Protective role of cortistatin in pulmonary inflammation and fibrosis

Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and pulmonary fibrosis remain major causes of morbidity, mortality and a healthcare burden in critically ill patient. There is an urgent need to identify factors causing susceptibility and for the design of new therapeutic agents. Here, we evaluate the effectiveness of the immunomodulatory neuropeptide cortistatin to regulate pulmonary inflammation and fibrosis in vivo.


| INTRODUCTION
Despite major treatment efforts made over the past decades, acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), characterized by refractory hypoxia, severe inflammation, increased vascular permeability and diffuse alveolar damage, remains a major cause of morbidity and mortality in critically ill patients (Matthay et al., 2017). ARDS can occur as a result of different clinical conditions, such as infections, pulmonary contusion and inhalation injury, that directly damage the pulmonary epithelial and endothelial cells and compromise alveolar-capillary barrier. ARDS is caused and sustained by an uncontrolled inflammatory activation characterized by massive release of cytokines and chemokines, diffuse lung oedema, inflammatory cell infiltration and disseminated coagulation.
In this sense, evidence indicates that the massive pulmonary infiltration (neutrophils and macrophages) and the subsequent inflammatory cytokine storm are closely related to secondary complications such as lung injury/ARDS, multiorgan failure and ultimately poor prognosis in the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic (Mehta et al., 2020;Zhou et al., 2020).
Moreover, in patients who develop ARDS, the progression of ALI to pulmonary fibrosis portends a fatal outcome, with severe disruption of lung function and elevated mortality . As in other cases of pulmonary fibrosis which are caused by persistent infection, oxidative stress and inflammatory insults, the injury to alveolar epithelial cells activates pulmonary fibroblasts, promoting their transformation to extracellular matrix-producing myofibroblasts (Wynn, 2011). These findings highlight the urgent need to develop safe and effective therapeutic agents with capacity to limit both inflammatory and fibrotic responses in injured lung. Moreover, due to the heterogeneous progression and severity of disease in patients with ALI/ARDS, it is critical to identify factors and genes that predispose/protect from the development of the most severe forms of lung injury and progressive lung fibrosis.  is a cyclic neuropeptide belonging to the somatostatin family that has emerged as a potent immunomodulatory agent (Gonzalez-Rey & Delgado, 2008) with capacity to protect against exacerbated inflammatory and autoimmune responses in various experimental models of sepsis, rheumatoid arthritis, colitis, myocarditis and multiple sclerosis (Delgado-Maroto et al., 2017;Gonzalez-Rey, Chorny, Del Moral, et al., 2007;Gonzalez-Rey, Varela, Sheibanie, et al., 2006;Souza-Moreira et al., 2013). These effects are exerted through the regulation of a plethora of inflammatory cytokines and chemokines, and by deactivating macrophages and lymphocytes, indicating that it is a multitargeted and safe modulator of the cytokine storm in various tissues. However, the endogenous role of cortistatin in the modulation of immune response has been scarcely investigated, with some paradoxical effects found in cortistatin-deficient animals (Qiu et al., 2020;Souza-Moreira et al., 2013). Moreover, its role in ALI and fibrotic disorders is completely unknown, although some data point out to a potential antifibrotic action of this neuropeptide. Thus, cortistatin receptors (somatostatin (SST) 5 receptors and ghrelin receptor GHSR) are expressed in fibroblasts, and other SST receptor agonists have been described that exert antifibrotic responses in various tissues, including lung (Borie et al., 2008;Egger et al., 2014;Tug et al., 2013). Therefore, cortistatin could converge immunomodulatory and antifibrotic properties that synergistically might contribute to ameliorate inflammatory and fibroproliferative disorders in the lung. In this study, we will evaluate the therapeutic potential of cortistatin in two well-established experimental models of ALI and pulmonary fibrosis, as well as the immune and antifibrotic mechanisms involved.
We will also investigate the role of cortistatin as a potential endogenous protective factor in the progression to severe ALI and pulmonary fibrosis in mice that are partially or totally deficient in this neuropeptide.

What is already known
• Acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis remain major causes of morbidity/mortality worldwide.
• Cortistatin is an immunomodulatory neuropeptide with potential to regulate inflammation and fibrosis in several tissues.

