Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Acute Lung Injury Caused by Severe Burn via Secreting TSG-6 and Inhibiting Inflammatory Response

doi:10.21203/rs.3.rs-127492/v1

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Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Acute Lung Injury Caused by Severe Burn via Secreting TSG-6 and Inhibiting Inflammatory Response

https://doi.org/10.21203/rs.3.rs-127492/v1

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published 18 Feb, 2022

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Objectives To investigate whether hUC-MSCs attenuated severe burn-induced ALI and the effects were based on TSG-6 secreted from hUC-MSCs.

Method Rat model was established and evaluated as follows：Anires2005 animal pulmonary function tester for pulmonary function; micro-CT scanner for lung imaging manifestations; Cytokine expression was measured by ELISA assay, both inflammatory cells infiltration and lung injury were assessed by immunohistochemistry assay.

Results In vitro, TSG-6 levels in serum from burn group were significantly increased than that of the sham group. In vivo, TSG-6 levels of lung tissues and serum in burn+ hUC-MSCs group were significantly increased than that of in the burn group. Higher parameters of the airway resistance（Ri, Re, etc）were markedly decreased and the disordered lung texture and funicular density shadows were significantly improved after hUC-MSCs administration. Both in lung tissues and serum, increased levels of pro-inflammatory cytokines（TNF-α, IL-1β, IL-6）were remarkably decreased but anti-inflammatory cytokine IL-10 increased after hUC-MSCs administrations (p<0.05). These significant positive effects after hUC-MSCs transplantation did not occur in the Burn+siTSG-6 group.

Conclusions Intra-tracheal implantation of hUC-MSCs had been an effective treatment for severe burn-induced ALI via promoting TSG-6 secretion and inhibiting inflammatory reaction in lung tissue.

Stem Cell & Developmental Cell Biology

Pulmonology

hUC-MSCs

TSG-6

ALI

inflammation

burns

Acute lung injury (ALI) and its severe manifestations acute respiratory distress syndrome (ARDS) are common forms of hypoxic respiratory failure in patients with a mortality rate of 25–40% [1]. ALI/ARDS also is the major complication and common cause of mortality in severe burned patients [2]. While treatments such as intensive care and mechanical ventilation continue to develop, the mortality among patients of severe burn-induced ALI/ARDS remains up to 40%~60% [1, 3]. Growing evidences from experimental and clinical studies have shown that the systemic inflammatory response played critical roles in the development and mortality of ALI/ARDS [4–6]. Thus, preventing inflammatory response has been developed as a potential therapeutic strategy for attenuating ALI/ARDS. Currently, mesenchymal stromal cells (MSCs) are increasingly recognized as a promising candidate therapy for ALI/ARDS [7]. However, the specific mechanism has not been thoroughly scrutinized. Several studies have shown that transplantation of MSCs can reduce systemic inflammation, attenuate lung injury, as well as improve survival in models of ALI [8–11]. Unfortunately, there are no animal models of severe burn-induced ALI in these studies. The human umbilical cord-MSCs (hUC-MSCs) emerged recently as an attractive source of MSCs for clinical application. The hUC-MSCs not only share the same features such as multi-lineage differentiation, paracrine functions and immunomodulatory properties as well as of all MSCs, but also have some unique advantages, such as non-required for bone marrow matching, low immunogenicity, higher self-renewal capacities, and for accelerating injury tissue repair processes [12–13]. In the past decade, they had been widely adopted in diseases' research of preclinical and clinical, including cardiovascular disease [14], preeclampsia symptoms [15], multiple sclerosis [16], cerebral palsy [17], Knee osteoarthritis [18], autoimmune connective tissue diseases such as systemic lupus erythematosus [19],inflammatory bowel disease [20]༌periodontal regeneration [21]༌and had shown promising results mainly due to their pro-angiogenic and anti-inflammatory properties. However, little is known about hUC-MSCs in the prevention of severe burn-induced ALI. Our previous experiments had demonstrated that severe burn could bring systemic inflammatory reaction. Meanwhile, hUC-MSCs could alleviate the systemic inflammatory reaction of severe burn through secreting a variety of bioactive factors [22]. Among the bioactive factors secreted by hUC-MSCs, the multipotent anti-inflammatory protein tumor necrosis factor (TNF) -a–stimulated gene/protein 6 (TSG-6) was a key natural modulator of inflammation, which is taken as a biomarker to predict efficacy of human MSCs in modulating sterile inflammation in vivo [23]. TSG-6 has been found to be efficacious in a wide range of disease models, including respiratory disease models. Our previous study further testified the potential role and mechanism of the hUC-MSCs was TSG-6 which secreted by hUC-MSCs attenuating severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling way [22]. Above discoveries prompted us to further investigate whether hUC-MSCs attenuate severe burn-induced ALI via paracrine secreting TSG-6 which inhibiting inflammatory reaction in lung tissue. Within this study, we investigate the therapeutic potential and mechanism of hUC-MSCs on severe burn-induced ALI in a rat model.

