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Induced hepatic stellate cell integrin, α8β1, enhances cellular contractility and TGFβ activity in liver fibrosis

Abstract No effective therapy exists for fatal fibrosis. New therapeutic targets are needed for hepatic fibrosis because the incidence keeps increasing. The activation and differentiation of fibroblasts into myofibroblasts that causes excessive matrix deposition is central to fibrosis. Here, we investigated whether (and which) integrin receptors for matrix proteins activate hepatic stellate cells (HSCs). First, integrin α‐subunits were investigated systematically for their expression over the course of HSC activation and their distribution on fibroblasts and other systemic primary cells. The most upregulated in plate culture‐activated HSCs and specifically expressed across fibroblast linages was the α8 subunit. An anti‐α8 neutralizing mAb was evaluated in three different murine fibrosis models: for cytotoxic (CCl4 treatment), non‐alcoholic steatohepatitis‐associated and cholestatic fibrosis. In all models, pathology and fibrosis markers (hydroxyproline and α‐smooth muscle actin) were improved following the mAb injection. We also CCl4‐treated mice with inducible Itga8−/−; these mice were protected from increased hydroxyproline levels. Furthermore, ITGA8 was upregulated in specimens from 90 patients with liver fibrosis, indicating the relevance of our findings to liver fibrosis in people. Mechanistically, inhibition or ligand engagement of HSC α8 suppressed and enhanced myofibroblast differentiation, respectively, and HSC/fibroblast α8 activated latent TGFβ. Finally, integrin α8β1 potentially fulfils the growing need for anti‐fibrotic drugs and is an integrin not to be ignored. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
The incidence of non-alcoholic steatohepatitis (NASH) with fibrosis is growing, without any effective therapies. Hepatic stellate cell (HSC) activation and myofibroblast differentiation are central to hepatic fibrosis and occur upon deposition of extracellular matrix [1]. Integrins are major extracellular matrix receptors, so we reasoned that integrins induced on HSCs could play a critical role in this process. In the current study we systematically examined integrin α-subunit expression over the course of activation of HSCs and found that the α8 subunit was most dramatically upregulated. α8 forms a heterodimer with β1 [2] and is expressed as α8β1 in smooth muscle cells and fibroblasts [3]. α8β1 is one of the eight integrins that recognize the tripeptide Arg-Gly-Asp (RGD), an integrin recognition motif that is present in the pro-peptide of TGFβ1 and TGFβ3. Integrin binding to this motif is critical for integrin-mediated TGFβ activation [4]. We therefore evaluated the functional significance of this integrin in multiple in vivo models and provide the mechanisms by which this integrin could modulate the behavior of activated HSCs.

Materials and methods
HSCs were isolated from C57BL/6 mice and Wistar rats. Murine HSCs were used for expression studies and rat HSCs for the validation. Mechanistic studies employed rat HSCs to avoid possible allogenic effects from a mixture of murine HSCs. Sufficient α8 expression was confirmed before each experiment to avoid potential variability in the time course of α8 induction during activation of HSCs. Animal use and euthanasia protocols were reviewed and approved by the Animal Committees of Hiroshima University or University California San Francisco. For the use of human tissue, informed consent was provided by patients in accordance with the Declaration of Helsinki and approval from the Hiroshima University Institutional Review Board. Chicken neutralizing anti-α8 recombinant mAb, YZ3, which reacts with most mammals, was generated in our laboratory [5] and characterized (see supplementary material, Figure S1). A chimeric form of YZ3 with mouse IgG 1 κ-Fc was used throughout this study. All antibodies in this study are summarized in supplementary material, Table S1.
The initial global α8 knockout mouse line made in 1997 was characterized by kidney agenesis and perinatal death [6]. Contribution of α8β1 to nephrogenesis can be modified by 'stochastic factors', as the penetrance of the phenotype was only approximately 50% [6]. To reduce the possible compensation for loss of α8β1 function, we employed temporally inducible global deletion, Itga8 flox/flox ;Rosa26-Cre ER , so that expression of α8β1 remains normal before induction of fibrosis. The shorter interval should avoid the effects of life-long genetic compensation that may have affected results in previous studies in other organs [7][8][9].
Details of cells and culturing, antibodies, animals, RT-qPCR, flow cytometry, experimental fibrosis, hydroxyproline assay, measurement of areas stained by Masson's Role of integrin α8β1 in liver fibrosis 367 trichrome or α-smooth muscle actin (αSMA) immunostaining, human liver tissues, recombinant nephronectin proteins, western blotting, immunofluorescence, gel contraction assay, TGFβ activation assay and statistical analyses are provided in supplementary material, Supplementary materials and methods. Figure 2 Legend on next page.