What does this study add
• Treatment with cortistatin ameliorates inflammation and fibrosis progression in two preclinical models of lung injury.
• Cortistatin acts as a potent endogenous inhibitor of pulmonary inflammation and fibrosis.
Both male and female mice (20-to 24-g body weight, 8-10 weeks old) were used in all experiments described in this study, and no differences were found between sexes. All animals were housed in a controlled-temperature/humidity environment (22 ± 1 C, 60-70% relative humidity) in individual cages (10 mice per cage, with wood shaving bedding and nesting material), with a 12-h light/dark cycle (lights on at 7:00 AM) and fed with rodent chow (Global Diet 2018, Harlan) and tap water ad libitum. Mice were allowed to acclimatize to the experimental room for 1 h before experiments. Mice were randomly assigned to the different experimental groups. Experiments were designed to make sample sizes relatively equal. However, this was not possible in some experiments due to the differential mortality rates occurring between genotypes and response to bleomycin. None of the animals were excluded from the study. Power calculations were performed using the software G*Power (www.gpower.hhu.de, RRID: SCR_013726) to ensure that adequate group sizes were used for the studies detailed below. For in vivo animal models, we calculated a minimum size of five to eight mice per group in order to have a power >0.95 of detecting approximately a 30% change, assuming an SD of 30% at a significance level of P < 0.05, expecting an effect size of 1.82 for ANOVA tests. In primary cell cultures, for effect sizes between 3.1 and 4, experiments were repeated at least four times to obtain P < 0.05 and a power >0.95.

| Materials
Unless otherwise indicated, all purchased reagents used in this study were from Sigma-Aldrich (St. Louis, MO, USA). Bleomycin sulphate (with specific activity of 1.6-2.0 UÁmg À1 , from Sigma-Aldrich, cat#B-8616) was dissolved in saline solution (0.9% NaCl) at 1 mgÁml À1 (1.8 UÁml À1 ) and stored at À20 C and was diluted in saline at the indicated doses immediately before its injection in animals. Mouse Bubendorf,Switzerland, was dissolved in ddH 2 O and stored at À80 C at a dose of 0.1 mM and was diluted in ddH 2 O (used as vehicle) at the indicated dose and volume immediately before its use in the experimental models. LPS (from Escherichia coli serotype 055:B5, Sigma-Aldrich, cat#L-2880) was dissolved in saline at 2 mgÁml À1 and stored at À20 C until its use.

| Induction of acute lung injury (ALI)
To investigate the effect of cortistatin deficiency in the severity of ALI, Cort+/+, Cort+/À and CortÀ/À mice were infused intranasally

| Induction of experimental lung fibrosis
To investigate the effect of cortistatin deficiency in severity of lung fibrosis, bleomycin was administered intratracheally (at 1.8-UÁkg À1 mouse, in 50-μl volume) to anaesthetized (i.p., ketamine 80 mg, xylazine 10-mgÁkg À1 mouse) Cort+/+, Cort +/À and CortÀ/À mice. When indicated, other doses of bleomycin (from 1.2-to 5-UÁkg À1 mouse) were assayed (see Figure 5). To investigate the therapeutic effect of cortistatin in lung fibrosis, Cort +/+ mice were injected intratracheally with bleomycin (3.2-UÁkg À1 mouse, in 50-μl volume) and treated three times per week with vehicle or cortistatin via a local intranasal pathway (at 50-pmol cortistatin per mouse, in 20-μl volume) or a systemic intraperitoneally pathway (at 1 nmol cortistatin per mouse, in 200-μl volume), starting immediately (protective acute regime) or 5 days (therapeutic regime) after bleomycin injection. Mice injected intratracheally with saline, instead of bleomycin, were used as basal controls of reference. When indicated, cyclosomatostatin and [D-Lys 3 ]-GHRP-6 were infused i.n. (10 μg per mouse, in 20-μl volume onto the nares, approximately 500-μgÁkg À1 mouse) 30 min before every cortistatin injection. Survival and body weight were daily monitored for 3 weeks. On different times after bleomycin injection, animals were killed by carbon dioxide and BALFs and lungs were isolated and analysed for leukocyte infiltration, cytokine contents, vascular permeability, histopathological signs and fibrotic markers (collagen content and gene expression) as described below.
Images were acquired in an Axio Scope.A1 microscope (Carl Zeiss, Germany) using 5Â and 10Â objectives and 10Â ocular and analysed with Zen 2011 Light Edition software (Carl Zeiss). All histopathological analysis and determinations were performed in a blinded manner by at least two independent researchers in whole-lung sections.
ALI-induced histopathology was scored in H&E-stained lung sections determining the extent of inflammatory cell infiltration on alveolar walls, alveolar haemorrhage and alveolar septum congestion, using a semiquantitative scale from 0 (normal and no focal inflammatory infiltrates) to 4 (severe infiltration and damage in lung structure).
Bleomycin-induced pulmonary fibrosis was scored in Masson's trichrome-stained lung sections according to a semiquantitative scale (0 to 4) evaluating alveolar thickness, damage of lung structure and fibrosis extension (Ashcroft et al., 1988):-0, normal lung or minimal fibrous thickening of alveolar or bronchial walls, 1, moderate thickening of the wall, with less than 25% of fibrotic area, but without obvious damage to lung architecture, 2, formation of fibrous bands, fibrous masses in 25-50% of lung area, and definitive damage of lung structure, 3, severe distortion of the structure and large fibrous areas (>50% of a cross-section involved) and 4, total fibrous obliteration of the field. Results show the mean value of at least three nonoverlapping randomly selected areas per lung section (with 5Â objective) and three representative sections per mouse (discarding at least 200 μm between sections).

