Exposure to cigarette smoke exacerbates polyhexamethylene guanidine-induced lung fibrosis in mice

Abstract

Cigarette smoke (CS) is the leading cause of chronic pulmonary diseases, including lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. In this study, we aimed to investigate the effects of repeated CS exposure on polyhexamethylene guanidine (PHMG)-induced pulmonary fibrosis in mice. A single intratracheal instillation of 0.6 mg/kg PHMG enhanced the immune response of mice by increasing the number of total and specific inflammatory cell types in the bronchoalveolar lavage fluid. It induced histopathological changes such as granulomatous inflammation/fibrosis and macrophage infiltration in the lungs. These responses were upregulated upon exposure to a combination of PHMG and CS. In contrast, a 4-hr/day exposure to 300 mg/m³ CS alone for 2 weeks by nose-only inhalation resulted in minimal inflammation in the mouse lung. Furthermore, PHMG administration increased the expression of fibrogenic mediators, especially in the pulmonary tissues of the PHMG + CS group compared with that in the PHMG alone group. However, there was no upregulation in the expression of inflammatory cytokines following exposure to a combination of PHMG and CS. Our results demonstrate that repeated exposure to CS may promote the development of PHMG-induced pulmonary fibrosis.

INTRODUCTION

Cigarette smoke (CS) is the leading cause of various fatal diseases, particularly smoking-related lung diseases. Numerous studies have demonstrated the causal relationship between CS and lung diseases, and the negative effects of CS on the lungs have been well established (Baumgartner et al., 1997; Takahashi et al., 2010; Sundar et al., 2014). Idiopathic pulmonary fibrosis (IPF) is a type of lung disease that results in fibrosis of an unknown etiology and is defined as a chronic and progressive fibrosing interstitial pneumonia, with the associated mortality rate of 50% within 3–5 years after diagnosis (Raghu et al., 2011). The increased risk of IPF is associated with various factors, and CS may most strongly relate to IPF development (Baumgartner et al., 1997; Taskar and Coultas, 2006; King et al., 2011).

The etiology of pulmonary fibrosis is often an irregular response in the wound healing process. When a tissue is damaged, inflammatory cells are released to eliminate the foreign substances and repair the damage. The recruitment of these cells to the injury sites is mediated by various inflammatory cytokines (Moldoveanu et al., 2009), and these cytokines promote the proliferation of fibroblasts and differentiation to myofibroblasts. The extracellular matrix (ECM), which is regulated by myofibroblasts, repairs the damaged tissues via wound contraction and tissue regeneration (Reinke and Sorg, 2012). However, excessive accumulation of ECM components owing to persistent inflammation or dysregulation of the repair process triggers fibrogenesis (Wynn, 2011; Todd et al., 2012). Therefore, although the association between inflammation and fibrosis development is still debated, immune cells and inflammatory cytokines are considered key players in the progression of fibrosis.

Polyhexamethylene guanidine (PHMG) is a water-soluble chemical that is widely used as a biocidal agent with a high activity against both gram-positive and gram-negative bacteria (Gilbert and Moore, 2005; Oulé et al., 2012). This macromolecule was previously known to have low toxicity in humans because of its characteristic incompatibility with mammalian cell membranes (Worley and Sun, 1996; Aleshina et al., 2001). However, it has been reported that the use of PHMG as a component of humidifier disinfectants may have severe negative effects on human lungs when inhaled (CDC Korea, 2011). Similarly, in animal experiments, treatment with PHMG increased the risk of inflammatory lung damage and provoked the subsequent fibrogenic impairment (Song et al., 2014; Kim et al., 2016a).

In this study, we investigated whether PHMG-induced lung damage could be exacerbated by exposure to CS, by evaluating the expression of inflammatory and fibrogenic mediators.

MATERIALS AND METHODS

Test articles

The reference 3R4F research cigarettes were purchased from Kentucky Tobacco Research and Development Center (Lexington, KY, USA). PHMG solution was gifted by SK Chemicals (Seongnam, Korea).

