Inhibitors of class I histone deacetylases attenuate thioacetamide‐induced liver fibrosis in mice by suppressing hepatic type 2 inflammation

Abstract

Background and Purpose

Chronic liver diseases feature excessive collagen and matrix protein deposition or crosslinking that characterises fibrosis, leads to scar tissue, and disrupts liver functions. There is no effective treatment. This study investigated whether treatment with selective histone deacetylase (HDAC) inhibitors might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis in mice.

Experimental Approach

Thioacetamide (TAA) was used to induce hepatic inflammation, fibrosis, and liver damage in female C57BL/6 mice, similar to the clinical features of chronic human liver disease. We used eight inhibitors of different human HDAC enzymes to probe histological (IHC and TUNEL), biochemical and immunological changes (flow cytometry, qPCR, Legendplex, and elisa) in pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression.

Key Results

Inhibitors of class I, but not class II, HDAC enzymes potently suppressed chronic hepatic inflammation and fibrosis in mice, attenuating accumulation and activation of IL‐33‐dependent, but not IL‐25‐dependent, group 2 innate lymphoid cells (ILC2) and inhibiting type 2 inflammation that drives hepatic stellate cells to secrete excessive collagen and matrix proteins.

Conclusions and Implications

The results show that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA‐induced liver fibrosis in mice. The specific HDAC enzymes identified here may be key promoters of inflammation in chronic liver fibrosis.

Abbreviations

HDAC

histone deacetylase

HSCs

hepatic stellate cells

iILC2

inflammatory group 2 innate lymphoid cell

ILC2

group 2 innate lymphoid cell

nILC2

natural group 2 innate lymphoid cell

TAA

thioacetamide

What is already known

• Type 2 driven immunity plays an important role in tissue repair and liver fibrosis.

• A causal link between IL‐33, ILC2 activation, and hepatic inflammation and fibrosis is well established.

What this study adds

• Class I selective HDAC inhibitors attenuate liver fibrosis in female C57BL/6 mice.

• This attenuation involved suppression of hepatic type 2 inflammation.

What is the clinical significance

• Some HDAC inhibitors are currently used in the clinic to treat certain human cancers.

• Class I selective HDAC inhibitors could be evaluated against chronic liver fibrosis in clinical trials.

1. INTRODUCTION

Liver fibrosis is estimated to affect 30% of the world's adult population and is caused by non‐alcoholic fatty liver disease, alcoholic steatohepatitis, hepatitis B or C virus infection, autoimmune and biliary diseases, and iron overload (Schuppan & Kim, 2013). Irrespective of aetiology, liver fibrosis develops from persistent hepatocellular stress, leading to cellular injury and chronic inflammation. This culminates in pathological tissue remodelling and increased deposits of extracellular matrix proteins, leading to decreased liver function and cirrhosis (Hams, Bermingham, & Fallon, 2015; Pellicoro, Ramachandran, Iredale, & Fallowfield, 2014; Schuppan & Kim, 2013). The common sequence of events in response to persistent hepatocellular stress includes hepatocyte cell death, increased expression and release of alarmins and chemokines/cytokines, recruitment of monocytes and other immune cell populations, activation of resident hepatic macrophages and stellate cells, as well as formation of a provisional matrix and remodelling of the granulomatous scar (Gieseck, Wilson, & Wynn, 2018; Krenkel & Tacke, 2017; Pellicoro et al., 2014). When this inflammation‐driven wound healing response for combatting hepatocellular stressors is not terminated, inflammation persists unabated and can lead to chronic liver fibrosis, long‐term liver damage and cirrhosis (Gieseck et al., 2018; Pellicoro et al., 2014).

Immune cells mediate important protective functions in the liver, but uncontrolled or aberrant immune cell infiltration and activation are now recognised as initiating and propagating human liver fibrosis and cirrhosis (Czaja, 2014; Greuter, Malhi, Gores, & Shah, 2017; Musso, Cassader, & Gambino, 2016). There is growing evidence for a critical role of type 2 driven immunity in tissue repair and liver fibrosis (Gieseck et al., 2018; Hart et al., 2017; Vannella et al., 2016). For example, type 2 inflammation in the liver is characterised by overexpression of cytokines, including IL‐5, IL‐9 and IL‐13, that drive inflammation and fibrogenesis by downstream recruitment and activation of effector cells, such as eosinophils, macrophages, and hepatic stellate cells (HSCs) (Gieseck et al., 2018; Hart et al., 2017; Vannella et al., 2016). Group 2 innate lymphoid cells (ILC2) that respond to hepatocyte stress‐related alarmins, such as IL‐25 and IL‐33, are a particularly important source of these key type 2 cytokines in the human fibrotic livers (Jeffery et al., 2017). Sustained activation of ILC2s and release of type 2 cytokines can stimulate downstream activation of infiltrating and tissue‐resident eosinophils, macrophages, and HSCs that are critical to fibrosis progression (Hams et al., 2015; Hart et al., 2017; Krenkel & Tacke, 2017; Weiskirchen & Tacke, 2017). Distinct subsets of ILC2 cells are now recognised as either inflammatory (iILC2) or natural (nILC2) ILC2 cells, differing in their responses to IL‐25 or IL‐33 (Huang et al., 2015; Huang & Paul, 2016; Koyasu, 2015), but their importance in liver injury and fibrosis are currently unclear. Targeting type 2 immunity is attracting substantial attention as a potential anti‐fibrotic and anti‐inflammatory strategy for treating a variety of human disease conditions (Hams et al., 2015).

Histone deacetylases (HDACs) are a diverse family of enzymes that mediate a range of inflammatory conditions through effects on immune cells, particularly macrophages and T cells (Shakespear, Halili, Irvine, Fairlie, & Sweet, 2011). The mechanism of action of HDAC inhibitors in cytokine suppression in these cells remains uncertain but is likely to involve effects on modulating transcription factor activity, immune cell metabolism as well as other epigenetic or post‐translational immunomodulatory effects (Das Gupta, Shakespear, Iyer, Fairlie, & Sweet, 2016; Shakespear et al., 2018). HDACs have also recently been involved in some fibrotic disorders (Iyer et al., 2010; Pang & Zhuang, 2010; Park et al., 2014; Van Beneden, Mannaerts, Pauwels, Van den Branden, & van Grunsven, 2013).

The precise HDAC isoenzymes involved in the initiation and progression of liver inflammation and fibrosis are unknown. There are experimental limitations in using gene deletion approaches for dissecting the roles of individual HDAC isoenzymes in driving liver inflammation and fibrosis. Aside from their enzymic deacetylase activity, many HDAC isoforms play non‐enzymic roles in the nucleus via protein–protein interactions, and genetic deletion of some of these HDACs in mice is either embryonically lethal or leads to early mortality after birth due to developmental defects. A few weak, non‐specific, inhibitors of HDAC enzymes given intraperitoneally at very high doses have been found to cause changes in some murine models of fibrosis and other chronic inflammatory diseases (Aher, Khan, Jain, Tikoo, & Jena, 2015; Iyer et al., 2010; Park et al., 2014; Van Beneden et al., 2013). No potent HDAC isoenzyme class‐selective inhibitors have been evaluated in liver inflammation and fibrosis. We hypothesised that selective HDAC inhibition might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis. To test this proposition, we synthesised a panel of eight inhibitors of human HDACs. Two inhibitors (Vorinostat and Panobinostat) are already registered medicines used clinically to treat some human lymphomas, and they inhibit most of the 11 known zinc‐containing HDAC enzymes (Gupta, Reid, Iyer, Sweet, & Fairlie, 2012). Another inhibitor (AR42) is a similar broad‐spectrum (pan) inhibitor of the same HDAC enzymes and is in clinical trials for certain cancers (Sborov et al., 2017). The five other compounds are more selective inhibitors of subsets of the zinc‐containing HDAC isoenzymes (class I—Entinostat [Cantley et al., 2015; Gupta et al., 2012], NW21 [Cantley et al., 2015], and BRD4884 [Wagner et al., 2015]; class IIa— PG100 [Gupta, 2012]; class IIb— PG50 [Gupta et al., 2010]).