| Bronchoalveolar lavage fluid (BALF) collection and analysis
A cannula (21G) was inserted into the trachea, and ice-cold PBS/100-mM EDTA (0.8 ml) was instilled twice into the lung. BALF was harvested and centrifuged (400 g, 8 min, 4 C). Cell pellets were resuspended in PBS and used to determine total cell numbers using a standard haemocytometer and to analyse the percentage of neutrophils, macrophages and T lymphocytes in BALF by flow cytometry as described below (Tager et al., 2008). Alternatively, cell populations were examined by counting at least 200 cells on Wright-Giemsa-stained BALF CytoSpin preparations. The supernatants collected from BALFs were used to determine the levels of cytokines and chemokines using sandwich ELISAs (see above for specific capture and biotinylated antibodies) following the manufacturer's recommendations, to measure total protein content using a BCA Protein Assay Kit (Pierce/Thermo Fisher) and to determine the levels of mouse albumin using an ELISA kit (Abcam, Cambridge, UK, cat#ab207620).

| Measurement of pulmonary myeloperoxidase activity
Oxidative stress and neutrophil infiltration in the lung was also monitored by measuring myeloperoxidase activity by using a method reported previously (Gonzalez-Rey, . In brief, left lung lobules were homogenized at 50 mgÁml À1 in phosphate buffer (50 mM, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide. Samples were frozen, thawed three times and centrifuged (30,000 g, 20 min). The supernatants were diluted at 1:30 with assay buffer consisting in 50-mM phosphate buffer pH 6.0 with 167-μgÁml À1 o-dianisidine and 0.0005% H 2 O 2 , and the colorimetric reaction was measured at 450 nm between 1 and 3 min (ΔA 450 ) in a spectrophotometer (VersaMax Microplate Reader, from Molecular Devices, San Jose, CA, USA). Myeloperoxidase activity per gram of wet lung was calculated as follows: 13.5 Â ΔA 450 ∕ lung weight. The coefficient 13.5 was empirically determined such that 1-U myeloperoxidase activity is the amount of enzyme that will reduce 1 μmol H 2 O 2 min À1 .

| Measurement of pulmonary vascular permeability
Pulmonary vascular permeability was quantified by measuring albumin and protein contents in BALFs (see above) and using the Evans blue dye extravasation assay (Tager et al., 2008). Briefly, Evans blue dye (20 mgÁkg À1 ) was injected through mouse tail vain 3 h before kill. At the time of killing, blood was collected into a heparinized syringe by cardiac puncture and mice were then perfused with PBS through the right ventricle to remove intravascular dye from the lungs. Lungs were dissected and homogenized, and Evans blue dye was extracted by the addition of 2 vol of formamide followed by incubation overnight at 60 C. After centrifugation (5000 g, 30 min), the absorption of Evans blue in lung supernatants and plasma was measured at 620 nm and corrected for the presence of haem pigments as follows: A 620 À (1.426 Â A 740 + 0.030). We calculated an Evans blue index as the ratio of the amount of dye in the lungs to the plasma dye concentration. Pulmonary vascular permeability was also monitored through the ratio between lung weight measured immediately after its excision (wet weight) and lung weight after 5 days in an oven at 60 C (dry weight).
Data were acquired until at least 20,000 events were collected from a live gate using forward/side scatter plots and 7-aminoactinomycin D staining. Percentages of CD64+ macrophages, CD3+ T lymphocytes and Ly6G+ neutrophils were analysed in a gated CD45+ cell population using FlowJo v9 software (RRID:SCR_008520) and the differential number of each cell subpopulation in BALF was calculated by multiplying this percentage by the total number of collected BALF cells.

| Measurement of collagen content in tissues
The collagen content in lungs of mice was measured using the hydroxyproline assay (Reddy & Enwemeka, 1996). Briefly, right lung lobes were hydrolysed in 6-N HCl (approximately 100 mg tissue ml À1 ) at 95 C for 20 h and shaking. After centrifugation (13,000 g, 15 min, 20 C), supernatants were diluted to reach a final concentration of 4-N HCl, transferred to 96-well plates and oxidized with 1.2% chloramine-T/10% propanol in citrate acetate buffer pH 6.5 (720-mM sodium acetate, 1% acetic acid, 200-mM citric acid and 680-mM sodium hydroxide) for 25 min at 20 C and shacking. Ehrlich's reagent (1 vol, 15% p-dimethylaminobenzaldehyde in propanol/perchloric acid 2:1, vol:vol) was added to wells and incubated at 60 C during 1 h. Absorbance at 550 nm was measured in a spectrophotometer and extrapolated to a standard hydroxyproline curve. Collagen content was calculated by multiplying the hydroxyproline measurements by 7.40 (a coefficient according to the fact that hydroxyproline represents 13.5% of amino acids in collagen sequence) and then expressed in μg relative to the weight of tissue.