Animals

Seven-week-old male Balb/C mice (n = 20) were purchased from Orient Bio Inc. (Seongnam, Korea) and were housed in environmentally controlled animal facilities, maintained at 22 ± 3°C, under relative humidity of 50% ± 20%, light intensity of 150–300 Lux, and light/dark cycle of 12/12 hr. The ventilation in the animal room was refreshed 10–20 times/hr. The mice were provided pelleted food (PMI Nutrition International, Richmond, IN, USA), and UV-irradiated (Steritron SX-1; Daeyoung Inc., Seoul, Korea) and filtered (through a 1-µm filter) tap water ad libitum. The mice were used in the experiments after 6 days of acclimation. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (IACUC #1612-0441).

Treatments and experimental groups

The mice were randomly divided into the following four groups (n = 5/group): vehicle control (VC), PHMG, CS, and PHMG + CS. Depending on the group, the animals received either filtered air (air control) or 300 μg total particulate matter /L of diluted CS, generated using a CSM 2080 30-port smoking machine (CH Technologies, Westwood, NJ, USA) according to the ISO 3308 regimen (35 mL puff volume, 2 sec puff duration, 60 sec between puffs, and no vent blocking). On day 0, the mice in the PHMG and PHMG + CS groups received a single intratracheal instillation of PHMG (0.6 mg/kg, 50 µL in saline), whereas the mice in the VC and CS groups received saline via the same route. The mice were exposed to CS via nose-only inhalation for 4 hr per day for 14 consecutive days. The body weight of mice was measured before the first instillation and on days 0 (first CS exposure), 1, 3, 6, 9, and 13. On day 14, the terminal body weight of the mice was recorded. Subsequently, the mice were euthanized by isoflurane overdose. The left lung lobe was dissected free of the connective tissue and weighed in a closed container. The weighed left lung lobe was fixed in 10% neutral-buffered formalin.

Bronchoalveolar lavage fluid (BALF) preparation

To collect BALF, the tracheas of mice were cannulated after exsanguination, and the right lung was lavaged three times with phosphate-buffered saline (PBS) (injection volume: 14 mL/kg terminal body weight (TBW)). Subsequently, the total cell count in 100 µL of BALF was determined using an automated cell analyzer (NucleoCounter^® NC-250™; ChemoMetec, Allerod, Denmark). In addition, 200 μL of the BALF was centrifuged (Shandon Cytospin 4; Thermo Fisher Scientific, Waltham, MA, USA) at 172 × g for 10 min and loaded on a cytospin-prepared slide (Shandon Cytospin 4; Thermo Fisher Scientific). The glass slides were stained with Diff-Quik (Sysmex, Hyogo, Japan). Differential cell counts were determined manually at 1000 × magnification using a light microscope (BX51; Olympus, Tokyo, Japan), by counting 200 cells per slide. The remaining BALF was centrifuged at 2,300 × g for 10 min, and the cell-free supernatants were collected and stored at −80°C until enzyme-linked immunosorbent assay (ELISA).

Lung tissue sampling and processing

After collecting the BALF, the right lung was removed, snap-frozen in liquid nitrogen, and stored at −80°C. For the cytokine measurements, the frozen lung tissues were homogenized in ice-cold PBS containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The homogenates were centrifuged at 13,400 × g for 20 min at 4°C to remove debris and insoluble material. The supernatants obtained were used to determine the total protein concentration and perform the ELISA.

Enzyme-linked immunosorbent assay

The expression levels of mouse interleukin 6 (IL-6), interleukin-1β (IL-1β), and C-C motif chemokine ligand 17 (CCL17) in the BALF and supernatants of lung tissue homogenates were measured using the Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions, and normalized to the total protein concentration. First, 96-well microplates were coated with the diluted capture antibody and incubated overnight at 25°C. The solution in each well was aspirated, and the wells were washed with wash buffer three times. Thereafter, to prevent non-specific protein binding, blocking buffer was added to each well. Then, the plates were incubated at 25°C for 1 hr. Subsequently, samples or standards were added to each well and incubated at 25°C for 2 hr. Detection antibody was then added to each well, and the plates were incubated at 25°C for 2 hr. The working solution of streptavidin-horseradish peroxidase (HRP) was added to each well, and the plate was incubated at 25°C for 20 min in the dark. The substrate solution was added to each well, and the plate was incubated at 25°C for 20 min in the dark. Finally, after confirming a change in color, stop solution was added to each well, and the microplate was gently mixed by tapping. The optical density of sample in each well was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). All standards and samples were examined in duplicate.

