Cathelicidin Treatment Silences Epithelial–Mesenchymal Transition Involved in Pulmonary Fibrosis in a Murine Model of Hypersensitivity Pneumonitis

Pulmonary fibrosis is becoming an increasingly common pathology worldwide. Unfortunately, this disorder is characterized by a bad prognosis: no treatment is known, and the survival rate is dramatically low. One of the most frequent reasons for pulmonary fibrosis is hypersensitivity pneumonitis (HP). As the main mechanism of pulmonary fibrosis is a pathology of the repair of wounded pulmonary epithelium with a pivotal role in epithelial–mesenchymal transition (EMT), we assumed that EMT silencing could prevent disease development. Because of several biological features including wound healing promotion, an ideal candidate for use in the treatment of pulmonary fibrosis seems to be cathelicidin. The aim of the studies was to understand the influence of cathelicidin on the EMT process occurring during lung fibrosis development in the course of HP. Cathelicidin’s impact on EMT was examined in a murine model of HP, wherein lung fibrosis was induced by chronic exposure to extract of Pantoea agglomerans (SE-PA) by real-time PCR and Western blotting. Studies revealed that mouse exposure to cathelicidin did not cause any side changes in the expression of investigated genes/proteins. Simultaneously, cathelicidin administered together or after SE-PA decreased the elevated level of myofibroblast markers (Acta2/α-smooth muscle actin, Cdh2/N-cadherin, Fn1/Fibronectin, Vim/vimentin) and increased the lowered level of epithelial markers (Cdh1/E-cadherin, Ocln/occludin). Cathelicidin provided with SE-PA or after cessation of SE-PA inhalations reduced the expression of EMT-associated factors (Ctnnd1/β-catenin, Nfkb1/NFκB, Snail1/Snail, Tgfb1/TGFβ1 Zeb1/ZEB1, Zeb2/ZEB2) elevated by P. agglomerans. Cathelicidin’s beneficial impact on the expression of genes/proteins involved in EMT was observed during and after the HP development; however, cathelicidin was not able to completely neutralize the negative changes. Nevertheless, significant EMT silencing in response to cathelicidin suggested the possibility of its use in the prevention/treatment of pulmonary fibrosis.


Introduction
Hypersensitivity pneumonitis (HP) or extrinsic allergic alveolitis (EAA) is a heterogenic group of interstitial lung diseases in which the chronic inhalation of a wide variety of organic dust provokes in susceptible subjects a hypersensitivity reaction with inflammation in the terminal bronchioles, the pulmonary interstitium, and the alveolar tree. This inflammation often organizes into granulomas and may progress to pulmonary fibrosis, which leads to the elimination of pathologically changed areas of lung tissue from by downregulation of E-cadherin, occludins, cytokeratin, and aquaporins, while α-SMA, vimentin, collagens, N-cadherin, fibronectin, and desmin were upregulated [21][22][23][24]. Snai1, a direct transcriptional repressor for the E-cadherin gene, was demonstrated as a target for EMT-promoting signaling pathways [25]. Recently, additional transcription factors ZEB1 and ZEB2 were identified as E-cadherin repressors and mediators of the EMT [26]. Our earlier studies [27], conducted in mice strain C57BL/6J chronically exposed to the antigen of Pantoea agglomerant (well-documented etiological factor of HP) [5], have also shown downregulation of epithelial markers (Cdh1, Cldn1, Jup, Ocln) and upregulation of myofibroblasts markers (Acta2, Cdh2, Fn1, Vim) in lung tissue in response to bacterial antigen treatment. Furthermore, the mentioned alterations in gene expression typical for EMT correlated with an increase in fibrosis markers (hydroxyproline, collagens) as well as histological changes characteristic for lung fibrosis development [27]. Considering the role of EMT in pulmonary fibrosis [24,[28][29][30], including the above-mentioned studies conducted by the authors that indicated EMT significance for the HP development in a murine model [27], it seemed reasonable to base HP therapy on inhibiting/counteracting EMT. Because of pleiotropic activities, an ideal candidate for the proposed therapeutic strategy seemed to be cathelicidin.
