Compound JNJ7777120 (JNJ; 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), a potent and selective H₄R antagonist, has been used at a concentration of 2.5 mg/kg of body weight (b.wt.). This compound was kindly provided by Dr. Robin Thurmond (Janssen Research & Development, USA). Compound VUF8430, (VUF; 2-[(aminoiminomethyl)amino] ethyl carbamodithioic acid ester, S-(2-guanidylethyl)-isothiourea), an H₄R agonist, (Sigma-Aldrich, San Louis, MO, USA), was used at a concentration of 2 mg/kg b.wt. Bleomycin (Merck-Millipore, Burlington, MA, USA) was used at a concentration of 0.05 IU for each mouse and dissolved in 50 μl of saline, in order to obtain the bleomycin-induced model of lung fibrosis. The drug doses and frequency of administrations were selected according to previous papers (Rosa et al., 2014; Lucarini et al., 2016).

Male wild-type (WT) C57BL/6 mice, approximately weighing 25–30 g and 8–9 weeks old, purchased from a commercial dealer (Harlan, Udine, Italy), and male C57BL/6 PARP-1 deficient mice (PARP-1^−/−) (Wang et al., 1995), comparable in mice strain, age, and weight with WT mice, were used for the experiments. All the animals were fed with standard diet and water at libitum and housed under a 12 h light/dark photoperiod at 22°C for at least 48 h before the experiments.

The study protocol complied with the recommendations of the European Economic Community (86/609/CEE) and the Declaration of Helsinki on animal experimentation and was approved by the animal Ethical and Care Committee of the University of Florence (Florence, Italy) and by the Italian Health Ministry (Authorization n 874/2017-PR). Experiments were performed at the Centre for Laboratory Animal Housing and Experimentation (CeSAL) at the University of Florence. The ARRIVE guidelines were considered (McGrath et al., 2010).

Seventy mice (30 WT and 40 PARP-1^−/−) were anesthetized with zolazepam/tiletamine (Zoletil, 50/50 mg/ml, Virbac Srl, Milan, Italy; 50 μg/g, in 100 μl of saline, i.p.); 50 of them (20 WT and 30 PARP-1^−/−) were intra-tracheally treated with bleomycin (0.05 IU in 50 μl of saline), and the other 20 (10 WT and 10 PARP-1^−/−) were intra-tracheally treated with 50 μl of saline (referred to as non-fibrotic negative controls, Naive).

Ten bleomycin-treated WT and 10 bleomycin-treated PARP-1 deficient mice received two daily intra-peritoneal (i.p.) injections of 100 μl of JNJ solution (2.5 mg/kg; i.p.), after bleomycin administration and for the next 21 days. These are referred to as Bleomycin+JNJ treated groups. Ten bleomycin treated PARP-1^−/− mice received two daily intra-peritoneal injection of 100 μl of VUF solution (2 mg/kg; i.p.), after bleomycin administration and for the next 21 days. These are referred to as Bleomycin+VUF treated group. Ten WT mice and 10 PARP-1^−/− mice were treated only with vehicle (PBS) and referred to as fibrotic positive controls (Bleomycin+Vehicle).

At the end of the treatment period, all mice were subjected to the measurement of airway resistance to inflation and static lung compliance, functional parameters for fibrosis-induced lung stiffness. The measurement is performed using a constant volume mechanical ventilation method with constant number of breaths in a minute; the static compliance determination is performed applying a positive end-expiratory pressure of 3 cm H₂O, to mimic spontaneous ventilation (Pini et al., 2010, 2012; Manni et al., 2016). Briefly, a 22-gauge cannula (Venflon 2; Viggo Spectramed, Windlesham, UK, 0.8 mm diameter) was inserted into the trachea of the anesthetized mouse. The animal was ventilated with a small-animal respirator (Ugo Basile, Comerio, Italy) adjusted to deliver a tidal volume of 0.8 ml at a rate of 20 strokes/min. A high-sensitivity pressure transducer (settings: gain 1, chart speed 25 mm/sec.; P75 type 379; Harvard Apparatus Inc., Holliston, MA, USA) connected to a polygraph (Harvard Apparatus Inc. Edenbridge, UK; settings: gain 1, chart speed 25 mm/s) was used to register the changes in lung resistance to inflation, defined as the pressure at airway opening (PAO). Changes in lung resistance to inflation, expressed as millimeters on the chart and registered for at least 3 min, were carried out not less than 40 consecutive tracings of respiratory strokes and then averaged. For static lung compliance determination, multiple linear regression was used to fit pressure and volume in each individual mouse to the linear model of the lung (Manni et al., 2016).

