Research ArticleFIBROSIS

Type 3 cytokines IL-17A and IL-22 drive TGF-β–dependent liver fibrosis

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Science Immunology  26 Oct 2018:
Vol. 3, Issue 28, eaar7754
DOI: 10.1126/sciimmunol.aar7754

Type 3 injury

Chronic liver injury caused by infection, toxins, or other inflammatory conditions can lead to liver fibrosis. Several cytokines have been linked to liver fibrosis, and here, Fabre et al. define a role for the type 3 cytokines interleukin-17A (IL-17A) and IL-22 in driving transforming growth factor–β (TGF-β)–dependent fibrosis. They observed elevated levels of IL-17A and IL-22 in intrahepatic lymphocytes from patients with hepatitis. Hepatic stellate cells treated with IL-22 showed enhanced p38 mitogen-activated protein kinase–dependent TGF-β signaling. Liver-resident neutrophils and mast cells were identified as the primary sources of IL-17 in humans, and mouse studies showed that blockade of IL-17A and IL-22 could reduce fibrosis. Together, these findings define a role for type 3 cytokines in driving fibrosis during liver injury.

Abstract

Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (Tregs), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17+CD4+/FOXP3hiCD4+ ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor–β signaling in HSCs in a p38 mitogen-activated protein kinase–dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.

INTRODUCTION

Chronic liver injury and inflammation induced by viral infections, toxins, and metabolic and autoimmune conditions lead to progressive fibrosis, cirrhosis, and liver cancer (1). Transforming growth factor–β (TGF-β) signaling is the key fibrogenic signal that activates hepatic stellate cells (HSCs), the main producer of extracellular matrix in the liver, and is modulated by proinflammatory mediators such as cytokines that act on HSCs and liver immune cells (1).

The inflammatory response can be divided into three types (2). Type 1 inflammation, characterized by production of interleukin-1β (IL-1β), IL-6, tumor necrosis factor–α (TNF-α), and interferon-γ (IFN-γ), is proinflammatory and correlates with liver inflammation but is antifibrogenic (3, 4). Type 2 inflammation, characterized by production of IL-4, IL-5, IL-10, IL-13, IL-25, and IL-33, is associated with reduced hepatic inflammation but correlates with progression of liver fibrosis (4). The current theory is that the imbalance between type 1 and type 2 inflammation is the major driver of fibrosis in the liver (1). Type 3 inflammation is characterized by production of IL-17A, IL-17F, IL-22, and IL-26 produced by neutrophils, mast cells, group 3 innate lymphoid cells (ILC3), and T helper 17 (TH17) and TH22 cells (2). Type 3 cytokines play an important physiological role in tissue homeostasis but can be pathogenic (5). Dysregulated type 3 immunity is associated with abnormal tissue repair, chronic inflammatory diseases, and cancer in the gut and the lung (5).

IL-17A has profibrogenic functions through recruitment of proinflammatory monocytes, increased production of TGF-β, and enhanced TGF-β responses in HSCs (610). IL-22 is hepatoprotective during acute liver injury (11, 12), but its function during chronic injury is controversial. Opposite effects of IL-22 were reported in the lung and the liver depending on the duration of injury and etiology (1315). IL-22 promotes liver cancer development in individuals with cirrhosis, which suggests that its protective function can be detrimental in the long term (16, 17). The balance between IL-17A and IL-22 also contributes to pathogenesis, but the molecular mechanisms are unknown (15). Thus, the role of type 3 inflammation in liver disease and fibrosis progression remains elusive.

Regulatory (FOXP3hiCD4+) T cells (Tregs) dampen activation and pathogenesis of proinflammatory cells in several inflammatory conditions (18, 19). The ratio between Tregs and other inflammatory cells is one of the key determinants of the progression of inflammatory diseases (18). IL-10, a cytokine that can be produced by Tregs, induces apoptosis of activated HSC (20, 21). It is therefore possible that Tregs could limit fibrosis progression through the secretion of IL-10. However, Tregs may have a dual role because they are enriched in fibrotic livers and may protect activated HSCs from natural killer (NK) cell–mediated killing, thus allowing them to achieve their full fibrogenic potential (22, 23). During chronic liver injury, Tregs induce aberrant B cell responses that may contribute to pathogenesis through the expression of CD40L (24).

Here, we investigated the contribution of the type 3 cytokines IL-17A and IL-22 to the progression of liver fibrosis. Using primary human intrahepatic lymphocytes (IHLs) and HSCs, liver biopsy specimens, advanced microscopy, gene expression, and knockout (KO) mice, we observed that dysregulation of IL-17A+CD4+/FOXP3hiCD4+ ratio and increased type 3 cytokines, especially IL-22, was associated with fibrosis progression. Neutrophils and mast cells were the main producers of IL-17A in situ. In vitro, we demonstrate a profibrogenic function of IL-22 through enhancement of TGF-β signaling in HSCs in a mitogen-activated protein kinase (MAPK)–dependent manner. Lack of IL-22 signaling in vivo or pharmacological inhibition of IL-22 and IL-17 reduced hepatic fibrosis.

RESULTS

Increased intrahepatic IL-17A and IL-22 is a common signature of advanced liver fibrosis

To investigate whether type 3 cytokines are implicated in liver fibrosis, we profiled the cytokines produced by IHLs from liver biopsies of patients with viral hepatitis (VH) and nonviral hepatitis (NVH) (table S1). A METAVIR score was used to stratify patients into moderate (F0-F2, n = 21) or advanced (F3-F4, n = 9) fibrosis. Because of the limited number of cells, IHLs were expanded in vitro and then polyclonally stimulated, and supernatants were used to quantify type 1 (IFN-γ, IL-6, and TNF-α), type 2 (IL-4, IL-5, IL-9, IL-10, and IL-13), and type 3 (IL-17A, IL-17F, IL-21, and IL-22) cytokines using the LEGENDplex assay. Type 1 cytokines were predominantly produced by IHLs independent of etiology or fibrosis score, suggesting a common signature of hepatitis (Fig. 1, A and B). Type 2 cytokines were increased in NVH compared with VH (Fig. 1, A and B) and correlated with progression of fibrosis and transition from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) as we have reported (25). Type 3 cytokines were also elevated in patients with advanced liver fibrosis independent of the etiology (Fig. 1, A and B). There was a significant increase of both IL-17A (VH: mean = 680 pg/ml for F0-F2 versus 3754 pg/ml for F3-F4; P = 0.0011; NVH: mean = 370 pg/ml for F0-F2 versus 1719 pg/ml for F3-F4; P = 0.02) and IL-22 (VH: mean = 630 pg/ml for F0-F2 versus 2349 pg/ml F3-F4; P = 0.0017; NVH: mean = 129 pg/ml for F0-F2 versus 1240 pg/ml for F3-F4; P = 0.007) with advanced fibrosis (Fig. 1C).

