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Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in phosgene-induced lung injury
Biochemical and Biophysical Research Communications 514 (2019) 586e592
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in phosgene-induced lung injury Kaili Ye a, b, c, Daikun He a, b, c, Yiru Shao a, b, c, Ning Xu a, b, c, Chaoyuan Jin a, b, c, Lin Zhang a, b, c, Jie Shen a, b, c, * a b c
Department of Intensive Care Unit, Center of Emergency and Intensive Care Unit, Jinshan Hospital, Fudan University, Shanghai, China Department of Intensive Care Unit, Medical Research Center of Chemical Injury, Jinshan Hospital, Fudan University, Shanghai, China Department of Intensive Care Unit, Medical Center of Radiation Injury, Jinshan Hospital, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2019 Accepted 27 April 2019 Available online 4 May 2019
Exogenous mesenchymal stem cells (MSCs) affect lung cells via cytokines as well as vesicles and activate the Notch signaling pathway thus affecting the proliferation of endogenous stem cells to repair damaged tissue. Club cells are endogenous lung stem cells whose proliferation is also closely related to the Notch signaling pathway. The club cell secretory protein (CCSP) has anti-inflammatory and anti-oxidative properties. This study aimed to investigate whether exogenous MSCs affect the function of club cells in an injured lung and whether these effects are related to the Notch signaling pathway. CCSP levels in bronchoalveolar lavage fluid (BALF) and serum were evaluated using enzyme-linked immunosorbent assay (ELISA) and the average fluorescence intensity (AFI) of CCSP in club cells was determined using flow cytometry. Immunohistochemistry and immunofluorescence were used to visualize club cells and proliferative club cells. The expression of important Notch signaling pathway components including Notch1~4, c-myc, Hey1 and Hes1 were also assessed. LY3039478 (LY), a specific inhibitor of the Notch signaling pathway, was applied. After MSCs intervention, CCSP levels decreased, and club cell AFI increased, indicating that the secretion of club cells had weakened. The expression of Notch1, Notch2, cmyc, Hey1, Hes1 increased, accompanied by an increase in the number of proliferative club cells. Furthermore, MSCs enhanced the proliferation of club cells, while LY suppressed this phenomenon. In summary, MSCs reduced the secretion of club cells. And MSCs enhanced the proliferation of club cells partly via activating the Notch signaling pathway, which promoted lung injury repair. © 2019 Elsevier Inc. All rights reserved.
Keywords: Mesenchymal stem cell Club cell Club cell secretory protein Notch signaling pathway Lung injury
1. Introduction Phosgene is a lively, colorless, toxic gas. Exposure to phosgene can cause acute lung injury (ALI) and even acute respiratory distress syndrome (ARDS). Phosgene inhalation can cause damage of lung epithelial/endothelial cells and inflammatory response [1]. The mortality rate of phosgene-induced lung injury, which has no current treatment, is high. Researchers have discovered a variety of endogenous lung stem cells. Endogenous stem cells play an important role in endogenous
* Corresponding author. Center of Emergency & Intensive Care Unit, Medical Center of Chemical Injury and Medical Research Centre for Chemical Injury, Emergency and Critical Care, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai, 201508, PR China. E-mail address: [email protected] (J. Shen). https://doi.org/10.1016/j.bbrc.2019.04.182 0006-291X/© 2019 Elsevier Inc. All rights reserved.
repair of lung injury. Club cells are a type of endogenous stem cells found in the lungs, mainly in the terminal and respiratory bronchioles. Under normal circumstances, in humans, club cells account for 11% and 22% of the terminal and respiratory bronchiole epithelium, respectively, and for 15% and 44% of the number of proliferating cells in these regions [2]. In mice, the distal airway epithelium is mainly composed of cells expressing CCSP, and the number of CCSP-positive cells may exceed 80%. Additionally, club cells are cuboid cells that contain secretory vesicles. Club cell secretory protein (CCSP) is a specific secretory protein present in the cytoplasm and cell membrane of club cells and is the most abundant protein in airway surface fluid. Therefore, CCSP can be used as a marker to identify club cells [3]. Club cells have anti-inflammatory effects and have been shown to be regenerated in lung injury, repairing damaged lung tissue [4]. A study showed that club cells improved inflammation in silica-induced lung injury [5]. Other studies found that club cells
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played a key role in host defense, airway barrier, protein secretion, and substance metabolism [6]. CCSP has anti-inflammatory and anti-oxidant effects [3] while serum CCSP levels are a sensitive indicator of lung injury [7,8]. According to pathological features of ALI/ARDS, the control of inflammation and repair of lung epithelial/endothelial cells are crucial for treatment. Club cells were chosen as our research focus for two reasons. On the one hand, CCSP secreted by club cells plays a role in regulating local inflammation [3], and serum CCSP can be used as an indicator for monitoring lung injury repair. On the other hand, club cells, as a type of stem cells, can self-proliferate in damaged lung tissue and restore the integrity of the airway epithelium. In addition, club cells can differentiate into ciliated cells and alveolar epithelial cells [4,9]. MSCs-based therapy is a novel treatment for lung injury as the protective effects of MSCs on damaged lung tissue have been preliminarily studied. Differentiation, immune regulation, and paracrine signaling of MSCs all have positive effects on lung tissue repair [10]. Our previous study found that bone marrow-derived MSCs improved lung inflammation and lung permeability in a phosgeneinduced lung injury model [11]. Other studies demonstrated that exogenous MSCs affected lung cells (including intravascular endothelial cells, T lymphocytes, B lymphocytes and macrophages) via cell contact or secretory vesicles. In an animal model of bronchopulmonary dysplasia, MSCs promoted the proliferation of bronchoalveolar stem cells (a type of lung endogenous stem cell that can differentiate into club cells) and contributed to the reconstruction of lung epithelial structure [12]. In addition to this study, few previous studies have investigated whether exogenous MSCs have an effect on lung endogenous stem cells. The Notch signaling pathway plays a key role in cell differentiation, proliferation and apoptosis. In mammals, the Notch signaling pathway contains four cognate receptors (Notch1, Notch2, Notch3 and Notch4) that bind to ligands to produce different biological effects. After binding of the extracellular domain of Notch receptors to ligands, cleavage of the Notch receptor is induced, releasing the Notch intracellular domain (NICD). A literature review found that the expression of constituent proteins and downstream genes in the Notch signaling pathway in damaged lung tissues varied [13,14]. In a lung injury model induced by smoke inhalation, MSCs promoted angiogenesis and Notch1 expression in lung tissue [15]. The proliferation and differentiation of MSCs [16] and club cells [17] are closely related to the Notch signaling pathway. In summary, the internal and external repair processes of lung tissue are inextricably linked to the Notch signaling pathway. Based on these findings, we believe that MSCs are highly likely to affect club cells via the Notch signaling pathway in the injured lung tissue. This study explored whether exogenous MSCs affected club cells in a phosgene-induced lung injury model, and whether an effect on proliferation was achieved through the Notch signaling pathway. 