Histopathological examination

The isolated left lung tissue was fixed in 10% neutral-buffered formalin and embedded in paraffin. Thereafter, the paraffin-embedded tissue blocks were sliced into 4-μm thick sections using a microtome (Leica RM2145, Nussloch, Germany). The tissue sections were then stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO, USA) for histological analysis and Masson’s trichrome (MT; Sigma-Aldrich) for examining fibrotic changes. Histological analysis of the paraffin-embedded lung tissue sections was conducted by fluorescent microscopy (LSM780; Carl Zeiss Jena, Oberkochen, Germany), and the severity of fibrosis was scored on a scale ranging from 0 to 8, using the Ashcroft scoring system for each mouse. The quantitative analysis of collagen deposition in MT-stained tissue sections was performed at the Pathology Research Group of the Korea Institute of Toxicology.

Western blotting

The frozen lung samples were homogenized in radio-immuno-precipitation assay buffer (Pierce Biotechnology, Rockford, IL, USA), containing a protease inhibitor cocktail. Homogenates were centrifuged at 13,400 × g for 20 min at 4°C, and the supernatants were collected. The total protein concentration in the supernatants was determined using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred on to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with tris-buffered saline with 0.1% Tween^® 20 (TBS-T), containing 5% bovine serum albumin (BSA) for 1 hr at 25°C. The membranes were then incubated overnight with diluted primary antibodies in TBS-T, containing 5% BSA at 4°C, with shaking. The membranes were then washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies in TBS-T, containing 5% BSA, at 25°C for 1.5 hr. Thereafter, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hr. The blots were then washed with TBS-T and examined using chemiluminescence reagents (Pierce Biotechnology), according to the manufacturer’s instructions. Anti-transforming growth factor beta 1 (TGF-β1) and anti-fibronectin antibodies were purchased from Abcam (Cambridge, UK), and anti-β-actin antibody and all secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The intensity of bands was quantified via densitometry using ImageJ software, and the results were normalized to the concentration of β-actin.

Real-time quantitative polymerase chain reaction (qRT-PCR)

The total RNA was extracted from the frozen lung tissues using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s recommendations. RNA concentrations were quantified using a NanoDrop ND-2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE, USA), at 260 nm. Complementary DNA was synthesized from the isolated total RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The sequences of mouse gene-specific primers used in this study were as follows: CCL2 forward, 5′-TTGTCACCAAGCTCAAGAGAGA-3′; CCL2 reverse, 5′-GAGGTGGTTGTGGAAAAGGTAG-3′; CCL6 forward, 5′-AGGCTGGCCTCATACAAGAA-3′; CCL6 reverse, 5′-TCCCCTCCTGCTGATAAAGA-3′; CCL17 forward, 5′-TGCTTCTGGGGACTTTTCTG-3′; CCL17 reverse, 5′-TGGCCTTCTTCACATGTTTG-3′; CXCL1 forward, 5′-GCTGGGATTCACCTCAAGAA-3′; CXCL1 reverse, 5′-TGGGGACACCTTTTAGCATC-3′; IL-10 forward, 5′-ATTTGAATTCCCTGGGTGAGAAG-3′; and IL-10 reverse, 5′-CACAGGGGAGAAATCGATGACA-3′. Inflammatory cytokine mRNA expression levels were determined by qRT-PCR, using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The total reaction volume (20 µL) contained 10 µL of Power SYBR^® Green Master Mix (Applied Biosystems), 0.5 mg of cDNA, and 100 nM of each primer. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 45 cycles at 95°C for 10 sec, 57°C for 30 sec, and 72°C for 10 sec. Relative gene expression was calculated using the 2^-ΔΔCt method, where C_t = threshold cycle.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Significant differences among the groups were evaluated using the analysis of variance, followed by Bonferroni’s multiple comparison test. In addition, a significant difference between two groups was judged according to the least significant difference test. All statistical analyses were performed using Prism 5.0 (GraphPad Software, La Jolla, CA, USA), and results with p < 0.05 were considered statistically significant.