Fig. 1 Type 3 cytokines are a signature of advanced liver fibrosis and drive activation of HSCs.

Expanded IHLs (2 × 106 cells/ml) were stimulated with anti-CD3/CD28 for 3 days. Supernatants were collected and screened by LEGENDplex for type 1 (TH1), 2 (TH2, TH9), and 3 (TH17) cytokines. (A) Stacked bars comparing the mean production of TH1, TH2, TH9, and TH17 cytokines by IHLs of patients with VH and NVH with moderate (F0-F2) and advanced (F3-F4) fibrosis, METAVIR scale. (B) Heatmaps comparing fold change in type 1, 2, and 3 cytokines of VH and NVH patients with moderate or advanced fibrosis (C) Quantification of TH17 cytokines (IL-17A, IL-17F, IL-21, and IL-22) produced by IHLs. (D) Fold change in expression of the profibrotic genes COL1A1, ACTA2, TIMP1, TGFB1, CXCL10, and CCL20 after stimulation of LX2 cells with F3-F4 supernatant (n = 7) with or without anti–IL-17RA (50 ng/ml), rIL-22BP (50 ng/ml), or anti–TGF-β (1 μg/ml). Error bars, means ± SEM. *P < 0.05, **P < 0.01 (ANOVA followed by a post hoc Tukey and Kruskal-Wallis tests were used for multiple and dual comparisons, respectively).

To further validate that IL-17A, hereinafter termed IL-17, and IL-22 are common markers of liver fibrosis, we consulted publicly available microarray data comparing moderate and advanced fibrotic livers in two studies on NASH (26) and hepatitis C virus (HCV) (27), respectively. We performed gene set enrichment analysis (GSEA) using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases focusing on the top 15 up-regulated pathways between moderate and advanced fibrosis [P < 0.05; false discovery rate (FDR), <25%; fold change (FC), >1.3]. IL-22 signaling was in the top five modulated pathways in both studies (P = 0.000006 NASH and P = 0.0305 HCV; fig. S1). Collectively, these results suggest a key role for IL-22 in advanced liver fibrosis.

Type 3 cytokines produced by IHLs promote activation of HSCs

Next, we investigated whether IL-17 and IL-22 enhanced fibrosis by modulating activation of HSCs. We used IHL supernatants from seven patients with advanced fibrosis and high levels of IL-17 and IL-22 to stimulate the human HSC cell line LX2 for 48 hours and then examined the expression of the profibrogenic genes ACTA2 [α–smooth muscle actin (α-SMA)], COL1A1, TGFB1, and TIMP1 and the chemokines CXCL10 and CCL20 involved in recruitment of type 3 immune cells (Fig. 1D). We observed high expression of profibrotic genes in stimulated cells (Fig. 1D). Neutralization of IL-17 (top panels) with anti–IL-17RA, IL-22 (middle panels) with recombinant IL-22 binding protein (rIL-22BP), or TGF-β (bottom panels) with anti–TGF-β reduced expression of these profibrogenic genes. These results not only validated the previously reported profibrotic function of IL-17A (69) but also highlighted a pathogenic role for IL-22 during liver fibrosis through enhanced HSC activation. The profibrogenic functions of IL-17 and IL-22 were dependent on TGF-β signaling as neutralization of TGF-β inhibited activation of LX2 cells.

Increased frequency of TH17 cells in advanced liver fibrosis

To determine whether a dysregulated type 3 response is implicated in liver fibrosis progression, we examined the frequency of TH17 cells, a hallmark of type 3 inflammation, by flow cytometry directly ex vivo in peripheral blood mononuclear cells (PBMCs) and IHLs from VH (n = 19) or NVH (n = 15) patients with different degrees of fibrosis (table S1). Because of the limited number of cells, we could not permeablize them to stain for the TH17-specific transcription factor (TF) RAR-related orphan receptor gamma-t (RORγt). Thus, TH17 cells were identified as CD161+CD26+CCR6+ CD4 T cells (Fig. 2A and fig. S2A) (28, 29). We did not observe substantial changes in the frequency of TH17 cells in PBMCs (fig. S2B) but a significant increase (P = 0.00173) in livers with advanced fibrosis independent of etiology (Fig. 2B). The frequency of intrahepatic TH17 cells did not correlate with liver injury measured by serum alanine amino transferase (ALT; fig. S2C), suggesting that they do not directly contribute to hepatocyte injury but rather enhance fibrosis.

Fig. 2 Dysregulated intrahepatic T

H17/Treg ratio in advanced liver fibrosis. (A) Representative flow cytometry plots of intrahepatic TH17 cells identified as CD161+CD26+CCR6+ CD4 T cells. (B) Frequency of TH17 cells directly ex vivo in the IHLs of patients with moderate (F0-F2, black circles) or advanced liver fibrosis (F3-F4, red circles) with VH (closed circles) or NVH (open circles). (C) Detection of IL-17–producing cells (white arrows) and Tregs (red arrows) by IF staining of IL-17 (yellow), FOXP3 (red), and CD4 (green) on FFPE liver sections; representative images from F0-F2 and F3-F4 VH participants are shown (200× magnification). (D) Representative tissue heatmaps (scale, blue = 0 to red = 5 cells/50 μm) of IL-17–producing (IL-17+CD4) and TH17 (IL-17 CD4+) cells from moderate and advanced fibrosis. (E and F) Total density (counts per square millimeter) of intrahepatic IL-17–producing cells (E), TH17 cells (F), from patients with moderate (black circles) and advanced (red circles) fibrosis with VH (closed circles) and NVH (open circles). (G) Ratio of IL-17–producing cells and (H) TH17 cells in scar/normal parenchyma. (I) Total density (counts per square millimeter) of intrahepatic Tregs, (J) TH17/Treg ratio and (K) TH17/Treg ratio in the scar. Scale bars, 100 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Kruskal-Wallis test). DAPI, 4′,6-diamidino-2-phenylindole.