2. Materials and methods 2.1. Animals Animal procedures followed the guidelines for laboratory animal management and use of China. And the experimental protocol was approved by the Ethics Committee of Jinshan Hospital affiliated to Fudan University. A total of 108 SPF grade male SD rats weighing 180e220 g each, provided by the Experimental Animal Center of Naval Medical University, Shanghai, China were used. The rats were housed at a constant temperature and with independent ventilation systems (12:12 h light and dark cycle). The rats were allowed to move freely and eat normally during the experiment. The rats were anesthetized through intraperitoneal injection of 20% carbamate
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solution (dose 1.0 g/kg), and were sacrificed at 6, 24 and 48 h after phosgene exposure. 2.2. Phosgene-induced lung injury Phosgene was prepared as described previously [18]. The final concentration of phosgene in the poisoning chamber was 8.33 g/ m3. Animals were exposed to phosgene for 5 min and then transferred to a room with fresh air. 2.3. Mesenchymal stem cells Bone marrow-derived mesenchymal stem cells of male SD rats (KALANG Technology Co., Ltd., Shanghai, China) were cultured in a 37 C incubator (5% CO2). After 24 h, MSCs were completely adherent. The culture solution was changed and culture was continued. When MSCs substantially covered the surface of the flask, trypsin was added to digest intercellular proteins. MSCs were then transferred into a centrifuge tube. After centrifugation, the supernatant was discarded. The MSCs were resuspended by adding a certain amount of PBS. The number of cells was adjusted to 1 106 per 50 mL PBS. The resuspended MSCs were immediately instilled into the lungs via the trachea. 2.4. Specific inhibitor of the Notch signaling pathway LY3039478 (LY; Selleck, Houston, USA), a potent Notch signaling pathway-specific inhibitor of intracellular Notch receptor domain cleavage, was used. LY was dissolved in a solvent of DMSO, polyethylene glycol, and double distilled water (volume ratio of 2:20:78) and a solution at a concentration of 4 mg/mL was prepared. For the groups treated with the inhibitor, LY solution was intraperitoneally injected 2 h before phosgene exposure at a dose of 10 mg/kg. 2.5. Experimental grouping Rats were randomly divided into the following six groups (n ¼ 6 per group at each time point): (1) control group (Air), rats always exposed to fresh air; (2) phosgene exposure group (PH), rats exposed to phosgene for 5 min, then transferred into fresh air immediately; (3) phosgene þ PBS group (PH þ PBS) rats exposed to phosgene for 5 min, then transferred to fresh air immediately, and 50 mL PBS (without MSCs) slowly instilled into the trachea after anesthesia; (4) phosgene þ MSCs group (PH þ MSCs) rats exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) slowly instilled into the trachea after anesthesia; (5) phosgene þ MSCs þ solvent group (PH þ MSCs þ SO) rats intraperitoneally pre-injected with a certain volume of the mixed solvent (i.e. mixed solution of DMSO, polyethylene glycol, double distilled water), according to the weight of each rat 2 h before phosgene exposure. The rats were exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) was slowly instilled into the trachea after anesthesia; (6) phosgene þ MSCs þ inhibitor group (PH þ MSCs þ LY) rats intraperitoneally pre-injected with a certain volume of LY solution (10 mg/kg) 2 h before phosgene exposure. The rats were exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) into the trachea after anesthesia. 2.6. Immunohistochemistry The middle lobes of the rats were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4e5 mm slices.
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The tissue was then dewaxed and hydrated. And endogenous peroxidase was blocked. The antigen was then repaired, exposed and non-specific binding sites were blocked. After adding the primary antibody (CCSP, 1:400, Santa Cruz, Heidelberg, Germany), the tissue was placed in a wet box at 4 C, overnight. A secondary antibody was added and re-staining of nuclei was executed with hematoxylin stain. The tissue was finally observed under an optical microscope (Olympus, Tokyo, Japan).
(diameter range: 100e200 mm) were selected from each slide. The number of CCSP and Ki-67 double positive cells and the total number of nuclei of tracheal epithelial cells were counted. For each section, the ratio of CCSP and Ki-67 double positive cells were calculated by dividing the average number of CCSP and Ki-67 double positive cells by the average number of nuclei.
2.7. Immunofluorescence 2.8. Enzyme linked immunosorbent assay After the tissue slices were dewaxed and hydrated, the antigen was repaired and exposed. Primary antibodies (CCSP, 1:500, Abcam, Cambridge, UK; Ki-67, 1:300, Bioss, Beijing, China) were employed and the tissue was incubated in a wet box at 4 C, overnight. The slides were washed and the secondary antibodies (Servicebio, Wuhan, China) were added and the tissue was incubated for 1 h at 20 C. DAPI was used to dye the nuclei. Lung tissue was observed under a fluorescence microscope (Nikon, Tokyo, Japan). The specific method was as follows: under 400-fold magnification, on each slice, six tracheal cross sections of a similar size and shape
The CCSP levels in BALF and serum were tested. After anesthetization, 6 mL of blood was collected from each rat. The left lung of the rat was ligated and after tracheal intubation, 3 mL of 4 C PBS was used to repeatedly lavage the left lobe three times. More than 80% of the lavage fluid was recovered. BALF and blood were centrifuged at 1500 rpm for 10 min at 4 C, and the supernatant was retained. CCSP levels in BALF and serum were determined using a rat CCSP ELISA kit (KALANG Technology Co., Ltd., Shanghai, China), according to the manufacturer's recommended protocol.
Fig. 1. CCSP levels and the average fluorescence intensity (AFI) of CCSP in club cells. CCSP levels in BALF (A) and serum (B) and CCSP AFI in club cells (C) in the lower lobe were shown. Results were expressed as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ## p < 0.01, compared with the PH group; & p < 0.05, && p < 0.01, compared with the PH þ PBS group.