| Isolation and culture of primary fibroblasts
Lung lobules were collected from Cort+/+ and Cort+/À mice (10 weeks old) and mechanically dissected in small pieces using sterile scalpels. Tissue fragments were digested in DMEM/F12 medium (Gibco) supplemented with 100-UÁml À1 penicillin/streptomycin, 2-mM L-glutamine and 140-UÁL À1 Liberase (Thermolysin Low, from Roche, Basel, Switzerland) at 37 C with shaking. After 60 min, digested tissues were centrifuged (525 g, 5 min, 20 C) and cell pellets were washed three times with complete DMEM/F12 medium (supplemented with 15% FBS, 100-UÁml À1 penicillin/streptomycin and 2-mM L-glutamine) and then cultured in complete DMEM/F12 medium in 75-cm 2 Nunc flasks (Nunc/Thermo Fisher), at 37 C, 5% CO 2 . After 3-7 days of culture, medium was replaced by MEMα complete medium (MEMα supplemented with 15% FBS, 100-UÁml À1 penicillin/streptomycin and 2-mM L-glutamine, all from Gibco) and adhered fibroblasts were cultured until 80% confluence, harvested by adding trypsin-EDTA solution (Sigma-Aldrich, cat#T4049) and maintained at 5 Â 10 5 cells per flask (in 175-cm 2 Nunc flasks) at 37 C, 5% CO 2 until their use. To evaluate gene expression by realtime qPCR, 4 Â 10 4 fibroblasts were cultured in six-well Nunc plates, cultured until 80% confluence, synchronized to G 0 phase by incubation in free-FBS MEMα (overnight at 37 C, 5% CO 2 ) and then cultured in complete MEMα in the absence or presence of TGF-β1 (10 ngÁml À1 , PeproTech) for 24 h. When indicated, cortistatin was added at 100 nM to cultures simultaneously with TGF-β1 stimulation. To evaluate Smad2/3 nuclear translocation by immunofluorescence analysis, 10 3 fibroblasts were cultured until 80% confluence in glass coverslips, which were inserted in 24-well Nunc plates, Migration of fibroblasts was determined using an in vitro wound healing assay. In brief, 10 3 fibroblasts were seeded in Culture-Inserts 2 Well in μ-Dish 35 mm (Ibidi, Gräfelfing, Germany) and then cultured to confluence. After cell synchronization, the inserts were removed and complete MEMα medium was added. At different time points, wells were observed in an Olympus microscope and images were acquired at 100Â magnification under phase-contrast mode. The percentage of unhealed wound area was quantified using Fiji ImageJ software and the MRI Wound Healing Tool (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/ Wound_Healing_Tool).

| Determination of gene expression by realtime PCR
Total RNA was isolated from lung lobes by tissue homogenization

| Data and statistical analysis
All experiments are randomized and blinded. All data are expressed as mean ± SD, unless when specified (i.e. Figure 6). To control for In accordance with journal policy, statistical analysis was performed only when a minimum of n = 5 independent samples was acquired.
We analysed data for statistical differences between groups using the   Figure 2d) in BALFs of Cort+/À and CortÀ/À mice. Moreover, pulmonary macrophages isolated from Cort+/À and CortÀ/À mice with ALI significantly produced more inflammatory TNF-α than macrophages isolated from BALF of Cort+/+ mice (Figure 2e). Importantly, exogenous administration of cortistatin to Cort+/À mice reversed the severe ALI phenotype that we observed in these animals ( Figure 3). Moreover, in vitro treatment with cortistatin impaired the enhanced TNF-α production by BALF macrophages isolated from Cort +/À and CortÀ/À mice with ALI ( Figure 2e). These findings indicate that a partial deficiency of cortistatin predisposes to develop exacerbated acute pulmonary inflammatory responses and severe lung damage after exposition to bacterial endotoxins.