RESULTS

Effects of CS and PHMG exposure on mortality and body and lung weights

As shown in Fig. 1A, the survival rate of mice did not change in the VC, CS alone, and PHMG alone groups. However, exposure to the combination of PHMG and CS affected the mortality of mice on day 9, and the survival rate decreased by 20%. In the PHMG group, the overall body weight of the mice decreased from day 1 to 3. From day 6, the mice in the PHMG group showed a gradual increase in body weight. Similar to the PHMG alone group, the mice in the PHMG + CS group showed a considerable decrease in body weight from day 1 to 3; however, recovery of body weight was not observed until the final day (Fig. 1B). The absolute and relative lung weights were significantly elevated in mice in the PHMG group compared with those in the VC group. Although there was no statistical significance, the absolute and relative weights of the lungs were increased in mice in the PHMG + CS group compared with those in the PHMG group. There were no significant differences in the body and lung weights of mice in the CS alone group compared with those in the VC group (Fig. 2).

Fig. 1

Effect of CS on mortality rate and body weight of PHMG-treated mice. Mouse survival rate (A) and body weight (B) were monitored in the different experimental groups for 14 days. Data are expressed as mean ± SD (n = 4–5 mice/group).

Fig. 2

Effect of CS on lung weight of PHMG-treated mice. (A) Lung absolute weight was measured on day 14 following the administration of CS, PHMG, and PHMG + CS. (B) Relative left lung weight was calculated as the ratio of left lung weight (mg) to body weight (g). Data are presented as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group.

Effects on the total and differential cell counts in the BALF

The total cell count significantly increased in the BALF of mice in the PHMG and PHMG + CS groups compared with that in the VC and CS alone groups (Fig. 3A). The number of macrophages significantly increased in the BALF of mice in the PHMG and PHMG + CS groups compared with that in the VC group (Fig. 3B). The number of neutrophils was significantly elevated in the BALF of mice treated with PHMG + CS compared with that in the other groups (Fig. 3C). The number of lymphocytes tended to increase in the BALF of mice in the PHMG and PHMG + CS groups compared with that in the VC group, but the difference was not statistically significant (Fig. 3D). The ratio of neutrophils to the total cell count was the highest in the BALF of mice in the PHMG + CS group (Fig. 3E).

Fig. 3

Effect of CS on BALF cell count (total and differential) in PHMG-treated mice. The number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) was determined in the BALF of the VC-, CS-, PHMG-, and PHMG + CS-treated mice. (E) Differential cell ratios are presented as a percentage of total cells. Data are expressed as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group.

Effects of CS and PHMG exposure on the expression of inflammatory cytokines

The levels of IL-6 in the BALF and IL-1β and CCL17 in the supernatant of lung homogenate were considerably increased in the PHMG and PHMG + CS groups compared with that in the VC group; however, the difference was not statically significant (Fig. 4). Exposure to CS alone affected the levels of IL-1β and CCL17, but not IL-6 (Fig. 4). Additionally, the mRNA expression of inflammatory cytokines (CCL2, CCL6, CCL17, CXCL1, and IL-10) was measured in the lung tissue. The expression of CCL2 was significantly increased in the PHMG and PHMG + CS groups compared with that in the VC group (Fig. 5A). In contrast, the expression of CCL6 and CXCL1 was significantly elevated in the CS alone group but decreased in the PHMG and PHMG + CS groups (Fig. 5B and 5D). The expression of CCL17 was significantly increased in the CS and PHMG groups compared with that in the VC group, but there was no significant difference between the VC and the PHMG + CS group (Fig. 5C). The anti-inflammatory cytokine, IL-10, was substantially upregulated only in the PHMG group (Fig. 5E).

