Potential conflict of interest: Nothing to report.
Interleukin (IL)-22 is known to play a key role in promoting antimicrobial immunity, inflammation, and tissue repair at barrier surfaces by binding to the receptors, IL-10R2 and IL-22R1. IL-22R1 is generally thought to be expressed exclusively in epithelial cells. In this study, we identified high levels of IL-10R2 and IL-22R1 expression on hepatic stellate cells (HSCs), the predominant cell type involved in liver fibrogenesis in response to liver damage. In vitro treatment with IL-22 induced the activation of signal transducer and activator of transcription (STAT) 3 in primary mouse and human HSCs. IL-22 administration prevented HSC apoptosis in vitro and in vivo, but surprisingly, the overexpression of IL-22 by either gene targeting (e.g., IL-22 transgenic mice) or exogenous administration of adenovirus expressing IL-22 reduced liver fibrosis and accelerated the resolution of liver fibrosis during recovery. Furthermore, IL-22 overexpression or treatment increased the number of senescence-associated beta-galactosidase-positive HSCs and decreased alpha-smooth muscle actin expression in fibrotic livers in vivo and cultured HSCs in vitro. Deletion of STAT3 prevented IL-22-induced HSC senescence in vitro, whereas the overexpression of a constitutively activated form of STAT3 promoted HSC senescence through p53- and p21-dependent pathways. Finally, IL-22 treatment up-regulated the suppressor of cytokine signaling (SOCS) 3 expression in HSCs. Immunoprecipitation analyses revealed that SOCS3 bound p53 and subsequently increased the expression of p53 and its target genes, contributing to IL-22-mediated HSC senescence. Conclusion: IL-22 induces the senescence of HSCs, which express both IL-10R2 and IL-22R1, thereby ameliorating liver fibrogenesis. The antifibrotic effect of IL-22 is likely mediated by the induction of HSC senescence, in addition to the previously discovered hepatoprotective functions of IL-22. (HEPATOLOGY 2012;56:1150–1159)
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Microbial infection activates the innate and adaptive immune responses, which, in turn, control infection and promote tissue repair. For example, bacterial infection results in the activation of different immune cells that produce interleukin (IL)-22, which plays an important role in controlling bacterial infection through the up-regulation of antimicrobial proteins. IL-22 also promotes tissue repair by up-regulating a variety of genes expressed in epithelial cells, such as hepatocytes.1-3 The action of IL-22 is mediated by binding to the receptors, IL-10R2 and IL-22R1, which activates signal transducer and activator of transcription (STAT) 3.1-3 IL-10R2 is ubiquitously expressed, whereas IL-22R1 is believed to be expressed exclusively in the epithelial cells of various organs.1-3 In the liver, hepatocytes express IL-22R1 and IL-10R2. By ligating these receptors in a heterodimer, IL-22 promotes hepatocyte survival and proliferation, resulting in liver repair.4, 5 However, the effect of IL-22 on liver fibrogenesis remains unknown.
Liver fibrosis is a consequence of chronic liver injury and is characterized by an accumulation of extracellular matrix (ECM) proteins and the activation of hepatic stellate cells (HSCs).6-8 Subsequent to liver injury, HSCs become activated, express alpha-smooth muscle actin (α-SMA), and produce large amounts of collagen.6-8 There has been tremendous progress in discovering the regulatory mechanisms that control the activation of HSCs during liver fibrogenesis, including inflammatory cells (e.g., Kupffer cells and natural killer [NK] cells), growth factors, cytokines, and chemokines.6-8 Additionally, the senescence of activated HSCs is also an important step in limiting the fibrogenic response to tissue damage.9, 10 After becoming senescent, activated HSCs stop proliferation and express reduced levels of ECM components, but increase levels of ECM-degrading enzymes.9, 10 Deletion of the important cell-cycle regulator, p53, reduces HSC senescence, leading to extensive liver fibrosis.9 Moreover, many cytokines, such as IL-6 and IL-8, and their downstream signaling molecules STAT5 and suppressor of cytokine signaling (SOCS) 1 have been shown to promote cellular senescence in many cell types11, 12; however, their roles in HSC senescence have not been reported. In the current study, we demonstrate, for the first time, that HSCs express high levels of IL-10R2 and IL-22R1. Furthermore, we provide evidence suggesting that IL-22 induces HSC senescence through the activation of STAT3, SOCS3, and p53 pathways, thereby inhibiting liver fibrosis.