Dysregulated TH17/Treg ratio in scars is associated with advanced fibrosis

Next, we examined the in situ density and localization of IL-17–producing (IL-17+CD4) and TH17 (IL-17+CD4+) cells by immunofluorescence (IF) on formalin-fixed, paraffin-embedded (FFPE) liver sections (Fig. 2C and fig. S2D). To avoid counting bias, we scanned entire sections and performed automated counting using Visiomorph image acquisition and processing software (Visiopharm). Liver sections were stained with Sirius red to differentiate scar from normal parenchyma and to delineate the region of interest for further analysis (fig. S3). As expected, we observed increased immune infiltrate in fibrotic livers (Fig. 2, C to F). We detected higher density of IL-17+ (P < 0.0001, n = 28) and IL-17+CD4+ cells (P < 0.0001, n = 28) in livers with advanced compared with moderate fibrosis (Fig. 2, C and D to F). Despite the use of different markers to identify TH17 cells ex vivo (CD161+CD26+CCR6+) and in situ (IL-17+CD4+), and the possibility that the functional readout used in situ may not be sensitive enough to identify resting cells or low producers of IL-17, the ex vivo frequency of TH17 cells measured by flow cytometry correlated with the in situ numbers of TH17 cells quantified by IF (P = 0.031, r = 0.17, n = 17) in a subset of patients for whom paired samples were available. This validated the use of cell surface markers to identify TH17 cells (fig. S2E). We then generated tissue heatmaps for IL-17–producing or TH17 cells to further confirm that type 3 responses were increased in advanced fibrosis and within the fibrotic/scar region (Fig. 2D). IL-17–producing cells accumulated in the scar region of patients with advanced fibrosis, suggesting a direct fibrogenic role (Fig. 2, G and H). Similar to our flow cytometry data, there was no correlation between the frequencies of neither IL-17–producing nor TH17 cells and serum ALT (fig. S2, F and G).

Tregs limit fibrosis through secretion of IL-10 and inhibition of other inflammatory cells including TH17 cells (18). Therefore, we examined the frequency of Tregs (FOXP3+CD4+) in liver sections (Fig. 2C). We observed increased numbers of intrahepatic Tregs (P = 0.0008, n = 28) in patients with advanced compared with moderate fibrosis (Fig. 2I). The number of intrahepatic Tregs correlated with serum ALT (P = 0.028, r = 0.23, n = 27; fig. S2H). This suggests that Tregs may be recruited to the liver as a mechanism to control ongoing hepatocyte damage. However, it has been proposed that the imbalance between TH17 and Tregs contributes to the pathogenesis of several inflammatory diseases (18) and therefore may influence fibrosis progression. To answer this question, we examined the IL-17+CD4+ (TH17)/Treg ratio in liver sections. We observed a tendency (P = 0.103, n = 28) for increased TH17/Treg ratio in livers with advanced compared with moderate fibrosis (Fig. 2J). When we delineated the scar versus parenchyma, we observed a significant increase in the TH17/Treg ratio (P = 0.032, n = 28) in the scar region (Fig. 2K), suggesting that dysregulation of the type 3 response is associated with enhanced fibrosis.

Neutrophils and mast cells are the main producers of IL-17 in the liver

Although we observed increased frequencies of TH17 cells (Fig. 2B) and a dysregulated TH17/Treg ratio in the livers of patients with advanced fibrosis (Fig 2, J and K), the majority of IL-17–producing cells in situ were negative for CD4. This suggested that TH17 cells may not be the main IL-17 producers in the tissue. The majority of IL-17–producing cells had multilobulated nuclei consistent with a neutrophilic phenotype. A second population was always located in the stroma and had a granulocytic morphology, suggesting that they may be mast cells. To characterize these cells, we recruited a new cohort of four patients with moderate and seven with advanced fibrosis (total n = 11). We examined IL-17–producing cells (IL-17+), neutrophils (CD66b+), and mast cells (tryptase+) by IF on FFPE liver sections (Fig. 3A). We observed a significant increase in the density of IL-17–producing cells (P = 0.012) in patients with advanced fibrosis, validating our previous observations (Fig. 3, B and E). The majority of IL-17–producing cells were CD66b+ neutrophils, which represented from 48 to 96% of total IL-17–producing cells (Fig. 3, A and C). IL-17 was stored inside the neutrophils as shown by confocal microscopy (fig. S4C). We also found that IL-17–producing neutrophils (IL-17+Ly6G+) were the main source of IL-17 after liver injury in mice (fig. S4A), consistent with the report by Tan et al. (30) using flow cytometry. The second dominant population was tryptase+ mast cells, which represented 2 to 40% of total IL-17–producing cells (Fig. 3, A and D). Advanced fibrosis was associated with increased density of IL-17+tryptase+ mast cells [P = 0.0061, a mean density of 2.4 (F0-F2) versus 14.61 (F3-F4)], and IL-17+CD66b+ neutrophils [P = 0.2; mean density, 21 (F0-F2) versus 56.1 (F3-F4)] (Fig 3, A, B, and E to H).

Fig. 3 Neutrophils and mast cells are the sources of IL-17 during liver fibrosis.

(A) Detection of IL-17A–producing neutrophils (cyan arrows) and IL-17–producing mast cells (magenta arrows) by IF of IL-17 (yellow), CD66b (cyan), and tryptase (magenta) on FFPE liver sections, representative images from F0-F2 and F3-F4 NVH are shown (200× magnification). (B) Representative tissue heatmaps (scale, blue = 0 to red = 5 cells/50 μm) of total IL-17–producing cells (IL-17+), IL-17–producing neutrophils (IL-17+CD66b+), and IL-17–producing mast cells (IL-17+ tryptase+) from moderate and advanced fibrosis. (C and D) Frequency of IL-17+ neutrophils, IL-17+ mast cells, and IL-17+CD66btryptase cells represented as percentage of total IL-17+ counts in the livers of patients with different degrees of fibrosis. (E to H) Total density (counts per square millimeter) of intrahepatic IL-17–producing cells (E), IL-17+ neutrophils (F), IL-17+ mast cells (G), and IL-17+CD66btryptase cells (H) from patients with moderate (black circles) and advanced (red circles) fibrosis with VH (closed circles) and NVH (open circles). Scale bars, 100 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01 (Kruskal-Wallis test).