Fig. 4

Effect of CS exposure on levels of inflammatory cytokines in BALF and lung tissue of PHMG-treated mice. IL-6 (A), IL-1β (B), and CCL17 (C) levels (pg/mg) in the BALF and lung tissue, as determined by ELISA. Data are expressed as mean ± SD (n = 4–5 mice/group) from three independent experiments. ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group.

Fig. 5

Effect of CS exposure on the mRNA expression of inflammatory cytokines in lungs of PHMG-treated mice. Gene expression was determined by qRT-PCR and normalized to GAPDH expression as an internal control. Values are shown as fold changes relative to those of the VC group. Data are expressed as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group.

Histopathological changes in the lung tissues

The lung tissues of mice in the PHMG and PHMG + CS groups showed severe lung lesions compared with those in the VC group (Fig. 6A). In addition, quantitative analysis of the Ashcroft score showed that granulomatous inflammation/fibrosis was significantly increased in the PHMG and PHMG + CS groups compared to the VC and CS groups, and the severity in the PHMG + CS group was higher than that in the PHMG alone group (Fig. 6B). Similarly, the accumulation of foamy macrophages in the alveoli also tended to increase in the PHMG + CS group, but this increase was not significant (data not shown). Collagen deposition was highly condensed in the PHMG group compared with that in the VC and CS groups (Fig. 7A). Although there was no statistical difference, collagen deposition tended to increase in the PHMG + CS group than in the PHMG alone group (Fig. 7B). In contrast, only minimal pathological changes were noted in mice in the CS group (Figs. 6 and 7).

Fig. 6

Histopathological changes in PHMG-treated mouse lungs exposed to CS. Representative images showing the histopathological changes in distinct lung sections stained with H&E. The granulomatous inflammation/fibrosis (black arrow) in the terminal bronchioles and alveolar duct, and infiltration of alveolar macrophages (white arrow) were found in the PHMG- and CS-treated mice. Notice the more pronounced lung lesions in the PHMG and PHMG + CS groups (A). A comparison of Ashcroft scores among the experimental groups (B). Scale bars represent 50 μm. Data are expressed as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group, ^cP < 0.05 vs. mice in the PHMG group.

Fig. 7

Effect of CS on degree of collagen deposition in PHMG-treated mouse lungs. Representative micrographs of the lung tissue sections with the most visible damage are presented. Collagen deposition in the lung sections was assessed by Masson’s trichrome (MT) staining. The lung sections of the exposed groups were analyzed and compared with those of the VC group with a normal structure. Data are expressed as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group.

Effects of CS and PHMG exposure on the expression of fibrogenic mediators

To assess the development of fibrosis, the protein expression of fibrogenic mediators (TGF-β1 and fibronectin) was detected in the lung tissues. Figure 8 shows that treatment with PHMG alone significantly elevated the levels of TGF-β1. In addition, the level of fibronectin protein in the lung tissue was also increased by treatment of PHMG alone, but there was no statistical difference. On the other hand, treatment with PHMG + CS significantly increased the expression of both mediators compared with the CS or PHMG alone treatment.

Fig. 8

Effect of CS exposure on the accumulation of fibrosis-related mediators in PHMG-treated mouse lungs. Lung TGF-β1 (A) and fibronectin (B) protein levels were determined by western blotting. Protein expression levels were quantified and normalized to the expression of β-actin. Values are shown as fold changes relative to the VC group and are expressed as mean ± SD (n = 4–5 mice/group). ^aP < 0.05 vs. mice in the VC group, ^bP < 0.05 vs. mice in the CS group, and ^cP < 0.05 vs. mice in the PHMG group.