Cathelicidins belong to a large, conserved group of antimicrobial peptides and represent an important part of innate immunity [31]. These host defense peptides directly kill bacteria as well as some enveloped viruses, parasites, and fungi by perturbing their cell membranes [32,33]. Furthermore, they can also neutralize the biological activities of endotoxin [34,35]. Cathelicidins increase the natural abilities of immune cells to fight infection in several different ways, including attraction and recognition of pathogens, enhancement of phagocytosis, and stimulation of production and release of pro-inflammatory compounds [32,[36][37][38][39][40]. Cathelicidins also accelerate epithelial cell proliferation, migration, and promotion of wound closure which, all together, play an important role in the maintenance of tissue homeostasis by supervising regenerative processes [41][42][43]. It needs to be highlighted that recent studies by the authors revealed the beneficial impact of cathelicidin on the development of lung fibrosis in the course of HP, which was associated with restoring the balance in quantity of immune cells (NK cells, macrophages, lymphocytes: Tc, Th, Treg, B), cytokine production (IFNγ, TNFα, TGFβ1, IL1β, IL4, IL5, IL10, IL12α, IL13), and synthesis of extracellular matrix components (hydroxyproline, collagens) [44]. Cathelicidin treatment also effectively protected lung tissue structure from pathological changes induced by antigen of P. agglomerans (HP trigger) [44]. Despite the fact that many advantageous biological activities of cathelicidin have been discovered, the presented study is the first to focus on understanding the influence of cathelicidin on the EMT process occurring during lung fibrosis development in the course of HP. Thus, the aim of the presented study was the identification of the molecular mechanism responsible for the antifibrotic properties of cathelicidin previously described by our research team [44].

Cathelicidin Restored the Balance in the Expression of Genes Responsible for EMT
Changes in the expression of genes involved in epithelial-mesenchymal transition in lung tissue homogenates obtained from mice chronically exposed to cathelicidin and/or saline extract of P. agglomerans were examined by real-time PCR (Figure 1, Table 1). The study revealed that chronic exposure of mice to cathelicidin did not cause any changes in the expression of all investigated genes. On the contrary P. agglomerans treatment induced alterations in the gene expression characteristic for epithelial-mesenchymal transition: downregulation of epithelial markers (Cdh1, Ocln), as well as significant upregulation of mesenchymal markers (Cdh2, Acta2, Fn1, Vim). The decrease in epithelial markers, on average, was 21.6% after 14 days and 40.6% after 28 days of SE-PA exposure, while the increase in mesenchymal markers, on average, was 26.7% and 39.1% at the mentioned time points, respectively. The level of mRNA coding transcription factors responsible for EMT (Snail1, Zeb1, Zeb2) was also upregulated by SE-PA exposure; however, statistically significant changes were noted in the case of Snail1 at both investigated time points (increase by 35.4% and 82.1%, respectively), and just after 28 days of inhalation with SE-PA in the case of Zeb1 and Zeb2 (average increase by 48.5%). Expression of representatives of signaling pathways leading to mesenchymal differentiation (Ctnnd1, Nfkb1, Tgfb1) in response to P. agglomerans reached the following levels: 1.236, 1.249, and 1.698 (2 weeks of inhalations) and 1.415, 1.565, and 2.255 (4 weeks of inhalations). The levels of almost all the investigated mRNA observed in mice 14 days after cessation of SE-PA chronic exposure were quite similar to the data obtained from mice only after 28 days of inhalation with P. agglomerans; the exception was Ocln, whose expression increased by 27.3% during 2 weeks without exposure. Cathelicidin administered together with SE-PA and after SE-PA treatment significantly increased the expression level of Cdh1 and Ocln lowered by P. agglomerans. Expression of other investigated mRNAs recorded in the homogenates of lungs collected from mice treated with both CRAMP and SE-PA was lower compared with samples obtained from animals exposed to bacterial extract; nevertheless, differences observed in the case of Zeb1 and Zeb2 in both tested time points, as well as changes recorded in the case of Vim, Nfkb1, and Tgfb1 after 14 days of exposure were not statistically significant. Statistically significant differences in the expression of Cdh2, Acta2, Fn1, Snail, and Ctnnd1 recorded during comparison "SE-PA + CRAMP 14d." vs. "SE-PA 14d.", on average, were 13.1%, while in the expression of Cdh2, Acta2, Fn1, Vim, Snail, Ctnnd1, Nfkb1, and Tgfb1 recorded during comparison of "SE-PA + CRAMP 