High density of IL-22–producing cells is associated with advanced liver fibrosis

As we observed increased IL-22 levels and IL-22 signaling in our initial screen (Fig. 1, A to C, and fig. S1), we investigated whether advanced fibrosis was also associated with a higher density of IL-22–producing cells in situ. We examined the in situ density and localization of IL-22–producing cells by IF in livers from VH (n = 18) and NVH (n = 19; Fig. 4A). We found higher density of both IL-22–producing cells and IL-22+CD4+ cells in participants with advanced fibrosis (n = 15) when compared with moderate fibrosis (n = 22), independent of the etiology (Fig. 4, B to D). Again, the majority of IL-22 producers were not CD4 T cells. They had either a lymphoid or a neutrophilic morphology. Visual inspection suggested that they were neutrophils. This could not be validated in humans due to lack of sufficient tissue, but we were able to validate this neutrophilic phenotype in situ in mice by costaining for IL-22 and Ly6G (fig. S4, A and B). Further validation of this neutrophilic phenotype in humans is required.

Fig. 4 Increased density of IL-22–producing cells is associated with advanced fibrosis.

(A) Detection of IL-22–producing cells (white arrows) by IF of IL-22 (magenta) and CD4 (green) on FFPE liver sections, representative images from one F0-F2 and one F3-F4 VH participant are shown (200× magnification). (B) Representative tissue heatmaps (scale, blue = 0 to red = 5 cells/50 μm) of IL-22+ CD4 and IL-22+ CD4+ cells from moderate and advanced fibrosis. (C and D) Total density (counts per square millimeter) of intrahepatic IL-22+ CD4 cells (C) and IL-22+ CD4+ (D) from patients with moderate (black circles) and advanced (red circles) fibrosis VH (closed circles) and NVH (open circles). (E and F) Ratio of IL-22+ CD4 cells (E) and IL-22+ CD4+ cells (F) between scar and normal parenchyma. Scale bars, 100 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Kruskal-Wallis test).

IL-22–producing cells were located in the parenchyma (mean scar/parenchyma ratio = 0.89) of patients with moderate fibrosis, which suggests a hepatoprotective function in this group (Fig. 4, E and F) (11). However, IL-22–producing cells were increasingly located in the scar of patients with advanced liver fibrosis (mean scar/parenchyma ratio = 1.78; Fig 4, E and F). The scar/parenchyma ratio was higher for IL-22–producing as compared with IL-17–producing cells (1.78 versus 0.68), suggesting that the IL-22 pathway is associated with fibrosis progression and may play distinct roles during different stages of fibrosis.

IL-22 enhances TGF-β signaling in HSCs independent of TGF-β-RII expression

To define the profibrotic function of IL-22, we stimulated primary human HSCs with either a low (1 ng/ml) or a high (40 ng/ml) dose of IL-22 for 48 hours and investigated whether it will induce HSC activation. HSCs treated with TGF-β (2.5 ng/ml, TGF-βhi) were used as a positive control. IL-22 alone was not sufficient to induce HSC activation as demonstrated by comparable mRNA expression levels of ACTA2, COL1A1, TIMP1, and TGFB1 (Fig. 5A) in treated versus untreated cells. This was validated by low expression of α-SMA detected by IF (Fig. 5, B and C) and Western blot (Fig. 5D). However, when combined with a low dose of TGF-β (0.1 ng/ml, TGF-βlo) that was not sufficient by itself to activate HSCs (6), IL-22 induced activation of HSCs as shown by increased α-SMA expression detected by IF (Fig. 5, B and C) and confirmed at the mRNA (Fig. 5A) and protein level (Fig. 5D). To confirm that IL-22 enhances TGF-β responses, we repeated our costimulation in the presence of LY2109761, an inhibitor of TGF-β signaling, and observed reduced expression of fibrogenic genes in HSCs (fig. S5, A and B). As we have previously demonstrated that IL-17 can enhance fibrosis by stabilizing cell surface expression of TGF-β-RII (6), we examined whether IL-22 would have a similar effect. IL-22 did not alter cell surface expression of TGF-β-RII (Fig 5E) but enhanced phosphorylation of SMAD2/3 in TGF-βlo response in IL-22–stimulated HSCs (Fig. 5F). These results suggested that IL-22 enhances TGF-β responsiveness in HSCs but not through direct action on the TGF-β receptor.

Fig. 5 IL-22 enhances activation of HSCs in response to TGF-β.

Primary HSCs were stimulated for 48 hours with TGF-βhi (2.5 ng/ml, positive control), low TGF-βlo (0.1 ng/ml), IL-17 (40 ng/ml), IL-22 (40 ng/ml), combination of IL-17 with TGF-βlo, combination of IL-22 with TGF-βlo, and vehicle (negative control). (A) Profibrotic genes expression of COL1A1, ACTA2, TGFB1, and TIMP1 quantified by quantitative reverse transcription polymerase chain reaction from three different donors (each donor is represented by one symbol). Replicates of three independent experiments are shown. (B) Representative IF of α-SMA (green) expression after 48-hour stimulation. (C) Fold change in α-SMA mean fluorescence intensity (MFI), mean of three independent experiments. (D) Protein expression of α-SMA and tissue inhibitor of metalloproteinases 1 (TIMP-I) after stimulation with TGF-βhi (2.5 ng/ml, positive control), suboptimal TGF-βlo (0.1 ng/ml), IL-22lo (1 ng/ml), IL-22hi (40 ng/ml), combination of IL-17 with TGF-βlo, combination of IL-22 with TGF-βlo, and vehicle (negative control) by Western blot. (E) Cell surface expression of TGF-β-RII on primary HSCs after stimulation with IL-22 and combination of IL-22 with TGF-βlo. (E) Fold change in the expression of TGF-β-RII from three independent experiments. (F) Representative IF of phosphorylated SMAD2/3 after 15-min stimulation with TGF-βhi or TGF-βlo in primary HSCs prestimulated for 24 hours with or without IL-22. Scale bars, 20 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA followed by a post hoc Tukey test).