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2.9. Real-time reverse transcription-polymerase chain reaction
3. Results
An appropriate amount of tissue was finely ground. An RNA extraction solution was created using chloroform, isopropanol and 75% ethanol. The concentration and purity of RNA was detected and the RNA and primers were used to perform reverse transcription and obtain cDNA, which was used to prepare a reaction system for amplification. Using the DDCt method normalized with GAPDH, relative expression levels of genes were determined. The primer sequences used were listed in the supplemental table.
3.1. MSCs affected the secretion of club cells
2.10. Flow cytometry The AFI of club cells was measured in this experiment. The entire left inferior lobe was collected from treated rats and 2 mL of PBS was added. The tissue was then ground thoroughly to prepare a single-cell suspension. Diluted primary antibody (CCSP, 1:100, Santa Cruz, Heidelberg, Germany) was added and the sample was incubated overnight at 4 C. After washing, the mixture was incubated with the secondary antibody (Biyuntian, Shanghai, China) at 4 C for 1 h in the dark. The sample was then centrifuged at 1500 rpm for 10 min; the supernatant was removed, and resuspended in PBS after washing. Each sample was tested by flow cytometer (BD, Franklin Lakes, USA).
2.11. Statistical analysis All data in this experiment were analyzed using GraphPad (version 5.0). Analysis of variance (ANOVA) was used to detect whether the differences were statistically significant, while unpaired t-test was used to compare differences between two groups. P < 0.05 was considered to be statistically significant.
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This study evaluated whether the secretion of club cells was changed after phosgene exposure and MSCs intervention. The results showed that there was no significant difference in CCSP expression among the lung tissue from four treatment group (Air, PH, PH þ PBS and PH þ MSCs groups) at 6, 24 and 48 h (negative results are not shown). At 6, 24 and 48 h, compared to the Air group, BALF and serum CCSP levels of the PH group increased. However, after the MSCs intervention (PH þ MSCs group) BALF and serum CCSP levels decreased, compared with that of the PH group (Fig. 1A). At 6, 24 and 48 h, compared to the PH and PH þ PBS groups, club cells were dark brown as shown through immunohistochemistry and the AFI of the club cells was enhanced as shown through flow cytometry in the PH þ MSCs group (Figs. 1B and 2). These results indicated that MSCs intervention weakened the secretion of club cells in the lung tissue exposed to phosgene. 3.2. MSCs affected the proliferation of club cells Changes in the proliferation of club cells were evaluated after phosgene exposure and MSCs intervention. We found that compared to the Air group, club cells in the tracheas were damaged and fell into the tracheal cavities after phosgene exposure at 6, 24 and 48 h. At 48 h, compared with the PH and PH þ PBS groups, the number of club cells in the tracheal cavities of the PH þ MSCs group were significantly decreased (Fig. 2). As shown with immunofluorescence double labelling, at 6, 24 and 48 h, the numbers of proliferative club cells (i.e., CCSP and Ki-67 double positive cells) in the middle lobe of the PH group rats increased compared with that of the Air group. Compared to the PH and PH þ PBS groups, the
Fig. 2. Immunohistochemistry of the middle lobe of the lung. Immunohistochemistry results with CCSP as the antibody were shown. Brown detached club cells were visible (red sun markers) in the tracheal lumens. Scale bar: 50 mm. Magnification: 400x. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 3. Immunofluorescence of the middle lobe and the number of CCSP and Ki-67 double positive cells. The immunofluorescence results (D) were shown. Antibodies used were Ki-67 (red channel) and CCSP (green channel). Proliferative cells (red arrow), club cells (green arrow), and proliferative club cells (yellow arrow) were labeled in the figure. DAPI (blue channel) was used to locate cell nuclei. Scale bar: 50 mm. Magnification: 400x. The analysis of CCSP and Ki-67 double positive cells was shown (E). Results are expressed as mean ± SD (n ¼ 6). Statistical differences are indicated as follows: ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the PH group; & p < 0.05, && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$$ p < 0.001, compared with the PH þ MSCs group; @@@ p < 0.001, compared with the PH þ MSCs þ SO group. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
number of proliferative club cells in the PH þ MSCs group further increased (Fig. 3C and D). These results indicated that in phosgeneinduced lung injury, MSCs intervention enhanced the proliferation of club cells. 3.3. Activation of the Notch signaling pathway The expression of important components of the Notch signaling pathway (including Notch1~4, c-myc, Hey1 and Hes1) in lung tissue of each group was monitored. It was shown that at 6, 24 and 48 h, compared to the Air group, the expression of Notch1, c-myc, Hey1 and Hes1 increased, the expression of Notch1 decreased and the expression of Notch3 and Notch4 did not change significantly. Compared with the PH and PH þ PBS groups, the expression of Notch1, Notch2, c-myc, Hey1 and Hes1 of lung tissue in the PH þ MSCs group increased at 6, 24 and 48 h. It suggested that the Notch signaling pathway in lung tissue was further activated after MSCs intervention. At 6, 24 and 48 h, the number of proliferative club cells in the PH þ MSCs group further increased compared to the PH and PH þ PBS groups (Figs. 3 and 4). These results indicated that MSCs might promote the proliferation of club cells through the Notch signaling pathway.
3.4. Application of Notch signaling pathway inhibitor We further validated whether changes in the proliferation of club cells were associated with the Notch signaling pathway by applying LY, a specific Notch signaling pathway-specific inhibitor. At 6, 24 and 48 h, the expression of Notch1, Notch2, c-myc, Hey1, and Hes1 in injured lung tissue decreased after the application of LY, indicating that the effect of LY was positive and as expected (Fig. 4). Compared to the PH þ MSCs and PH þ MSCs þ SO groups, the number of proliferative club cells in the PH þ MSCs þ LY group decreased significantly (Fig. 3). It indicated that the MSCs-induced proliferation of club cells was weakened by LY. These results indicated that in phosgene-induced lung injury, MSCs at least partially activated the Notch signaling pathway to further enhance the proliferation of club cells. 4. Discussion Our results showed that in phosgene-induced lung injury, the intervention of exogenous MSCs reduced the secretion and enhanced the proliferation of club cells. MSCs enhanced the proliferation of club cells by further activating the Notch signaling
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Fig. 4. Expression of major constituent proteins in the Notch signaling pathway. The expression of Notch1, Notch2, c-myc, Hey1 and Hes1 were shown. Results were expressed as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; ## p < 0.01, ### p < 0.001, compared with the PH group; && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$ p < 0.01, $$$ p < 0.001, compared with the PH þ MSCs group; @@ p < 0.01, @@@ p < 0.001, compared with the PH þ MSCs þ SO group.