Ad, adenovirus; ALT, alanine aminotransferase; α-SMA, alpha-smooth muscle actin; Bcl-2, B-cell lymphoma 2; BrdU, bromodeoxyuridine; caSTAT3, constitutively activated STAT3; CHX, cycloheximide; ECM, extracellular matrix; ERK1/2, extracellular signal-related kinase 1/2; GFP, green fluorescent protein; HMGA1, high-mobility group AT hook protein 1; HSCs, hepatic stellate cells; hHSCs, human HSCs; IL, interleukin; IL-22TG mice, IL-22 liver-specific transgenic mice; KIR, kinase inhibitory region; mHSCs, mouse HSCs; MMP-9, matrix metalloproteinase-9; mRNA, messenger RNA; NK, natural killer; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; p-p53ser15, phosphorylated p53 at serine 15; RT-PCR, reverse-transcriptase polymerase chain reaction; pSTAT3, phosphorylated STAT3; SA-β-Gal, senescence-associated β-galactosidase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; STAT3Hep−/−, hepatocyte-specific STAT3 knockout mice; TIMP, tissue inhibitor of metalloproteinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild type.
Materials and Methods
C57BL/6 mice and SOCS3flox/flox mice were purchased from the Jackson Laboratory (Bar Harbor, ME). IL-22 transgenic (IL-22TG) mice and hepatocyte-specific STAT3 knockout (STAT3Hep−/−) mice were described previously.13 To induce hepatic fibrosis, mice were treated intraperitoneally with 2 mL/kg body weight of 10% CCl4 (Sigma-Aldrich, St. Louis, MO) for 8 weeks. Animals were sacrificed at 1 or 5 days after the last injection. All animal experiments were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.
Analysis of HSC Senescence.
HSC senescence in fibrotic livers or in cultured HSCs was determined by the detection of SA-β-Gal (senescence-associated β-galactosidase) activity using an SA-β-Gal staining kit (Cell Signaling Technology, Danvers, MA). Briefly, frozen liver sections or adherent cells were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 minutes, washed with PBS containing 1 mM of MgCl2, and stained overnight in PBS containing 1 mM of MgCl2, 1 mg/mL of X-Gal, 5 mM of potassium ferricyanide, and 5 mM of potassium ferrocyanide. Sections were counterstained with eosin. SA-β-Gal-positive areas were measured in at least three low-power (×100) microscope fields using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Bethesda, MD).
Data are expressed as the mean ± standard error of the mean (n = 6-10). To compare values between two groups, the Student's t test was used. A P value <0.05 was considered significant. Most of the experiments were repeated in three or four independent trials with similar results, and representative images are included in this article.
All other materials and methods are described in the Supporting Materials and Methods.
IL-22 Predominately Activates STAT3 in HSCs That Express High Levels of IL-22R1 and IL-10R2.
IL-22R1 messenger RNA (mRNA) expression was detected in quiescent and activated mouse HSCs (mHSCs), and these levels were comparable to IL-22R1 mRNA levels in hepatocytes (Fig. 1). IL-22R1 mRNA expression increased further after treatment with IL-22 in cultured HSCs (Fig. 1B). Expression of IL-10R2 mRNA, which is also required for IL-22 signaling, was detected in HSCs as well as in hepatocytes and Kupffer cells (Fig. 1A). Additionally, western blotting analyses revealed the expression of IL-22R1 protein in primary mHSCs, which was slightly increased after IL-22 treatment (Fig. 1C). Fluorescence-activated cell sorting analyses detected IL-22R1 protein expression on the surface of primary mHSCs, and comparable expression levels were observed in HSCs from wild-type (WT) and IL-22TG mice (Supporting Fig. 1A,B). Finally, the expression of IL-22R1 and IL-10R2 mRNA was also detected in primary human HSCs (hHSCs) from 3 human donors and in the hHSC cell line, LX2 (Fig. 1D).
