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Potential conflict of interest: Nothing to report.
This work was supported by the Inserm (National Institute of Health and Medical Research), the Université Paris-Est Créteil, by grants for young investigators from Université Paris-Est Créteil (to F.L.), and from the Agence Nationale de la Recherche (ANR retour Post-doc; to F.L.). A.G. was the recipient of a fellowship from the French Ministry of Education and Research.
Interleukin (IL)-17 is a proinflammatory and fibrogenic cytokine mainly produced by T-helper (Th)17 lymphocytes, together with the hepatoprotective and antifibrogenic cytokine, IL-22. Cannabinoid receptor 2 (CB2) is predominantly expressed in immune cells and displays anti-inflammatory and antifibrogenic effects. In the present study, we further investigated the mechanism underlying antifibrogenic properties of CB2 receptor and explored its effect on the profibrogenic properties of IL-17. After bile duct ligation (BDL), the hepatic expression of Th17 markers and IL-17 production were enhanced in CB2−/− mice, as compared to wild-type (WT) counterparts, and correlated with increased fibrosis in these animals. In contrast, IL-22-induced expression was similar in both animal groups. Inhibition of Th17 differentiation by digoxin lowered Th17 marker gene expression and IL-17 production and strongly reduced liver fibrosis in CB2−/− BDL mice. In vitro, differentiation of CD4+ naïve T cells into Th17 lymphocytes was decreased by the CB2 agonist, JWH-133, and was associated with reduced Th17 marker messenger RNA expression and IL-17 production, without modification of IL-22 release. The inhibitory effect of JWH-133 on IL-17 production relied on signal transducer and activator of transcription (STAT)5 phosphorylation. Indeed, STAT5 phosphorylation and translocation into the nucleus was enhanced in JWH133-treated Th17 lymphocytes, and the addition of a STAT5 inhibitor reversed the inhibitory effect of the CB2 agonist on IL-17 production, without affecting IL-22 levels. Finally, in vitro studies also demonstrated that CB2 receptor activation in macrophages and hepatic myofibroblasts blunts IL-17-induced proinflammatory gene expression. Conclusion: These data demonstrate that CB2 receptor activation decreases liver fibrosis by selectively reducing IL-17 production by Th17 lymphocytes via a STAT5-dependent pathway, and by blunting the proinflammatory effects of IL-17 on its target cells, while preserving IL-22 production. (Hepatology 2014;58:296–306)
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Liver fibrosis is the common exacerbated wound-healing response associated with chronic liver injury. The immune system plays an important role in fibrosis, because persistent inflammation precedes and perpetuates the fibrogenic process. Indeed, it is now well documented that various immune subsets, including monocytes/macrophages, neutrophils, and lymphocytes govern fibrosis progression and regression by controlling hepatic myofibroblast (MFB) accumulation.[1-3] CD4+ T-helper (Th) lymphocytes (Th1, Th2, and Th17) are major regulators of liver immune response and control liver fibrosis progression. According to their phenotype, Th cells can promote or inhibit the fibrogenic process. A predominant Th1 response, characterized by a high production of type 2 interferon-gamma (IFN-γ), results in the inhibition of liver fibrosis subsequent to MFB apoptosis.[4, 5] In contrast, Th2 polarization promotes liver fibrosis, in particular, by production of interleukin (IL)−13. Though the Th1/Th2 paradigm galvanized the immunology field for several years, a newly described subset of Th cells, T-helper 17 (Th17), has been recently identified in liver of patients with biliary cirrhosis, alcoholic cirrhosis, or hepatitis C virus infection.