pathway, thus promoting the repair of injured tissue. In our experiment, MSCs attenuated the secretion of club cells. This might be due to the anti-inflammatory, anti-oxidative cytokines (similar to CCSP) secreted by MSCs, resulting in a reduced “demand” for CCSP for the repair of lung injury. Under normal circumstances, a small quantity of CCSP in the airways enters the bloodstream due to the presence of the blood-gas barrier in the lung. In lung damage caused by phosgene, more CCSP entered blood circulation due to increased secretion of CCSP and destruction of lung barrier function, resulting in elevated CCSP levels in serum. After MSCs intervention, the decrease in serum CCSP was due to a reduction of BALF-derived CCSP and MSCs-induced improvement in lung permeability [11]. Therefore, to some extent, the decrease in serum CCSP indicated the repairing effect of MSCs on lung injury. Studies have shown that CCSP is a good biomarker of epithelial damage [19]. After exposure to asbestos [20] and ozone [21], CCSP levels in serum were often associated with lung epithelial damage. In asthmatic patients, serum CCSP was associated with the involvement of small airway [22]. CCSP levels can indicate a change in lung permeability and are associated with treatment outcomes. In this experiment, serum CCSP changed after phosgene exposure and MSCs intervention. However, whether CCSP would be a good biomarker in phosgene-induced lung injury requires further investigation. We used lung homogenates (including all lung interstitial cells and airway epithelial cells) to assess changes in the Notch signaling pathway. Our results showed that club cells proliferated in response to the activation of the Notch signaling pathway. However, we were unable to determine whether the changes in the Notch signaling pathway were restricted to the airway epithelium. In future studies, isolation and in vitro culture of relevant cells will help to further evaluate evidence. Co-culture of club cells with
MSCs in vitro will further reveal the effects of MSCs on club cells with increased accuracy. Endogenous stem cells are greatly advantageous for directional differentiation into damaged cells in the repair of lung injury, but endogenous repair is often difficult to regulate. Moreover, the lung injury microenvironment where endogenous stem cells are located can be changed and then repair cannot be guaranteed. Studies have found that MSCs played a role in exogenous damage repair through paracrine, differentiation and other methods [10]; control of cell number and cell function is better. Both internal and external repair have their own strengths. If these two repair methods are combined, better treatment results can be obtained. Previously, a few studies regarding the effects of exogenous stem cells on endogenous stem cells have been published but the mechanism involved has never been explored. Our study found that MSCs instilled in the airways affected endogenous club cells in multiple ways (including changes in secretion and proliferation). This influence on the proliferation was related to the changes of the Notch signaling pathway. In the future, key regulators related to the Notch signaling pathway can be overexpressed in MSCs, promoting the function of club cells and further enhancing the repair of lung injury, and thereby increasing clinical efficacy. This research examined the link between endogenous and exogenous repair pathways in the lungs, which may help clinical innovation and may provide a new direction for the treatment of acute lung injury.
Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81471850), Shanghai Public Health System (2017-1) and the Research project of Shanghai Health and Family Planning Commission (No. JSZK2015A01). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.182. References [1] J. Borak, W.F. Diller, Phosgene exposure: mechanisms of injury and treatment strategies, J. Occup. Environ. Med. 43 (2001) 110e119. [2] J.E. Boers, A.W. Ambergen, F.B. Thunnissen, Number and proliferation of clara cells in normal human airway epithelium, Am. J. Respir. Crit. Care Med. 159 (1999) 1585e1591. [3] A.P. Wong, A. Keating, T.K. Waddell, Airway regeneration: the role of the Clara cell secretory protein and the cells that express it, Cytotherapy 11 (2009) 676e687. [4] S.D. Reynolds, A.M. Malkinson, Clara cell: progenitor for the bronchiolar epithelium, Int. J. Biochem. Cell Biol. 42 (2010) 1e4. [5] K. Yatera, Y. Morimoto, H.N. Kim, et al., Increased expression of matrix metalloproteinase in Clara cell-ablated mice inhaling crystalline silica, Environ. Health Perspect. 109 (2001) 795e799. [6] C.H. Dean, R.J. Snelgrove, New rules for club development: new insights into human small airway epithelial Club cell ontogeny and function, Am. J. Respir. Crit. Care Med. 198 (2018) 1355e1356. [7] H. Stenberg, E. Wadelius, S. Moitra, et al., Club cell protein (CC16) in plasma, bronchial brushes, BAL and urine following an inhaled allergen challenge in allergic asthmatics, Biomarkers 23 (2018) 51e60. [8] J.A. Kropski, R.D. Fremont, C.S. Calfee, et al., Clara cell protein (CC16), a marker of lung epithelial injury, is decreased in plasma and pulmonary edema fluid from patients with acute lung injury, Chest 135 (2009) 1440e1447. [9] D. Zheng, B. Soh, L. Yin, et al., Differentiation of Club cells to alveolar epithelial