The effects of IL-22 on the signaling pathways in HSCs are shown in Fig. 1E. IL-22 exposure significantly activated STAT3 in all samples, with peak effects observed at 30-60 minutes. Activated STAT3 levels returned to basal levels by 120 minutes. IL-22 also induced extracellular signal-related kinase 1/2 (ERK1/2) activation in primary mHSCs and, to a lesser extent, in hHSCs and LX2 cells. Furthermore, IL-22-dependent STAT3 activation in HSCs was further confirmed by immunostaining for phosphorylated STAT3 (pSTAT3) in the nuclei of HSCs (Supporting Fig. 1C,D).
IL-22 Inhibits HSC Apoptosis Without Affecting HSC Proliferation In Vitro.
IL-22 has been shown to promote hepatocyte survival and proliferation4; therefore, we examined the potential antiapoptotic and mitogenic effects of IL-22 on HSCs. The nuclear morphology of HSCs revealed a significant increase in apoptosis after a 4-hour incubation with cycloheximide (CHX) that was markedly reduced in IL-22 pretreated HSCs (Fig. 2A and Supporting Fig. 2). The antiapoptotic function of IL-22 in HSCs was further demonstrated by a reduction in CHX-mediated induction of caspase-3 and -7 activity and cleaved caspase-3 expression in HSCs after IL-22 treatment (Fig. 2A,B). Furthermore, Fig. 2C shows that serum and platelet-derived growth factor (PDGF), but not IL-22 treatment, increased bromodeoxyuridine (BrdU) incorporation in HSCs (Fig. 2C), indicating that IL-22 does not affect HSC proliferation. Finally, the expression of antiapoptotic proteins, such as pSTAT3 and B-cell lymphoma 2 (Bcl-2), was markedly increased, whereas expression of the mitogenic protein, cyclin D1, was slightly elevated in HSCs after IL-22 exposure (Fig. 2D).
IL-22TG Mice Are Resistant to Chronic CCl4 Treatment-Induced Liver Fibrosis, but Not Liver Injury.
Our results support a model in which IL-22 protects against HSC apoptosis in vitro; therefore, we examined whether IL-22 also prevents HSC apoptosis in vivo. Although hepatic and serum IL-22 levels were not significantly elevated in CCl4-treated mice, hepatic IL-22 levels were markedly increased in viral hepatitis patients.13-15 To identify the effect of high IL-22 levels on liver fibrosis, we used CCl4 to induce liver fibrosis in IL-22TG mice, which overexpress IL-22 in the liver13 and mimic the elevated IL-22 levels associated with viral hepatitis. Both WT and IL-22TG mice were treated with CCl4 for 8 weeks (Supporting Fig. 3A), and then mice were sacrificed and the extent of HSC apoptosis and liver fibrosis was analyzed. HSC apoptosis (α-SMA+/terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]+) decreased in the liver of IL-22TG mice, when compared to WT mice (Fig. 3A,B). This suggests that IL-22 promotes HSC survival in vivo, which led us to hypothesize that IL-22 may exacerbate liver fibrosis. However, to our surprise, the degree of liver fibrosis was lower in IL-22TG mice, when compared to WT mice (Fig. 3A-C and Supporting Fig. 3B). After CCl4 treatment, we observed reduced areas of α-SMA and Sirius Red staining as well as a reduction in the expression of α-SMA protein and collagen mRNA in IL-22TG mice, when compared to WT mice. Finally, the percentage of 5- and 1-day Sirius Red areas was significantly lower in the IL-22TG mice, when compared to WT mice, during the fibrosis resolution stage (Supporting Fig. 3C), indicating that IL-22TG mice resolved hepatic fibrosis much faster than WT mice.