| Deficiency in cortistatin exacerbates pulmonary fibrosis in bleomycin-challenged mice
We further investigated the role played by cortistatin in a wellcharacterized model of pulmonary fibrosis induced by intratracheal injection of bleomycin, which shares significant similarities with human idiopathic pulmonary fibrosis (IPF) and has been widely used for studying pulmonary fibrogenesis and evaluating the effect of therapeutic antifibrotic strategies (Kolb et al., 2020). In this model, as occurred with exposition to infection, toxins or radiation, the antineoplastic drug bleomycin causes alveolar epithelial injury that induces the release of profibrotic cytokines/growth factors (i.e. TGF-β1, TNFα and CTGF), which activate lung fibroblasts and their subsequent transformation into αSMA-expressing myofibroblasts that are responsible of the excessive extracellular matrix protein deposition that characterizes the fibrotic lung. We first found that mice lacking cortistatin showed significantly earlier and higher mortality after bleomycin challenge, relative to wild-type animals ( Figure 4). Significant was the fact that bleomycin at doses as low as 1.2-1.8 UÁkg À1 , which did not compromise survival of Cort+/+ mice, increased the mortality rate above 50% in Cort+/À and CortÀ/À mice. In order to investigate the clinical markers and mechanisms involved in this susceptibility to bleomycin, we further used a dose of 1.8 UÁkg À1 . Exposure of Cort+/À and CortÀ/À mice to this dose of bleomycin resulted in a significant loss of body weight (up to 20%) and death rates ranging from 75% to 80% ( Figure 5a). Histopathological examination of Masson's trichromestained pulmonary sections showed that, whereas lungs of bleomycinchallenged Cort+/+ mice had moderate thickening of the alveolar walls, less than 25% of fibrotic area and no obvious damage in lung architecture, mice that are partially or fully deficient in cortistatin had lungs with large fibrous areas (above 60%), in many cases with total fibrous obliteration of the field, showing severe distortion of pulmonary structure (Figure 5b). We observed this increased fibrosis score in cortistatin-deficient mice as early as 7 days after bleomycin instillation, coinciding with the drop in survival (Figure 5a,b). Moreover, the exacerbated bleomycin-induced lung fibrosis observed in Cort+/À and CortÀ/À mice was accompanied by an excessive early leukocyte infiltration (mainly composed by neutrophils and macrophages) and

| Cortistatin-deficient fibroblasts show increased fibrogenic responses
To investigate whether endogenous cortistatin directly regulates fibrosis, independently of its immunoregulatory effects, we evaluated the fibrogenic responses of primary pulmonary fibroblasts isolated from wild-type and cortistatin-deficient mice. Because in vivo experiments demonstrated that partially deficient and totally deficient mice for cortistatin showed similar exacerbated pulmonary fibrosis in response to bleomycin, and as individuals with partial deficiency in this neuropeptide will be more frequent within the human population than those fully deficient, we focused in vitro experiments on   (c) The number of total cells, macrophages, neutrophils and T lymphocytes in bronchoalveolar lavage fluids (BALFs) (n = 8 mice per group), myeloperoxidase (MPO) activity in lung extracts (n = 6 mice per group) and BALF cytokine contents (n = 6 mice per group) were determined at the indicated times. (d) Lung oedema was determined by measuring protein contents in BALFs (n = 8 mice per group) and Evans blue extravasation index in lungs (n = 5 mice per group) isolated at the indicated times. (e) Markers of lung fibrosis were determined at the indicated times by measuring collagen contents and TGF-β1 levels in lung protein extracts (n = 6 mice per group), CTGF mRNA expression in lungs (n = 12 mice per group) and αSMA-positive immunofluorescence in lung sections (n = 5-6 mice per group, scale bar: 100 μm). Dashed horizontal lines correspond to values obtained from naïve mice (n = 6). Results are the mean ± SD with dots representing individual values of biologically independent animals. *P < 0.05 versus Cort +/+ mice Thus, the activation and subsequent nuclear translocation of Smad2/3 was significantly increased in Cort+/À fibroblasts (Figures 6c and   S1A). Moreover, the basal levels of activated phosphorylated forms of various protein kinases, including Akt, p38 MAPK and ERK1/2, were markedly increased in cortistatin-deficient fibroblasts, showing similar or even higher activation levels than those showed by TGF-β1-activated Cort+/+ fibroblasts (Figures 6c and S1B). Furthermore, in comparison with wild-type fibroblasts, cortistatin-deficient pulmonary fibroblasts showed accelerated and increased migratory responses in a wound healing assay (Figure 6d). However, lack of cortistatin did affect neither fibroblast growth nor viability ( Figure S2). We finally assessed its antifibrotic effect by adding cortistatin to cultures of TGF-β1-activated Cort+/+ and Cort+/À fibroblasts. As expected, treatment with cortistatin significantly reduced the expression of profibrotic markers and extracellular matrix components, including αSMA, CTGF, Col1a2 and fibronectin, in activated lung fibroblasts ( Figure 6e). Therefore, these findings indicate that cortistatin could act as an endogenous break of activation, migration and differentiation of fibroblasts.