hUC-MSCs Preparation

All experimental procedures were in accordance with the international guidelines for biomedical research in experimental animals issued by the committee of international organizations of medical scientific (CIOMS). We approved the certification of the institutional animal care and used committee of the Fourth Medical Center of PLA General Hospital, and obtained the informed consent of all subjects. For umbilical cord blood collection, we obtained approval from IACUC and consent from subjects with consent forms.

The hUC-MSCs were extracted from discarded umbilical cord donated with consensus of 5 full-term gestation healthy fetuses via caesarean delivery, expanded in vitro, and characterized as previously described [24]. The hUC-MSCs from passage 5 were used through the experiment [22].

Ex vivo evaluation of TSG-6 release from hUC-MSCs by serum of burn animals

The hUC-MSCs were plated into 6-well plates with a cell density of 5000 cells/cm² and incubated with 3 ml mesenchymal stem cell medium-serum free (MSCM-sf) (ScienCell Research Laboratories, San Diego, CA) for 1 day, which were subsequently cultured continuously with 3 mL of MSCM-sf containing 10% sham serum or burn serum at 24 hours post severe burn. At this time-point, the levels of inflammatory mediators, oxidative stress factors, and other factors were very high. TSG-6 secretion from hUC-MSCs after 10% burn serum continuous culture, and the supernatant of the cells was assayed by ELISA kit.

hUC-MSCs were transfected with siRNA for TSG-6

We selected the lentiviral expression vector containing the TSG-6 siRNA sequence (GeneChem, Shanghai, China) for specific targeting TSG-6, including Lenti-TSG-6-siRNA, and its negative control vector, Lenti-GFP-siRNA. TSG-6 siRNA lentivirus vectors were generated by ligating the vector pGC-LV. TSG-6 siRNA oligonucleotide sequences are:

Forward, 5’-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3’; Reverse, 5’-AATTCAAAAATTCTCCGAACGTGTCACGTTCT CTTGAAACGTGACACGTTCGGAGAA-3’.

The sequences of control siRNA are:

Forward, 5’-CCGGGCAAGCTGACCCTGAAGTTCATCTCGAGATGAACTTCAGGGTCACGTTGCTTTTTG-3’. Reverse, 5’-AATTCAAAAAGCAAGCTGACCCTGAAGTTCATCTCGAGATGAACTTCAGGGTCACGTTGC-3’.

The recombinant lenti-virus was produced by cotransfecting HEK293T cells with pGC-LV-GFP-siRNA or pGC-LV-TSG-6-siRNA, pHelper 1.0, and pHelper 2.0 plasmid using Lipofectamine 2000 (GeneChem, Shanghai, China). hUC-MSCs were transfected with the prepared lentivirus (Lenti-TSG-6-siRNA or Lenti-GFP-siRNA). The hUC-MSCs were plated into either 6-well plates or T-75 culture bottles with a cell density of 5000 cells/cm² and incubated with 3 ml or 10 ml MSCM-sf without antibiotics for 1 day. And then, these cells were transfected in 1 ml medium of Lenti-TSG-6-siRNA or Lenti-GFP-siRNA according to the protocol provided by the manufacturer. After 12 hours, the medium was changed, and these cells were incubated with MSCM-sf without antibiotics for 3–4 days sequentially. The efficiency of TSG-6 gene knockdown in hUC-MSCs were evaluated using GFP-fluorescence observation and Western blot. Then hUC-MSCs in T-75 culture bottles were harvested with 0.25% trypsin, and resuspended at 5 × 10⁶ cells in 1 mL sterile PBS for the subsequent implantation.

Animals

All studies adhered to procedures consistent with the International Guiding Principles for Biomedical Research Involving Animals issued by the Council for the International Organizations of Medical Sciences (CIOMS), and all of the experimental protocols were approved by the Institutional Animal Care and Use Committee at the Fourth Medical Center of PLA General Hospital and the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. Six-week-old male Wistar rats (180–220 g) were obtained from the local animal facility, housed at the Institute of Animal Experiments of the Forth Medical Center of PLA General Hospital in stables with a temperature of 22° C, a relative humidity of 55% and a day/night cycle of 12/12 hours, with food and water ad libitum throughout the experiment.