[7, 8] The Th17 phenotype is characterized by the production of its signature cytokine, IL-17, as well as other interleukins, such as IL-21 and IL-22. Differentiation of Th17 cells occurs upon combined stimulation with transforming growth factor beta (TGF-β), IL-6, and IL-21 in mice, is controlled by two transcription factors, RAR-related orphan receptor (ROR-γt; RORc in humans) and ROR-α, and the phenotype is stabilized by IL-23 binding to the IL-23 receptor. IL-17 signals through the IL-17 receptor virtually expressed in all cell types by activation of nuclear factor kappa B and signal transducer and activator of transcription (STAT)3, and promotes recruitment and activation of neutrophils as well as induction of proinflammatory genes in immune cells, particularly in macrophages. IL-22 belongs to the IL-10 cytokine family and plays a major role in host defense by enhancing production of antimicrobial peptides. IL-22 signals through STAT3 after binding to an IL-22 receptor with restricted expression in epithelial cells. In the liver, IL-22 displays hepatoprotective effects and stimulates hepatic regeneration. More recently, IL-17 and IL-22 have also emerged as key regulators of organ fibrosis. Indeed, profibrogenic effects of IL-17 have been described in lung and heart and were attributed to either enhanced inflammation or direct activation of myofibroblast profibrogenic functions.[13, 14] In contrast, antifibrogenic effects of IL-22 have been reported in lung. In the liver, recent data have also demonstrated the profibrogenic effects of IL-17 and the antifibrogenic properties of IL-22 in several experimental models.[10, 16]
Cannabinoid receptor 2 (CB2) is a G-protein-coupled receptor, which is a constituent element of the endocannabinoid system that also comprises CB1 receptor and endogenous lipidic ligands (the endocannabinoids, anandamide and 2-arachydonoyl-glycerol). CB2 receptor is abundantly expressed in immune cells, including natural killer (NK), NKT, B, and T lymphocytes as well as monocytes and macrophages and generally displays potent anti-inflammatory properties. In the liver, CB2 receptor is induced after an acute or chronic injury, in both immune and liver fibrogenic cells.[19-21] Several studies have demonstrated that endogenous activation of this receptor limits liver injury through anti-inflammatory effects. Indeed, CB2 receptor inhibits the inflammatory process associated with acute ischemia/reperfusion, concanavalin A–induced hepatitis, alcohol-induced steatosis, and hepatocyte injury. Moreover, we have described that CB2 receptor displays antifibrogenic properties. Recent data have shown immunosuppressive properties of CB2 receptor on Th17 cells. In the present study, we therefore explored whether inhibition of IL-17 response may contribute to the antifibrogenic effects of CB2 receptors in the liver. We show that CB2 receptor expressed by Th17 cells participates in its antifibrogenic properties by selectively inhibiting IL-17-induced immune response, without affecting IL-22-mediated beneficial effects.
Materials and Methods
Male mice (8-12 weeks old) were used in this study. IL-17-deficient mice in a C57BL/6 background were generously provided by Prof. Yoichiro Iwakura (Tokyo University, Tokyo, Japan). CB2-deficient mice in a C57BL/6 background were kindly provided by Prof. Andreas Zimmer (University of Bonn, Bonn, Germany). Wild-type (WT) C57BL/6J mice were purchased from Janvier (Le Genest-St-Isle, France). All animals were housed and fed ad libitum in a pathogen-free animal facility and used in accordance with protocols approved by the French ethical committee (COMETH; authorization no.: 10/11/09-03B) and under the supervision of authorized investigators.
Bile Duct Ligation
Bile duct ligation (BDL) and section was performed, as previously described, and sham animals underwent laparotomy. Animals were sacrificed 3, 6, or 14 days after surgery. When indicated, mice were daily intraperitoneally injected with digoxin (40 µg/g; Sigma-Aldrich, Lyon, France) or vehicle (0.8% dimethyl sulfoxide) for 2 weeks, starting 1 day after surgery. After sacrifice, blood was collected and the liver was either fixed in buffered formalin or snap-frozen in liquid nitrogen. Experiments were performed on at least 4 animals per group and per time point.