α8 expression in HSCs and fibroblasts
Mouse HSCs grown on plastic for 14 days showed increased Itga8 ( Figure 1A; p = 0.0171) and Itga11 mRNA expression. A similar increase in Itga8 expression was observed in rat HSCs up to 27-fold and four-fold at the mRNA (see supplementary material, Figure S2A; 27-fold, p < 0.0001) and the protein (see supplementary material, Figure S2B) level, respectively. Notably, α8 protein was not detected in quiescent HSCs at days 1 and 3. Integrin α6, which is constitutively expressed in HSCs [10], was used as a control. As culture-activated HSCs mimic the fibrotic response [11], we explored α8 expression in HSCs in response to in vivo CCl 4 treatment. Western blotting of whole liver lysate showed marked α8 upregulation. Immunofluorescence showed barely detectable α8 in normal livers and an increase in the periportal area colocalizing with PDGFRβ ( Figure 1B). Furthermore, α8 was upregulated in HSCs isolated from CCl 4 -treated mice ( Figure 1C). Next, we analyzed α8 expression from databases analyzing fibroblasts and related linages. In 144 primary cells from various human tissues [12], ITGA8 mRNA was expressed across 15 fibroblast lines but was less abundant in other cell types ( Figure 1D), in contrast to integrin β6 or αv subunits. FACS analyses showed constitutive α8 expression in fibroblasts from the lung, heart, and kidney (see supplementary material, Figure S2C). Taken together, α8 was found to be expressed in HSCs/fibroblast linages, and was induced by activation of HSCs in vitro and in vivo.

In vivo role of α8 in fibrosis
The effects of neutralizing anti-α8 mAb YZ3 [5] were evaluated in mouse models of cytotoxic (CCl 4 treatment), NASH-associated (choline-deficient, L-amino aciddefined, high-fat diet), and cholestatic (bile duct ligation) liver fibrosis. In all models, morphologic evidence of fibrosis was attenuated, and elevated hydroxyproline content and αSMA protein or mRNA expression were reduced by inhibition of α8β1 (Figure 2A and supplementary material, Figure S3), which were validated by quantification of the area stained by Masson's trichrome or αSMA (see supplementary material, Figure S4). We used CCl 4 treatment in mice with global tamoxifen (Tam)-inducible loss of α8 and found protection against increased hydroxyproline content. Cre-recombination efficiency appeared to be excellent and unaffected by CCl 4 treatment in mTmG reporter mice ( Figure 2B) and the recombination was confirmed by identifying the expected version of α8 protein truncated by 69 residues by western blotting (see supplementary material, Figure S5). We then analyzed ITGA8 expression in liver tissues from 90 human patients and found expression increased in fibrotic livers compared with F0 controls (p < 0.0001, Figure 2C).