DISCUSSION

CS is considered a major risk factor for various diseases because it contains numerous hazardous chemicals classified as toxic or carcinogenic (Church and Pryor, 1985; Eldridge et al., 2015). In particular, accumulating evidence from epidemiological and experimental studies suggests that CS is significantly associated with the development of fatal lung diseases, including lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. Furthermore, environmental exposure to hazardous chemicals such as PHMG leads to severe pulmonary damage. Song et al. (2014) reported that single intratracheal instillation of PHMG caused lung inflammation, leading to pulmonary fibrosis, and promoted lymphocyte depletion, causing thymic atrophy. Moreover, Kim et al. (2016a, 2016b) reported that inhalation of PHMG may induce pulmonary fibrosis by epithelial cell damage via oxidative stress and lead to excessive wound healing via the expression of inflammatory cytokines. To the best of our knowledge, there is no toxicological study on the effects of repeated exposure to CS on PHMG-induced lung damage. Therefore, in the present study, we confirmed the exacerbated effects of CS exposure on the lung damage induced by PHMG.

To determine the adverse effects of exposure to PHMG and CS, we observed body and lung weights and survival rate of the treated mice. A previous study demonstrated a 10% loss in body weight after the instillation of 0.6 mg/kg PHMG, and mortality of mice was observed in the group treated with 3.0 mg/kg PHMG (Song et al., 2014). Similar to the results of Song et al., in this study, the instillation of PHMG (0.6 mg/kg) decreased the body weight of mice, and from day 6, the reduced weight gradually recovered in the final stage. In contrast, repeated exposure to CS in mice treated with the same concentration of PHMG showed a decrease in body weight, followed by no recovery. In addition, in contrast to the group exposed to PHMG or CS only, the survival rate of mice decreased by 20% in the CS + PHMG group. Furthermore, the absolute and relative lung weights significantly increased in mice exposed to both PHMG and CS. Our findings indicate that repeated exposure to CS aggravates the lung damage caused by the instillation of PHMG, and it may be associated with the subsequent changes in the body and lung weights.

Inflammation plays a key role in most interstitial lung diseases, and various immune cells such as macrophages, neutrophils, and lymphocytes are actively involved in the inflammatory response (Wynn, 2008; Moldoveanu et al., 2009). Exposure to CS is known to increase the number of immune cells in the BALF (Seagrave et al., 2004), and this increases the risk of tissue damage via the release of toxic mediators such as proteolytic enzymes and reactive oxygen species (Bhalla et al., 2009). However, a previous study reported that in PHMG-induced pulmonary fibrosis, there was no increase in the number of immune cells in response to inhaled substances (Kim et al., 2018). To evaluate inflammation associated with the pathogenesis of pulmonary fibrosis caused by exposure to CS and PHMG, we evaluated the changes in the expression of immune cells and their mediators. In the present study, the number of total cells and macrophages was significantly increased in the BALF of mice in the PHMG and PHMG + CS groups compared with that in the VC and CS only groups. In particular, the influx of neutrophil and lymphocyte was higher in the BALF of mice in the PHMG + CS group than in the PHMG group; however, the difference was not statically significant. We then evaluated the changes in the expression of inflammatory cytokines in the BALF and lung tissue. Numerous inflammatory cytokines trigger inflammation by recruiting immune cells to the site of injury, and they are associated with the subsequent development of fibrosis (Coker and Laurent, 1998; Bringardner et al., 2008; Wilson and Wynn, 2009; Borthwick et al., 2013). In the present study, the expression of inflammatory cytokines such as IL-6 and IL-1β was increased in the PHMG + CS group compared with that in the CS group. IL-6 is known to be released in the early stages of acute inflammation and is involved in the generation and diffusion of chronic inflammation by promoting the regulation of cytokine production by endothelial cells (Barnes et al., 2011). It has also been shown that bleomycin (BLM)-induced disruption of IL-6 in lung injury may contribute to a delay in the onset of the fibrotic stage (Kobayashi et al., 2015). IL-1β is also a major cytokine involved in the initiation and persistence of inflammation. Several studies using gene transfer in mice have shown that enhanced expression of IL-1β induces acute lung injury and leads to chronic fibrosis (Kolb et al., 2001; Ganter et al., 2008). In addition, IL-1β regulates the expression of the macrophage-derived cytokine CCL2 in the acute exacerbation of IPF (Schupp et al., 2015) and enhances the production of the neutrophil attractant CXCL1 (Lappalainen et al., 2005). Results from the present study showed that the expression of CCL2 was significantly increased in the lung tissue of mice exposed to PHMG or PHMG + CS. In contrast, the expression of CXCL1 was significantly increased in only the CS group with no significant changes observed in the PHMG or PHMG + CS groups. (Fig. 5D). Previous studies have reported that treatment with PHMG and BLM significantly increased the expression of CCL6 and CCL17 in the mouse lungs (Kim et al., 2018). CCL6 has been reported to play a key role in the pathogenesis of lung inflammation and tissue remodeling by IL-13 (Ma et al., 2004), and CCL17 is elevated in the lung tissue with pulmonary fibrosis and in IPF induced by BLM (Belperio et al., 2004). However, in our study, while CCL6 and CCL17 mRNA expression was increased in the CS only and/or the PHMG groups, neither were significantly increased in the PHMG + CS group. The histopathological analysis confirmed severe lung injury with inflammation and subsequent fibrosis induced by treatment with PHMG + CS, which enhanced the severity of granulomatous inflammation/fibrosis compared to the PHMG only group. The histopathological changes revealed that exposure to the combination of PHMG and CS enhanced the severity of granulomatous inflammation/fibrosis compared with the exposure to PHMG only. In addition, the accumulation of foamy macrophages in the alveoli was increased in the PHMG + CS group compared with that in the PHMG only group. These results revealed that the exposure to the combination of PHMG and CS promoted the development of severe lung injury, however, inflammatory cytokines may not be involved in the aggravated effects of repeated exposure to CS on PHMG-induced damage.