Next, we investigated whether the observed reduction in liver fibrosis in IL-22TG mice was the result of the hepatoprotective effects of IL-22.4 Although IL-22TG mice were completely resistant to Con A–induced liver injury,13 surprisingly, serum alanine aminotransferase (ALT) levels were comparable between IL-22TG and WT mice after CCl4 treatment (Supporting Fig. 3D). In addition, the expression of hepatic cytochrome P450 2E1, a key enzyme responsible for the metabolization of CCl4 in the liver, was not up-regulated in IL-22TG mice (Supporting Fig. 3E). These results suggest that the decreased hepatic fibrosis observed in IL-22TG mice was neither caused by a reduction in CCl4 metabolism nor by liver injury.
To further understand the mechanisms underlying the reduction in fibrosis in IL-22TG mice, HSC senescence, a key step in limiting liver fibrosis,9, 10 was examined. More SA-β-Gal+ cells accumulated in the fibrotic scar tissue of liver sections from CCl4-treated IL-22TG mice, when compared to liver tissues from WT mice (Fig. 3D). Serial coimmunostaining using α-SMA antibody with SA-β-Gal staining or with another senescence marker, high-mobility group AT hook protein 1 (HMGA1),16 showed that the expression of SA-β-Gal and HMGA1 colocalized with α-SMA (Supporting Fig. 3F). These results indicate that IL-22TG mice have a higher number of senescent HSCs, when compared to WT mice after chronic CCl4 treatment.
Administration of IL-22 Adenovirus Accelerates Spontaneous Liver Fibrosis Resolution.
To examine the effect of IL-22 on liver fibrosis resolution, CCl4-treated mice were challenged with adenovirus (Ad)-IL-22 or control Ad-GFP (green fluorescent protein) immediately after the final CCl4 injection (Supporting Fig. 4A). The number of apoptotic HSCs was lower in Ad-IL-22-treated mice, when compared to Ad-GFP-treated mice (Fig. 4A,B). Despite reduced HSC apoptosis, liver fibrosis resolution was faster in Ad-IL-22-treated mice than in WT mice, as demonstrated by lower levels of α-SMA expression, Sirius Red staining, α-SMA protein, and collagen mRNA in Ad-IL-22-treated mice 5 days post-CCl4 treatment (Fig. 4A-C and Supporting Fig. 4B).
Immunohistochemistry staining revealed an increase in the number of SA-β-Gal+ cells in livers of Ad-IL-22-treated mice than in the livers of Ad-GFP-treated mice, and these cells tended to reside within fibrotic scar tissue (Fig. 4D). Moreover, the administration of Ad-IL-22 up-regulated the expression of matrix metalloproteinase-9 (MMP-9) and proinflammatory genes, but down-regulated tissue inhibitor of metalloproteinase (TIMP) expression (Supporting Fig. 4C), which is consistent with a senescence-associated secretory phenotype.9 Finally, we also isolated HSCs and performed SA-β-Gal staining in vitro to further confirm that IL-22 promotes HSC senescence. The data in Supporting Fig. 4D show that approximately 60% of HSCs from Ad-IL-22-treated mice were positive for SA-β-Gal staining, whereas only 20% of HSCs from Ad-GFP-treated mice were positive.
Because liver regeneration affects liver fibrosis,17 we examined hepatocyte proliferation in CCl4-treated mice after IL-22 administration. Ad-IL-22 injection for 5 days markedly increased hepatocyte proliferation, whereas such treatment for 24 hours resulted in no differences (Supporting Fig. 4F). To further determine whether the hepatoprotective and mitogenic functions of IL-22, which are mediated by the activation of STAT3 in hepatocytes,4 also contribute to the IL-22-mediated inhibition of liver fibrosis, we used STAT3Hep−/− mice. Treatment with Ad-IL-22 ameliorated liver fibrosis in STAT3Hep−/− mice, but the degree of inhibition was lower than in WT mice (Supporting Fig. 4F,G). This suggests that IL-22 inhibits liver fibrosis through hepatocyte STAT3-dependent and -independent mechanisms.