IL-22 enhances TGF-β signaling in HSCs through activation of the p38/MAPK pathway

To identify the pathways activated by IL-22 that result in an enhanced TGF-β response, we performed RNA sequencing (RNA-seq) analysis on primary human HSCs from three different donors stimulated with IL-22 and TGF-βlo and compared the modulated pathways to TGF-βlo– and TGF-βhi–treated cells normalized to untreated cells. In contrast to a previous report suggesting that IL-22 induces senescence and limits proliferation of HSCs (14), our GSEA revealed that IL-22 induces a prosurvival proliferative phenotype (fig. S6). GSEA of TFs showed up-regulation of a TF cluster regulated by p38, including MEF-2, a known regulator of fibrosis and TGF-β signaling (31), in IL-22 + TGF-βlo–treated compared with TGF-βhi–treated cells (Fig. 6A). We then validated that IL-22 induces phosphorylation of p38 in primary human HSCs (Fig. 6B). Chemical inhibition of p38 (SB203538, 10 μM) in IL-22 + TGF-βlo–treated primary HSCs led to reduced HSCs activation as shown by decreased α-SMA expression (Fig. 6, C and D) but had no impact on TGF-βhi–treated cells. Treatment of HSCs with SB203538 reduced phosphorylation of SMAD2/3 in response to a suboptimal dose of TGF-β and IL-22 stimulation of HSCs (Fig. 6E). This indicated that IL-22 enhances TGF-β signaling in HSCs via activation of the p38/MAPK pathway.

Fig. 6 IL-22 activates p38/MAPK pathway to enhance TGF-β signaling in HSCs.

(A) GSEA of TFs in primary HSCs from three different donors stimulated for 48 hours with TGF-βlo, TGF-βhi, or IL-22 with TGF-βlo normalized to vehicle-treated cells. KEGG database was used (P < 0.05; FDR, <25%; FC, >1.3). (B) Representative IF and Western blot of phosphorylated p38 after 15-min stimulation with IL-22. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Representative IF of α-SMA (green) expression after 48-hour stimulation in the presence or absence of SB203538 (10 μM, chemical inhibitor of p38). Representative pictures from three independent experiments and three independent donors are shown. Fold change in α-SMA MFI, mean of three independent experiments. DMSO, dimethyl sulfoxide. (D) α-SMA expression after 48-hour stimulation in the presence or absence of SB203538 quantified by Western blot. (E) Phosphorylation p-SMAD2/3 after 15-min stimulation with either TGF βlo or TGF-βhi after prestimulation with or without IL-22 in the presence or absence of SB203538 quantified by Western blot. Scale bars, 20 μm.

IL-22 signaling is not required for control of hepatic inflammation during chronic liver injury

To characterize the function of IL-22 during chronic liver injury, we treated 6- to 8-week-old male and female (matched ratio between groups) IL-22RA1 KO C57BL/6 mice and wild-type (WT) littermates with either carbon tetrachloride (CCl4) or thioacetamide (TAA) for 12 weeks to model high and mild hepatic inflammation, respectively. At 12 weeks, we observed increased liver size, liver/body mass ratio, and splenomegaly after CCl4 treatment (fig. S7, A to C). Serum ALT was also increased in both models (figs. S7D and S8B). There was no significant difference in serum ALT levels between IL-22RA1 KO and WT treated mice, suggesting that IL-22 does not exert hepatoprotective functions in this chronic injury model as previously reported during acute injury (11). We also observed increased frequency of activated Tregs (CD39+CTLA4+) after injury in both IL-22RA1 KO and WT mice (fig. S9, A and B) and a positive correlation between Tregs and serum ALT similar to our human data (fig. S9C). The frequency of Tregs also correlated with the hydroxyl-proline liver content (fig. S9D) suggesting a profibrotic function of these cells, as previously reported (22, 23).

IL-22 signaling enhances liver fibrosis during chronic hepatic injury

Next, we sought to determine whether lack of IL-22 signaling in vivo during chronic liver injury will affect fibrosis. WT mice exhibited overt liver fibrosis that was reduced in IL-22RA1 KO. Expression levels of profibrogenic genes col1a1, timp1, tgfb1, and loxl2 as well as genes associated with activated HSCs acta2 and tgfbrii were reduced in the liver of IL-22RA1 KO compared with WT in CCl4 and TAA models (Fig. 7, A and G, and fig. S8A). We confirmed this phenotype at the protein level as shown by decreased expression of α-SMA, desmin, and collagen type I deposition (Fig. 7, B and D to G) measured by IF, Sirius red staining, and hydroxy-proline liver content, respectively. WT showed severe bridging fibrosis and thick collagen network as compared with the IL-22RA1 KO (Fig. 7D). This correlated with the reduced expression of loxl2 (Fig. 7A). We observed 40% reduction in collagen deposition in IL-22RA1 KO compared with WT after both CCl4 and TAA treatment (Fig. 7, E and F, and fig. S8, C and D). We also observed significant increase (P < 0.0001) of IL-22 in both WT and IL-22RA1 KO (Fig. 7A and fig. S10A). Last, we confirmed that IL-22–producing cells were present in the liver of treated mice by IF. In both WT and IL-22RA1 KO, IL-22–producing cells were increased in the liver and located in close proximity of HSCs (Fig. 7C). As described above, we observed two morphologically distinct IL-22–producing cellular populations of either neutrophilic or lymphocytic morphology. We further characterized the lymphocytic IL-22–producing cells by flow cytometry and found that the majority were CD3-negative, NK1.1+Nkp46+RORγt+ cells, suggesting that they may be ILC3 (fig. S10, A and B) (32). There were no major differences in the distribution of immune cell subsets between IL-22RA1 KO and WT, suggesting that the reduction in fibrosis is due to the absence of IL-22 signaling and not due to alterations in inflammatory cell frequencies.

Fig. 7 IL-22 enhances liver fibrosis during chronic liver injury.

