP, and alfpCre gp130Y757F/LoxP mice. Three animals per group were treated in parallel by i.p. injection of recombinant IL-6 (IL-6 rα) (200 μg/kg) or NaCl. Liver RNA was isolated, and gene expression was compared. Most of the examples shown are differentially regulated by gp130-STAT signaling. Glycine receptor (Glycine R) and cytochrome p450 (p450) represent examples that are regulated by gp130-ras-MAPK signaling. The change of gene expression level is marked by increasing color intensity. LBP, LPS binding protein.
Treatment with SAA2 and KC protects against Con A–induced liver damage. We analyzed in more detail the regulation of saa and kc and their potential roles during Con A–induced liver failure. Both genes are selectively induced via IL-6–gp130–STAT3. The analysis of SAA serum expression showed that pronounced transcriptional induction of the saa gene strictly depended on IL-6–gp130–STAT3 signaling in hepatocytes. In order to confirm the array analysis, we also investigated hepatic mRNA expression (data not shown) and serum levels of SAA and found that both were elevated only in wild-type and alfpCre gp130Y757F/LoxP mice (Figure 7A).
Acute-phase protein SAA2 and the chemokine KC are IL-6–gp130–STAT–induced, liver-protective proteins. (A) SAA2 serum levels after IL-6 (200 μg/kg) injection. Each time point represents the serum of 3–5 animals. Filled squares represent wild-type mice, open squares show alfpCre gp130LoxP/LoxP mice, filled triangles mark alfpCre gp130Y757F/LoxP mice, and open triangles represent alfpCre gp130ΔSTAT/LoxP. (B) Pretreatment with recombinant SAA (0.8 mg/kg) leads to a significant reduction of AST levels in the time course of Con A–induced hepatitis in wild-type mice. Either SAA (dotted line) or NaCl (solid line) was injected intravenously 1 hour before induction of Con A–induced hepatitis. (C) Serum KC levels after IL-6 injection. Filled squares represent wild-type mice, open squares show alfpCre gp130LoxP/LoxPmice, filled triangles mark alfpCre gp130Y757F/LoxP mice, and open triangles represent alfpCre gp130ΔSTAT/LoxP. (D) IL-6 treatment results in a significant reduction of PMN infiltration during Con A–induced hepatitis. Four hours after Con A injection, the influx of PMNs was counted on H&:E-stained paraffin sections by an experienced liver pathologist. The arrows mark liver infiltrating PMNs. hPF, high powered field. (E) KC (40 μg/kg) treatment leads to a significant reduction of AST levels during Con A–induced hepatitis. Eight mice per group were treated with either KC (dotted line) or NaCl (solid line) 1 hour before Con A injection. §P < 0.001 and *P < 0.05 vs. corresponding control groups at the same time point.
SAA is known to interact with metalloproteinases and to influence the activity of immune cells (33). Therefore, we studied the impact of SAA on Con A–induced hepatitis by pretreating wild-type animals with recombinant SAA 1 hour before Con A injection. SAA injection resulted in a significant reduction in the increase of AST levels compared with those in vehicle-treated control animals, indicating that SAA may have a protective effect for Con A–induced liver damage (Figure 7B).
KC controls chemotaxis and activation of polymorph nuclear cells (PMNs) in areas of inflammation. Serum KC expression was also strictly dependent on an intact IL-6–gp130–STAT3–signaling axis in hepatocytes (Figures 6 and 7C). No hepatic KC regulation was found in alfpCre gp130ΔSTAT3/LoxP and alfpCre gp130LoxP/loxP animals. Earlier results showed that the preexistence of high KC concentration in the serum leads to decreased PMN infiltration during inflammation (34). This effect is caused by reduced sensitivity of downstream pathways of the corresponding chemokine receptor (34). We thus analyzed the influence of IL-6 pretreatment on PMN infiltration during Con A–induced hepatitis. IL-6 treatment resulted in a significant reduction of PMN infiltration 4 hours after Con A injection (Figure 7D). This time coincided with the maximum of PMN infiltration. In order to further investigate the relevance of this observation, recombinant KC was injected before Con A administration. KC had a protective effect on Con A–induced T cell–mediated liver failure. The increase in transaminases was significantly blocked in the animals treated with KC (Figure 7E). Additionally, KC treatment leads to 100% survival during Con A–induced hepatitis compared with 70% in NaCL-treated animals.