| Treatment with cortistatin ameliorates bleomycin-induced pulmonary inflammation and fibrosis
Our previous results indicate that cortistatin has a critical role in the regulation of pulmonary inflammation and fibrosis and that administration of cortistatin is a potential strategy for the prevention and F I G U R E 6 Cortistatin (CST)-deficient fibroblasts show exacerbated profibrotic responses. (a) Gene expression of cortistatin by unstimulated and TGF-β1-activated mouse primary lung fibroblasts (n = 3 cultures) and by lungs isolated from naïve or bleomycin (Bleo)-treated mice (n = 5 mice per group). (b-d) Primary lung fibroblasts were isolated from wild-type (Cort+/+) and partially deficient (Cort+/À) mice for cortistatin were cultured in the absence (À) or presence (+) of TGF-β1 (10 ngÁml À1 ) stimulation. (b) αSMA and CTGF gene expression was determined after 24-h culture (13 unstimulated cultures and nine stimulated cultures). (c) Nuclear translocation of activated Smad2/3 was determined by immunofluorescence analysis and the levels of activated phosphorylated Akt, p38 MAPK and ERK1/2 were analysed by western blot after 1 h of culture (n = 4-5 independent experiments, in triplicates). See Figure S1 for representative immunofluorescence images and western blots. (d) Fibroblast migration activity was measured at different time points using an in vitro wound healing assay (n = 6 independent cultures). (e) Fibroblasts isolated from Cort+/+ and Cort+/À lungs were stimulated with TGF-β1 in the absence (À) or presence (+) of cortistatin, and the gene expression of αSMA, CTGF, collagen Iα2 (Col1a2) and fibronectin was determined after 24-h culture (n = 5-6 independent cultures). Results are the mean ± SEM with dots representing individual values of independent experiments. Data in (b), (c) and (e) are expressed as fold change relative to the mean of unstimulated Cort+/+ fibroblasts. *P < 0.05 treatment of lung injury-induced fibrosis. The systemic injection of cortistatin at the early stage significantly prevented the profound body weight loss, high mortality and severe pulmonary fibrosis that were induced by the administration of high-dose bleomycin ( Figure 7a). These protective effects correlated with inhibition of pulmonary inflammation, injury and vascular leak (Figure 7a). Importantly, cortistatin also therapeutically attenuated bleomycin-induced mortality and the severity of pulmonary fibrosis at the later stages following bleomycin instillation (Figure 7b). Indeed, treatment of animals with cortistatin beginning 5 days after bleomycin challenge, once that pulmonary inflammation was fully established, significantly improved fibrosis score, reduced collagen deposition and decreased the presence of αSMA-expressing myofibroblasts in lung (Figure 7b). We found similar therapeutic efficiencies using both systemic (i.p.) and local (i.n., at 20-fold lower doses) routes of administration (Figure 7b).
Noteworthy from a therapeutic point of view is the fact that cortistatin treatment was able to reverse the susceptibility to suffer severe pulmonary fibrosis in mice that had a partial deficiency in cortistatin after their exposition to low doses of bleomycin ( Figure 8).
These results suggest that cortistatin-based therapies could impair pulmonary inflammation and attenuate the established pulmonary fibrosis.

| Involvement of somatostatin and ghrelin receptors in the therapeutic effect of cortistatin in pulmonary inflammation and fibrosis
We finally investigated the receptors through which cortistatin could exert its anti-inflammatory and antifibrotic activities in vivo. Previous evidence demonstrates the capacity of cortistatin to bind, between others, to SST receptors and ghrelin receptor , supports that both receptors play major roles in the immunomodulatory effect of cortistatin in several organs (Gonzalez-Rey & Delgado, 2008) and indicates that signalling through SST receptors and ghrelin receptor exerts antifibrotic actions other tissues (Borie et al., 2008;Egger et al., 2014;Tug et al., 2013).