(A) Representative profibrotic gene expression of timp1, col1a1, acta2, tgfb1, loxl2, lrat, tgfbrii, and il22 normalized to ribosomal 28s expression in the livers of WT littermates (n ≥ 7, dark red) and IL-22RA1 KO mice (n = 6, light red) after 12-week treatment with CCl4. (B) Representative IF of α-SMA (green) and desmin (red) of WT littermates (top panel) and IL-22RA1 KO (bottom panel) after chronic CCl4 injury. (C) Representative IF of IL-22 (cyan) and desmin (red) of WT littermates (top panel) and IL-22RA1 KO (bottom panel) after chronic CCl4 injury (200× magnification). (D) Collagen deposition was evaluated by Sirius red staining. (E) Quantification of Sirius red staining after 12-week treatment (n = 37, 8 vehicle WT, 12 CCl4, 7 vehicle KO, and 10 CCl4) with CCl4 (100× magnification). (F) Hydroxy-proline content of the liver. (G) Heatmap of fold change in expression of profibrotic genes, Sirius red staining, and hydroxy-proline content. Scale bars, 100 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA followed by a post hoc Tukey and Kruskal-Wallis tests were used for multiple and dual comparisons, respectively).

Inhibition of the type 3 cytokines, IL-22 and IL-17, with AHR and RORγt antagonists reduces liver fibrosis

RORγt and aryl hydrocarbon receptor (AHR) are master regulators of type 3 responses (33). RORγt is important for the differentiation of type 3 immune cells and controls production of IL-17, whereas production of IL-22 is largely dependent on expression of the TF AHR (3335). These two pathways have become interesting targets for therapeutic intervention in various inflammatory conditions (36). Thus, we investigated whether inhibition of IL-22 and IL-17 production in vivo could reduce liver fibrosis. WT mice were treated for 4 weeks with CCl4 or TAA. At 2 weeks, mice started receiving daily intraperitoneal injections of either the AHR antagonist (CH-223191, 10 mg/kg) or the RORγt antagonist (GSK805, 10 mg/kg), whereas control mice received vehicle (Fig. 8A). Treatment was well tolerated and we did not observe any toxicity in the heart, lung, or kidney by histological analysis (fig. S11). We observed reduction in the mRNA levels of the profibrogenic genes col1a1, acta2, timp1, tgfb1, and loxl2 in the liver (Fig. 8B and fig. S12A). We then quantified the desmin-positive area and the liver density of IL-22+ cells. Mice treated with either the AHR or RORγt antagonist had a significant reduction of both desmin-positive area (closed circles) and the liver density of IL-22+ cells (open circles; Fig. 8C). Intrahepatic neutrophils and IL-17+ cells were also reduced in mice treated with the RORγt antagonist (fig. S13). Furthermore, collagen deposition in the liver was reduced as shown by Sirius red and hydroxy-proline assays (Fig. 8, D to F, and fig. S12, B to D), respectively. In conclusion, modulation of IL-22– and IL-17–producing cells using AHR and RORγt antagonists during chronic liver injury reduced the degree of liver fibrosis (Fig 8G).

Fig. 8 Inhibition of IL-22–producing cells with AHR or RORγt antagonists reduces liver fibrosis.

(A) Experimental timeline of therapeutic intervention during CCl4 injury with AHR antagonist (salmon red) or RORγt antagonist (light red). (B) Representative profibrotic gene expression of timp1, col1a1, acta2, tgfb1, loxl2, and lrat normalized to ribosomal 28s expression in the liver of WT (n = 12, dark red) and WT treated with AHR antagonist (10 mg/kg daily for 2 weeks, n = 11, salmon red) or RORγt antagonist (10 mg/kg daily for 2 weeks, n = 10, light red) after 4-week treatment with CCl4. (C) Representative IF of IL-22 (cyan) and desmin (red) of WT (right panel), WT treated with AHR antagonist (middle panel), or RORγt antagonist after chronic CCl4 injury (200× magnification). IL-22+ cell density (counts per square millimeter, open circles) and desmin-positive area (closed circles) were quantified in whole-liver sections. (D) Collagen deposition was evaluated by Sirius red staining (100× magnification). (E) Quantification of Sirius staining after 4-week treatment (n = 37, 6 vehicle WT, 12 CCl4 WT, 11 CCl4 WT treated with AHR antagonist, and 10 CCl4 WT treated with RORγt antagonist) with CCl4. (F) Hydroxy-proline content of the liver. (G) Proposed model: Liver fibrosis progression is associated with dysregulated IL-17–producing cells/Treg ratio leading to increased production of the type 3 cytokines IL-17 and IL-22. Both cytokines enhance TGF-β responses in HSCs. IL-17 increases TGF-β receptor in HSCs in a c-Jun N-terminal kinase (JNK)–dependent manner. IL-22 activates the p38/MAPK pathway increasing phosphorylation of SMAD3 and therefore HSCs activation. Scale bars, 100 μm. Error bars, means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA followed by a post hoc Tukey and Kruskal-Wallis tests were used for multiple and dual comparisons, respectively).

DISCUSSION

We demonstrated that type 3 inflammation is a major driver of fibrosis during chronic liver injury. We observed dysregulation of type 3 responses in fibrotic livers, with increased production of IL-17 and IL-22. This was confirmed by gene signatures in publicly available microarray data. We have also identified neutrophils and mast cells as the main producers of IL-17 in situ. Neutrophils and mast cells are implicated in the progression of fibrosis in many organs due to their capacity to secrete profibrogenic factors such as reactive oxygen species, LOXL2 TGF-β, and platelet-derived growth factor (37). IL-17–producing neutrophils were already reported to be profibrogenic during experimental hepatitis and in one cohort of HCV-infected patients (30, 38). Mast cells have been associated with liver fibrosis progression, interact with HSCs, and are one of the “core” fibrogenic populations in the body (39). IL-17–producing mast cells are associated with disease progression in rheumatoid arthritis and psoriasis (40, 41), suggesting that they may have a similar function in the liver. IL-17–producing cells accumulated in the scar region independent of the etiology, suggesting an active role in the fibrogenic process (Fig. 2, G and H). Although TH17 cells were not the principal source of IL-17 in situ, they may enhance recruitment of innate effectors through induction of proinflammatory chemokines by endothelial cells (7). It is also possible that TH17 cells are active early during inflammation but become inhibited in situ with advanced fibrosis.