28d." vs. "SE-PA 28d.", on average, were 21.1%. The cathelicidin administration after 28 days of mice exposure to SE-PA also decreased the elevated by P. agglomerans treatment level of all tested mesenchymal markers (on average by 11.7%), as well as factors involved in EMT (on average by 22.0%). It has to be noted that cathelicidin administered after cessation of SE-PA treatment better restored the balance in the mRNA levels of Snail, Zeb1, and Zeb2; on the contrary, alterations induced by P. agglomerans in the expression of other investigated genes better neutralized CRAMP together with bacterial extract. However, significant differences (at least 5%) were noted in the cases of Cdh2, Ctnnd1, Fn1, and Tgfb1. Expression of gene coding epithelial markers, mesenchymal markers, and factors involved in epithelial-mesenchymal transition. Gene expression was investigated in homogenates of lungs collected from untreated mice (control) and animals exposed to investigated compounds for 14, 28, or 42 days using the real-time PCR method. Results are presented as the mean of relative mRNA amount ± SD. Each research group consisted of 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed in 3 replications. Statistical significance from a one-way ANOVA test followed by Tukey's post hoc test: compared to untreated mice at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); SE-PA + CRAMP 14 d/28 d vs. SE-PA 14 d/28 d (comparison within Figure 1. Alterations in the expression of genes involved in epithelial-mesenchymal transition (EMT) in response to cathelicidin (CRAMP) and/or saline extract of Pantoea agglomerans (SE-PA) treatment. Expression of gene coding epithelial markers, mesenchymal markers, and factors involved in epithelial-mesenchymal transition. Gene expression was investigated in homogenates of lungs collected from untreated mice (control) and animals exposed to investigated compounds for 14, 28, or 42 days using the real-time PCR method. Results are presented as the mean of relative mRNA amount ± SD. Each research group consisted of 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed in 3 replications. Statistical significance from a oneway ANOVA test followed by Tukey's post hoc test: compared to untreated mice at p < 0.05 (*),

Cathelicidin Eliminated Negative Changes in the Expression of Proteins Associated with EMT
Changes in the expression of proteins involved in epithelial-mesenchymal transition were investigated by Western blotting in homogenates of lung tissue collected from mice treated with cathelicidin and/or saline extract of Pantoea agglomerans ( Figure 2, Table 2). Western blots revealed that cathelicidin used alone did not impact the expression of proteins associated with EMT: E-cadherin, N-cadherin, β-catenin, fibronectin, NFκB, vimentin, occludin, Snail, α-smooth muscle actin (α-SMA), TGFβ, ZEB1, ZEB2. On the contrary, chronic exposure of mice to saline extract of P. agglomerans significantly decreased the expression of epithelial markers (E-cadherin, occludin) and distinctly increased the level of mesenchymal markers (N-cadherin, fibronectin, vimentin, α-SMA). Expression of epithelial markers were lowered, on average, 48.8% and 56.4% after 14 and 28 days of SE-PA exposure, respectively. Simultaneously, the expression of mesenchymal markers grew, in response to 14 and 28 days of mice treatment with P. agglomerans, on average by 53.7% and 101.4%, respectively. Furthermore, a distinct increase in transcription factors responsible for EMT (Snail, ZEB1, ZEB2) and key members of signaling pathways involved in EMT (β-catenin, NFκB, TGFβ) was also observed in SE-PA-treated mice. The most significant changes were recorded in the case of TGFβ, in which expression on the 14th and 28th days of the experiment reached 257.3% and 376.0% of the control, respectively. Equally important changes were observed in the case of Snail and β-catenin, whose expressions increased to 224.2% and 247.6% of the control at the above-mentioned time points, respectively. It has to be noted that the expression of all investigated proteins recorded at 28 days of SE-PA treatment and 14 days after cessation of SE-PA chronic exposure was maintained at a similar level. Cathelicidin administered together with SE-PA and after cessation of SE-PA treatment significantly increased the expression of epithelial markers lowered by inhalations with P. agglomerans, and the most significant improvement was observed in the case of occludin, the expression of which after 14 days of CRAPM + SE-PA exposure approached the level of the control. The expression of almost all mesenchymal markers (except vimentin in time point of 14th days) elevated by the bacterial extract was significantly lower in the lung samples collected from mice treated simultaneously with SE-PA and