Here, we have investigated these eight HDAC inhibitors (Figure S1) for anti‐fibrotic activity in a mouse model of thioacetamide (TAA)‐induced liver injury. This model reproduces most of the clinical features of severe progressive human liver fibrosis, including chronic inflammation, oxidative stress, centrilobular necrosis and apoptosis, together with a well‐characterised pro‐fibrotic response and excessive peri‐central fibrosis with bridging that ultimately leads to cirrhosis (Wallace et al., 2015). We found that inhibitors of class I HDAC enzymes were anti‐fibrotic in TAA‐induced mouse liver injury. They attenuated hepatocyte death and specifically down‐regulated alarmin and type 2 cytokine‐driven pro‐inflammatory and pro‐fibrotic responses, including the suppression of ILC2 activation. These findings identify inhibition of specific class I HDAC enzymes as a promising new anti‐inflammatory approach to intervention in the progress of chronic liver disease.

2. METHODS

2.1. Animals

All animal care and experimental studies were approved by the University of Queensland Animal Ethics Committee under project IMB/509/15/UQ. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. Four‐week‐old female C57BL/6 mice were purchased from Animal Resources Centre, Australia, and housed under specific pathogen–free conditions, on a 12‐hr controlled day/night cycle with food and water available ad libitum in the Institute for Molecular Bioscience, Australia. Mice were randomly assigned to each experimental group (five mice per group). The experimenters were not blinded to the treatments given to the animals. Pan‐HDAC inhibitor Vorinostat (25 mg·kg⁻¹ body weight p.o.) or Panobinostat (10 mg·kg⁻¹ p.o.), class I HDAC inhibitor Entinostat (10 mg·kg⁻¹ p.o.), class IIa HDAC inhibitor PG100 (25 mg·kg⁻¹ p.o.), class IIb HDAC inhibitor PG50 (25 mg·kg⁻¹ p.o.), or HDAC 1 and HDAC 2 selective inhibitors NW21 (10 mg·kg⁻¹ p.o.) or BRD4884 (10 mg·kg⁻¹ p.o.) was orally administered daily in 100‐μl olive oil. The anti‐TGFβ antibody (mu1D11‐mG1K‐aTGFB, 10 mg·kg⁻¹ i.p.) was given 3 times per week in 100‐μl injection saline. The required dose for each experimental compound was selected based on previous pharmacokinetic studies and observed efficacy for these compounds in other in vivo rodent models of disease in our laboratory. The well‐being of the mice was consistent with our expectations for responses to TAA administration. Daily administration of the experimental drugs alone for 6 or 12 weeks did not show any overt signs of toxicity or distress to mice, which were healthy. Mice were killed by CO₂ inhalation at the end of each experiment.

2.2. TAA‐induced liver injury and fibrosis

TAA (Sigma Aldrich) solution was prepared fresh for each experiment. For acute TAA‐induced liver injury, TAA was administered at 300 mg·kg⁻¹ body weight i.p. in 0.9% saline and experiments conducted after 24 hr (1 day). Control female C57BL/6 mice received an appropriate volume of 0.9% saline. For chronic TAA‐induced liver fibrosis, female C57BL/6 mice were administered daily with 300 mg·L⁻¹ TAA in drinking water for either 6 or 12 weeks to induce chronic liver injury and fibrosis. Control mice received regular drinking water. This model of TAA‐induced liver fibrosis is used interchangeably in both male and female C57BL/6 mice with no major differences in disease pathology (Wallace et al., 2015). In this study, we used female mice because induction of fibrosis via this protocol was well established in our laboratory, and we had institutional ethics approval for females only at the time. The livers were perfused terminally with 10 ml of PBS via the portal vein in situ prior to tissue collection to minimise blood contamination. Portions of the liver were either fixed in 10% neutral buffered formalin and embedded in paraffin or snap‐frozen and stored at −80°C for RNA and protein isolation.

2.3. Histology and microscopic investigation

Paraffin sections were stained with haematoxylin–eosin (H&E) for inflammation and necrosis or Picrosirius red (PSR) for fibrosis. Staging of liver fibrosis with PSR staining and focal inflammation scoring with H&E staining were adapted from a modified Ishak system (Ishak et al., 1995). Scoring of all histology samples was performed blinded. Staging of liver fibrosis is defined as follows: 0, no fibrosis; 1, some portal tract fibrotic ± short fibrous septa; 2, most portal tract fibrotic ± short fibrous septa; 3, most portal tract fibrotic with occasional portal‐portal (P‐P) bridging; 4, portal tract fibrotic with marked P‐P and portal‐central (P‐C) bridging; 5, marked P‐P and/or P‐C bridging with occasional nodules (incomplete cirrhosis); 6, cirrhosis (probable or definite). Focal inflammation scores are defined as follows: 0, absent; 1, one focus or less per 10× objective (ob); 2, two to four foci per 10× ob; 3, five to 10 foci per 10× ob; 4, more than 10 foci per 10× ob.

2.4. SMA staining

The antibody‐based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology. For SMA immunofluorescence staining, antigen unmasking was performed by boiling slides in sodium citrate buffer (10‐mM Tri‐sodium citrate, 0.05% Tween, pH 6.0) for 10 min. Slides were then incubated in blocking buffer (5% FCS, 0.3% Triton X‐100 in PBS). Incubation in primary monoclonal antibody was performed overnight using rabbit anti‐SMA (Cell Signaling Technology, cat no. 19245, 1/200 dilution in blocking buffer, D4K9N, RRID:AB2734735), followed by incubation with secondary antibody (anti‐rabbit Alexa‐647, Life Technologies, 1/400 dilution) for 1 hr. Slides were subsequently stained with DAPI for 10 min, after which they were mounted using Dako mounting media (Dako) and sealed.

2.5. TUNEL staining

For liver apoptosis analysis, we used the Click‐iT^® Plus TUNEL Assay for In Situ Apoptosis (Thermofisher Scientific, USA), according to the manufacturer's instructions. Positive cells were quantified using FIJI/ImageJ 1.42q software (U.S. National Institutes of Health, Bethesda, USA). All microscopic images were obtained using an Olympus BX51 upright microscope with Olympus DP71 12Mp colour camera, utilising DP Capture and DP Manager software packages (Olympus, Japan). Researchers who performed histological analysis and scoring were blinded to the sample identity.

2.6. Cytokine analysis

Liver tissues (50 mg) were homogenised in RIPA buffer (50‐mM Tris–HCl, 150‐mM sodium chloride, 1‐mM EDTA, 1% NP‐40, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate) with a protease inhibitor cocktail (100×; Cell Signaling Technology, USA). IL‐2, IL‐5, IL‐9, IL‐13, IL‐21, IL‐17A, IL‐17F, IL‐6, and TNF protein levels were then detected in tissue supernatants using the Legendplex Mouse Th cytokine Capture Beads multi‐analyte flow assay kits (Biolegend), as per manufacturer's instructions. Data were collected on a FACSCanto II flow cytometer (Becton Dickinson) and analysed using Legendplex Data Analysis V7.1 software (Biolegend). IL‐25, IL‐33, IL‐1β, MMP2, MMP9, and TIMP1 protein levels were measured by elisa according to the manufacturer's protocol. The sensitivity of the assay (or where unavailable, the lowest standard used) is provided in parentheses: IL‐1β (8 pg·ml⁻¹), IL‐25 (16 pg·ml⁻¹; ThermoFisher Scientific), IL‐33 (15.6 pg·ml⁻¹), MMP9 (14 pg·ml⁻¹; R&D Systems), MMP2 (25 pg·ml⁻¹), and TIMP1 (30 pg·ml⁻¹; Abcam).

2.7. Liver enzymes analysis

Serum samples were collected via heart puncture immediately after euthanasia. Aspartate aminotransferase (AST) and alanine transaminase (ALT) were determined using a Beckman Coulter AU analyser (California, USA).