We observed increased numbers of FOXP3hiCD4+ Tregs in livers with advanced fibrosis and frequency of Tregs correlated with elevated serum ALT levels (Fig. 2G and fig. S2H). This is consistent with observations in mice (24) and humans (23) suggesting that Tregs are recruited to the liver to regulate ongoing inflammation and damage. However, distribution of Tregs was not uniform because we observed dysregulation of the TH17/Treg ratio in the scar compared with normal parenchyma in patients with advanced fibrosis (22, 23). Because TGF-β is essential for the differentiation of both TH17 and Tregs (42) and given the reported plasticity between these two cell subsets (19), it is possible that liver inflammation mediated by TNF-α, IL-1β, and IL-6 indirectly promotes differentiation of Tregs into TH17 cells and therefore promotes fibrosis. TGF-β may also favor inhibition of TH17 cells by Tregs during chronic liver injury (43). This may explain our inability to detect IL-17 production by TH17 cells in situ despite their high frequency. It is also possible that Tregs accelerate to liver fibrosis progression through inhibition of the antifibrogenic function(s) of NK cells (22, 23).

IL-22 was the cytokine most significantly up-regulated in advanced fibrosis in clinical samples (Fig. 1 and fig. S1). Whether IL-22 exerts pro- or antifibrogenic functions on HSCs is controversial (44). Some studies reported that in vitro IL-22 induces senescence of HSCs through induction of p21 or β-catenin after 24 hours but protects against apoptosis (14, 45). In contrast, our RNA-seq analysis (at 48 hours) demonstrated that IL-22 did not induce senescence but rather proliferative and antiapoptotic signatures and degradation of p21 in primary HSCs (fig. S6). This is consistent with reports in the pancreas and liver (46, 47). This can be explained by different stimulation times. It is also possible that increased proliferation of HSCs in response to IL-22 may cause them to reach cellular confluence by 24 hours and up-regulate cell cycle arrest molecules such as p21/p53 (48) that are then degraded by 48 hours. We also demonstrated that IL-22 by itself did not activate HSCs but it enhances TGF-β signaling in a p38-dependent mechanism (Fig. 5). Other studies have reported direct activation of pancreatic stellate cells or LX2 cells by IL-22 (46, 47). However, they used higher IL-22 concentrations (100 ng/ml versus 1 and 40 ng/ml) and reported activation of different sets of genes. In our hands, IL-22 + TGF-βlo–treated HSCs exhibited a gene signature associated with MEF-2 activation (Fig. 6A). MEF-2 is a p38-dependent TF important for activation of HSCs and regulation of TGF-β signaling (31, 49). This may reflect a dual and concordant effect of IL-22 and TGF-β that may promote optimal tissue repair under normal or acute injury conditions, but elevated levels and sustained signaling may enhance the pathogenic effects of both cytokines during chronic injury. Last, lack of IL-22 signaling in vivo led to reduced fibrosis and decreased activation of HSCs (Fig. 7B). IL-22–producing cells were found in proximity of HSCs, suggesting that they contribute to their activation by enhancing TGF-β signaling (Fig. 7C). In vivo, most IL-22–producing lymphocytes were CD3-negative NK1.1+ NKp46+ cells expressing high levels of RORγt (fig. S10), suggesting that they are ILC3s (50). These results differ from Kong et al. (14) that used a liver-specific transgenic mouse model, where IL-22 levels were high and IL-22 signaling was most likely directed to hepatocytes that might not be physiological. We demonstrated that, in humans and mice, IL-22–producing cells were located in the scar. This suggests that the localization and cellular targets of IL-22 (epithelium versus fibroblast) may shift the balance from an anti- to a profibrogenic/inflammatory function.

In conclusion, we demonstrated a profibrogenic function of IL-22 through enhancement of TGF-β signaling in a p38/MAPK-dependent mechanism. Our data suggest that dysregulation of type 3 inflammation during chronic liver injury is one of the key mechanisms of fibrogenesis in humans, and we identified neutrophils and mast cells as major producers of IL-17 in this setting. Morphological examination and flow cytometry suggest that IL-22 is also produced by multiple sources, potentially by neutrophils and ILC3 but further characterization is needed. Additional research examining the kinetics of recruitment and/or activation of the different IL-17– and IL-22–producing cells in the liver after injury is essential. Similarly, the role of Tregs in regulating type 3 response during liver fibrosis is not well understood. Last, in acute liver injury, lack of IL-22BP, which leads to abnormal IL-22 signaling, induces pathological inflammatory responses and necrosis (51). The in vivo regulation of IL-22 by IL-22BP during chronic liver injury remains an open question and should be examined in future studies. We have provided a proof of concept that targeting IL-17 and IL-22 with antagonists of TFs such as AHR and RORγt can limit liver fibrosis, but additional research with prolonged treatment and different treatment regimens applied during various stages of liver injury should be tested to validate the potential use of this strategy in treatment of liver fibrosis.

MATERIALS AND METHODS

Study design

The goal of this study was to investigate the role of the type 3 cytokines IL-17 and IL-22 in liver fibrosis. We used clinical samples (IHLs and FFPE liver sections) from participants undergoing diagnostic liver biopsies at the Centre hospitalier de l’Université de Montréal (CHUM) with different liver disease etiologies to identify, quantify, and define localization of IL-17– and IL-22–producing cells in the liver using flow cytometry and advanced imaging techniques. Samples were blinded for the degree of liver fibrosis throughout the initial immunological analysis and were all scored using the METAVIR score by an independent pathologist then unblinded at the final analysis. The fibrogenic role of IL-17 and IL-22 was confirmed in vitro using primary HSC and in vivo using IL-22RA KO mice and pharmacological inhibitors. The number of independent experiments and inclusion of individuals are described in the figure legends and in table S1. Data points were excluded only in the case of technical or processing errors that caused poor quality control of the sample.

Study participants

Peripheral blood and fresh and FFPE human liver specimens were obtained from participants after informed consent. This study was approved by the institutional ethics committee (protocol SL09.228) and performed in accordance with the Declaration of Helsinki. Participants’ clinical characteristics and demographics are listed in table S1.