Experimental Groups

Wistar rats were randomly divided into 5 groups: 1). Sham; 2). Burn; 3). Burn + hUC-MSCs (burn and implantation with hUC-MSCs); 4).Burn + Vehicle (burn and implantation with hUC-MSCs transfected with Lenti-GFP-siRNA); 5). Burn + siTSG-6 (burn and implantation with hUC-MSCs transfected with Lenti-TSG-6-siRNA). Rats in each group were divided equally into three subgroups of 6 rats according to the indicated time points of d1, d3 and d7 for samples collected.

Animal Treatment

Rats were anesthetized with Avertin (20 mg/ml, 300 mg/kg) (2, 2, 2-tribromoethanol, Sigma, USA) via intra-peritoneal injection. The dorsal and abdominal hair was completely shaved. Rat model with 50% TBSA full-thickness burn injury were established via placing the backside and abdomen into hot water (94 °C) for 12 s and 6 s, respectively(22, 25, 26). Intra-peritoneal injections of balanced salt solution (40 ml/kg) were immediately administered to prevent shock in all of groups. We adopted 20-gauge central catheter as tracheal catheter, while rats were restrained on the 45°inclined operating board with their head facing the operator. The upper incisors of the rat were fixed with cotton thread to the nails at one end of the operating board, so the head of the rat was raised and we inserted the laryngoscope into its mouth. The glottis of the rat can be seen in the light of laryngoscope. The tracheal catheter was quickly inserted into the airway when glottis was opened (27, 28). The rats in the Burn + hUC-MSCs group immediately received intra-tracheal implantation of 5 × 106 (1 ml) hUC-MSCs after burn. The rats in the Burn + Vehicle and Burn + siTSG-6 groups received intra-tracheal implantation of 5 × 106 (1 ml) hUC-MSCs transfected with Lenti-GFP-siRNA or Lenti-TSG-6-siRNA at the same time. The rats in the Sham and Burn groups received intra-tracheal implantation of 1 mL PBS. All rats in each group were clinically evaluated. The burn wound and rats in sham group were treated as previously described [22]. Blood and lung tissue samples were collected at each indicated time points. After anesthesia with the above anesthetics, rats’ blood was extracted from the inferior vena cava. And then, rats were euthanized with an overdose intravenous of pentobarbital sodium.

At each indicated time points, blood samples from the inferior vena cava were extracted, lung tissue samples were collected. And then, rats were euthanized with an overdose intravenous of pentobarbital sodium.

Specimen collection and detection

Both the lung tissue and blood samples in all groups were taken from the aortaventralis at d1, d3 and d7 after injury. Subsequently, one piece of lung specimen was stored in liquid nitrogen for TSG-6 detection using ELISA kit, as well as another piece was fixed using 4% paraformaldehyde for immunohistochemical analysis and H.E. staining. The detection range of the kit is from 0.312 ng/ml to 20 ng/ml. 100 mg lung tissue was rinsed with PBS, homogenized in 1 ml of PBS and stored overnight at -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 µg, 2–8 °C. The supernate was removed and assayed immediately. Standard: Pipette 250 µl of Sample Diluent into each tube. Use the stoc solution to produce a 2-fold dilution series. Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard (20 ng/ml). Sample Diluent serves as the standard zero.

Pulmonary function test

Pulmonary function test (PFT) can provide a simple noninvasive method of assessing airway compromise. On d1, d3 and d7 after burn, pulmonary function was measured by AniRes2005 animal lung function detector, and then data were assayed using AniRes2005 software (Beijing Bestlab High-Tech Co., Ltd, China). After anesthesia, trachea was separated and an inverted T-shaped incision was cut. Rats were placed in a scanning container of the Anires animal lung function analysis system. The trachea cannula was inserted and fixed. End of trachea cannula was connected with a ventilator for animals. The operating interface of Anires2005 was set with tidal volume of 5 mL /kg, respiratory ratio of 15:10, and air pressure of 10 cm H₂O. The changes of airway pressure curve were observed to indirectly acquire the changes of lung volume, and the airway pressure and lung capacity curves were adjusted to be synchronized with the animal ventilator. The airway resistance curve was smooth, then various indicators of lung function were obtained.

Arterial blood gas analysis

Arterial blood samples from the aortaventralis were analyzed using a blood-gas analyzer (Siemens RAPIDLab 1265 System, Tarrytown, NY).