CRAMP as well as animals treated with defense peptide for the next 14 days after cessation of antigen administration. The most spectacular restoration of the level of mesenchymal markers altered by SE-PA was observed in the case of α-SMA, the expression of which after 14 days of cathelicidin administration together with SE-PA dropped to the level registered in untreated mice; the differences between the compared research groups (SE-PA + CRAMP 14d vs. SE-PA 14d) was 38.5%. The beneficial effect of cathelicidin given together with or after P. agglomerans treatment was also observed in the expression of factors involved of EMT, the level of which significantly decreased, compared to the data recorded in lung samples collected from mice treated with SE-PA. Among the investigated factors, the most significant improvement was observed in the expression of TGFβ, the level of which decreased by 115.8% (SE-PA + CRAMP 14d vs. SE-PA 14d), 187.4% (SE-PA + CRAMP 28d vs. SE-PA 28d), and 185.0% (SE-PA 28d + CRAMP 14d vs. SE-PA 28d + untreated 14d). The second factor with the highest amplitude of changes was ZEB1, the level of which decreased by 124.3% (SE-PA + CRAMP 28d vs. SE-PA 28d) and 103.7% (SE-PA 28d + CRAMP 14d vs. SE-PA 28d + untreated 14d); however, the difference of 16.7% in the expression of ZEB1 observed between "SE-PA + CRAMP 14d" and "SE-PA 14d" was not statistically significant. The beneficial impact of cathelicidin administered together or after P. agglomerans exposure on the expression of investigated proteins altered by SE-PA was quite similar in most of the performed experiments. Nevertheless, in the case of Snail and β-catenin, cathelicidin worked much better if given together with SE-PA; on the contrary, the defense peptide administered after cessation of SE-PA exposure better restored the balance in the expression of ZEB2, NFκB, and N-cadherin.

Discussion
Despite the well-described EMT role in pulmonary fibrosis [24,[27][28][29][30]45], as well as several pieces of data indicating cathelicidin involvement in the EMT process [41][42][43]46], there is no evidence regarding the cathelicidin influence of EMT in the course of HP development. The current study fills this gap of knowledge, describing the cathelicidin impact on the expression of genes and proteins involved in EMT under developing lung fibrosis in the course of HP. The study was conducted in created and validated by our research group the murine model of HP, wherein pulmonary fibrosis was induced by an extract of Pantoea agglomerans administered daily for 28 days to fibrosis mice strain C57BL/6J [47][48][49][50][51]. Pathological changes observed in mice in response to chronic exposure to the antigen of P. agglomerans were similar to the clinical picture of HP, which was confirmed on the genome and proteome level as well as in the microscopic image of lung tissue and in the characteristics of the immune response. Furthermore, earlier studies by our team also revealed that depending on the time of exposure, successive stages of disease development can be obtained: acute with a strong inflammatory response (7-14 days of exposure) and chronic with significant signs of fibrosis (28 days of exposure) [47][48][49][50][51]. It should be emphasized that according to our best knowledge, the mentioned model is the only research model of HP that, under laboratory conditions, reproduces the environmental exposure to organic dust causing lung fibrosis. For this reason, the use of this model in the presented study increases the chances of effective translation of the obtained results into clinical practice, thus accelerating the introduction of a new HP therapeutic strategy. An additional reason for selecting the mentioned model for the current study was the previously mentioned data [27], which demonstrated the relationship between pulmonary fibrosis induced by mice chronic exposure to P. agglomerans with the antigen ability to provoke epithelial-mesenchymal transition type 2, which is the focus of this study. First of all, the study revealed that the chronic exposure of mice to cathelicidin did not cause any changes in the expression of all investigated molecules, neither the genes nor their proteins products: Acta2/α-smooth muscle actin, Cdh1/E-cadherin, Cdh2/N-cadherin, Ctnnd1/β-catenin, Fn1/fibronectin, Nfkb1/NFκB, Vim/vimentin, Ocln/occludin, Snail1/Snail, Tgfb1/TGFβ, Zeb1/ZEB1, Zeb2/ZEB2. On the contrary, SE-PA treatment induced alterations characteristic for EMT: downregulation of epithelial markers (Cdh1/E-cadherin, Ocln/occludin) and significant upregulation of mesenchymal markers (Cdh2/N-cadherin, Fn1/fibronectin, Vim/vimentin, Acta2/α-smooth muscle actin). Chronic exposure of mice to P. agglomerans also increased the level of transcription factors responsible for the EMT process (Snail1/Snail, Zeb1/ZEB1, Zeb2/ZEB2) and selected members of signaling pathways, leading to mesenchymal differentiation (Ctnnd1/β-catenin, Nfkb1/NFκB, Tgfb1/TGFβ). The most significant changes were noted in the expressions of Tgfb1/TGFβ and Snail1/Snail. The observed changes highlight the key roles of these two molecules in the execution of the EMT program in response to SE-PA exposure. TGFβ has been shown to play a pivotal role in pulmonary fibrosis, not only through induction of EMT in alveolar epithelial cells and its ability to attract and stimulate proliferation of fibroblasts and myofibroblasts but also as potent inducers of ECM production, including collagen and other matrix proteins [28,29,52,53]. Since Snail is an immediate-early response gene for TGFβ, the observed significant increase in its expression seems to be logical. Indeed, most EMT studies revealed Snail induction in response to TGFβ and furthermore demonstrated a correlation between the elevated level of this transcription factor with the repression of genes coding epithelial markers, as well as concomitant activation of mesenchymal gene expression [54][55][56]. While the amplitudes of changes in the level of expression of other investigated genes in response to SE-PA were similar, analysis of protein expression indicated two additional molecules (ZEB1 and β-catenin) that, in addition to the TGFβ and Snail, were proven to be very sensitive to the action of the tested bacterial extract. As in the case of the Snails, the expression of transcription factor ZEB1 is activated by TGFβ and directly represses the expression of epithelial marker genes, increases expression of the mesenchymal markers, and additionally promotes cell migration [29,54,[57][58][59]. β-catenin is physiologically part of a major cell-surface adhesion complex, but if released from there becomes an important part of Wnt signaling, the involvement of which in EMT has been proven in many studies [29,30,57]. Released β-catenin interacts with transcription factors LEF1 or TCF and as a complex translocates into the nucleus and regulates the transcription of several genes associated with EMT, e.g., decreasing the expression of E-cadherin gene, increasing the expression of genes coding vimentin and fibronectin, and stimulating the production of matrix metalloproteinases [30,57,60].
The conducted studies revealed that cathelicidin administered with SE-PA or after 28 days of P. agglomerans exposure restored the balance in the expression of genes and proteins disturbed by mice exposure to the mentioned extract. CRAMP increased the lowered by SE-PA level of Cdh1/E-cadherin and Ocln/occludin. Cathelicidin particularly effectively neutralized the negative effect of P. agglomerans on the expression of Ocln/occludin-14 days of CRAMP treatment together with SE-PA or after cessation of SE-PA inhalations increased the expression of the investigated molecule to the level observed in untreated mice. Additionally, CRAMP administered with bacterial extract or after SE-PA exposure decreased the elevated by SE-PA level of Cdh2/N-cadherin, Acta2/α-SMA, Fn1/fibronectin, and Vim/vimentin. Nevertheless, complete neutralization of the alteration induced by SE-PA was observed in the case of α-SMA after 14 days of mice exposure to both CRAMP and SE-PA. The similar beneficial impact of the tested peptide on α-SMA expression was noted in mice after 14 days of CRAMP treatment preceded by 28 days of SE-PA inhalations. Alterations in the expression of factors associated with EMT observed in mice chronically exposed to SE-PA were also leveled by cathelicidin treatment. Nevertheless, the effectiveness of cathelicidin treatment depends on the way of cathelicidin administration. The investigation of gene expression revealed three different patterns of CRAMP response: (1) Snail1 and Ctnnd1: significant differences in the expression after 14 and 28 days of animals exposure to SE-PA used alone and together with CRAMP; (2) Nfkb1 and Tgfb: significant differences in the expression after 28 days of animals exposure to SE-PA used alone and together with CRAMP; (3) differences in the expression of Zeb1 and Zeb2 after 14 and 28 days of animals exposure to SE-PA used alone and together with CRAMP were not significant. Nevertheless, alteration in the expression of all investigated factors was restored when CRAMP was provided after cessation of SE-PA inhalations. Results obtained from Western blotting revealed that mice inhalation with CRAMP after 28 days of SE-PA exposure as well as CRAMP administration for 4 weeks together with SE-PA significantly lowered the pathological level of all investigated factors. Furthermore, the expression of Snail, ZEB2, β-catenin, and TGFβ was also improved in mice exposed to both CRAMP and P. agglomerans antigene for 14 days.