IL-22 Promotes Activated HSC Senescence and Inhibits HSC Activation In Vitro.
Next, we used cultured HSCs to test whether IL-22 has a direct effect on HSC activation and senescence. IL-22 exposure decreased α-SMA and collagen-(I) mRNA and protein levels in HSCs cultured for 4 or 7 days (Fig. 5A,B), suggesting that IL-22 inhibits HSC activation. Moreover, our results suggest that IL-22 promotes HSC senescence. IL-22 treatment increased the number of SA-β-Gal+ HSCs, but decreased telomerase activity in HSCs (Fig. 5C and Supporting Fig. 5A,B). Additionally, reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of senescence-associated inflammatory genes showed that IL-22 treatment up-regulated the expression of MMP-9, IL-6, and macrophage inflammatory protein 2, but down-regulated TIMP1 expression (Fig. 5D). Finally, IL-22 exposure up-regulated the expression of several cellular senescence-associated proteins, including phosphorylated p53 at serine 15 (p-p53ser15), p53, p21, and SOCS3 (Fig. 5E). In contrast, IL-22 challenge failed to promote mouse hepatocyte senescence (Supporting Fig. 5C).
IL-22 Induces HSC Senescence Through a STAT3-Dependent Mechanism.
To test the role of STAT3, a key downstream transcription factor of IL-22, in IL-22 induction of HSC senescence, we generated STAT3−/− HSCs. STAT3 protein deletion was confirmed by western blotting (Fig. 6A). IL-22 treatment up-regulated SA-β-Gal activity and the expression of p-p53ser15, p53, and p21 in WT HSCs, but not in STAT3−/− HSCs (Fig. 6B,C and Supporting Fig. 6A).
Additionally, we generated constitutively activated STAT3 (caSTAT3)+ cells that overexpress caSTAT3 to further determine the function of STAT3 in HSC senescence. The expression of Flag-caSTAT3 was confirmed by western blotting (Fig. 6D). Infection with Ad-Flag/caSTAT3 markedly decreased α-SMA protein expression (Fig. 6D), but increased SA-β-Gal staining in HSCs (Fig. 6E and Supporting Fig. 6B). Moreover, the introduction of Flag-caSTAT3 increased the expression of p-p53ser15, p53, and p21 in HSCs (Fig. 6F).
SOCS3 Is Required for IL-22-Induced Senescence and p53 Activation in HSCs.
SOCS1 has been shown to induce cellular senescence by binding to p53.18 Furthermore, the structure of SOCS3 is similar to SOCS1, both of which have a kinase inhibitory region (KIR) capable of binding to p53.18 This led us to hypothesize that SOCS3, a STAT3-regulated gene, may contribute to the IL-22-mediated induction of HSC senescence. IL-22 exposure up-regulated the expression of SOCS3 mRNA and protein in HSCs (Fig. 7A,B). To investigate the importance of SOCS3 in IL-22-induced senescence, we generated SOCS3−/− HSCs and confirmed SOCS3 deletion by real-time PCR (Fig. 7C). IL-22 treatment induced the accumulation of a higher number of SA-β-Gal+ HSCs in WT HSCs, when compared to SOCS3−/− HSCs, indicating that SOCS3−/− HSCs are refractory to IL-22-induced senescence (Fig. 7D and Supporting Fig. 7). Furthermore, the deletion of SOCS3 abrogated the IL-22-mediated induction of p53 and its target genes (Fig. 7E).