Results are expressed as mean ± standard error of the mean (SEM), and statistical significance was determined by a two-tailed Student t test and one- or two-way analysis of variance, as appropriate, using PRISM 4.0 software. Data were considered significantly different for P < 0.05.
IL-17 Production and Liver Fibrosis Are Enhanced in CB2-Deficient Mice After BDL
We first investigated whether CB2 receptor may inhibit fibrogenesis through indirect inhibitory effects on T lymphocytes and compared the expression of Th1, Th2, and Th17 markers and associated cytokines in CB2-deficient and WT mice at days 3, 6, and 14 after BDL. WT and CB2-deficient BDL mice showed comparable expression of Th1 or Th2 marker and no modification of related hepatic IFN-γ and IL-4 cytokine production (Fig. 1). In contrast, CB2-deficient mice displayed enhanced messenger RNA (mRNA) expression of the Th17 markers, Rorα, Rorγt, and Il23r, and increased hepatic IL-17 production, as compared to WT counterparts, at 6 and 14 days after BDL (Fig. 1). Interestingly, hepatic IL-22 levels were similarly increased in BDL CB2-deficient and WT animals (Fig. 1). Up-regulation of Th17 immune response was associated with enhanced fibrosis in CB2-deficient BDL mice, as shown by an increase in Sirius Red staining, higher induction of collagen I mRNA, and enhanced αsma mRNA and protein expression, as compared to WT-BDL mice (Fig. 2A). Interestingly, WT-BDL mice displayed enhanced endocannabinoid-CB2-related tone, as shown by enhanced Cnr2 mRNA expression, increased expression of 2-arachydonoylglycerol (2-AG)-synthetizing enzymes Daglα and Daglβ, and decreased expression of 2-AG-degrading enzyme Mgll (Fig. 2B). CB2 deficiency did not modify the expression of Daglα and Daglβ or that of Mgll (Fig. 2B). We next directly addressed the role of IL-17 in BDL-induced liver fibrosis, using IL-17-deficient mice. Sirius Red staining was reduced in IL-17-deficient BDL mice, as compared to the WT group (Fig. 3A). In addition, IL-17-deficient mice displayed decreased alpha smooth muscle actin (α-SMA) staining and reduced αsma mRNA expression, when compared to WT animals (Fig. 3A). Liver injury was also reduced in IL-17-deficient BDL mice, as shown by reduced necrotic areas and lower serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes (Fig. 3B). Altogether, these results demonstrate that IL-17 deficiency protects mice from BDL-induced liver fibrosis and injury.
IL-17 Contributes to Enhanced Fibrosis in CB2-Deficient Mice
To determine whether enhanced IL-17 expression in CB2−/− mice contributes to increased fibrogenic response, we used digoxin, an RORγt antagonist that inhibits CD4+ T-cell differentiation toward Th17 lymphocytes. As expected, in vitro treatment of isolated CD4+ T cells with digoxin markedly diminished IL-17 and IL-22 production (Fig. 4A). Furthermore, Sirius Red and α-SMA staining, together with expression of collagen I, αsma, and Tgfβ mRNA, were significantly decreased in digoxin-treated WT and CB2-deficient-BDL mice, when compared to vehicle counterparts (Fig. 4B-D). Comparison of liver fibrosis parameters in the two groups of digoxin-treated animals showed that fibrosis remained significantly higher in CB2-deficient BDL animals, as compared to WT counterparts (Fig. 4B-D). Hypoxia-inducible factor (HIF)−1α is a transcriptional factor involved in BDL-induced liver fibrosis, and its expression is down-regulated in digoxin-treated Hep3B cells. Therefore, we wondered whether antifibrotic effects of digoxin could be mediated through inhibition of Hif-1α in Th17 cells. In vivo, increased hepatic Hif-1α expression occurred similarly in both BDL WT and CB2−/− mice (Supporting Fig. 2A) and was not modified under digoxin treatment (Supporting Fig. 2B). Furthermore, Hif-1α-induced expression in Th17 cells remained unmodified in the presence of digoxin (Supporting Fig. 2C).