| DISCUSSION
Inflammation and wound healing are two physiological processes aimed at restoring normal tissue structure and function after an insult or injury. However, they can be more damaging than the insult itself if uncontrolled, excessive or prolonged. In the lung, a dysregulated inflammation causes excessive leukocyte accumulation and increased permeability of endothelial and alveolar epithelial barriers. A wound healing response that has gone out of control after lung injury causes pulmonary fibrosis, which is characterized by progressive loss of alveolar structure, disruption of the epithelial-endothelial barrier, activation of fibroblasts and their differentiation to myofibroblasts, excessive deposition of extracellular matrix and tissue remodelling.
Far of restoring host pulmonary homeostasis, aberrant inflammatory and fibrotic responses, in some cases being part of the same cascade, contribute to the pathogenesis of severe lung disorders, such as ALI/ARDS and IPF. A precise balance of inflammatory/fibrogenic versus immunomodulatory/antifibrotic factors must exist to tune adequately these responses. The identification of the factors that limit or reverse both processes is critical for understanding the pathophysiology and identifying new therapeutic targets for these disorders. In this study, by using two well-characterized experimental models of ARDS and pulmonary fibrosis, we point out to cortistatin as an endogenous protective factor. We found that a deficiency in cortistatin predisposes for developing exacerbated inflammatory and fibrotic responses in injured lungs after exposition to bacterial endotoxins or chemotherapeutic drugs, even at low doses, and to subsequently suffer more severe disease progression and increased mortality. Moreover, our data show that a treatment with cortistatin is able to mitigate these pathological processes.
We envision the involvement of various non-excluding and complementary cellular and molecular mechanisms that could explain the protective effect of cortistatin in pulmonary inflammation and fibrosis ( Figure 10). First, previous reports demonstrated the antiinflammatory activity of cortistatin on macrophages and described its protective effect in murine models of sepsis and endotoxemia, acting mainly by regulating a wide range of inflammatory mediators (Gonzalez-Rey, Gonzalez-Rey, Varela, Sheibanie, et al., 2006). Here, we have confirmed that in the lung cortistatin down-regulates the production of various inflammatory cytokines and chemokines by activated BALF macrophages. Further, and importantly, we found that infiltrating pulmonary macrophages, that are deficient in cortistatin produced excessive levels of cytokines that are responsible for the pathophysiology of ALI/ARDS. This effect could be initiated in an autocrine/paracrine manner, as macrophages express both cortistatin (Dalm et al., 2003;Gonzalez-Rey, Varela, Sheibanie, et al., 2006;Markovics et al., 2012), as well as both somatostatin and ghrelin receptors, which are involved in the antiinflammatory activity of cortistatin .
Indeed, we found that blockade of any of these receptors, mainly the ghrelin receptor, impaired the protective action of cortistatin in ALI. We found that pulmonary fibroblasts isolated from cortistatindeficient mice showed overactivated TGF-β1-signalling pathways, including Smad2/3, Akt and MAPKs (p38 and ERK1/2), that drive the expression of genes (for example, collagen, CTGF and αSMA) that are involved in pathological fibrosis (Wynn, 2011). Again, this effect could be mediated in a paracrine fashion, because fibroblasts express both cortistatin and its receptors (Borie et al., 2008;Egger et al., 2014;this study;Tug et al., 2013). In this respect, we observed Despite the enormous progression made in the identification of the pathogenic mechanisms involved in the initiation and progression of these diseases, they are an enduring problem in respiratory and critical medicine that remains therapeutically unresolved, as they remain a major cause of morbidity and mortality worldwide (Matthay et al., 2017). This urgent need has acquired a global dimension lately during the pandemic COVID-19, with the association of severe ARDS F I G U R E 8 Exogenous administration of cortistatin (CST) reversed the exacerbated fibrogenic phenotype observed in cortistatin-deficient mice. Lung fibrosis was induced in partially deficient mice (Cort+/À) for cortistatin and treated with vehicle or cortistatin as indicated in the scheme. Bleomycin (Bleo)-challenged Cort+/+ mice were used as controls of reference. Mortality, histopathological signs of fibrosis (5-11 mice per group), pulmonary collagen content (7-8 mice per group) and the presence of αSMA-positive myofibroblasts (five mice per group) were determined in lungs isolated at the indicated time points. Scale bars: 100 μm. Results are the mean ± SD with dots representing individual values of biologically independent animals. *P < 0.05 and pulmonary fibrosis which has a poor prognosis in SARS-CoV-2-infected patients (Mehta et al., 2020). Due to the redundancy and complexity of the cytokine and fibrogenic network, the multitargeted action of cortistatin as an immunomodulatory agent on a plethora of mediators of the cytokine storm offers obvious advantages versus other therapies based on neutralization of a single F I G U R E 9 Involvement of somatostatin receptors and ghrelin receptor in the therapeutic effect of cortistatin (CST) in acute lung injury (ALI) and pulmonary fibrosis. (a) ALI was induced in partially deficient mice for cortistatin (Cort+/À) by intranasal injection of LPS and then treated intraperitoneally with vehicle or cortistatin. Saline, a SST 1-5 antagonist (cyclosomatostatin, cycloSOM), or a ghrelin receptor (GHSR) antagonist ([D-Lys 3 ]-GHRP-6) was injected intranasally (i.n.) 30 min before cortistatin injection as indicated in the scheme. Leukocyte infiltration, and protein and cytokine contents were determined in bronchoalveolar lavage fluids (BALFs) isolated 24 h after LPS-induced ALI (n = 7 mice per group). (b) Lung fibrosis was induced in Cort+/À mice with bleomycin and then treated with vehicle or cortistatin as indicated in the scheme. Saline, cycloSOM or [D-Lys 3 ]-GHRP-6 was injected i.n. 30 min before cortistatin injection. Histopathological signs of fibrosis and the contents of collagen were determined in lungs collected 10 days after bleomycin challenge (5-8 mice per group). Results are the mean ± SD with dots representing individual values