After PAO measurements, the animals were killed with lethal dose of anesthetic drugs. The whole left lungs were removed and fixed with 4% paraformaldehyde in PBS for histological analysis. The right lungs were weighed, quickly frozen, and stored at −80°C. For biochemical measurements, the samples were thawed at 4°C, homogenized on ice in 50 mM Tris-HCl buffer (180 mM KCl and 10 mM EDTA, pH 7.4), and then centrifuged for 30 min at 10,000 g at 4°C, unless otherwise reported. The homogenized supernatants were collected.

Six μm thick histological sections were cut from the paraffin-embedded lung samples. In order to minimize artefactual differences in the staining process, all sections were stained in a single session. A digital camera connected to a light microscope equipped with an ×20 objective was used to randomly take photomicrographs of the histological slides. Computer-aided densitometry was performed to obtain quantitative assessment of the stained sections. The free-share ImageJ 1.33 image analysis program¹ was used to measure optical density (OD) and surface area. For each measured parameter, values are means ± SEM of the OD measurements (arbitrary units) of individual mouse (five images each) from every experimental groups (tested blind).

The assessment of lung collagen was obtained by staining the histological sections with a simplified Azan method for collagen fibers (Smolle et al., 1996), omitting azocarminium and orange G to reduce parenchymal tissue background. OD measurements of the aniline blue-stained collagen fibers were performed after selection of a correct threshold to eliminate aerial air spaces and bronchial/alveolar epithelium (Formigli et al., 2007). To confirm the assessment of lung collagen, the Picrosirius red staining was carried out; the sections were stained in 0.1% Direct Red 80/Sirius Red F3B (Sigma-Aldrich) in saturated picric acid at room temperature for 1 h, and then they were differentiated in 0.5% acetic acid, prior to dehydration, clearing, and mounting (Lattouf et al., 2014; Marcos-Garcés et al., 2017).

Moreover, lung tissue sections were stained with hematoxylin and eosin or with periodic acid-Schiff (PAS) staining for mucins in order to obtain morphometry of smooth muscle layer thickness and bronchial goblet cell number, respectively, both key markers of airway remodeling. Digital microphotographs of small-sized bronchi were taken randomly. The thickness of the bronchial smooth muscle layer was measured on the digitized images using the above-mentioned software. Total bronchial epithelial cells and PAS-stained goblet cells were counted on bronchial cross-section profiles, and the percentage of goblet cells was calculated.

For determination of PARylated protein content in the lung, tissues were homogenized in RIPA buffer plus a cocktail of protease inhibitors and centrifuged at 12,000 g for 5 min. Supernatants were collected, and total protein levels were measured using Micro BCA Protein Assay (ThermoFisher Scientific, Waltham, MA, USA). Thirty μg of proteins were used for Western blot analysis, with a mouse monoclonal anti-PAR(10H) antibody (Alexis Biochemicals, Florence, Italy) diluted 1:1,000 in PBS-T containing 5% non-fat dry milk. The PAR(10H) monoclonal antibody recognizes poly(ADP-ribose) synthesized by PARP enzymes. A suitable peroxidase-conjugated secondary antibody (diluted 1:5,000 in PBS-T containing 5% non-fat dry milk) was used to determine the binding of the primary antibody. The loading transfer of equal amounts of proteins was controlled by reblotting the membrane with an anti-β-actin antibody (diluted 1:20,000 in 5% non-fat dry milk-PBS-T, Sigma-Aldrich). Bands were visualized by enhanced chemiluminescence (Luminata Crescendo Western HRP substrate, Merck Millipore) and quantified by densitometric analysis with the ImageJ software.