Taken together, these data demonstrate that CB2 receptor invalidation leads to enhanced liver fibrosis by increasing IL-17 production, in addition to previously reported IL-17-independent pathways.
CB2 Receptor Activation Inhibits IL-17, but Not IL-22, Production by Th17 Lymphocytes
To clarify the effect of CB2 receptor activation on IL-17 production and Th17 gene expression, we next performed experiments in cultured Th17 lymphocytes. Addition of the CB2 agonist, JWH-133, to CD4+ undergoing Th17 differentiation for 5 days strongly reduced the number of IL-17+ cells, as shown by flow cytometry (FCM) analysis (Fig. 5A), and selectively decreased IL-17 production while sparing IL-22 levels (Fig. 5B). Importantly, the inhibitory effects of JWH-133 were lost in Th17 lymphocytes from CB2-deficient mice (Fig. 5B). Moreover, JWH-133 significantly reduced mRNA expression of Il17, Il23r, and Rorγt by Th17 lymphocytes, without affecting induction of Il22 mRNA expression (Fig. 5C).
T-Cell CB2 Receptor Activation Inhibits IL-17 Production by a STAT5-Dependent Pathway
Recent studies have shown that STAT5 plays a central role in the inhibition of IL-17 production. Therefore, we hypothesized that the decrease in IL-17 production by CB2 agonists may be mediated by increased STAT5 activity. Indirect immunofluorescence (IF) confocal microscopy experiments were performed in Th17 cells exposed to JWH-133. There was no phospho-STAT5 staining detectable in nondifferentiated CD4+ T cells, and a weak signal was found in the nucleus of JWH-133-treated cells (Fig. 6A). However, under Th17 cell differentiation, a strong nuclear phospho-STAT5 labeling was detected in JWH-133-treated cells, whereas the signal was exclusively restrained to the cytoplasmic compartment in vehicle-treated cells. Likewise, these results were further confirmed by western blotting analysis, showing a strong increase in STAT5 phosphorylation in Th17 cells incubated with JWH-133, whereas no increase was observed in vehicle-treated cells (Fig. 6B). We next investigated the effect of STAT5 inhibition on IL-17 production by Th17 lymphocytes exposed to JWH-133. Pharmacological inhibition of STAT5 blunted JWH-133-induced STAT5 phosphorylation (Fig. 6B) and strongly reduced the inhibitory effect of JWH-133 on IL-17 production, without affecting IL-22 levels (Fig. 6C). These data demonstrate that CB2 receptor activation selectively reduces IL-17 production by Th17 lymphocytes through a STAT5-dependent signaling pathway.
CB2 Activation Reduces IL-17-Induced Inflammatory Response on Macrophages and MFBs
We have previously described that CB2 receptor is expressed in Kupffer cells (KCs) and MFBs.[19, 24] The presence of IL-17 receptor has also been recently described in the same cells.[8, 10] Therefore, we investigated whether in addition to directly inhibiting IL-17 production by Th17 lymphocytes, CB2 receptor may also counteract the functional properties of IL-17 on its target cells. Treatment of RAW264.7 macrophages with IL-17 promoted their polarization toward a proinflammatory M1 phenotype, as shown by increased expression of M1 markers, including Tnfα, Il6, and Il1β or Ccl2 and Ccl4, and decreased the expression of M2 genes, including Clec7a, Mrc2, and Arg1 (Fig. 7A). Exposure of RAW264.7 macrophages to the CB2 agonist, JWH-133, strongly inhibited the induction of M1 genes by IL-17, but did not affect the inhibitory effects of IL-17 on M2 genes (Fig. 7A). Parallel experiments in MFBs demonstrated that IL-17 induces expression of several proinflammatory genes, including Il1β, Il6, and Ccl2, without affecting profibrogenic gene expression (Collagen I, Mmp2, Tgfβ, and Timp1; Fig. 7B). IL-17-induced proinflammatory response was blunted by JWH-133 (Fig. 7B). Taken together, these data demonstrate that CB2 receptor activation counteracts the proinflammatory response of IL-17 on its target cells.