Histological analyses

After fixation with 4% paraformaldehyde for 1 week at room temperature, the specimens were embedded in paraffin and sectioned into five-micrometer-thick sections. Then, slices were deparaffined with dimethylbenzene, rehydrated, and then stained with H&E in accordance with standard procedures. Infiltration of neutrophil and macrophage were detected via incubating with specific antibodies (MPO, CD68; from R&D Systems, Minneapolis, MN), the corresponding secondary antibody and the PAP (peroxidase–anti-peroxidase) complex in turn, and finally stained with DAB (3,3′-diaminobenzidine). Myeloperoxtidase (MPO) exists in the cytoplasm of neutrophils and can be used as an indicator of neutrophils in lung tissue. The positive reaction of neutrophils is brown-yellow colorization. CD68 is a macrophage specific antigen which is a direct evidence for the identification of macrophages. The positive reaction of macrophages is brown colorization. Randomly, five different visual fields under a light microscope (400ⅹ) of each respective section were chosen and CD86 and MPO positive cells were counted by an experienced scientist in a blinded manner.

Western blotting

Protein from hUC-MSCs was isolated using RIPA buffer (Sigma Life Science), containing PMFS (Sigma Life Science) and complete Mini, Protease inhibitor cocktail (Roche). Cells were centrifuged at 3000 g for 10 min to collect the nuclei and then, the supernatant were further centrifuged at 17,000 g for 20 min to separate the mitochondrion. Cytoplasm were diluted in Laemmli buffer and denaturated at 95 °C for 10 min. Protein was quantified using a commercial bicinchoninic acid (BCA) kit (BCA Protein Assay Kit; Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of protein were subjected to SDS-PAGE gel as previously described [29], and anti-TSG-6 antibody (1:1,000) and anti-β-actin antibody (1:20,000) (R&D Systems, Minneapolis, MN) were used for protein expression assay.

Enzyme-linked immunosorbent assay (ELISA)

100 mg lung tissue was rinsed and homogenized, then stored overnight at -20 °C. The samples were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 µg, 2–8 °C. The supernatant was removed. The double-antibody sandwich ELISA kits (eBioscience, USA) were used to detect the levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, MPO and TSG-6 in lung tissues and serum according to the manufacturer’s protocols.

Micro-CT Scan detection

Here we detected the lung imaging in representative rats of 5 groups using a 3D geometric morphometric approach with x-ray micro-CT scan data. All scan data were set up at the end of the respiratory cycle. The interval of each CT image was set at 1.6 mm.

Statistical analysis

All data were expressed as the mean ± SD and analyzed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). ANOVA followed by Tukey Cicchetti test for multiple comparisons or non-parametric Kruskal-Wallis test, as appropriate was used for statistical analysis. Statistical significance level was set at p < 0.05.

TSG-6 levels in supernatant/lung tissues/serum and TSG-6 knockdown efficiency

To investigate whether TSG-6 secreted from hUC-MSCs influenced its therapeutic effect or not, we firstly examined the levels of TSG-6 secretion from hUC-MSCs after treatment with serum of sham or burn. Compared with the sham group, TSG-6 levels in supernatant of burn serum were significantly increased in vitro at 24 hours post burn (shown in Fig. 1.A). hUC-MSCs cultured in vitro were transfected with Lenti-GFP-siRNA or Lenti-TSG-6-siRNA. As shown in Fig. 1.D, representative photographs demonstrated successful transfection with Lenti-GFP-siRNA or Lenti-TSG-6-siRNA. Data from Western blotting showed that TSG-6 expression was significantly decreased after Lenti-TSG-6-siRNA transfection than that of the vehicle group (shown in Fig. 1.E).

In vivo,TSG-6 levels of burn + hUC-MSCs group in lung tissues were significantly increased compared with burn group at d1, d3 and d7 post severe burn (shown in Fig. 1.B). Meanwhile༌TSG-6 levels of burn + hUC-MSCs group in serum were significantly increased compared with the burn group in vivo (shown in Fig. 1.C). TSG-6 levels of lung tissues and serum reached the peak at the 3th day in burn + hUC-MSCs groups.