Mice

All experimental procedures were approved by the Institutional Animal Care and Use Committee. IL-22RA1 KO were rederived using sperm from the Mutant Mouse Resource and Research Center (University of California, Davis, CA) and C57/BL6 mice from the Jackson laboratory (Sacramento, CA). Six- to 8-week-old male and female IL-22RA1 KO and WT littermates were treated with CCl4 (Sigma-Aldrich, Oakland, ON) resuspended in corn oil [0.5 ml/kg two intraperitoneal (i.p.) injections per week], TAA (Sigma-Aldrich) in phosphate-buffered saline (PBS) (200 mg/kg i.p., three times per week), or vehicle (corn oil or PBS). For the therapeutic intervention, WT mice were placed on CCl4 (0.5 ml/kg i.p., twice per week) or increasing doses of TAA [three i.p. injections per week at 100 mg/kg i.p. (week 1), 200 mg/kg (week 2), and 300 mg/kg (weeks 3 and 4)] for 4 weeks and treated from weeks 2 to 4 daily with either the AHR antagonist CH-223191 (10 mg/kg i.p. in corn oil) or the RORγt antagonist GSK805 (10 mg/kg i.p. in corn oil) using vehicle as a control. Mice were terminally euthanized with sodium pentobarbital (400 mg/kg) and 2% xylocaine. All animals were housed under specific pathogen-free conditions.

Immunofluorescent staining and image analysis

FFPE sections were deparaffinized and dehydrated. Antigen retrieval was performed using sodium citrate (pH 6) for 10 min at high temperature and pressure. Sections frozen in optimal cutting temperature (OCT) compound were stored at −80°C and warmed up to room temperature. All sections were blocked with either human serum in 1% bovine serum albumin (BSA) in PBS or in serum-free blocking agent (Dako, Santa Clara, CA). To reduce autofluorescence, sections were incubated in 0.1 mM glycine for 10 min, then incubated with primary antibodies overnight in 1% BSA, 0.1% Triton X-100 in PBS at 4°C, and then washed five times for 5 min in PBS, 0.1% Triton X-100 solution. Secondary antibody incubation was performed in 1% BSA, 0.1% Triton X-100 in PBS solution at room temperature for 1 hour. Sections were mounted in Slowfade Gold mounting media with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Fremont, CA, USA).

Statistical analysis

All data were analyzed using GraphPad Prism 6 and 7 for generation of heatmap (GraphPad Software, La Jolla, CA). Differences between two groups were determined by Mann-Whitney, whereas differences between groups were determined by analysis of variance (ANOVA), followed by Tukey post hoc test. Kruskal-Wallis tests were used when group sizes were too different and if the data did not meet the assumption of homogeneity of variance. Correlations were tested using Spearman’s rank correlation.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/3/28/eaar7754/DC1

Materials and Methods

Fig. S1. Top 15 regulated pathways associated with fibrosis in NASH and HCV.

Fig. S2. Identification of IL-17–producing, TH17, and Treg cells by flow cytometry in fresh liver biopsies and by IF in FFPE liver biopsies.

Fig. S3. Image processing and definition of region of interest.

Fig S4. Neutrophils represent the majority of IL-17 and IL-22 producing cells in the liver of humans and mice during chronic hepatitis.

Fig. S5. IL-22 enhances TGF-β responses in primary human HSCs independent of TGF-β receptor expression.

Fig. S6. IL-22 induces a proliferative and antiapoptotic phenotype in primary human stellate cells.

Fig. S7. IL-22 signaling is not required for control of hepatic inflammation during chronic liver injury.

Fig. S8. IL-22 enhances liver fibrosis during chronic TAA-mediated hepatic injury.

Fig. S9. Chronic liver injury induces activation of regulatory T cells in both WT and IL-22RA1 KO.

Fig. S10. Phenotypic characterization of intrahepatic IL-17– and IL-22–producing cells during chronic liver injury.

Fig. S11. Histology of the heart, lung, and kidney after therapeutic intervention with antagonist of AHR and RORγt.

Fig. S12. Inhibition of IL-22–producing cells with AHR or RORγt antagonists reduces liver fibrosis in TAA-induced injury.

Fig. S13. Treatment with RORγt antagonist reduces the number of IL-17–producing neutrophils and correlates with fibrosis reduction.

Fig. S14. Full Western blots from main figures.

Table S1. Patients’ characteristics and demographics.

Table S2. Raw data and statistics for all figures.

REFERENCES AND NOTES

Acknowledgments: We dedicate this work to the memory of G. Pomier-Layrargues, an outstanding hepatologist and mentor who helped us start this project. We thank the study participants and research nurses. We acknowledge the flow cytometry, bioinformatics, molecular biology and cell imaging platforms, and the animal facility at the CRCHUM for excellent technical assistance. Funding: This study was supported by grants from the Canadian Institutes of Health Research (CIHR; HEO-115696), the Canadian Liver Foundation, Fonds de recherche du Québec–Santé (FRQS) AIDS and Infectious Disease Network (Réseau SIDA-MI), and the Canadian Network on Hepatitis C (CanHepC). CanHepC is funded by a joint initiative from CIHR (NHC-142832) and the Public Health Agency of Canada. T.F. received doctoral fellowships from CIHR and CanHepC. M.F.M. received fellowships from the Université de Montréal, Bourse Gabriel Marquis, and the FRQS. M.B. is the Novartis/Canadian Liver Foundation Hepatology Research Chair at the Université de Montréal. Author contributions: T.F. and M.F.M. designed and performed experiments and analyzed data including statistical analysis. G.S. performed all the pathological scoring of liver biopsies and provided input on all the pathological aspects. J.-P.G. analyzed RNA-seq data. B.W. and J.-P.V. recruited participants and provided clinical data. M.B. coordinated, supervised, and provided valuable input on all clinical aspects. T.F., M.B., and N.H.S. conceptualized and designed the study. N.H.S. supervised the work and obtained funding. T.F. and N.H.S. wrote the manuscript. All authors reviewed and approved the manuscript. Competing interests: B.W. received funds and consultancy fees from Gilead Biosciences for a clinical research protocol. The other authors declare that they have no competing interests. Data and materials availability: RNA-seq raw data were submitted to the NCBI-GEO database, accession number GSE119047. The link will become public once the manuscript is accepted.
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