2.8. Isolation of hepatic immune cells

Non‐parenchymal cells were isolated by finely dissociating the liver tissue using surgical scissors then passing the fragments through a stainless steel strainer with the addition of 14 ml of HBSS containing 1 mg·ml⁻¹ type 4 collagenase (Worthington Biochemical Corporation, USA), 1 μg·ml⁻¹ DNase I (Sigma), 100 U·ml⁻¹ penicillin, and 100 μg·ml⁻¹ streptomycin (Irvine et al., 2015). The cell suspension was filtered through a 40‐μM filter (Becton Dickinson) and incubated at 37°C for 5 min, prior to the addition of 10‐mM EDTA and a further 5‐min incubation period. The lysate was washed twice in cold PBS containing 2% FBS (FACS buffer) and resuspended in 25 ml of 33% Percoll in PBS (isotonic) and centrifuged at 600× g for 15 min with no brake. The supernatant was discarded and the pellet washed twice and resuspended in FACS buffer (Irvine et al., 2015).

2.9. Flow cytometry analysis and antibodies

Freshly isolated hepatic leukocytes (1 × 10⁶) were blocked on ice with anti‐mouse CD16/32 (Biolegend, USA) for 10 min before 30 min of staining using the following antibodies. Antibodies were purchased from BioLegend (USA): AF488‐conjugated anti‐CD45 (1/400 dilution, 30‐F11), FITC‐conjugated anti‐Ly6G (1/200 dilution, 1A8), FITC‐conjugated anti‐CD3 (1/200 dilution, 17A2), FITC‐conjugated anti‐CD2 (1/200 dilution, RM2.5), FITC‐conjugated anti‐CD19 (1/200 dilution, 6D5), FITC‐conjugated anti‐FCεRI (1/200 dilution, MAR‐1), FITC‐conjugated anti‐Ter‐119 (1/200 dilution, TER‐119), FITC‐conjugated anti‐CD11b (1/200 dilution, M1/70), PE‐conjugated anti‐Ly6c (1/800 dilution, HK1.4), APC cy7‐conjugated anti‐MHCII (1/100 dilution, M5/114.15.2), APC cy7‐conjugated anti‐CD90.2 (1/200 dilution, 30‐H12), BV421‐conjugated anti‐CD11b (1/400 dilution, M1/70), PerCP‐conjugated anti‐F4/80 (1/50 dilution, BM8), PerCP/Cy5.5‐conjugated anti‐ST2 (1/50 dilution, DIH9), PE/Cy7‐conjugated anti‐CD19 (1/400 dilution, clone 6D5), PE‐conjugated anti‐CD3ε (1/400 dilution, 145‐2C11), PerCP‐conjugated anti‐CD4 (1/200 dilution, GK1.5), BV421‐conjugated anti‐CD8a (1/100 dilution, 53‐6.7), BV421‐conjugated anti‐KLRG1 (1/100 dilution, 2F1/KLRG1), BV500‐conjugated anti‐NK1.1 (1/200 dilution, PK136), and BV500‐conjugated anti‐CD45 (1/100 dilution, 30‐F11). Live cells were differentiated using Fixable Viability Dye eFluor^® 660 (eBioscience, USA) according to manufacturer's protocol. Canto II flow cytometer (BD Biosciences) and FlowJo software version 10 (Tree Star, Ashland, OR) were used to phenotype immune cell populations. The absolute number of each cell type was calculated by multiplying the frequency by the total number of viable hepatic leukocytes per liver sample.

2.10. Real‐time qPCR analysis

Total RNA was isolated from homogenised tissue samples using the Isolate II RNA Mini Kit (Bioline, Australia) according to the manufacturer's instructions. RNA was treated with DNase to remove genomic DNA contamination. RNA concentration and quality (by measuring absorbance at λ = 260/280 and 260/230 nm) was determined using a nanodrop spectrophotometer (Thermo Scientific, USA); 5 μg of RNA was converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Australia) and Oligo (dT)1218 primer (Invitrogen, Australia) according to manufacturer's instructions. Real‐time qPCR was measured on a ABI PRISM 7900HT (Applied Biosystems); each target gene was normalised to housekeeping Hprt mRNA, and fold change was calculated relative to control sample (Sham) using the 2^−ΔΔCt formula. All samples were run in duplicates. Primer sequences commercially designed by Sigma Aldrich are listed in Table S1.

2.11. Data and statistical analysis

The data and statistical analysis in this study comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. All experimental results are expressed as means ± SEM. The data were plotted and analysed using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego CA). Statistically significant differences were assessed using one‐way ANOVA. Dunnett's post hoc test was then performed if F achieved P < .05 and when there was no significant variance inhomogeneity. Statistical significance is designated as *P < .05.

2.12. Materials

The formal chemical names and references describing the synthesis of HDAC inhibitors used in this study are as follows: Vorinostat, N1‐hydroxy‐N8‐phenyloctanediamide, and Panobinostat, (E)‐N‐hydroxy‐3‐(4‐(((2‐(2‐methyl‐1H‐indol‐3‐yl)ethyl)amino)methyl)phenyl)acrylamide, both prepared by the methods in the supporting information of Wang et al. (2011); AR42, (S)‐N‐hydroxy‐4‐(3‐methyl‐2‐phenylbutanamido)benzamide (Sborov et al., 2017); Entinostat, pyridin‐3‐ylmethyl (4‐((2‐aminophenyl)carbamoyl) benzyl)carbamate (Gediya, Belosay, Khandelwal, Purushottamachar, & Njar, 2008); PG50, tert‐butyl (S)‐(1‐(cyclopentylamino)‐8‐(hydroxy amino)‐1,8‐dioxooctan‐2‐yl)carbamate (Gupta et al., 2010); PG100, (R,S)‐6‐(1,3‐dioxoisoindolin‐2‐yl)‐N‐hydroxy‐2‐phenylhexan amide (Gupta, 2012); NW21, (S)‐2‐(4‐(dimethylamino)benzamido)‐N8‐hydroxy‐N1‐(quinolin‐8‐yl)octanediamide (Kahnberg et al., 2006); BRD4884, N‐(4‐amino‐4′‐fluoro‐[1,1′‐biphenyl]‐3‐yl)tetrahydro‐2H‐pyran‐4‐carboxamide (Wagner et al., 2015). Known selectivity profiles for Entinostat (inhibition of HDAC activity IC50 values: HDAC1, 0.18 μM; HDAC2, 1.16 μM; HDAC3, 2.31 μM; HDAC4, >10 μM; HDAC6, >10 μM; HDAC7, >10 μM, and HDAC8, >10 μM; Cantley et al., 2015; Gupta et al., 2012); NW21 (IC50: HDAC1, 0.02 μM; HDAC2, 0.04 μM; HDAC3, 0.3 μM; HDAC4, >10 μM; HDAC6, 0.88 μM; HDAC7, >1 μM, and HDAC8, >3 μM; Cantley et al., 2015; Gupta et al., 2012); BRD4884 (IC50: HDAC1, 0.03 μM; HDAC2, 0.06 μM; HDAC3, 1.09 μM; HDAC4, >33 μM; HDAC5, >33 μM; HDAC6, >33 μM; HDAC7, >33 μM; HDAC8, >33 μM; and HDAC9, >33 μM; Wagner et al., 2015).