cells in vitro, Sci. Rep. 7 (2017) 41661. [10] M. Hayes, G. Curley, B. Ansar, et al., Clinical review: stem cell therapies for acute lung injury/acute respiratory distress syndrome - hope or hype? Crit. Care 16 (2012) 205. [11] J. Chen, Y. Shao, G. Xu, et al., Bone marrow-derived mesenchymal stem cells attenuate phosgene-induced acute lung injury in rats, Inhal. Toxicol. 27 (2015) 254e261. [12] K.A. Tropea, E. Leder, M. Aslam, et al., Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia, Am. J. Physiol. Lung Cell Mol. Physiol. 302 (2012) 829e837. [13] H. Han, G. Gong, X. Bai, et al., Inhibition of notch signaling protects mouse lung against zymosan-induced injury, Shock 40 (2013) 312e319. [14] M.D. McGraw, J.S. Rioux, R.B. Garlick, et al., From the cover: impaired proliferation and differentiation of the conducting airway epithelium associated with bronchiolitis obliterans after sulfur mustard inhalation injury in rats, Toxicol. Sci. 157 (2017) 399e409. [15] F. Zhu, J. Wang, X. Qiu, et al., Smoke inhalation injury repaired by a bone marrow-derived mesenchymal stem cell paracrine mechanism: angiogenesis involving the Notch signaling pathway, J. Trauma Acute Care Surg. 78 (2015) 565e572. [16] J.M. Díaz-Tocados, C. Herencia, J.M. Martínez-Moreno, et al., Magnesium chloride promotes osteogenesis through Notch signaling activation and expansion of mesenchymal stem cells, Sci. Rep. 7 (2017) 7839. [17] Y. Xing, A. Li, Z. Borok, et al., NOTCH1 is required for regeneration of Clara cells during repair of airway lnjury, Stem Cell. 30 (2012) 946e955. [18] D. He, Y. Shao, L. Zhang, et al., Adenovirus-delivered angiopoietin-1 suppresses NF-kB and p38 MAPK and attenuates inflammatory responses in phosgene-induced acute lung injury, Inhal. Toxicol. 26 (2014) 185e192. [19] R. Helleday, B. Segerstedt, B. Forsberg, et al., Exploring the time dependence of serum clara cell protein as a biomarker of pulmonary injury in humans, Chest 130 (2006) 672e675. [20] M. Petrek, C. Hermans, V. Kolek, et al., Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos, Biomarkers 7 (2002) 58e67. [21] F. Broeckaert, A. Clippe, R. Wattiez, et al., Lung hyperpermeability, Clara-cell secretory potein (CC16), and susceptibility to ozone of five inbred strains of mice, Inhal. Toxicol. 15 (2003) 1209e1230. [22] S. Bommart, G. Marin, N. Molinari, et al., Club cell secretory protein serum concentration is a surrogate marker of small-airway involvement in asthmatic patients, J. Allergy Clin. Immunol. 140 (2017) 581e584.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in phosgene-induced lung injury Kaili Ye a, b, c, Daikun He a, b, c, Yiru Shao a, b, c, Ning Xu a, b, c, Chaoyuan Jin a, b, c, Lin Zhang a, b, c, Jie Shen a, b, c, * a b c
Department of Intensive Care Unit, Center of Emergency and Intensive Care Unit, Jinshan Hospital, Fudan University, Shanghai, China Department of Intensive Care Unit, Medical Research Center of Chemical Injury, Jinshan Hospital, Fudan University, Shanghai, China Department of Intensive Care Unit, Medical Center of Radiation Injury, Jinshan Hospital, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2019 Accepted 27 April 2019 Available online 4 May 2019
Exogenous mesenchymal stem cells (MSCs) affect lung cells via cytokines as well as vesicles and activate the Notch signaling pathway thus affecting the proliferation of endogenous stem cells to repair damaged tissue. Club cells are endogenous lung stem cells whose proliferation is also closely related to the Notch signaling pathway. The club cell secretory protein (CCSP) has anti-inflammatory and anti-oxidative properties. This study aimed to investigate whether exogenous MSCs affect the function of club cells in an injured lung and whether these effects are related to the Notch signaling pathway. CCSP levels in bronchoalveolar lavage fluid (BALF) and serum were evaluated using enzyme-linked immunosorbent assay (ELISA) and the average fluorescence intensity (AFI) of CCSP in club cells was determined using flow cytometry. Immunohistochemistry and immunofluorescence were used to visualize club cells and proliferative club cells. The expression of important Notch signaling pathway components including Notch1~4, c-myc, Hey1 and Hes1 were also assessed. LY3039478 (LY), a specific inhibitor of the Notch signaling pathway, was applied. After MSCs intervention, CCSP levels decreased, and club cell AFI increased, indicating that the secretion of club cells had weakened. The expression of Notch1, Notch2, cmyc, Hey1, Hes1 increased, accompanied by an increase in the number of proliferative club cells. Furthermore, MSCs enhanced the proliferation of club cells, while LY suppressed this phenomenon. In summary, MSCs reduced the secretion of club cells. And MSCs enhanced the proliferation of club cells partly via activating the Notch signaling pathway, which promoted lung injury repair. © 2019 Elsevier Inc. All rights reserved.
Keywords: Mesenchymal stem cell Club cell Club cell secretory protein Notch signaling pathway Lung injury
1. Introduction Phosgene is a lively, colorless, toxic gas. Exposure to phosgene can cause acute lung injury (ALI) and even acute respiratory distress syndrome (ARDS). Phosgene inhalation can cause damage of lung epithelial/endothelial cells and inflammatory response [1]. The mortality rate of phosgene-induced lung injury, which has no current treatment, is high. Researchers have discovered a variety of endogenous lung stem cells. Endogenous stem cells play an important role in endogenous
* Corresponding author. Center of Emergency & Intensive Care Unit, Medical Center of Chemical Injury and Medical Research Centre for Chemical Injury, Emergency and Critical Care, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai, 201508, PR China. E-mail address: [email protected] (J. Shen). https://doi.org/10.1016/j.bbrc.2019.04.182 0006-291X/© 2019 Elsevier Inc. All rights reserved.