α8β1-mediated myofibroblast differentiation
To investigate the functional consequences of increased α8 for liver fibrosis, we inhibited or induced ligand engagement of α8β1 in vitro. HSCs grown on plastic upregulated fibrosis markers, Acta2 (αSMA), Col1a1 (collagen type I α1 chain) and EDA (fibronectin extradomain A), and Acta2 was reduced by α8β1 inhibition ( Figure 3A), whereas the inhibition had no effect on Col1a1 and EDA (see supplementary material, Figure S6A). To explore the relevance of the Acta2 induction to α8β1, HSCs were plated on to a ligand for α8β1, nephronectin, a basement membrane protein identified based on its function as an α8β1 ligand. α8β1 binds specifically only to nephronectin [13]. In serum-free 24 h culture, Acta2 was induced on nephronectin, and reduced by α8-mAb, whereas expression of Co1a1 or EDA was unaffected ( Figure 3B). The ligand engagement-induced Acta2 expression and its inhibition by the mAb was recapitulated in fibroblasts (see supplementary material, Figure S6B). Plating the fibroblasts on nephronectin also induced αSMA immunofluorescence and formation of actin stress fibers ( Figure 3C). Furthermore, nephronectin enhanced contraction of collagen gels by fibroblasts, which was reduced by α8 inhibition (p = 0.0004; Figure 3D). A collagen gel assay using the fibroblasts showed no contraction in 0.5% FCS medium, regardless of the presence of nephronectin, but with TGFβ (10 ng/ml) supplementation, contraction was detected and greatly enhanced by nephronectin, and this enhancement was reduced by α8 inhibition. The nephronectin concentrations used for each cell type were selected to be within the range of those required for specific interaction with α8β1 (see supplementary material, Figure S6C). These results indicate that α8β1-mediated signal enhances fibroblast/HSC contractility and myofibroblast differentiation.

Role of integrin α8β1 in liver fibrosis 369
TGFβ activation by α8β1 on HSCs As several RGD-binding integrins have been reported to activate TGFβ [14] ( Figure 4A), we examined α8β1 for this ability. Although β6-transfected SW480 cells clearly activated TGFβ, α8-SW480 did not ( Figure 4B, left). As, in contrast to αvβ6, α8β1 is expressed in fibroblasts, but not epithelial cells in vivo ( Figure 1D), we employed α8-expressing HSCs. Luciferase activity was detected and, notably, distinctly reduced with α8-mAb by approximately 35% (p < 0.0001; Figure 3B, right). The TGFβ activation required actin polymerization as it was abolished by cytochalasin D, and we suspect is explained  Figure S7). Lung and heart fibroblasts expressing high levels of Acta2 like HSCs, displayed α8β1-mediated TGFβ activation ( Figure 3C). α8β1 thus activates TGFβ when it is expressed by HSCs and fibroblasts.

Discussion
HSC α8β1 integrin appears to contribute broadly to liver fibrosis as blockade or deletion of this integrin inhibits fibrosis in four different settings. In vitro studies suggest that α8β1 probably contributes to fibrosis by enhancing myofibroblast differentiation and by TGFβ activation of HSCs.
Our findings illuminated previously unknown characteristics of α8β1: de novo expression in activated HSCs in vitro and in vivo, upregulation of αSMA, and TGFβ activation on HSCs and fibroblasts. These findings fit well with previous observations of Itga8 expression in other contractile cells, including airway [15] and gastric [16] smooth muscle cells, arrector pili [17] and sensory hair cells [18]. Recent single-cell RNA-sequencing data from NASH mice revealed exclusive Itga8 expression in HSCs in liver cells [19]. Moreover, another group identified a class of murine HSCs that undergo myofibroblast Role of integrin α8β1 in liver fibrosis 371 differentiation, in which hierarchical clustering characterized the transition from a quiescent to a collagenproducing phenotype, with upregulation of pro-fibrogenic genes, including Col1a1, Col1a2, Col3a1, and Lox [20]. Interestingly, the upregulated genes include only one integrin, Itga8. Cell matrix communication is characterized by redundant ligand-receptor interactions. The partial effects of anti-α8 mAb found in this study for culture-induced Acta2 expression, gel contraction, and TGFβ activation could be attributed to contributions of other integrins. Nonetheless, inhibition of α8β1 in vivo potently inhibited liver fibrosis. The molecular mechanisms by which HSC activation leads to induction of Itga8 and α8β1 engagement induces myofibroblast differentiation should be the focus of future studies.
The increased ITGA8 expression in patients with hepatic fibrosis suggests that our findings are relevant to liver fibrosis in people. Because α8 expression is minimal in healthy liver, the relevance of this integrin as a driver of liver fibrosis has been largely overlooked. However, our findings that the specific expression of α8β1 in activated HSCs is critical for induction of a contractile phenotype and TGFβ activation make this integrin an attractive therapeutic target.   Table S1. Antibodies used in this study Table S2. PCR primer sequences Role of integrin α8β1 in liver fibrosis 373