2.13. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

3. RESULTS

3.1. Certain HDAC inhibitors can attenuate progression of chronic murine liver inflammation and fibrosis

Daily oral administration of TAA to mice over 6 weeks induced a well‐characterised pro‐fibrotic response, resulting in a pronounced deposition of extracellular matrix proteins such as collagen, formation of bridging fibrosis and scaring in the liver, and the development of cirrhosis. Animals treated with TAA for 6 weeks displayed decreased body weight gain compared to control mice (Figure S2). Daily oral administration of a pan‐HDAC inhibitor, either Vorinostat at 25 mg·kg⁻¹ or Panobinostat or AR42 at 10 mg·kg⁻¹, to mice for 6 weeks significantly attenuated the TAA‐induced increase in collagen deposition, fibroplasia, and bridging fibrosis in terms of both PSR‐positive area and Ishak fibrosis scores, compared with data from untreated mice given TAA and vehicle only (Figure 1a–c). Treatment with Vorinostat or Panobinostat or AR42 reduced total TAA‐induced necrotic areas and inflammatory foci in the livers and prevented changes to the hepatic sinusoidal and lobular architecture (Figures 1a and 2a,b). All three HDAC inhibitors prevented TAA‐induced increased serum AST and ALT concentrations (Figure S3). Longer term daily treatment with Vorinostat at 25 mg·kg⁻¹ was also well tolerated and effective over 12 weeks of TAA treatment that produced even more severe fibrosis and cirrhosis in controls (Figure S4). Among the more selective HDAC inhibitors, only Entinostat, which inhibits HDAC1–3 enzymes (Hu et al., 2003), completely attenuated the increased liver collagen deposition, fibroplasia, and bridging fibrosis together with a decrease in total necrotic areas and inflammatory foci observed in mice after 6 weeks of TAA treatment (Figure 1a–c). Neither PG100 nor PG50, which are selective inhibitors of class IIa and class IIb enzymes, respectively, were able to prevent TAA‐induced fibrosis (Figure 1a–c). Together, these results indicated that inhibitors of class I HDACs are effective in attenuating chronic TAA‐induced murine liver collagen deposition and hepatic fibrosis.

Figure 1.

Pan and class I selective HDAC inhibitors attenuate chronic TAA‐induced liver fibrosis in mice. (a) Representative images (100×) of liver sections stained with PSR (Picrosirius red) to assess collagen deposition. Scale bar = 50 μm. (b) Quantitative morphometry of collagen positive area (PSR) using Aperio Imagescope. (c) Fibrosis stage was determined using the Ishak scoring method. (d–g) Hepatic mRNA expression for (d) connective tissue growth factor (Ctgf), (e) Tgfβ1, (f) tissue inhibitor of metalloproteinase‐1 (Timp1), and (g) Mmp9 using real‐time qPCR. C57BL/6 mice were dosed daily for 6 weeks with Vorinostat (Vori, 25 mg·kg⁻¹ p.o.), Panobinostat (Pano, 10 mg·kg⁻¹ p.o.), AR42 (10 mg·kg⁻¹ p.o.), Entinostat (Enti, 10 mg·kg⁻¹ p.o.), PG100 (25 mg·kg⁻¹ p.o.), or PG50 (25 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated, ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; TAA, thioacetamide

Figure 2.

Pan and class I selective HDAC inhibitors attenuate chronic TAA‐induced liver inflammation in mice. (a) Representative images (200×) of liver sections stained with haematoxylin and eosin (H&E) to assess hepatic inflammation. Scale bar = 25 μm. (b) Hepatic inflammation was quantitatively scored using Ishak's inflammatory loci scoring method. (c) Quantification of total hepatic CD45⁺ cell number. (d) Representative gating strategy for hepatic CD45⁺ cells after excluding dead cells using viability dyes and doublets using SSC‐H&W and FSC‐H&W. C57BL/6 mice were dosed daily for 6 weeks with Vori (25 mg·kg⁻¹ p.o.), Pano (10 mg·kg⁻¹ p.o.), AR42 (10 mg·kg⁻¹ p.o.), Enti (10 mg·kg⁻¹ p.o.), PG100 (25 mg·kg⁻¹ p.o.), and PG50 (25 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Enti, Entinostat; Pano, Panobinostat; TAA, thioacetamide; Vori, Vorinostat

Further, we assessed the expression of key genes and proteins implicated in liver fibrogenesis, such as connective tissue growth factor (CTGF), TGFβ1, tissue inhibitor of metalloproteinase‐1 (TIMP1), MMP2, and MMP9. Consistent with the increased collagen deposition, treatment with TAA for 6 weeks significantly enhanced the expression of most of these profibrogenic mediators in the liver compared to sham mice (Figures 1d–g and 3a–e). Similar to results on collagen deposition and bridging fibrosis, only Vorinostat, Panobinostat, AR42, and Entinostat inhibited the expression of these profibrogenic mediators in the liver compared to vehicle‐treated TAA mice (Figures 1d–g and 3a–e). In relation to inflammation, flow cytometry analyses (Figure S5) revealed that Vorinostat, Panobinostat, and AR42, but not PG100 and PG50, significantly reduced the numbers of CD45⁺ hepatic immune cells relative to mice treated with TAA alone (Figure 2c,d). Entinostat was even more effective with almost completion reversal of TAA‐induced accumulation of CD45⁺ immune cells. In summary, inhibitors of class I HDAC enzymes, either pan‐HDAC inhibitors or a class I selective inhibitor (Entinostat), were able to attenuate chronic TAA‐induced liver injury and fibrosis.

Figure 3.

Pan and class I selective HDAC inhibitors attenuate chronic TAA‐induced liver fibrosis in mice. (a–c) Hepatic protein expression for (a) MMP2, (b) MMP9, and (c) tissue inhibitor of metalloproteinase‐1 (TIMP1) using elisa. (d, e) Ratio of hepatic MMP2/TIMP1 (d) and MMP9/TIMP1 (e) protein expression. C57BL/6 mice were dosed daily for 6 weeks with Vorinostat (Vori, 25 mg·kg⁻¹ p.o.),Panobinostat (Pano, 10 mg·kg⁻¹ p.o.), AR42 (10 mg·kg⁻¹ p.o.), Entinostat (Enti, 10 mg·kg⁻¹ p.o.), PG100 (25 mg·kg⁻¹ p.o.), or PG50 (25 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; TAA, thioacetamide

3.2. Selective inhibitors of HDAC 1 and 2 enzymes attenuate chronic murine liver inflammation and fibrosis

The inhibitor of class I HDACs, Entinostat, selectively inhibits HDACs 1–3 in vitro (Gupta et al., 2012), suggesting that the anti‐fibrotic efficacy of this inhibitor in mice treated with TAA is likely to be mediated through inhibition of one or more of the class I HDAC enzymes. To date, very little is known about HDAC1 and HDAC2 in chronic liver disease, while two previous studies have reported that liver‐specific knockout of HDAC3 in mice spontaneously leads to increased lipogenic gene expression, severe hepatosteatosis, and hepatocellular carcinomas (Sun et al., 2013). Hence, using two compounds (BRD4884 and NW21) that are selective inhibitors of HDAC1 and HDAC2, we next investigated the anti‐fibrotic potential of inhibiting both specific enzymes HDAC1 and HDAC2 in this mouse model of chronic TAA‐induced liver inflammation and fibrosis. Oral treatment of TAA mice with 10 mg·kg⁻¹ Entinostat served as positive control (Figure 4). Oral administration of BRD4884 or NW21 at 10 mg·kg⁻¹ for 6 weeks significantly attenuated the increased collagen deposition, fibroplasia, and bridging fibrosis observed in vehicle‐treated TAA mice (Figure 4a–c). The TAA‐induced increase in PSR‐positive area and Ishak fibrosis scores were both decreased in mice treated with BRD4884 and NW21, compared with vehicle (Figure 4b,c). TAA given for 6 weeks significantly enhanced the expression of Col1a1 mRNA in the fibrotic livers, while treatment with Entinostat, BRD4884, and NW21 prevented this increase, compared with vehicle‐treated TAA mice (Figure 4D). Similar to results for collagen deposition and bridging fibrosis, BRD4884 and NW21 also inhibited the expression of key profibrogenic mediators in the liver compared to vehicle‐treated TAA mice (Figure 5a–e). Furthermore, Entinostat, BRD4884, and NW21 prevented the increased inflammatory foci and CD45⁺ hepatic immune cell accumulation compared to vehicle‐treated TAA mice (Figure 4e–g). Also, treatment with Entinostat, BRD4884, and NW21 prevented TAA‐induced increased serum AST and ALT concentrations (Figure S3). Together, these results indicated that orally active inhibitors selective for the specific class I HDAC enzymes, HDAC1 and HDAC2, did attenuate TAA‐induced chronic liver inflammation and fibrosis.