We previously reported on the antifibrogenic properties of CB2 receptor in experimental models of liver fibrosis. These beneficial properties were related to both direct effects on MFB and to a reduction of inflammatory infiltration into the liver.[24, 31] The present study identifies CB2 receptor as a major negative selective regulator of the profibrogenic cytokine, IL-17. We also demonstrate that selective inhibition of IL-17 response is a key component of the antifibrogenic effects of CB2 receptors in the liver (Fig. 8).
Sustained inflammation is a major driving force in fibrosis development, and characterization of specific anti-inflammatory pathways is a critical issue for identification of antifibrogenic molecules. Our results show that IL-17 deficiency protects against BDL-induced fibrosis. These data are in line with recent studies describing the profibrogenic properties of IL-17 in different experimental models of liver, pulmonary, or myocardial fibrosis by direct effects on myofibroblasts or indirectly by release of proinflammatory and fibrogenic cytokines from macrophages.[10, 13, 14, 32] Interestingly, we also found that IL-17-deficient BDL mice are more resistant to liver injury than WT animals. Because hepatocyte apoptosis is an important initiating event in development of liver fibrosis, these data suggest that profibrogenic functions of IL-17 may also be consecutive to enhanced hepatocellular damage. However, although IL-17 receptor is expressed by hepatocytes, IL-17 does not directly promote hepatocyte apoptosis, suggesting that IL-17-induced hepatocellular injury may result from proapoptotic cytokine release from nonparenchymal cells. These data, combined with clinical studies showing that the number of IL-17-positive cells correlates with fibrosis severity in patients with chronic hepatitis B or alcoholic liver disease,[8, 34] strongly suggest that neutralization of IL-17 may represent an interesting antifibrogenic approach.
Recently, negative regulators of IL-17 have been identified, including interleukins such as IL-27 and IL-15 and nuclear receptor agonists, in particular, molecules activating peroxisome proliferator-activated receptor (PPAR)-γ and liver X receptor (LXR). Interestingly, IL-15 displays hepatoprotective properties and agonists of LXR or PPAR-γ show antifibrogenic and anti-inflammatory effects in the liver,[40, 41] but whether these beneficial effects rely on reduction of IL-17 production remains to be further investigated. Our data identify the CB2 receptor as a novel negative regulator of profibrogenic functions of IL-17. Indeed, we show that among the different T-helper phenotypes, the hepatic Th17 transcriptional program is selectively enhanced in CB2-deficient BDL mice and results in increased IL-17 production. Our in vitro studies further document these findings and demonstrate that activation of CB2 receptor by JWH-133 triggers direct inhibition of IL-17 production by isolated Th17 lymphocytes, the main source of IL-17 in the fibrotic liver. The suppressive role of CB2 receptor on IL-17 proinflammatory and fibrogenic functions is corroborated by in vivo experiments with digoxin, an inhibitor of Th17 differentiation, showing reduction of fibrosis in CB2-deficient mice. These data demonstrate that reduction in IL-17 production contributes to CB2-mediated antifibrogenic effects. However, Th17 cells may play a double-edged sword role during chronic liver injury because not only do they produce IL-17, but they also release the hepatoprotective and antifibrogenic cytokine, IL-22. Therefore, a critical issue when targeting Th17 lymphocytes is to favor inhibition of IL-17 while sustaining the beneficial properties of IL-22. Our data demonstrate that CB2 receptor agonist may fulfill these functions. Indeed, we demonstrate that activation of CB2 receptor in Th17 lymphocytes selectively constrains IL-17 production while sparing that of IL-22. In vivo studies further document these data and show that enhanced production of IL-17 in CB2-deficient BDL mice is not associated with modification of IL-22 levels. These findings, together with the observation that JWH-133 maintains Th17 viability (Supporting Fig. 1) suggest that CB2 receptor stimulation in Th17 cells may represent an innovative antifibrogenic approach by preserving the beneficial effects of IL-22 while dampening the deleterious consequences of IL-17 production.