repair of lung injury. Club cells are a type of endogenous stem cells found in the lungs, mainly in the terminal and respiratory bronchioles. Under normal circumstances, in humans, club cells account for 11% and 22% of the terminal and respiratory bronchiole epithelium, respectively, and for 15% and 44% of the number of proliferating cells in these regions [2]. In mice, the distal airway epithelium is mainly composed of cells expressing CCSP, and the number of CCSP-positive cells may exceed 80%. Additionally, club cells are cuboid cells that contain secretory vesicles. Club cell secretory protein (CCSP) is a specific secretory protein present in the cytoplasm and cell membrane of club cells and is the most abundant protein in airway surface fluid. Therefore, CCSP can be used as a marker to identify club cells [3]. Club cells have anti-inflammatory effects and have been shown to be regenerated in lung injury, repairing damaged lung tissue [4]. A study showed that club cells improved inflammation in silica-induced lung injury [5]. Other studies found that club cells
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played a key role in host defense, airway barrier, protein secretion, and substance metabolism [6]. CCSP has anti-inflammatory and anti-oxidant effects [3] while serum CCSP levels are a sensitive indicator of lung injury [7,8]. According to pathological features of ALI/ARDS, the control of inflammation and repair of lung epithelial/endothelial cells are crucial for treatment. Club cells were chosen as our research focus for two reasons. On the one hand, CCSP secreted by club cells plays a role in regulating local inflammation [3], and serum CCSP can be used as an indicator for monitoring lung injury repair. On the other hand, club cells, as a type of stem cells, can self-proliferate in damaged lung tissue and restore the integrity of the airway epithelium. In addition, club cells can differentiate into ciliated cells and alveolar epithelial cells [4,9]. MSCs-based therapy is a novel treatment for lung injury as the protective effects of MSCs on damaged lung tissue have been preliminarily studied. Differentiation, immune regulation, and paracrine signaling of MSCs all have positive effects on lung tissue repair [10]. Our previous study found that bone marrow-derived MSCs improved lung inflammation and lung permeability in a phosgeneinduced lung injury model [11]. Other studies demonstrated that exogenous MSCs affected lung cells (including intravascular endothelial cells, T lymphocytes, B lymphocytes and macrophages) via cell contact or secretory vesicles. In an animal model of bronchopulmonary dysplasia, MSCs promoted the proliferation of bronchoalveolar stem cells (a type of lung endogenous stem cell that can differentiate into club cells) and contributed to the reconstruction of lung epithelial structure [12]. In addition to this study, few previous studies have investigated whether exogenous MSCs have an effect on lung endogenous stem cells. The Notch signaling pathway plays a key role in cell differentiation, proliferation and apoptosis. In mammals, the Notch signaling pathway contains four cognate receptors (Notch1, Notch2, Notch3 and Notch4) that bind to ligands to produce different biological effects. After binding of the extracellular domain of Notch receptors to ligands, cleavage of the Notch receptor is induced, releasing the Notch intracellular domain (NICD). A literature review found that the expression of constituent proteins and downstream genes in the Notch signaling pathway in damaged lung tissues varied [13,14]. In a lung injury model induced by smoke inhalation, MSCs promoted angiogenesis and Notch1 expression in lung tissue [15]. The proliferation and differentiation of MSCs [16] and club cells [17] are closely related to the Notch signaling pathway. In summary, the internal and external repair processes of lung tissue are inextricably linked to the Notch signaling pathway. Based on these findings, we believe that MSCs are highly likely to affect club cells via the Notch signaling pathway in the injured lung tissue. This study explored whether exogenous MSCs affected club cells in a phosgene-induced lung injury model, and whether an effect on proliferation was achieved through the Notch signaling pathway. 2. Materials and methods 2.1. Animals Animal procedures followed the guidelines for laboratory animal management and use of China. And the experimental protocol was approved by the Ethics Committee of Jinshan Hospital affiliated to Fudan University. A total of 108 SPF grade male SD rats weighing 180e220 g each, provided by the Experimental Animal Center of Naval Medical University, Shanghai, China were used. The rats were housed at a constant temperature and with independent ventilation systems (12:12 h light and dark cycle). The rats were allowed to move freely and eat normally during the experiment. The rats were anesthetized through intraperitoneal injection of 20% carbamate
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solution (dose 1.0 g/kg), and were sacrificed at 6, 24 and 48 h after phosgene exposure. 2.2. Phosgene-induced lung injury Phosgene was prepared as described previously [18]. The final concentration of phosgene in the poisoning chamber was 8.33 g/ m3. Animals were exposed to phosgene for 5 min and then transferred to a room with fresh air. 2.3. Mesenchymal stem cells Bone marrow-derived mesenchymal stem cells of male SD rats (KALANG Technology Co., Ltd., Shanghai, China) were cultured in a 37 C incubator (5% CO2). After 24 h, MSCs were completely adherent. The culture solution was changed and culture was continued. When MSCs substantially covered the surface of the flask, trypsin was added to digest intercellular proteins. MSCs were then transferred into a centrifuge tube. After centrifugation, the supernatant was discarded. The MSCs were resuspended by adding a certain amount of PBS. The number of cells was adjusted to 1 106 per 50 mL PBS. The resuspended MSCs were immediately instilled into the lungs via the trachea. 2.4. Specific inhibitor of the Notch signaling pathway LY3039478 (LY; Selleck, Houston, USA), a potent Notch signaling pathway-specific inhibitor of intracellular Notch receptor domain cleavage, was used. LY was dissolved in a solvent of DMSO, polyethylene glycol, and double distilled water (volume ratio of 2:20:78) and a solution at a concentration of 4 mg/mL was prepared. For the groups treated with the inhibitor, LY solution was intraperitoneally injected 2 h before phosgene exposure at a dose of 10 mg/kg. 2.5. Experimental grouping Rats were randomly divided into the following six groups (n ¼ 6 per group at each time point): (1) control group (Air), rats always exposed to fresh air; (2) phosgene exposure group (PH), rats exposed to phosgene for 5 min, then transferred into fresh air immediately; (3) phosgene þ PBS group (PH þ PBS) rats exposed to phosgene for 5 min, then transferred to fresh air immediately, and 50 mL PBS (without MSCs) slowly instilled into the trachea after anesthesia; (4) phosgene þ MSCs group (PH þ MSCs) rats exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) slowly instilled into the trachea after anesthesia; (5) phosgene þ MSCs þ solvent group (PH þ MSCs þ SO) rats intraperitoneally pre-injected with a certain volume of the mixed solvent (i.e. mixed solution of DMSO, polyethylene glycol, double distilled water), according to the weight of each rat 2 h before phosgene exposure. The rats were exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) was slowly instilled into the trachea after anesthesia; (6) phosgene þ MSCs þ inhibitor group (PH þ MSCs þ LY) rats intraperitoneally pre-injected with a certain volume of LY solution (10 mg/kg) 2 h before phosgene exposure. The rats were exposed to phosgene for 5 min, then transferred into fresh air immediately, and 50 mL PBS (containing 1 106 MSCs) into the trachea after anesthesia. 2.6. Immunohistochemistry The middle lobes of the rats were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4e5 mm slices.
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The tissue was then dewaxed and hydrated. And endogenous peroxidase was blocked. The antigen was then repaired, exposed and non-specific binding sites were blocked. After adding the primary antibody (CCSP, 1:400, Santa Cruz, Heidelberg, Germany), the tissue was placed in a wet box at 4 C, overnight. A secondary antibody was added and re-staining of nuclei was executed with hematoxylin stain. The tissue was finally observed under an optical microscope (Olympus, Tokyo, Japan).
(diameter range: 100e200 mm) were selected from each slide. The number of CCSP and Ki-67 double positive cells and the total number of nuclei of tracheal epithelial cells were counted. For each section, the ratio of CCSP and Ki-67 double positive cells were calculated by dividing the average number of CCSP and Ki-67 double positive cells by the average number of nuclei.