Figure 4.

Selective inhibitors of HDAC 1 and 2 attenuate chronic TAA‐induced liver inflammation and fibrosis in mice. (a) Representative images (100×) of liver sections stained with PSR (Picrosirius red) to assess collagen deposition. Scale bar = 50 μm. (b) Quantitative morphometry of collagen positive area (Picrosirius red) using Aperio Imagescope. (c) Fibrosis stage was determined using the Ishak scoring method. (d) Hepatic mRNA expression for collagen 1a1 (Col1a1) using real‐time qPCR. (e) Representative images (200×) of liver sections stained with haematoxylin and eosin (H&E) to assess hepatic inflammation. Scale bar = 25 μm. (f) Hepatic inflammation was quantitatively scored using the Ishak inflammatory loci scoring method. (g) Representative gating strategy for hepatic CD45⁺ cells after excluding dead cell using viability dyes and doublet using SSC‐H&W and FSC‐H&W (left) and total hepatic CD45⁺ cell numbers (right). C57BL/6 mice were dosed daily for 6 weeks with Enti (10 mg·kg⁻¹ p.o.), BRD4884 (10 mg·kg⁻¹ p.o.), or NW21 (10 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Enti, Entinostat; TAA, thioacetamide

Figure 5.

Selective inhibitors of HDAC 1 and 2 attenuate chronic TAA‐induced liver fibrosis in mice. (a–c) Hepatic protein expression for (a) MMP2, (b) MMP9, and (c) tissue inhibitor of metalloproteinase‐1 (TIMP1) using elisa. (d, e) Ratio of hepatic MMP2/TIMP1 (d) and MMP9/TIMP1 (e) protein expression. C57BL/6 mice were dosed daily for 6 weeks with Enti (10 mg·kg⁻¹ p.o.), BRD4884 (10 mg·kg⁻¹ p.o.), or NW21 (10 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Enti, Entinostat; TAA, thioacetamide

3.3. Inhibitors of certain HDACs attenuate the local chronic inflammatory milieu in the fibrotic livers

Hepatic inflammation is a characteristic feature of all stages of chronic liver disease, including before and during fibrosis development (Gieseck et al., 2018). Here, we investigated whether selective inhibitors of class I HDAC enzymes target the local inflammatory milieu in the chronically injured liver. Daily administration of TAA to mice for 6 weeks induced an immune response characteristic of a mixed type 2 and type 3 immune response signature in the fibrotic livers (Figure 6a–i). The livers from vehicle‐treated TAA mice showed increased protein expression of both classical type 2 (IL‐2, IL‐5, IL‐9, IL‐13, and IL‐21; Figure 6a–e) and type 3 (IL‐17A, IL‐17F, IL‐6, and TNF; Figure 6f–i) inflammatory cytokines, as compared to the livers from sham‐treated mice. This inflammatory signature was accompanied by an increased accumulation of type 2 effector CD45⁺/Ly6G⁻/MHCII⁻/SSC^hi eosinophils (Harms, Morsey, Boyer, Fox, & Sarvetnick, 2012; Figure 6j), CD45⁺/Ly6G⁻/CD11b^int/ F4/80^hi Kupffer cells (Ramachandran et al., 2012; Figure 6k), CD45⁺/Ly6G⁻/CD11b^hi/ F4/80^int monocyte‐derived macrophages (Ramachandran et al., 2012; Figure 6l), CD45⁺/Ly6G⁻/CD11b^hi/F4/80^int/Ly6C^hi pro‐fibrotic macrophages (Ramachandran et al., 2012), CD45⁺/Ly6G⁻/CD11b^hi/F4/80^int/Ly6C^low restorative macrophages (Ramachandran et al., 2012; Figure 6m–o), and type 3 effector CD45⁺/Ly6G⁺/MHCII⁻ neutrophils (Potter & Harding, 2001; Figure 6p) in the TAA‐treated livers compared to sham animals.

Figure 6.

Selective inhibitors of class I HDAC enzymes attenuate chronic TAA‐induced hepatic pro‐inflammatory responses in mice. (a–e) Hepatic type 2 cytokines IL‐2, IL‐5, IL‐9, IL‐13, and IL‐21 were quantified using Legendplex. (f–i) Hepatic type 3 cytokines IL‐17A, IL‐17F, IL‐6, and TNF were quantified using Legendplex. (j–l) Total hepatic immune cells (j) CD45⁺/Ly6G⁻/MHCII⁻/SSC^hi eosinophils, (k) CD45⁺/Ly6G⁻/CD11b^int/F4/80^hi Kupffer cells, and (l) CD45⁺/Ly6G⁻/CD11b^hi/F4/80^low monocytes‐derived macrophages were quantified using flow cytometry. (m–o) Percentage of CD45⁺/Ly6G⁻/CD11b^hi/F4/80^low/Ly6C^hi or CD45⁺/Ly6G⁻/CD11b^hi/F4/80^low/Ly6C^low macrophages populations, total hepatic CD45⁺/Ly6G⁻/CD11b^hi/F4/80^low/Ly6C^hi macrophages, and CD45⁺/Ly6G⁻/CD11b^hi/F4/80^low/Ly6C^low macrophages were quantified using flow cytometry. (p) Total hepatic CD45⁺/Ly6G⁺/MHCII⁻ neutrophils were quantified using flow cytometry. C57BL/6 mice were dosed daily for 6 weeks with Pano (10 mg·kg⁻¹ p.o.), Enti (10 mg·kg⁻¹ p.o.), BRD4884 (10 mg·kg⁻¹ p.o.), and NW21 (10 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Enti, Entinostat; Pano, Panobinostat; TAA, thioacetamide

Daily oral treatment for 6 weeks with the pan‐HDAC inhibitor Panobinostat, or any of the three structurally different inhibitors (Entinostat, BRD4884, NW21) that are more selective for class I HDACs, strikingly attenuated this increased chronic type 2 and type 3 inflammatory signature, compared with vehicle treated TAA mice (Figure 6a–p). All four HDAC inhibitors decreased TAA‐induced expression of type 2 (IL‐2, IL‐5, IL‐9, IL‐13, and IL‐21; Figure 6a–e) and type 3 (IL‐17A, IL‐17F, IL‐6, and TNF; Figure 6F‐I) inflammatory cytokines compared to vehicle‐treated TAA mice. These HDAC inhibitors also decreased the TAA‐induced increased IL‐1β expression compared to vehicle‐treated TAA mice (Figure S6). This strong anti‐inflammatory response was also accompanied by a significant decrease in the accumulation of type 2 effector cells, such as CD45⁺/Ly6G⁻/MHCII⁻/SSC^hi eosinophils (Figure 6j) and CD45⁺/Ly6G⁻/CD11b^hi/F4/80^int/Ly6C^hi pro‐fibrotic macrophages (Figure 6n), together with reduced type 3 effector CD45⁺/Ly6G⁺/MHCII⁻ neutrophils (Figure 6p), in the livers of TAA mice treated with Panobinostat, Entinostat, BRD4884, or NW21 compared to vehicle. Importantly, CD45⁺/Ly6G⁻/CD11b^hi/F4/80^int/Ly6C^low restorative macrophages, which promote resolution of inflammation during fibrosis regression, were increased in the livers of TAA mice treated with Panobinostat, Entinostat, BRD4884, or NW21 compared to vehicle (Figure 6o). Furthermore, other hepatic immune cell populations that participate in propagating a chronic inflammatory milieu, such as CD4⁺ and CD8⁺ T cells, B cells, monocytes, NK cells, and NKT cells were also increased in the livers of 6 week TAA‐treated mice compared to those in sham controls (Figure S7). Treatment with Panobinostat, Entinostat, BRD4884, or NW21 attenuated this TAA‐induced increase in CD4⁺ and CD8⁺ T cells, B cells, and monocytes but did not significantly affect NK or NKT cells (Figure S7). Together with the cytokine profile, these results demonstrate that orally active inhibitors of class I HDAC enzymes can attenuate the mixed type 2 and type 3 inflammatory phenotype associated with TAA‐induced hepatic fibrosis in mice.