It should be emphasized that changes recorded on genome and proteome levels correspond with alterations in lung tissue morphology previously described by our team [44] (see the Supplementary Materials Figure S1). Similar to the presented data, histological examination of murine lungs stained with hematoxylin and eosin (H&E) or Masson trichrome (TRI) revealed the lack of any changes in tissue morphology after 14 and 28 days of animal exposure to cathelicidin. On the contrary, mice inhalations with SE-PA induced changes typical for HP; in particular, significant inflammatory response with interstitial infiltrations of lymphocyte and macrophage (mean score for inflammation in time points 14 and 28: 1.6 and 2.0) as well as signs of fibrosis (mean score for fibrosis in time points 14 and 28: 0.8 and 1.6) associated with abnormal collagen deposition, leading to thickening of alveolar walls, which intensified during the time of exposure. The mentioned alterations perfectly corresponded with the herein presented increase in expression of mesenchymal markers and transcription factors involved in EMT. Moreover, the histological examination also did not show statistically significant changes between lungs collected 14 days after cessation of mice chronic exposure to SE-PA to lungs obtained directly after 28 days of inhalation with P. agglomerans (SE-PA 28d + untreated 14d vs. SE-PA 28d). Furthermore, the evaluation of lung sections also revealed the beneficial impact of cathelicidin treatment on fibrosis development. Comparison data obtained from the following research groups: SE-PA + CRAMP 28d vs. SE-PA 28d as well as SE-PA 28d + CRAMP 14d vs. SE-PA 28d + untreated 14d, demonstrated a significant decrease in fibrosis scores by 36.8% and 38.9%, respectively. The above-mentioned antifibrotic properties of cathelicidin correspond with data obtained in presented studies, that revealed the beneficial impact of CRAMP treatment in the inhibition of the expression of both mesenchymal markers and factors involved in EMT, which have been pathologically elevated by P. agglomerans. It needs to be highlighted that the concordance of changes observed at the level of genes and proteins as well as in the histological evaluation indicated that the previously described beneficial properties of cathelicidin should be associated with its ability to modulate the course of EMT [44].
The presented results indicate CRAMP as an effective inhibitor of EMT associated with pulmonary fibrosis in HP, which is the first such evidence according to the best of our knowledge. Nevertheless, the ability of cathelicidin to inhibit pathological EMT was reported previously by Cheng et al., who showed that LL-37 inhibited TGFβ-induced gene expression of ACTA2 and VIM as well as protein expression of E-cadherin, Twist1, and Slug in human colon cancer HT-29 cells. Furthermore, Cheng et al. demonstrated in vivo that administration of cathelicidin expression viral vector or synthetic mCRAMP inhibited vimentin and E-cadherin expression and collagen deposition, leading to suppression of EMT and consequently to colon cancer development [46]. Additionally, Zheng et al. revealed the inhibition of cardiac fibrosis in diabetic mice heart treated with CRAMP, which was a consequence of an increase in endothelial markers (CD31, cadherin), a decrease in fibroblast markers (collagen I, collagen III, vimentin), and a reduction of transcription factors (Snial1, Snial2, Twist1, Twist2). Zheng et al. connected the anti-fibrotic CRAMP abilities with the silencing of signal transduction in the TGFβ/Smad pathway [61]. Despite the fact that some alterations in the expression of genes and proteins in response to CRAMP observed by Cheng [61]. Consequently, the presented study is the first report showing the influence of cathelicidin on EMT type 2. Furthermore, obtained data indicated that maintaining the physiological level of cathelicidin in the respiratory tract plays an important role in the supervision of regenerative processes and consequently prevention of pathological wound healing leading to fibrosis.