To investigate how SOCS3 modulates the activity of p53, we performed an immunoprecipitation assay with anti-p53 or anti-SOCS3 antibodies in HSCs. IL-22 treatment or caSTAT3 overexpression up-regulated both p53 and SOCS3 proteins (Fig. 7F). Immunoprecipitation assays showed that p53 and SOCS3 bound each other in HSCs, and that IL-22 treatment or caSTAT3 overexpression promoted the interaction between p53 and SOCS3.
In this study, we have demonstrated, for the first time, that HSCs, cells of nonepithelial origin, express IL-22R1 and IL-10R2. Through the ligation of both receptors, IL-22 induces HSC senescence, resulting in the inhibition of liver fibrosis. Furthermore, results from our mechanistic studies suggest that IL-22 induction of HSC senescence is mediated by the activation of a STAT3/SOCS3/p53-signaling axis (as summarized in Fig. 8).
IL-22R1 has been classically thought to be expressed exclusively in epithelial cells.1-3 Interestingly, our study demonstrates the detection of high levels of IL-22R1 mRNA and protein expression in quiescent and activated primary mHSCs, primary hHSCs, and the human HSC cell line, LX2. HSCs are thought to originate from mesodermal mesothelial cells/submesothelial cells19 and differ from hepatocytes and biliary epithelial cells, which are derived from the embryonic endoderm. Additionally, the expression of IL-22R1 was reported on colonic subepithelial myofibroblasts.20 Therefore, there is evidence that, in addition to epithelial cells, some nonepithelial cells, such as quiescent HSCs, activated HSCs/myofibroblasts, subepithelial myofibroblasts, and skin fibroblasts, also express IL-22R1.
Upon binding to IL-22R1 and IL-10R2, IL-22 promotes epithelial cell (e.g., hepatocyte) proliferation and survival.4 In the present article, we have demonstrated that IL-22 also promotes HSC survival, but induces HSC senescence, rather than stimulating HSC proliferation. Our study shows that the overexpression of IL-22 by either gene targeting (i.e., transgenic) or the exogenous administration of Ad-IL-22 increased the number of senescent HSCs within the fibrotic scars of the livers of CCl4-treated mice. Furthermore, we show that IL-22 challenge modulates the expression of “senescence-associated secretory phenotype” genes10 by up-regulating proinflammatory genes and MMP-9 and by down-regulating TIMP1/2 genes in the liver in vivo and in cultured HSCs in vitro. Finally, in vitro IL-22 treatment increased SA-β-Gal activity and the expression of the cellular senescence-associated genes, p53 and p21. The up-regulation of these genes likely contributes to IL-22-mediated HSC senescence, because the p53-p21 axis is known to inhibit the cell cycle.21-23
Our study also provided evidence suggesting that the IL-22-dependent up-regulation of p53 and p21 is mediated through STAT3 and SOCS3, resulting in HSC senescence. Although there is no evidence suggesting that STAT3 directly promotes cellular senescence, several STAT3 downstream target genes have been shown to induce cellular senescence, including p53, p21, and the SOCS family.18, 21-24 Our data in this article showed that the deletion of STAT3 abolished the IL-22-mediated induction of p53, p21, and HSC senescence, whereas the overexpression of caSTAT3 promoted HSC senescence (Fig. 6). This suggests that STAT3 plays an important role in IL-22-mediated HSC senescence through the induction of p53 and p21.
SOCS3 is an important feedback suppressor for STAT3 activation during normal cytokine signaling. Our results support another aspect of SOCS3 function, in that SOCS3 directly binds to p53, thus enhancing the expression of p53 protein and p53 target genes. The deletion of SOCS3 abolished the IL-22-mediated induction of p53 and p53-regulated genes (Fig. 7). Immunoprecipitation experiments between SOCS3 and p53 suggest that these proteins directly interact with each other. However, how SOCS3 activates p53 remains unknown. SOCS1 has been reported to form a ternary complex with p53 and ataxia telangiectasia mutated kinase through the KIR domain of SOCS1, followed by activating p53, increasing p53 protein expression, and, subsequently, promoting oncogene-induced senescence.18 SOCS3 also contains a KIR, which suggests that the KIR in SOCS3 might be responsible for SOCS3 binding to and activating p53 and resulting in HSC senescence.