of biologically independent animals. *P < 0.05 F I G U R E 1 0 Scheme illustrating the cellular and molecular mechanisms involved in the protective effect of cortistatin on pulmonary inflammation and fibrosis. (a) Structure of a healthy alveolus showing a cleared alveolar space and intact and thin epithelial-endothelial barrier and surfactant layer that allow normal gas exchange. (b) Bacterial infection (i.e. LPS), pneumonia or tissue damage by chemicals (i.e. bleomycin (Bleo)) may cause acute lung injury (ALI), which is characterized by neutrophil recruitment to the lung, with both alveolar (by resident macrophages) and systemic release of inflammatory cytokines (TNF-α, IL-1 and IL-6) and chemokines (CXCL2/MIP-2). Exaggerated alveolar inflammation and oxidative stress induce apoptosis/necrosis of epithelial and endothelial cells and damage the alveolar-capillary barrier, leading to the development of pulmonary oedema and hypoxemia. Moreover, the release of inflammatory cytokines and growth factors (TNF-α and TGF-β1) activates pulmonary fibroblasts and induces secretion/deposition of extracellular matrix components (i.e. collagen and fibronectin), generating an interstitial fibrotic scar that contributes to the impairment of gas exchange. (c) The subsequent course of ARDS is aggravated in the absence of cortistatin. In cortistatin-deficient mice, even after the exposition to low doses of injury-inducing agents, the exacerbated and persistent pulmonary inflammation and the progression to intra-alveolar fibrosis/scarring by hyperactivated fibroblasts and αSMA + -myofibroblasts avoid the reabsorption of alveolar oedema fluid and repair of the injured alveolar epithelium, do not allow recovery from respiratory failure and in some cases causes the death. (d) Cortistatin, which is produced endogenously in the lung by alveolar macrophages (Mɸ) and fibroblasts or provided exogenously from other tissues (i.e. infiltrating inflammatory cells and pulmonary circulation) or through cortistatin-based treatments, is able to (1) deactivate alveolar and infiltrating macrophages and reduce production of inflammatory cytokines and chemokines that damage alveolar structure and activate/attract neutrophils; (2) impair the production by macrophages of cytokines/growth factors that signal for fibrogenic responses in fibroblasts; and (3) reduce the activation of fibroblasts and their differentiation to myofibroblasts by acting through its receptors (CST-R) as a break of intracellular signal factors (Smad2/3, Akt and MAPKs) that are critical for gene expression of profibrogenic mediators (depicted in square box). Interestingly, fibrogenic activation impairs the secretion of cortistatin by fibroblasts, thus releasing this endogenous break molecule (i.e. monoclonal antibodies). Moreover, cortistatin-based therapies could limit the start of late-onset pulmonary fibrosis in patients with ARDS or COVID-19 in post-infection stages. It is important to mention that cortistatin has a favourable safety profile in humans and has demonstrated clinical efficacy in patients with Cushing's disease (Giordano et al., 2007). Furthermore, the interest of the pharmaceutical companies in developing cortistatin-based analogues with improved half-life in serum has increased lately and a recent report demonstrated their efficiency in inflammatory conditions (Rol et al., 2021). We have recently witnessed an example of repositioning of a therapy based on another immunomodulatory and antifibrotic neuropeptide, vasoactive intestinal peptide (VIP) or aviptadil (Chorny & Delgado, 2008;Prasse et al., 2010), for the treatment of ARDS in patients with severe COVID-19 (Scavone et al., 2020;clinical trial: NCT04311697).
Second, the fact that a simple partial deficiency in cortistatin could predispose patients for developing exacerbated inflammatory and fibrotic responses and could be used to anticipate the diagnostically the developmen of more severe forms of pulmonary disorders.
In this sense, this study opens the possibility of initiating further clinical research that could corroborate plasma cortistatin level as a biomarker of protection or prognosis in patients with ARDS or IPF.
If this is the case, our results also suggest that a treatment based on cortistatin injection would easily correct this deficiency and improve disease progression. Although the endogenous and environmental factors that could influence the body levels of cortistatin are mostly unknown, one could anticipate that these levels will change during our life under different circumstances and scenarios, and thus our susceptibility to suffer certain disorders. In any case, a percentage of the human population is heterozygous for cortistatin from birth, because cortistatin gene is located at chromosome 1p36.22 and monosomy of 1p36 is the most common subtelomeric terminal deletion syndrome (Jordan et al., 2015). Besides a spectrum of neurological and sensorial defects, these individuals develop cardiac fibrosis and cardiomyopathy, however no studies have described increased incidence of pulmonary disorders, despite their short life expectancy.
Finally, bleomycin is a chemotherapeutic drug used for treating many types of cancer. Unfortunately, a proportion of bleomycintreated patients develop severe side effects that are associated to appearance of pulmonary and skin fibrosis that mostly cause treatment to be withdrawn. Understanding that a deficiency in a factor like cortistatin could predispose patients to such adverse effects could help selection of the correct chemotherapy or try to correct the adverse effects by cortistatin-based treatments.
In summary, this study provides new insights into the function of cortistatin in pulmonary inflammation and demonstrates a novel role of this neuropeptide in fibrosis, which could be extrapolated beyond lung tissue. Overall, we demonstrate that deficiency in cortistatin could be considered a potential biomarker of susceptibility to suffer severe forms of pulmonary disorders including ARDS, IPF or pneumonia and provide a powerful rationale for the assessment of the efficacy of cortistatin or its stable analogues as therapeutic approaches to the treatment of diseases that are still unsolved from a clinical point of view. However, because the effects observed are based on preclinical mouse models of ALI/ARDS in the absence of viral or bacterial infection, and many pulmonary fibrotic disorders are of idiopathic nature, extrapolations to clinical practice have to be made with caution.