Reagents
Unless otherwise indicated, the chemicals used in the study were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) LLC. Murine cathelicidin (ISRLAGLLRKGGEKIGEKLKKI GQKIKNFFQKLVPQPE) was purchased from Novozym Polska s.c. Poznań Science and Technology Park, Poznań, Poland. The preparation of Pantoea agglomerans extract, as well as its main composition, has been described previously [27].

Animal Inhalations
Three-month-old female C57BL/6J mice were purchased from Mossakowski Medical Research Centre of the Polish Academy of Sciences in Warsaw, Poland. The conditions in which the animals were kept, as well as the procedure for their preparation for the study, has been described previously [44]. Mice were exposed to a finely dispersed aerosol of the saline extract of P. agglomerans (SE-PA; 10 mg/mL; 5 mL/single inhalation), cathelicidin (CRAMP; 1.44 µg/mL; 5 mL/single inhalation), or phosphate-buffered saline (PBS; 5 mL/single inhalation), administered separately or in combination ( Table 3). The mice were treated to each investigated factor for one hour daily for 14, 28, or 42 days. Inhalations were carried out using the Buxco Inhalation Tower (Data Sciences International, St. Paul, MN, USA) under the following conditions: airflow 1.5 L/min; pressure 0.5 cm H 2 O; room temperature; nebulization rate 84 µL/min. Before and after the indicated time of treatment, the animals were sacrificed by cervical dislocation with spinal cord rupture, after which lung samples were collected, frozen in liquid nitrogen, and stored at −80 • C until evaluation.

Evaluation of Gene Expression
Gene expression evaluation has been described previously [62]; nevertheless, what should be mentioned are the TaqMan Gene Expression Assays (sets of probes and primers) used in the research: Mm00725412_s1 for Acta2; Mm02619580_g1 for Actb; Mm01247357_m1 for Cdh1; Mm01162497_m1 for Cdh2; Mm01334599_m1 for Ctnnd1; Mm01256744_m1 for Fn1; Mm00476361_m1 for Nfkb1; Mm00500912_m1 for Ocln; Mm01178820_m1 for Tgfb1; Mm00441533_g1 for Snail1; Mm01333430_m1 for Vim; Mm00495564_m1 for Zeb1; Mm00497196_m1 for Zeb2. Relative expression was calculated using the efficiency method (relative advanced quantification) and normalized to the expression of Actb.

Statistical Analysis
The data were presented as the mean value and standard deviation (SD). Statistical analysis was performed using linear regression analysis, as well as the one way-ANOVA with Tukey's post hoc test, and column statistics were used for comparisons. Significance was accepted at p < 0.05.

Conclusions
In summary, the beneficial impact of cathelicidin treatment on the expression of genes/proteins involved in EMT was observed both during and after the development of hypersensitivity pneumonitis. Despite the fact that cathelicidin was not able to completely neutralize the negative changes induced by P. agglomerans, EMT silencing in response to CRAMP was significant. Furthermore, cathelicidin used alone did not cause any sidechanges in the expression of the investigated genes and proteins. It needs to be highlighted that the presented study is the first report showing the influence of cathelicidin on EMT type 2. Due to the importance of EMT for pulmonary fibrosis, the presented results suggest the possibility of using cathelicidin in the prevention and treatment of this pathological process. Unfortunately, exogenous cathelicidin was not able to eliminate the negative changes completely, perhaps because of the fact that CRAMP inhalations did not restore the physiological level of cathelicidin disturbed by the disease development. Nevertheless, in the face of the absence of a safe and successful strategy for the prevention and treatment of pulmonary fibrosis in the course of HP, even a slight beneficial effect of cathelicidin, especially in terms of the lack of side effects, deserves attention and presentation in order to create the basis for the development an effective therapeutic strategy in the future.