The levels of TGF-β, the main profibrotic cytokine involved in fibroblast activation, and two pro-inflammatory cytokines, IL-1β and TNF-α, were measured on aliquots (20 μl) of lung samples homogenated in PBS by using the FlowCytomix assay (Bender Medsystems GmbH, Vienna, Austria), following the protocol provided by the manufacturer. Briefly, suspensions of anti-TGF-β, IL-1β or TNF-α-coated beads were incubated with the supernatant samples and then with biotin-conjugated secondary antibodies and streptavidin-phycoerythrin. Fluorescence was read with a cytofluorimeter (CyFlow® Space, Partec, Carate Brianza, MB, Italy).

The levels of the anti-inflammatory cytokine IL-10 were measured on aliquots (100 μl) of lung homogenate supernatants by using the mouse IL-10 ELISA Ready-SET&Go!® assay (eBioscience, San Diego, USA), following the protocol provided by the manufacturer. Values are indicated as means ± SEM of 10 individual mice from each group and expressed as pg/μg of total proteins determined over an albumin standard curve.

Immunofluorescence analysis was performed as previously described (Lucarini et al., 2014). Briefly, histological sections of 5 μm thick were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0, Bio-Optica, Milan, Italy) for antigen retrieval. A pre-incubation in 1.5% bovine serum albumin (BSA) in PBS, pH 7.4 for 20 min at RT was necessary to minimize the unspecific binding; whereupon, the sections were incubated overnight at 4°C with rabbit monoclonal anti-αSMA antibody (1:200 ABCAM, USA) followed by goat anti-rabbit Alexa Fluor 488-conjugated IgG (1:300 Invitrogen, San Diego, CA, USA) for 2 h in the dark at RT. Negative controls were performed with non-immune rabbit serum substituted for the primary antibody. The counterstaining of nuclei was obtained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images were acquired with an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus, Milan, Italy). αSMA expression was quantified by densitometric analysis of fluorescence signal intensity, measured on digitized images using ImageJ software². Twenty regions of interest (ROI) were evaluated for each sample. Values are expressed as mean ± SEM of the OD measurements (arbitrary units) of individual mouse from the different experimental groups.

Lung DNA isolation was performed as previously described (Lodovici et al., 2000) with minor modifications. In brief, lung samples were homogenized in 1 ml of 10 mM PBS, pH 7.4, sonicated on ice for 1 min, added to 1 ml of 10 mM Tris-HCl buffer, pH 8, containing 10 mM EDTA, 10 mM NaCl, and 0.5% SDS, and incubated for 1 h at 37°C with 20 μg/ml RNase 1 (Sigma-Aldrich). Samples were incubated at 37°C overnight in the presence of 100 μg/ml proteinase K (Sigma-Aldrich). The mixture was extracted with chloroform/isoamyl alcohol (10:2, v/v). DNA was precipitated from the aqueous phase with 0.2 volumes of 10 M ammonium acetate, solubilized in 200 μl of 20 mM acetate buffer, pH 5.3, and denatured at 90°C for 3 min. The extract was supplemented with 10 IU of P1 nuclease (Sigma-Aldrich) in 10 μl and incubated for 1 h at 37°C with 5 IU of alkaline phosphatase (Sigma-Aldrich) in 0.4 M phosphate buffer, pH 8.8. All the procedures were performed in the dark. The mixture was filtered by an Amicon Micropure-EZ filter (Merck-Millipore), and 100 μl of each sample were used for 8-OHdG determination by using an ELISA kit (JalCA, Shizuoka, Japan), following the instructions provided by the manufacturer. The absorbance of the chromogenic product was measured at 450 nm. The results were calculated from a standard curve based on an 8-OHdG solution and expressed as ng of 8-OHdG/ng of total DNA.

Frozen lung samples were weighed and homogenized (10 μl/mg of tissue) in 0.2 M phosphate buffer solution (PBS), pH 6, supplemented with protease inhibitors (1 mM PMSF, 20 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mg/ml Pefabloc SC, and 2.5 μg/ml aprotinin, Sigma-Aldrich) and were centrifuged at 10,000 g at 4°C for 30 min. MPO was measured in the supernatants with a specific immunoassay kit (CardioMPO; PrognostiX, Cleveland, OH), according to the manufacturer’s instructions (Pini et al., 2010). Total protein concentration in the lung tissue samples was determined over an albumin standard curve. The results are expressed as picomoles/mg of protein. Values are means ± SEM of individual mice from different experimental groups.