Activation of STAT3 is critical for Th17 differentiation and IL-17 production. Strikingly, mice bearing a specific deletion of STAT3 in T lymphocytes show blunted IL-17 production, but no modification of IL-22 levels.[43, 44] These data pointed out that IL-17 and IL-22 are differentially regulated by STAT3-dependent and -independent pathways, respectively. They also suggested that targeting inhibition of STAT3 in Th17 lymphocytes may represent a promising mean to decrease IL-17 production while sparing IL-22 expression. Interestingly, recent studies have shown that STAT5 restrains STAT3-induced IL-17 gene expression by competing for the same binding sites in the IL-17 promoter. Our data show that CB2 receptor activation by JWH-133 promotes STAT5 phosphorylation and its translocation within Th17 nuclei, suggesting that STAT5 could also mediate the suppressive effect of CB2 receptor agonist on IL-17 production. Consistent with this hypothesis, the addition of a STAT5 inhibitor blunted the inhibitory effect of JWH-133 on IL-17 production without modifying IL-22 levels. These data reveal that STAT5 activation is a major signaling pathway in the inhibitory effects of CB2 agonists on IL-17 production.
Recent data have identified MFBs and KCs as IL-17 target cells.[8, 10] We found that IL-17 not only activates macrophages to produce proinflammatory cytokines, but also regulates the M1/M2 macrophage phenotype balance. Indeed, IL-17 promoted M1 polarization, but, at the same time, decreased the expression of alternative M2 markers. In addition, IL-17 enhanced the proinflammatory response of MFB, by up-regulating the expression of Il1β, Il6, Ccl2, and Tnfα, without affecting profibrogenic gene expression, in keeping with results obtained in human MFBs.[8, 34] Interestingly, KCs and MFBs also express CB2 receptor,[19, 24] suggesting that CB2 receptor activation could counteract proinflammatory response in these cells. Indeed, we found that JWH-133 down-regulates IL-17-induced macrophage activation by blunting induction of the M1 signature without affecting M2 response. These data are in line with our previous study and further argue for a major role of CB2 receptor as a regulator of the KC M1/M2 balance. In addition, JWH-133 strongly reduced the proinflammatory response elicited by IL-17 in MFBs. Therefore, in addition to reducing IL-17 production by Th17 cells, CB2 receptor activation in macrophages and MFBs counteracts the proinflammatory response of IL-17 on its target cells.
It is well established that pathogenesis of liver fibrosis results from a combination of events originating from parenchymal and nonparenchymal cells, including immune cells and MFBs. We have previously shown that the antifibrogenic effects of CB2 receptor result from antiproliferative and antiapoptotic properties on MFBs, hepatoprotective effects, and inhibition of the proinflammatory response of M1 KCs.[19, 24, 26] The present report identifies a novel pathway underlying the antifibrogenic properties of CB2 receptor, which relies on inhibition of IL-17-induced immune and fibrogenic responses in the liver. These data may also provide a rationale for the use of CB2 agonists in autoimmune and chronic inflammatory disease, in which development of selective IL-17-based therapies are anticipated.
The authors thank Prof. Andreas Zimmer for providing CB2-deficient mice and Prof. Yoichiro Iwakura for providing IL-17-deficient mice. The authors thank Meriem Garfa-Traoré for her expertise in confocal microscopy analyses (at Necker Hospital). The authors are grateful to Sophia Balustre for her help during in vivo experiments as well as Aurélie Guguin and Adeline Henry for their precious help in FCM experiments.