The effect of hUC-MSCs and TSG-6 knockdown on lung function/pulmonary metabolism function of rats after severe burn

We evaluated therapeutic influence of hUC-MSCs before and after TSG-6 knockdown on lung function parameters, such as inspiratory resistance (Ri), expiratory resistance (Re), compliance (CI), peak expiratory flow (PEF), vital capacity (VC) and maximal voluntary ventilation (MVV), and pulmonary metabolism function parameters, such as PaO₂ and PaCO₂ at d1, d3 and d7 after severe burn using AniRes2005 animals lung function detector and blood-gas analyzer, respectively. As shown in Table 1, these parameters affected the airway resistance, such as Ri and Re were significantly higher than those in the sham group, and they were markedly decreased after hUC-MSCs administration. But the therapeutic effects of hUC-MSCs with knockdown TSG-6 on the Ri and Re were dramatically reduced at d1, d3 and d7 after severe burn. In addition, we also found that values of the Ri and Re were at their highest at d1 after severe burn. However, the other parameters of lung function, CI, PEF, VC and MVV were significantly decreased compared with those of the sham group, and they were also remarkably improved after hUC-MSCs administration. Furthermore, the therapeutic effectiveness of hUC-MSCs with knockdown TSG-6 on these parameters were also significantly reduced at d1, d3 and d7 post severe burn.

As shown in Table 2, compared with those in the sham group, the values of PaO₂ in burn group were significantly decreased, which were markedly increased in the burn + hUC-MSCs and burn + Vehicle groups at d1 and d3 after severe burn (p < 0.05). In addition, the values of PaO₂ in the burn + siTSG-6 group were significantly lower than that in the burn + Vehicle group at d1 and d3 post severe burn (p < 0.05). Meanwhile, we also found that the values of PaO₂ at d7 post severe burn in all groups were very close (p > 0.05).

Table 2

Effects of TSG-6 on the values of PaO2 and PaCO2 induced by severe burn
Groups	1d PaO2 PaCO2		3d PaO2 PaCO2		7d PaO2 PaCO2
Sham	105.5 ± 4.16	39.45 ± 2.26	105.9 ± 5.85	43.66 ± 3.77	104.25 ± 3.72	42.5 ± 2.38
Burn	76.5 ± 2.01**	21.90 ± 1.88**	82.94 ± 2.61**	25.5 ± 3.22**	94.33 ± 3.01**	43.18 ± 3.09
Burn+ hUCMSC	96.10 ± 1.08^##	33.19 ± 2.64^##	98.55 ± 2.82^#	40.11 ± 1.21^##	100.26 ± 2.74^#	39.44 ± 2.52
Burn+ Vehicle	95.04 ± 2.92^##	32.95 ± 1.7^##	97.77 ± 2.97^##	39.07 ± 2.15^##	99.27 ± 3.33	39.18 ± 2.04
Burn+ siTSG-6	83.6 ± 2.22^@@	26.84 ± 0.81^@	89 ± 2.72	31.24 ± 3.21^@	96.39 ± 3.61	40.25 ± 2.83
Values are represented as Mean ± SD (n = 6), asterisk () and double asterisk (*) stand for p < 0.05 and p < 0.01 compared with sham group, respectively. Single # (#) and double # (##) stand for p < 0.05 and p < 0.01 compared with Burn group, respectively. Single @ (@) and double @ (@@) stand for p < 0.05 and p < 0.01 compared with Burn + Vehicle group, respectively.

The effect of hUC-MSCs and TSG-6 knockdown on lung imageological changes induced by severe burn

We evaluated therapeutic influence of hUC-MSCs and TSG-6 knockdown on imageological changes in lungs at d1, d3 and d7 post severe burn using micro-CT assay. As shown in Fig. 2, the lung texture of the sham group was clear and no abnormal density shadow was found. In the burn group, the texture of both lungs was disordered with some funicular density shadows. Density of hilum shadow increased and blood vessels expended with the edges of them being obscure and irregular at d1. Disordered lungs’ texture was gradually recovered at d3 and d7. Meanwhile the CT scan demonstrated higher density of lung in burn + siTSG-6 group than burn + hUC-MSCs group in d7, while the density of burn + siTSG-6 group better than burn group with small area around hilum of lung. The damage was significantly improved after hUC-MSCs administration at d7. The therapeutic effect of hUC-MSCs on the imageological changes of lung tissues were heavily reduced after TSG-6 knockdown (shown in Fig. 2).