Data were reported as mean values (±SEM) of individual average measures of the different animals per group, for each assay. Significance of differences among the groups was evaluated by one-way ANOVA followed by Newman-Keuls post hoc test for multiple comparisons. Calculations were made with Prism 5 statistical software (GraphPad Software, Inc., USA). A probability value p < 0.05 was considered significant.

Intra-tracheal administration of bleomycin causes an increase in airway stiffness leading to a clear-cut elevation of the pressure in airway opening (PAO) (Masini et al., 2005). Intra-tracheal delivery of bleomycin to rodents results in an intense inflammatory reaction within the first week, followed by the development of fibrosis by day 14, with maximal responses at day 21. Therefore, bleomycin administration caused a significant increase in airway stiffness, as judged by the elevation of PAO in the fibrotic positive controls (Bleomycin+Vehicle) compared with the non-fibrotic negative ones (Naïve), both in WT and PARP-1^−/− mice (Figure 1).

After 21 days, the treatment with JNJ significantly reduces PAO both in WT and in PARP-1^−/− mice, in comparison to the relative group of animals treated with Vehicle; while VUF treatment seems to be not significantly effective in reducing the bleomycin-induced airway stiffness (Figure 1). The selected doses were based on previous papers (Pini et al., 2012; Lucarini et al., 2016). These results confirm our previous observation that H₄R ligands could have beneficial effects in decreasing lung fibrosis in WT (Lucarini et al., 2016) and in PARP-1^−/− animals, indicating that the treatment with H₄R antagonists exerts its beneficial effects also in situation of PARP-1 deletion.

It has been demonstrated that bleomycin administration increases PARP activity in mouse lung tissues (Genovese et al., 2005). Moreover, previous studies provided evidence that PARylated protein levels, the major products of PARP activity, are significantly increased in lung homogenates of WT mice treated with bleomycin, while the treatment with HYDAMTIQ, a potent PARP-1/2 inhibitor, dose-dependently, prevents this PARylation (Lucarini et al., 2017).

To evaluate the effects of intra-tracheal administration of bleomycin on PARP activity, we investigated the formation of polyADP ribose (PAR) polymers in mouse lung tissues in WT and PARP-1^−/− mice. As shown in Figure 2 and in Supplementary Figure 1, Western blot analysis with anti-PAR antibodies report an increase in PARylated protein content in lung homogenates of WT and in PARP-1^−/− mice treated with bleomycin (Vehicle), compared to non-fibrotic controls (Naїve). Treatment with the H₄R antagonist reduces the PARylated protein content in WT animals. Interestingly, our results indicate a reduction of PARylated protein levels in the lung tissues of PARP-1^−/− mice treated with JNJ, compared to those treated with VUF, suggesting a positive role of H₄R antagonism in suppressing catalytic activity of PARP enzymes different from PARP-1.

Acute inflammatory events and deposition of collagen fibers cause a pathological remodeling of the parenchyma of the lungs. Morphological observation and computer-aided densitometry on Azan-stained sections (Figure 3A) reveal a significant increase in collagen deposition in the lungs of all bleomycin-treated animals (Vehicle) compared to the non-fibrotic negative controls (Naïve). As already published, in WT animals, H₄R antagonists reduce the deposition of collagen fibers in lungs compared to Vehicle (Rosa et al., 2014; Lucarini et al., 2016). The treatment with JNJ causes an important and significant reduction of the amount of lung collagen fibers in PARP-1^−/− mice compared to Vehicle group. A slight effect is reported in VUF treated mice. To confirm the assessment of lung collagen, the Picrosirius red was used as second staining method; this specific staining for collagen (Lattouf et al., 2014; Marcos-Garcés et al., 2017) confirms the results obtained with Azan staining (Figure 3B).

We also evaluated the bronchial remodeling by measuring the number of goblet cells and the thickness of the smooth muscle layer, key histological parameters of inflammation-induced adverse airway remodeling (Bai and Knight, 2005). These two parameters are increased after intra-tracheal injection of bleomycin, both in WT and PARP-1 deficient mice. The treatment with JNJ significantly reduces the percentage of PAS-positive goblet cells over bronchial epithelial cells (Figure 4A), as well as the thickness of the airway smooth muscle layer (Figure 4B). On the contrary, VUF administration has no effect on both histological parameters, confirming our previous published results that on the effect of H₄R antagonists on goblet cells and thickness of the smooth muscle layer in lungs, (Rosa et al., 2014; Lucarini et al., 2016).