Although many studies have suggested that senescent cells eventually succumb to p53-mediated cell death,25 our findings imply that IL-22 promotes HSC senescence and does not induce, but rather prevents, HSC apoptosis. We suspect that this may be because IL-22 activates STAT3, which not only promotes cell senescence through the induction of p53, but also acts as a key transcriptional factor for cell survival through the induction of the antiapoptotic genes, Bcl-2 and B-cell lymphoma extra large. Interestingly, other groups have also reported the simultaneous observation of resistance to apoptosis and senescence in human fibroblasts.26
The induction of activated HSC senescence plays an important role in limiting fibrogenic response, which is likely mediated by enhancing the in vivo clearance of senescent HSCs, presumably by NK cells, the down-regulation of collagen and TIMP expression, and the up-regulation of MMP expression.9, 10 Interestingly, in vitro treatment of HSCs with IL-22 not only induces HSC senescence, but also down-regulates α-SMA expression in these cells (Fig. 5). Because senescent HSCs are not associated with the down-regulation of α-SMA,10 the decreased α-SMA expression in response to IL-22 may not be directly the result of senescence, and a distinct mechanism may be involved, which will require further studies to elucidate.
In addition to the IL-22/STAT3 induction of HSC senescence, the well-documented hepatoprotective and mitogenic effects of IL-22/STAT3 in hepatocytes4, 5 may also contribute to the observed amelioration of liver fibrosis by IL-22. Indeed, the administration of Ad-IL-22 markedly inhibited liver fibrosis in STAT3Hep−/− mice, but the degree of inhibition was lower, when compared to that in WT mice, indicating that IL-22 inhibits liver fibrogenesis through hepatocyte STAT3-dependent and -independent mechanisms. Moreover, the administration of Ad-IL-22 increased liver regeneration (Supporting Fig. 4E), which is shown to ameliorate liver fibrosis and may contribute to the antifibrotic effect of IL-22.17 Although the hepatoprotective effects of IL-22 have been well documented,4, 5 IL-22TG mice displayed a similar extent of liver injury after chronic CCl4 treatment, when compared to WT mice (Supporting Fig. 3D). Thus, in our model of CCl4-induced liver injury, the IL-22-mediated inhibition of liver fibrosis is not completely mediated by its hepatoprotective effects. However, IL-22 has been shown to protect against hepatocellular damage in various models of liver injury.4, 5 Thus, the hepatoprotective function of IL-22 likely plays an important role in inhibiting liver fibrosis in these models.
It has been reported that hepatic IL-22 levels are elevated in viral hepatitis patients; but, the effect of IL-22 on liver injury and fibrosis in these patients remains obscure. We have previously shown that the number of IL-22+ lymphocytes positively correlates with the grade of inflammation and serum ALT or aspartate aminotransferase levels in viral hepatitis patients.13 Interestingly, a recent study has shown that hepatic IL-22 expression inversely correlates with the histological activity index and fibrosis stage in hepatitis B virus patients.14 These findings suggest that elevated hepatic IL-22 levels may play a compensatory role in preventing liver injury and fibrosis in viral hepatitis patients.
The authors thank Dr. Michitaka Ozaki (Hokkaido University, Sapporo, Japan) for providing the caSTAT3 adenovirus and also Drs. Mingquan Zheng and Jay K. Kolls (Louisiana State University, New Orleans, LA) for providing IL-22 adenovirus; and also thank Dr. Scott Friedman (Mount Sinai School of Medicine, New York) for providing the LX2 cells. They thank Dr. Howard Young (National Cancer Institute at Frederick, National Institutes of Health) for editing the manuscript for this article.