The effect of hUC-MSCs and TSG-6 knockdown on the structural damage and total inflammatory cells infiltrations in lung tissues after severe burn

In this part, we evaluated the therapeutic influence of hUC-MSCs and TSG-6 knockdown on the structural changes and infiltrated degrees of total inflammatory cells in lungs at d1, d3 and d7 post severe burn using H&E staining. As shown in Fig. 3, compared with the sham group, the structural damages, including alveoli fusion, interstitial hemorrhage, alveoli exudation and infiltrated degrees of total inflammatory cells in lung tissues of burn group were remarkably increased at d1. Meanwhile, these changes were less severe in burn + hUC-MSCs group at d1. Destroyed structure and inflammatory cells infiltrations after burn, such as neutrophil infiltration, interstitial edema, and pulmonary hemorrhage, were all improved gradually at d3 and d7. But we noted that burn + hUC-MSCs recovered faster than other groups. Moreover, we found that the therapeutic effect of hUC-MSCs on the structural damages and total inflammation was sharply decreased after TSG-6 knockdown (shown in Fig. 3).

The effect of hUC-MSCs and TSG-6 knockdown on neutrophil infiltrations in lung tissues after severe burn

We calculated on the neutrophil infiltrations of lung tissues induced by severe burn to evaluate the therapeutic influence of hUC-MSCs and TSG-6 knockdown on lung tissues at d1, d3 and d7 after severe burn using immunohistochemical staining. As shown in Fig. 4.A, compared with the sham group, neutrophil infiltrations degrees of lung tissues in burn group were remarkably increased, and they were markedly decreased by hUC-MSCs administrations (p < 0.05). Further, the data showed that therapeutic effect of hUC-MSCs on neutrophil infiltrations in lung was sharply decreased by TSG-6 knockdown (shown in Fig. 4.A). The quantitative analysis were reported in the corresponding histogram (p < 0.05) (shown in Fig. 4.B).

The effect of hUC-MSCs and TSG-6 knockdown on macrophage infiltrations in lung tissues after severe burn

We calculated the macrophage infiltrations of lung tissues induced by severe burn to evaluated differences of the therapeutic influence of hUC-MSCs and TSG-6 knockdown at d1, d3 and d7 after severe burn using immunohistochemical staining. As shown in Fig. 5.A, in comparison with that in sham group, macrophage infiltrations degrees of lung tissues in burn group were significantly increased, which were dramatically decreased after hUC-MSCs implantation (p < 0.05). Further, the data showed that therapeutic effect of hUC-MSCs on macrophage infiltrations in lung was sharply decreased by TSG-6 knockdown (shown in Fig. 5.A). The quantitative analysis were reported in the corresponding histogram (p < 0.05) (shown in Fig. 5.B).

The effect of hUC-MSCs and TSG-6 knockdown on inflammatory cytokines in lung tissues after severe burn

We also tested the levels of the pro-inflammatory cytokines of TNF-α, IL-1β, IL-6, MPO and anti-inflammatory cytokine IL-10 in lung tissues at d1, d3 and d7 after severe burn using ELISA. As shown in Fig. 6, compared with the sham group, the levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MPO in lung tissues of the burn group were remarkably increased, and they were all markedly decreased after hUC-MSCs administrations (p < 0.05). It’s worth noting that the level of anti-inflammatory cytokine IL-10 in Burn + hUC-MSCs group was significantly higher than that in burn group and sham group, and that in burn group was also higher than that in sham group (p < 0.05).

While data also indicated that therapeutic effect of hUC-MSCs knockdown TSG-6 obviously increased levels of the pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, MPO and decreased anti-inflammatory cytokine IL-10 in lung tissues (p < 0.05) (shown in Fig. 6.A-E).

During last few years, human MSCs as a cellular therapy for ALI/ARDS has ignited much interest of the researchers and the therapeutic effects have been demonstrated in different animal models [30–32]. Studies have shown that MSCs have therapeutic effects for various preclinical models of lung diseases via direct differentiation and paracrine action [33–35]. Indeed, there is a few of MSCs survive for more than one week after systemic administration, suggesting that the main effects of MSCs are probably mediated by paracrine mechanisms [36].

Patients with ALI caused only by severe burns or scald were not uncommon in the clinic, especially in children. In the present study, we established a severe burn-induced ALI in rat model to investigate the therapeutic effects of hUC-MSCs and its possible mechanism. We found that intra-tracheal implantation of hUC-MSCs increased the survival rates and significantly attenuated severe burn-induced ALI via inhibiting pulmonary inflammation, and the therapeutic effects of hUC-MSCs were strongly reduced when the expression of TSG-6 were inhibited via RNA interference. It indicated that the therapeutic effects of hUC-MSCs in severe burn-induced ALI rats were associated with the soluble factor TSG-6 that secreted by hUC-MSCs itself.