3.4. HDAC inhibitors reduce hepatic stellate cell activation in the fibrotic livers

In order to investigate whether Panobinostat and the more selective inhibitors of class I HDACs can prevent HSC activation that is downstream of type 2 inflammation in the injured livers, we examined the expression of α‐SMA at both the mRNA and protein levels in TAA‐treated mice. Chronic TAA administration led to increased α‐SMA expression in the liver at both the mRNA and protein levels (Figure 7a–c). Further, treatment with Panobinostat and the three different, structurally distinct, selective inhibitors of class I HDACs (Entinostat, BRD4884, and NW21) completely prevented the increase in TAA‐induced Acta2 mRNA and α‐SMA protein expression in the livers compared to vehicle‐treated TAA mice (Figure 7a–c). We also assessed mRNA levels of other markers of fibrogenesis and HSC activation such as Tgfβ1 and glial fibrillary acid protein (Gfap). TAA administration for 6 weeks significantly increased the expression of these genes, and treatment with Panobinostat, Entinostat, BRD4884, or NW21 prevented this effect (Figure 7d, e). Taken together, these results suggest that pan and selective inhibitors of class I HDACs also decrease activation of HSCs and fibrogenesis in the injured inflamed liver.

Figure 7.

Selective inhibitors of class I HDACs attenuate chronic TAA‐induced hepatic stellate cell activation in mice. (a) Hepatic mRNA expression for smooth muscle α actin (Acta2) using real‐time qPCR. (b) Quantitative morphometry of hepatic SMA positive area using Aperio Imagescope. (c) Immunohistochemical staining for smooth muscle α actin (SMA) in liver sections (200×). Scale bar = 25 μm. (d, e) Hepatic mRNA expression for (d) Tgfβ1 and (e) glial fibrillary acid protein (Gfap) using real‐time qPCR. C57BL/6 mice were dosed daily for 6 weeks with Pano (10 mg·kg⁻¹ p.o.), Enti (10 mg·kg⁻¹ p.o.), BRD4884 (10 mg·kg⁻¹ p.o.), or NW21 (10 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received olive oil as vehicle control. Mice were provided ad libitum access to drinking water containing TAA (300 mg·L⁻¹) for 6 weeks. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Enti, Entinostat; Pano, Panobinostat; TAA, thioacetamide

3.5. Inhibitors of class I HDACs attenuate acute and chronic IL‐33‐dependent nILC2 activation and liver injury in mice

Type 2 immunity plays a major role in the initiation and progression of a pro‐fibrotic phenotype in response to hepatocyte injury (Gieseck et al., 2018), while type 3 immunity after HSC activation is associated with high disease severity and contributes to fibrosis progression in the inflamed liver (Li et al., 2017; Sun et al., 2012). However, the relative abundance and importance of key type 2 cytokine‐producing CD45⁺/Lin⁻/Thy‐1⁺/ST2⁻/KLRG1^hi iILC2 and CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC2 in liver injury and fibrosis are unclear. Here, we firstly characterised the response of acute and chronic TAA‐induced liver injury on iILC2 and nILC2 accumulation and activation in the injured liver. We then investigated whether selective inhibitors of class I HDACs prevent initiation and progression of TAA‐induced liver injury, type 2 immunity, and fibrosis together with attenuating the apoptosis‐driven IL‐25‐/IL‐33‐dependent ILC2 inflammatory axis.

TAA administration for 6 weeks increased the formation of necrotic areas and apoptosis in the fibrotic livers and increased the number of TUNEL‐positive cells compared to sham‐treated mice (Figure 8a). Further, this increase in TUNEL‐positive cells correlated with a selective increase in IL‐33, but not IL‐25, protein concentration in the liver compared to the sham‐treated livers (Figure 8b,c). Similarly, in terms of type 2 cytokine‐producing ILC2s, chronic TAA administration selectively increased the accumulation of IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC2 cells compared to IL‐25‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁻/KLRG1^hi iILC2 cells (Figure 8d, e). As with the suppression of pro‐fibrotic type 2 cytokines (Figure 7), daily oral treatment with either Panobinostat or NW21 completely attenuated the increase in TAA‐induced TUNEL‐positive cells, IL‐33 expression (both at mRNA and protein levels) and type 2 cytokine‐producing IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC2 cells in the liver compared to vehicle‐treated TAA mice (Figures 8a–e and S8).

Figure 8.

Selective inhibitors of class I HDACs attenuate chronic TAA‐induced hepatic IL‐33‐dependent nILC2 activation in mice. (a) Representative images (200×) of liver sections stained for TUNEL‐positive cells (white arrows) to assess apoptosis, scale bar = 25 μm, and TUNEL‐positive cells (fold change) were quantified using Aperio Imagescope. (b, c) Liver protein concentrations of (b) IL‐25 and (c) IL‐33 were measured using elisa. (d, e) Total hepatic immune cells (d) CD45⁺/Lin⁻/Thy‐1⁺/ST2⁻/KLRG1^hi inflammatory ILC2 (iILC2) and (e) CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int natural type 2 innate lymphoid cells (nILC2) were quantified using flow cytometry. C57BL/6 mice were provided ad libitum access to either drinking water containing TAA (300 mg·L⁻¹) or regular water for 6 weeks. Pano (10 mg·kg⁻¹ p.o.) or NW21 (10 mg·kg⁻¹ p.o.) was dosed daily via oral gavage in olive oil for 6 weeks. Sham and TAA group mice received only olive oil as vehicle control. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Pano, Panobinostat; TAA, thioacetamide

To investigate whether IL‐33‐nILC2 infiltration is associated with the initiation of TAA‐induced pro‐fibrotic and pro‐inflammatory responses before established fibrosis (Figure S9), we used an acute 1‐day model of TAA‐induced liver injury. Similar to 6‐week TAA‐induced liver injury, acute 1‐day TAA administration induced a significant increase in TUNEL‐positive cells together with increased IL‐33 (both at mRNA and protein levels) and type 2 cytokine‐producing IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC cells (Figure 9a–e and S8) without affecting liver IL‐25 concentrations or IL‐25‐dependent iILC2 cells (Figure 9b, d). Both Panobinostat and NW21 completely attenuated the increase in acute TAA‐induced TUNEL‐positive cells, IL‐33 expression and IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC cells in the liver compared to vehicle‐treated TAA mice (Figure 9a–e and S8). Furthermore, in relation to an inflammatory response, the livers from vehicle‐treated acute TAA mice showed a selective increase in key type 2 cytokines (IL‐2, IL‐5, IL‐9, and IL‐13; Figure 10a–f) and type 2 effector cells, such as CD45⁺/Ly6G⁻/MHCII⁻/SSC^hi eosinophils (Figure 10c) and CD45⁺/Ly6G⁻/CD11b^hi/F4/80^int/Ly6C^hi pro‐fibrotic macrophages (Figure S10), without inducing a type 3 inflammatory response including key cytokines IL‐17A and IL‐17F (Figure 10g, h) compared with sham‐treated mice. Importantly, acute TAA administration did not affect other key type 2 cytokine‐producing hepatic immune cell populations compared to sham mice (Figure S10). Both Panobinostat and NW21 attenuated this increase in liver IL‐2, IL‐5, IL‐9, and IL‐13 concentrations as well as attenuating the increased eosinophilia and pro‐fibrotic macrophages observed in vehicle‐treated acute TAA mice (Figures 10a–f and S10). In summary, these results suggest that type 2, but not type 3, immunity is important in chronic TAA‐induced liver fibrosis and that selective inhibition of class I HDAC enzymes decreases chronic liver injury and fibrosis while also attenuating IL‐33‐nILC2‐mediated type 2 inflammation.

Figure 9.