TGF-β signaling is one of the most potent inducers of ECM via the proliferation, differentiation, and migration of myofibroblasts (Verrecchia and Mauviel, 2002). TGF-β regulates ECM component deposition by controlling the expression of components in the ECM, such as fibrillar collagens and fibronectin, and stimulating the expression of protease inhibitors that prevent enzymatic breakdown of the ECM (Verrecchia and Mauviel, 2002). Remodeling of the ECM is an important pathway for wound healing, but the dysregulation of remodeling results in excessive accumulation of the ECM components, which is a hallmark of pulmonary fibrosis (Wynn, 2011). Among the three TGF-β isoforms, TGF-β1 is the most predominant and is expressed during pulmonary fibrosis (Baecher-Allan and Barth, 1993; Khalil et al., 1996; Coker et al., 1997), and transient overexpression of active TGF-β1 using vector-mediated gene transfer leads to progressive pulmonary fibrosis in the lungs of wild-type mice (Bonniaud et al., 2004). The results of this study revealed that the expression of TGF-β1 was significantly increased in the lung tissue of mice treated with PHMG. Furthermore, repeated exposure of PHMG-treated mice to CS upregulated the expression of TGF-β1 compared with exposure to PHMG or CS alone. In addition, increased levels of collagen and fibronectin after exposure to a combination of PHMG and CS were observed in the lung tissue, as determined by MT staining and western blotting, respectively.

The results of this study confirmed that pulmonary fibrosis induced by the instillation of PHMG is exacerbated by repeated exposure to CS. However, an inflammatory response was not associated with these aggravated effects under the present experimental conditions. We speculate that the induction of inflammation-related cells and mediators occurred early in the CS exposure (2 weeks) and rapidly activated the fibrosis mechanism.

In conclusion, the results of the current study indicated that repeated exposure of the lungs to CS could accelerate the development of pulmonary fibrosis induced by PHMG. However, inflammation-related pathogenesis of pulmonary fibrosis remains unclear. Therefore, additional experiments such as time-course studies are necessary to confirm the mechanism of aggravation of PHMG-induced pulmonary fibrosis by exposure to CS.

ACKNOWLEDGMENTS

The authors would like to especially thank the technical staff of the Inhalation Toxicology Research Group at KIT for their technical support. This work was supported by the Ministry of Food and Drug Safety [grant number 21203MFDS318] and the Korea Institute of Toxicology, Republic of Korea [grant number KK-1904].

Conflict of interest

The authors declare that there is no conflict of interest.

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