We created the rat model with a classical pulmonary inflammatory state via severe burn [25–26, 37]. The severity of the lung injury was characterized by several pathological parameters, just as described in the diagram and table (shown in Fig. 2–5 and Table 1–2). All these pathological changes are closely tied to the human ALI, which indicates our model successfully duplicated human ALI. These parameters were all improved after intra-tracheal implantation of hUC-MSCs, which indicated the therapeutic benefits of hUC-MSCs implantation on severe burn-induced ALI. These findings proved that the intra-tracheal injection of hUC-MSCs attenuated lung injury in severe burn-induced ALI rats. Our results were consistent with some other previous studies, in which the administration of hUC-MSCs or BM-MSCs reduced systemic inflammation and attenuate LPS-induced ALI in rats [38].

ALI is an uncontrollable pulmonary inflammatory disease characterized by the release of cytokines and the neutrophil accumulation. Neutrophils are the primary cells being convened to the site of inflammation, and releasing the inflammatory cytokines. Macrophages are another predominant cells existing in either a pro-inflammatory (M1) or anti-inflammatory (M2) state, acting as a trigger of the inflammatory response, and contributing to both the initiation and resolution of inflammation. MSCs treatment could regulate macrophages to anti-inflammatory phenotype, and simultaneously enhance the phagocytic activity of macrophages [39]. In our study, the infiltration of neutrophil and macrophage in lung tissue in the burn group were remarkably increased and obviously reduced after treated with hUC-MSCs. This alleviative lung injury of hUC-MSCs once confirmed in Canine radiation-induced ALI model via reducing oxidative stress, inflammatory reactions, and TGF-β-Smad 2/3 pathway activation [40].

MPO is an enzyme stored in neutrophils and macrophages and released into extracellular fluid in the setting of inflammatory process. Since MPO is an important enzyme in the inflammatory process, there is an ongoing interest in the use of MPO as a biomarker for assessing the extent of inflammatory response in our present study. After a severe burn, a remarkable increase of MPO in lung tissues was detected in the burn group, and the peak level was shown at d1. As we expected, treatment with hUC-MSCs clearly reduced the MPO activity at d1, d3 and d7. Previously, Li et al. found that intravenously infused with hUC-MSCs significantly inhibited LPS-stimulated MPO activity in rat lung tissues [38]. Reduced MPO activity indicates an improvement of lung injury and confirmed the therapeutic effects of hUC-MSCs on burn-induced lung injury through promoting anti-inflammatory homeostasis.

The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 are primary mediators of the acute phase response. Previous studies showed that decreased neutrophil recruitment into the lung tissue and suppressed expression of TNF-α, IL-1β, and IL-6 can improve outcomes of ALI [41]. Several other studies have also shown that MSCs could decrease the plasma level of TNF-α and IL-1β through paracrine secretion [42]. Consistent with previous studies, increased level of these inflammatory cytokines were found in our model of severe burn-induced ALI. After severe burn, the concentration of TNF-α, IL-1β, and IL-6 significantly increased. The difference is simply that the peak level occurs at different times. For TNF-α and IL-1β, the peak level appeared on d1, while the peak level of IL-6 appeared on d3. When hUC-MSCs were intra-tracheal implanted after burn, the concentration of pro-inflammatory cytokines in lung tissues decreased significantly, which was mostly in agreement with previous studies demonstrating that BM-MSCs can reduce TNF-α and IL-6 secretion by lung macrophages via paracrine pathway or direct contact with host cells [42]. Our results also confirmed that hUC-MSCs were able to protect the lung from injury through reducing inflammatory response.

Interleukin-10 (IL-10), one of the most important anti-inflammatory cytokines, is known to reduce the synthesis of pro-inflammatory cytokines. IL-10 treatment is supposed to be a promising therapeutic strategy to reduce lung injury [42–44]. Some argued that the progression of ALI is associated with decreased expression and secretion of IL-10 [45]. But in our study, the IL-10 level rose in lung tissues after burn injury, and it is further markedly elevated after hUC-MSCs administration. Thus, protective effect of IL-10 in lung inflammation response, which had been well-described in previous studies, might partially explain the mechanism through which hUC-MSCs exerted their therapeutic effects on severe burn-induced ALI.