Selective inhibitors of class I HDACs attenuate acute TAA‐induced hepatic IL‐33‐dependent nILC2 activation in mice. (a) Representative images (200×) of liver sections stained for TUNEL‐positive cells (white arrows) to assess apoptosis, scale bar = 25 μm, and TUNEL‐positive cells (fold change) were quantified using Aperio Imagescope. (b, c) Liver protein concentrations of (b) IL‐25 and (c) IL‐33 were measured using Legendplex. (d, e) Total hepatic immune cells (d) CD45⁺/Lin⁻/Thy‐1⁺/ST2⁻/KLRG1^hi inflammatory ILC2 (iILC2) and (e) CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int natural type 2 innate lymphoid cells (nILC2) were quantified using flow cytometry. C57BL/6 mice were administered via intraperitoneal injection with either TAA (300 mg·kg⁻¹ in 0.9% saline) or 0.9% saline. Pano (10 mg·kg⁻¹ p.o.) or NW21 (10 mg·kg⁻¹ p.o.) was dosed once 1 hr before TAA injection with via oral gavage in olive oil. Sham and TAA group mice received only olive oil as vehicle control. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Pano, Panobinostat; TAA, thioacetamide

Figure 10.

Selective inhibitors of class I HDACs attenuate acute TAA‐induced hepatic type 2 inflammatory responses in mice. (a–f) Hepatic type 2 cytokines (a) IL‐2, (b) IL‐5, (d) IL‐9, (e) IL‐13, and (f) IL‐21 were quantified using Legendplex. (g, h) Hepatic type 3 cytokines (g) IL‐17A and (h) IL‐17F were quantified using Legendplex. (c) Total hepatic CD45⁺/Ly6G⁻/MHCII⁻/SSC^hi eosinophils were quantified using flow cytometry. C57BL/6 mice were dosed once 1 hr before TAA injection with Pano (10 mg·kg⁻¹ p.o.) and NW21 (10 mg·kg⁻¹ p.o.) via oral gavage in olive oil. Sham and TAA group mice received only olive oil as vehicle control. TAA (300 mg·kg⁻¹ body weight in 0.9% saline) was administered via intraperitoneal injection to induce acute liver injury after 1 day only. Sham and TAA group mice received vehicle (i.p. 0.9% saline) as control. Data shown are means ± SEM; n = 5 in each group. *P < 0.05, significantly different as indicated; ns, not significantly different; one‐way ANOVA with Dunnett's post hoc test. Cmpd, compound; Pano, Panobinostat; TAA, thioacetamide

4. DISCUSSION

Liver fibrosis is considered to be an inflammation‐driven, wound healing process that replaces apoptotic and necrotic hepatocytes with the deposition of extracellular matrix protein and collagen (Pellicoro et al., 2014; Trautwein, Friedman, Schuppan, & Pinzani, 2015). When this physiological process becomes dysregulated, it can lead to chronic inflammation, liver fibrosis, and cirrhosis, which is associated with an increased risk for developing hepatocellular carcinomas (Gieseck et al., 2018; Pellicoro et al., 2014). Despite many trials of anti‐inflammatory drugs, there are currently no effective drugs available to treat human liver fibrosis or cirrhosis, and the underlying pathological mechanisms of chronic liver fibrosis remain incompletely understood (Rudnick, 2017; Schuppan & Kim, 2013; Trautwein et al., 2015). HDAC inhibitors that target liver inflammatory pathways may represent an effective new approach to attenuating human liver disease, including liver fibrosis and cirrhosis progressing to liver cancer, although evidence for this approach is limited (Coradini & Speranza, 2005; Goto & Kato, 2017; Van Beneden et al., 2013). The individual HDAC isoforms involved and the molecular mechanisms by which HDAC inhibitors might regulate hepatic fibrogenesis remain elusive. Here, we show that inhibitors of certain HDAC enzymes have previously unknown effects on inflammation, inflammatory cells, and matrix deposition in chronic liver fibrosis. Our findings support a new concept that inhibition of selected class I HDAC enzymes can modulate IL‐33 and IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int natural group 2 innate lymphoid cells (nILC2s), together with ameliorating chronic hepatic type 2 driven inflammation and downstream HSC activation and fibrosis. The link between IL‐33, ILC2s and hepatic inflammation, and fibrosis is already established (Gieseck et al., 2018; McHedlidze et al., 2013). Some compounds that inhibit all or most of the 11 known zinc‐containing HDAC enzymes are already clinically approved as anti‐cancer drugs (Gupta et al., 2012), so there is an established pathway to the clinic for such inhibitors. International efforts to develop specific inhibitors of individual HDAC isozymes are very much still a work in progress, but results of the present study using experimental inhibitors of class I HDACs are promising for treatment of liver fibrosis.

In the present study, we examined three clinically approved drugs (Vorinostat, Panobinostat, and Entinostat), and four, more isozyme‐selective, though still experimental HDAC inhibitors (NW21, BRD4884, PG100, and PG50) for their capacity to curtail fibrosis in a murine model of chronic liver injury. Although the precise role of each of the 18 HDAC enzymes in the liver is currently unknown (Van Beneden et al., 2013), we find that inhibitors of class I (but not class II) HDAC enzymes can potently suppress type 2 cytokine‐driven liver inflammation and fibrosis in TAA‐induced hepatocellular stress and liver injury in mice. We show that some clinically approved pan‐HDAC inhibitors and selective inhibitors of HDAC1 and HDAC2 significantly decreased hepatocyte apoptosis and expression of IL‐33, an alarmin that activates liver CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC2 cells (McHedlidze et al., 2013; Weiskirchen & Tacke, 2017). These HDAC inhibitors selectively decreased the total number of IL‐33‐activated nILC2 cells, while also suppressing key type 2 inflammatory cytokines IL‐2, IL‐5, IL‐9, IL‐13, and IL‐21. This type 2 cytokine‐driven inflammatory response may increase infiltration and activation of eosinophils, monocytes/macrophages, and HSCs that drive fibrogenesis (Gieseck et al., 2018; Hams et al., 2015); all of which were also attenuated by oral treatment with pan‐ (Vorinostat, Panobinostat, and AR42) or class I selective‐ (Entinostat, BRD4884, and NW21) HDAC inhibitors for 6 weeks. One limitation of the present study was the use of a preventative treatment protocol to identify the precise HDAC isoenzymes involved in initiation and progression of liver inflammation and fibrosis. Further studies with selective inhibitors of class I HDACs using clinically relevant fibrosis reversal protocols in different models of liver fibrosis are now warranted.

The liver was historically viewed as a non‐immunological organ, primarily involved in homeostatic functions such as metabolic regulation, nutrient storage, and host detoxification (Robinson, Harmon, & O'Farrelly, 2016). However, inflammatory mechanisms are crucial in maintaining liver homeostasis, as well as protecting against pathogens, nutritional excess, tissue damage, and other external stressors (Czaja, 2014; Krenkel & Tacke, 2017; Robinson et al., 2016). The healthy liver contains a large repertoire of resident myeloid, lymphoid, and innate lymphoid immune cell populations, including macrophages, eosinophils, neutrophils, ILC2 cells, T cells, B cells, natural killer (NK), and NKT cells that, together with other non‐haematopoietic cells such as HSCs, play central roles in pathological inflammation in the liver. However, the key cells and molecular mechanisms that initiate and propagate chronic inflammation in liver fibrosis development are not completely elucidated. Inflammatory mediators, including type 2 cytokines IL‐5, IL‐9, and IL‐13, are critical effectors of chronic human liver fibrosis (Gieseck et al., 2018; Hams et al., 2015; Weiskirchen & Tacke, 2017). These type 2 cytokines can increase eosinophilia and regulate chronic macrophage and HSC activation, thereby leading to increased extracellular matrix production and fibrosis (Gieseck et al., 2018; Hams et al., 2015; Weiskirchen & Tacke, 2017). ILC2 cells are now commonly associated with a variety of fibrotic conditions in the lung and liver and have been identified as the primary source of type 2 cytokines that initiate and propagate organ fibrosis (Gieseck et al., 2018; Hams et al., 2015; Weiskirchen & Tacke, 2017). Together with type 2 cytokines, targeting ILC2 activation is emerging as a possible way to regulate certain allergic and chronic fibrotic disorders (Horsburgh, Todryk, Ramming, Distler, & O'Reilly, 2018).