An increased TGF-β expression and an amplified TGF-β signaling through Smad pathways contribute to the establishment and development of pulmonary fibrosis (Leask and Abraham, 2004; Chen et al., 2013; Chitra et al., 2015). Our results clearly indicate that TGF-β, a major pro-fibrotic cytokine, is increased in the vehicle groups, both in WT and PARP-1^−/− mice. Systemic administration of JNJ causes a significant decrease of the levels of TGF-β in WT as well as PARP-1^−/− animals, while the treatment with the H₄R agonist has no effect on the production of this cytokine (Figure 5A). These results demonstrate the anti-fibrotic activity of JNJ both in WT and in PARP-1^−/− mice.

Transforming growth factor-β signaling has been reported to regulate the expression of αSMA, a marker of fibroblast activation, and myofibroblasts differentiation (Martin et al., 2007; Conte et al., 2014). To investigate the expression of αSMA during the fibrotic process, we performed immunofluorescence analysis in mouse lung tissues. Our results show a significant increase of αSMA levels in bleomycin-exposed PARP-1^−/− animals (Figures 5B,C). Treatment with JNJ significantly reduces αSMA levels in lung tissue of PARP-1^−/− mice compared to the vehicle group. Systemic administration of the H₄R agonist provides no changes in αSMA lung levels. These results indicate that the use of an H₄R antagonist, in the absence of PARP-1 enzyme, reduces the activation of fibroblasts and myofibroblasts differentiation and consequently the development of progressive fibrotic disease in bleomycin-exposed animals.

Chronic inflammation is determined by pathological wound-healing response, resulting in the accumulation of permanent scar tissue at the site of injury and consequently in the development of progressive fibrotic disease (Wynn and Ramalingam, 2012). Based on this evidence, we evaluated the late (21 days after challenge) inflammatory response to bleomycin by measuring the pro-inflammatory cytokines IL-1β and TNF-α, in lung homogenates of WT and PARP-1^−/− mice. Bleomycin treatment increases the levels of IL-1β and TNF-α both in WT and PARP-1^−/− mice; of note, in PARP-1^−/− mice, this increase is less pronounced in comparison to WT mice. The systemic administration of JNJ significantly reduces the increase in both groups of animals, but in PARP-1^−/− mice, this reduction is more evident. The treatment with VUF is not effective in the reduction of inflammatory cytokine production (Figures 6A,B).

In order to confirm the anti-inflammatory activity of the H₄R antagonist, we evaluated the levels of interleukin-10 (IL-10), the most potent anti-inflammatory cytokine involved in resolution of different acute and chronic inflammatory diseases (Ajuebor et al., 1999). Our results report a significant increase in the production of IL-10 in WT and PARP-1^−/− mice treated with JNJ in comparison to PARP-1^−/− mice treated with vehicle or VUF (Figure 6C).

Our findings support the hypothesis that H₄R antagonism exerts anti-inflammatory and anti-fibrotic effects in a model of bleomycin-induced lung fibrosis.

Lung MPO is a peroxidase enzyme abundantly expressed in neutrophils and monocytes/macrophages granules, and it is considered a reliable marker for leukocyte accumulation in inflamed tissues (Mullane et al., 1985). Levels of MPO were very low in the Naïve groups of both WT and PARP^−/− mice; MPO levels increased significantly in the bleomycin-treated mice (Vehicle groups of WT and PARP^−/− mice). A significant decrease in MPO was demonstrated after treatment with JNJ in WT and PARP^−/− groups of animals in comparison to Vehicle; treatment with VUF has some non-significant effects (Figure 7A).

The determination of 8-OHdG, a biological marker of DNA damage under oxidative stress, demonstrates that it is significantly increased in bleomycin-exposed animals (Vehicle), compared with non-fibrotic negative ones (Naïve). Interestingly, our results show a significant reduction of 8-OHdG levels in WT and PARP-1^−/− animals treated with JNJ (Figure 7B), but not with VUF.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.