2.7. Immunofluorescence 2.8. Enzyme linked immunosorbent assay After the tissue slices were dewaxed and hydrated, the antigen was repaired and exposed. Primary antibodies (CCSP, 1:500, Abcam, Cambridge, UK; Ki-67, 1:300, Bioss, Beijing, China) were employed and the tissue was incubated in a wet box at 4 C, overnight. The slides were washed and the secondary antibodies (Servicebio, Wuhan, China) were added and the tissue was incubated for 1 h at 20 C. DAPI was used to dye the nuclei. Lung tissue was observed under a fluorescence microscope (Nikon, Tokyo, Japan). The specific method was as follows: under 400-fold magnification, on each slice, six tracheal cross sections of a similar size and shape
The CCSP levels in BALF and serum were tested. After anesthetization, 6 mL of blood was collected from each rat. The left lung of the rat was ligated and after tracheal intubation, 3 mL of 4 C PBS was used to repeatedly lavage the left lobe three times. More than 80% of the lavage fluid was recovered. BALF and blood were centrifuged at 1500 rpm for 10 min at 4 C, and the supernatant was retained. CCSP levels in BALF and serum were determined using a rat CCSP ELISA kit (KALANG Technology Co., Ltd., Shanghai, China), according to the manufacturer's recommended protocol.
Fig. 1. CCSP levels and the average fluorescence intensity (AFI) of CCSP in club cells. CCSP levels in BALF (A) and serum (B) and CCSP AFI in club cells (C) in the lower lobe were shown. Results were expressed as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ## p < 0.01, compared with the PH group; & p < 0.05, && p < 0.01, compared with the PH þ PBS group.
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2.9. Real-time reverse transcription-polymerase chain reaction
3. Results
An appropriate amount of tissue was finely ground. An RNA extraction solution was created using chloroform, isopropanol and 75% ethanol. The concentration and purity of RNA was detected and the RNA and primers were used to perform reverse transcription and obtain cDNA, which was used to prepare a reaction system for amplification. Using the DDCt method normalized with GAPDH, relative expression levels of genes were determined. The primer sequences used were listed in the supplemental table.
3.1. MSCs affected the secretion of club cells
2.10. Flow cytometry The AFI of club cells was measured in this experiment. The entire left inferior lobe was collected from treated rats and 2 mL of PBS was added. The tissue was then ground thoroughly to prepare a single-cell suspension. Diluted primary antibody (CCSP, 1:100, Santa Cruz, Heidelberg, Germany) was added and the sample was incubated overnight at 4 C. After washing, the mixture was incubated with the secondary antibody (Biyuntian, Shanghai, China) at 4 C for 1 h in the dark. The sample was then centrifuged at 1500 rpm for 10 min; the supernatant was removed, and resuspended in PBS after washing. Each sample was tested by flow cytometer (BD, Franklin Lakes, USA).
2.11. Statistical analysis All data in this experiment were analyzed using GraphPad (version 5.0). Analysis of variance (ANOVA) was used to detect whether the differences were statistically significant, while unpaired t-test was used to compare differences between two groups. P < 0.05 was considered to be statistically significant.
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This study evaluated whether the secretion of club cells was changed after phosgene exposure and MSCs intervention. The results showed that there was no significant difference in CCSP expression among the lung tissue from four treatment group (Air, PH, PH þ PBS and PH þ MSCs groups) at 6, 24 and 48 h (negative results are not shown). At 6, 24 and 48 h, compared to the Air group, BALF and serum CCSP levels of the PH group increased. However, after the MSCs intervention (PH þ MSCs group) BALF and serum CCSP levels decreased, compared with that of the PH group (Fig. 1A). At 6, 24 and 48 h, compared to the PH and PH þ PBS groups, club cells were dark brown as shown through immunohistochemistry and the AFI of the club cells was enhanced as shown through flow cytometry in the PH þ MSCs group (Figs. 1B and 2). These results indicated that MSCs intervention weakened the secretion of club cells in the lung tissue exposed to phosgene. 3.2. MSCs affected the proliferation of club cells Changes in the proliferation of club cells were evaluated after phosgene exposure and MSCs intervention. We found that compared to the Air group, club cells in the tracheas were damaged and fell into the tracheal cavities after phosgene exposure at 6, 24 and 48 h. At 48 h, compared with the PH and PH þ PBS groups, the number of club cells in the tracheal cavities of the PH þ MSCs group were significantly decreased (Fig. 2). As shown with immunofluorescence double labelling, at 6, 24 and 48 h, the numbers of proliferative club cells (i.e., CCSP and Ki-67 double positive cells) in the middle lobe of the PH group rats increased compared with that of the Air group. Compared to the PH and PH þ PBS groups, the
Fig. 2. Immunohistochemistry of the middle lobe of the lung. Immunohistochemistry results with CCSP as the antibody were shown. Brown detached club cells were visible (red sun markers) in the tracheal lumens. Scale bar: 50 mm. Magnification: 400x. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 3. Immunofluorescence of the middle lobe and the number of CCSP and Ki-67 double positive cells. The immunofluorescence results (D) were shown. Antibodies used were Ki-67 (red channel) and CCSP (green channel). Proliferative cells (red arrow), club cells (green arrow), and proliferative club cells (yellow arrow) were labeled in the figure. DAPI (blue channel) was used to locate cell nuclei. Scale bar: 50 mm. Magnification: 400x. The analysis of CCSP and Ki-67 double positive cells was shown (E). Results are expressed as mean ± SD (n ¼ 6). Statistical differences are indicated as follows: ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the PH group; & p < 0.05, && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$$ p < 0.001, compared with the PH þ MSCs group; @@@ p < 0.001, compared with the PH þ MSCs þ SO group. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
number of proliferative club cells in the PH þ MSCs group further increased (Fig. 3C and D). These results indicated that in phosgeneinduced lung injury, MSCs intervention enhanced the proliferation of club cells. 3.3. Activation of the Notch signaling pathway The expression of important components of the Notch signaling pathway (including Notch1~4, c-myc, Hey1 and Hes1) in lung tissue of each group was monitored. It was shown that at 6, 24 and 48 h, compared to the Air group, the expression of Notch1, c-myc, Hey1 and Hes1 increased, the expression of Notch1 decreased and the expression of Notch3 and Notch4 did not change significantly. Compared with the PH and PH þ PBS groups, the expression of Notch1, Notch2, c-myc, Hey1 and Hes1 of lung tissue in the PH þ MSCs group increased at 6, 24 and 48 h. It suggested that the Notch signaling pathway in lung tissue was further activated after MSCs intervention. At 6, 24 and 48 h, the number of proliferative club cells in the PH þ MSCs group further increased compared to the PH and PH þ PBS groups (Figs. 3 and 4). These results indicated that MSCs might promote the proliferation of club cells through the Notch signaling pathway.