ILC2 cells are activated by stress‐related alarmins, such as IL‐25, IL‐33, or their combination, resulting in increased local proliferation of these cells and increased production of IL‐5, IL‐9, and IL‐13 that activate downstream type 2 effector immune cells and HSCs (Gieseck et al., 2018; Hart et al., 2017; Vannella et al., 2016). Among these, the IL‐1 family cytokine IL‐33 has been identified as a key hepatocyte stress‐related cytokine that initiates and propagates chronic liver fibrosis (Liew, Girard, & Turnquist, 2016; Weiskirchen & Tacke, 2017). Both IL‐33 and its cell surface receptor ST2 have been causally linked to induce fibrosis in various disease states, including Crohn's disease, lung, and liver fibrosis (Hams et al., 2015). Further, IL‐33 has been shown to be elevated in the serum and livers of human patients with chronic liver fibrosis (McHedlidze et al., 2013; Tan et al., 2017). Persistent hepatocellular stress‐mediated release of IL‐33 from apoptotic and necrotic hepatocytes initiates the proliferation and activation of liver ILC2s via ST2 (McHedlidze et al., 2013). Sustained IL‐33‐mediated activation of ILC2s induces increased eosinophilia and chronic activation of macrophages and HSCs via increased type 2 cytokines, thereby contributing to the downstream development of fibrosis (McHedlidze et al., 2013). Apart from activating ILC2s, IL‐33 has also been shown to directly act on HSCs via ST2 to induce their proliferation and activation to potentiate liver fibrosis (Tan et al., 2017; Weiskirchen & Tacke, 2017). To date, very little is known about the effects of HDAC inhibitors on IL‐33 and ILC2 activation in the liver. A recent study has shown that HDAC inhibition by a pan‐HDAC inhibitor TSA attenuates ILC2 activation together with the production of type 2 cytokines in a mouse model of allergic inflammation and asthma (Toki et al., 2016). In the current study, we show that, among ILC2 cells, TAA‐induced liver injury selectively induces the increased accumulation of IL‐33‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁺/KLRG1^int nILC2s relative to IL‐25‐dependent CD45⁺/Lin⁻/Thy‐1⁺/ST2⁻/KLRG1^hi iILC2 cells. Further, we show for the first time that clinically used pan‐HDAC inhibitors, and especially inhibitors of specific class I HDACs (HDAC1 and 2), can suppress the early initiation events that propagate chronic liver fibrosis, including suppression of hepatocyte apoptosis, IL‐33 expression, IL‐33‐mediated nILC2 activation, and secretion of key type 2 cytokines IL‐2, IL‐5, IL‐9, IL‐13, and IL‐21. However, the exact molecular mechanisms by which HDAC inhibitors attenuate IL‐33 expression remain to be elucidated. The mechanisms could potentially involve HDAC inhibitors indirectly activating a key transcription factor, either at the transcriptional or post‐translational level, which directly governs IL‐33 gene expression. More broadly, we suggest that HDAC inhibitors elicit anti‐fibrotic responses in the liver by two sequential anti‐inflammatory mechanisms. Firstly, HDAC inhibition prevents both hepatocyte apoptosis‐triggered IL‐33 expression and the proliferation and activation of IL‐33‐dependent nILC2 cells during fibrosis initiation and progression, thereby reducing chronic type 2 driven liver inflammation. Secondly, they also reduce type 2 cytokine‐driven proliferation and activation of HSCs and pro‐fibrotic macrophages in the damaged liver, thereby attenuating the overall chronic inflammatory wound healing process after pro‐fibrotic stimulation. Thus, targeting inflammatory events at both the initiation and progression stages of disease with pan‐, and especially selective class I, HDAC inhibitors could be clinically valuable and allow for prevention, regression, and resolution of chronic human liver fibrosis.

In summary, we show here that several, structurally different, inhibitors of class I HDAC enzymes, including some clinically approved (albeit broad spectrum) HDAC inhibitors, suppress hepatic fibrosis and attenuate IL‐33‐dependent nILC2 activation, type 2 inflammation, and HSC activation in a mouse model of TAA‐induced severe liver injury. By contrast, other anti‐inflammatory and anti‐fibrotic compounds were not very effective inhibitors in this very aggressive model of chronic liver fibrosis (Figures S4 and S11). Our study indicates that inhibitors of class I, but not class II, HDACs can prevent the progression of chronic liver disease. Our results reveal that modulation of the local type 2 inflammatory milieu, inhibition of chronic natural ILC2 activation, and suppression of fibrogenesis in the liver by orally active, low molecular weight (MW), HDAC inhibitors may be a viable approach to targeting the sequential, multifactorial, and complex events that lead to chronic liver remodelling, inflammation, and fibrosis, independent of the underlying disease‐causing aetiology. Some HDAC inhibitors that we have studied here are already used in humans for the treatment of lymphomas, so it may be possible and expeditious to examine these drugs in another clinical setting for preventing chronic liver disease, particularly human liver fibrosis and cirrhosis. If successful, this could pave the way for more potent, more HDAC isozyme‐selective, compounds to be trialled as effective therapies for prevention and treatment of human liver fibrosis.

AUTHOR CONTRIBUTIONS

Z.L., R.L.F., and A.I. performed all mouse and cellular experiments; R.C.R. and P.K.G. synthesised and characterised the HDAC inhibitors; D.R. performed the SMA staining; A.C. performed the Picrosirius red staining; K.M.I. and E.E.P. contributed the key reagents/materials/analysis tools; Z.L., K.S., J.L.S., M.J.S., D.P.F., and A.I. conceived the study; Z.L., D.P.F., and A.I. designed the experiments, analysed the data, and wrote the manuscript; all authors contributed in editing the manuscript.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1. Chemical structures of anti‐fibrotic HDAC inhibitors.

Figure S2. Response of HDAC inhibitors on body weight changes in 6 wk TAA mice.

Figure S3. HDAC inhibitors attenuate TAA‐induced increased serum aspartate transaminase (AST) (A, B) and serum alanine transaminase (ALT) (C, D) in mice.

Figure S4. Long‐term treatment with Vorinostat, but not other anti‐inflammatory compounds, attenuated TAA‐induced progressive fibrosis and cirrhosis in mice.

Figure S5. Representative gating strategy to define hepatic CD45+ myeloid and lymphoid cells from TAA administered mice.

Figure S6. Pan and isozyme selective inhibitors of class I HDAC enzymes suppress IL‐1β expression at both the mRNA (A) and protein (B) levels in 6 wk TAA mice.

Figure S7. Treatment with Panobinostat, Entinostat, BRD4884 and NW21 attenuated chronic TAA‐induced increase in T cells, B cells and monocytes, but not NK or NKT cells, in mice. A‐F)

Figure S8. Pan and an isozyme selective inhibitor of class I HDAC enzymes suppress IL‐33 expression at the mRNA level in 1 d, 6 wk and 12 wk TAA mice.

Figure S9. Representative gating strategy to define hepatic group 2 natural and inflammatory innate lymphoid cells in TAA‐administered mice.

Figure S10. Acute 1 day TAA administration to mice increased hepatic inflammatory cell infiltration and macrophage numbers without affecting total T cells, B cells, NK cells or NKT cell numbers.

Figure S11. Treatment with anti‐TGFβ antibody reduced chronic TAA‐induced liver fibrosis but not inflammatory cells in mice.

Table S1. List of mouse real time qPCR primer sequences.

Loh Z, Fitzsimmons RL, Reid RC, et al. Inhibitors of class I histone deacetylases attenuate thioacetamide‐induced liver fibrosis in mice by suppressing hepatic type 2 inflammation. Br J Pharmacol. 2019;176:3775–3790. 10.1111/bph.14768