3.4. Application of Notch signaling pathway inhibitor We further validated whether changes in the proliferation of club cells were associated with the Notch signaling pathway by applying LY, a specific Notch signaling pathway-specific inhibitor. At 6, 24 and 48 h, the expression of Notch1, Notch2, c-myc, Hey1, and Hes1 in injured lung tissue decreased after the application of LY, indicating that the effect of LY was positive and as expected (Fig. 4). Compared to the PH þ MSCs and PH þ MSCs þ SO groups, the number of proliferative club cells in the PH þ MSCs þ LY group decreased significantly (Fig. 3). It indicated that the MSCs-induced proliferation of club cells was weakened by LY. These results indicated that in phosgene-induced lung injury, MSCs at least partially activated the Notch signaling pathway to further enhance the proliferation of club cells. 4. Discussion Our results showed that in phosgene-induced lung injury, the intervention of exogenous MSCs reduced the secretion and enhanced the proliferation of club cells. MSCs enhanced the proliferation of club cells by further activating the Notch signaling
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Fig. 4. Expression of major constituent proteins in the Notch signaling pathway. The expression of Notch1, Notch2, c-myc, Hey1 and Hes1 were shown. Results were expressed as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; ## p < 0.01, ### p < 0.001, compared with the PH group; && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$ p < 0.01, $$$ p < 0.001, compared with the PH þ MSCs group; @@ p < 0.01, @@@ p < 0.001, compared with the PH þ MSCs þ SO group.
pathway, thus promoting the repair of injured tissue. In our experiment, MSCs attenuated the secretion of club cells. This might be due to the anti-inflammatory, anti-oxidative cytokines (similar to CCSP) secreted by MSCs, resulting in a reduced “demand” for CCSP for the repair of lung injury. Under normal circumstances, a small quantity of CCSP in the airways enters the bloodstream due to the presence of the blood-gas barrier in the lung. In lung damage caused by phosgene, more CCSP entered blood circulation due to increased secretion of CCSP and destruction of lung barrier function, resulting in elevated CCSP levels in serum. After MSCs intervention, the decrease in serum CCSP was due to a reduction of BALF-derived CCSP and MSCs-induced improvement in lung permeability [11]. Therefore, to some extent, the decrease in serum CCSP indicated the repairing effect of MSCs on lung injury. Studies have shown that CCSP is a good biomarker of epithelial damage [19]. After exposure to asbestos [20] and ozone [21], CCSP levels in serum were often associated with lung epithelial damage. In asthmatic patients, serum CCSP was associated with the involvement of small airway [22]. CCSP levels can indicate a change in lung permeability and are associated with treatment outcomes. In this experiment, serum CCSP changed after phosgene exposure and MSCs intervention. However, whether CCSP would be a good biomarker in phosgene-induced lung injury requires further investigation. We used lung homogenates (including all lung interstitial cells and airway epithelial cells) to assess changes in the Notch signaling pathway. Our results showed that club cells proliferated in response to the activation of the Notch signaling pathway. However, we were unable to determine whether the changes in the Notch signaling pathway were restricted to the airway epithelium. In future studies, isolation and in vitro culture of relevant cells will help to further evaluate evidence. Co-culture of club cells with
MSCs in vitro will further reveal the effects of MSCs on club cells with increased accuracy. Endogenous stem cells are greatly advantageous for directional differentiation into damaged cells in the repair of lung injury, but endogenous repair is often difficult to regulate. Moreover, the lung injury microenvironment where endogenous stem cells are located can be changed and then repair cannot be guaranteed. Studies have found that MSCs played a role in exogenous damage repair through paracrine, differentiation and other methods [10]; control of cell number and cell function is better. Both internal and external repair have their own strengths. If these two repair methods are combined, better treatment results can be obtained. Previously, a few studies regarding the effects of exogenous stem cells on endogenous stem cells have been published but the mechanism involved has never been explored. Our study found that MSCs instilled in the airways affected endogenous club cells in multiple ways (including changes in secretion and proliferation). This influence on the proliferation was related to the changes of the Notch signaling pathway. In the future, key regulators related to the Notch signaling pathway can be overexpressed in MSCs, promoting the function of club cells and further enhancing the repair of lung injury, and thereby increasing clinical efficacy. This research examined the link between endogenous and exogenous repair pathways in the lungs, which may help clinical innovation and may provide a new direction for the treatment of acute lung injury.
Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81471850), Shanghai Public Health System (2017-1) and the Research project of Shanghai Health and Family Planning Commission (No. JSZK2015A01). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.182. References [1] J. Borak, W.F. Diller, Phosgene exposure: mechanisms of injury and treatment strategies, J. Occup. Environ. Med. 43 (2001) 110e119. [2] J.E. Boers, A.W. Ambergen, F.B. Thunnissen, Number and proliferation of clara cells in normal human airway epithelium, Am. J. Respir. Crit. Care Med. 159 (1999) 1585e1591. [3] A.P. Wong, A. Keating, T.K. Waddell, Airway regeneration: the role of the Clara cell secretory protein and the cells that express it, Cytotherapy 11 (2009) 676e687. [4] S.D. Reynolds, A.M. Malkinson, Clara cell: progenitor for the bronchiolar epithelium, Int. J. Biochem. Cell Biol. 42 (2010) 1e4. [5] K. Yatera, Y. Morimoto, H.N. Kim, et al., Increased expression of matrix metalloproteinase in Clara cell-ablated mice inhaling crystalline silica, Environ. Health Perspect. 109 (2001) 795e799. [6] C.H. Dean, R.J. Snelgrove, New rules for club development: new insights into human small airway epithelial Club cell ontogeny and function, Am. J. Respir. Crit. Care Med. 198 (2018) 1355e1356. [7] H. Stenberg, E. Wadelius, S. Moitra, et al., Club cell protein (CC16) in plasma, bronchial brushes, BAL and urine following an inhaled allergen challenge in allergic asthmatics, Biomarkers 23 (2018) 51e60. [8] J.A. Kropski, R.D. Fremont, C.S. Calfee, et al., Clara cell protein (CC16), a marker of lung epithelial injury, is decreased in plasma and pulmonary edema fluid from patients with acute lung injury, Chest 135 (2009) 1440e1447. [9] D. Zheng, B. Soh, L. Yin, et al., Differentiation of Club cells to alveolar epithelial
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