According the above findings, it can be inferred that hUC-MSCs administration improved ALI by balancing homeostasis of the cytokine network. Furthermore, we would like to know more about how hUC-MSCs did the work. In our previous study, we found the levels of TSG-6 levels were significantly elevated in the systemic inflammatory response in burn rats. TSG-6 expression is induced as a result of an inflammatory response. Our data showed that severe burn-induced ALI could up-regulate the expression of TSG-6, which was in agreement with our previous study [22]. The even higher level of TSG-6 in burn + hUC-MSCs group might relate with the TSG-6 secreted by hUC-MSCs in response to inflammatory signals. To test our hypothesis and investigate the role of TSG-6, TSG-6 knockdown was achieved via RNA interference by transfection with siRNA for TSG-6 in our ALI rat model. In the Burn + siTSG-6 group, the level of TSG-6 was significantly decreased. Meanwhile, pathological changes and inflammatory response were detected in Burn + siTSG-6 group and Burn + Vehicle group. These results showed that the efficacies of hUC-MSCs implantation, including improvement of lung function and pulmonary metabolism function, structure protection of lung tissues, reduction of inflammatory cells infiltrations, suppression of pro-inflammatory cytokines, and promoting expression of anti-inflammatory, were shown to be diminished by TSG-6 knockdown. Our data inferred that the anti-inflammatory properties of hUC-MSCs in severe burn-induced ALI are explained, at least in part, by activation of hUC-MSCs to secrete TSG-6. These researches further proved that TSG-6 is an inflammation associated secreted protein that has been implicated as having important and diverse tissue protective and anti-inflammatory properties [46]. Our results showed the observed beneficial effects of hUC-MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hUC-MSCs in the lung are explained, at least in part, by activation of hUC-MSCs to secrete TSG-6. TNFα-stimulated gene-6 (TSG-6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages were not fully understood. From our earlier report [22] and some literatures [47], we could found that TSG-6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of pro-inflammatory proteins (iNOS, IL-6, TNFα, IL-1β, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of pro-inflammatory transcription factors STAT1 and STAT3. Thus, TSG-6 functions by converting macrophages from a pro-inflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.

It was well known that delivered locally has the advantage of reaching the target organ directly with relatively small dosage, faster and stronger effectiveness, and slighter systemic adverse reactions. In clinical, physicians prefer to use medicine through intra-tracheal administration or aerosol inhalation for patients with lung diseases, especially for severe cases. So we chose the intra-tracheal implantation as the route of medication. While increasing the dose of drugs might increase the efficacy of the treatment, we couldn’t exclude systemic adverse reactions and more waste of resources caused by larger doses. Taking these factors into consideration, we selected the optimal dosage of hUC-MSCs (1 × 10⁶) as suggested in our previous study [48].

In our rat model, full-thickness burn injury was established via placing the backside and abdomen into hot water, not the chest, so ALI is caused by severe burn-induced excessive inflammations and serious metabolic disturbances rather than the full thickness eschars at the back and abdomen.

Though the 50% TBSA burn was severe, we managed to avoid the death of rat model. From the early period of this study, we performed consistent treatments as follow: keep warmth and then intra-peritoneal injections of balanced salt solution (40 ml/kg) were immediately administered to prevent shock. And burn wound was then treated with 1% tincture of iodine and kept dry to prevent infection.

To the best of our knowledge, this is the first report about the therapeutic evaluation of hUC-MSCs in severe burn-induced ALI. TSG-6 secreted from hUC-MSCs played a key role on severe burn-induced ALI via inhibiting inflammatory reaction in lung tissue. It suggests that intra-tracheal implantation of hUC-MSCs is an effective treatment for severe burn-induced ALI rat model. Meanwhile, knockdown of TSG-6 mRNA expression in hUC-MSCs did not completely abrogate the anti-inflammatory effects suggested some other unknown mechanisms were involved. It means additional experiments are required to determine the relative contribution of these factors to the beneficial effects of hUC-MSCs in the lung injury.

TNF-α: tumor necrosis factor α; TSG-6: TNF-a stimulated gene/protein 6; hUC-MSC: human umbilical cord-derived mesenchymal stem cell; ALI: acute lung injury; ELISA: enzyme linked immunosorbent assay; ARDS: acute respiratory distress syndrome; MSCM-sf: Mesenchymal Stem Cell Medium serum-free medium; GFP: green fluorescent protein; MPO: myeloperoxidase; CD68: cluster of differentiation 68; siRNA: small interference RNA; PMSF: Phenylmethanesulfonyl fluoride; IL: interleukin.

Acknowledgments

We thank Dr. Jing Yang，Xiao Li, Longlong Yang and Shunyi Nie for animal experimental support. We thank Mr. Leo Liu for language modification.

Statement of Ethics

The present study was approved by the Fourth Medical Center of PLA General Hospital Ethics Committees.

All experimental procedures were in accordance with the international guidelines for biomedical research in experimental animals issued by CIOMS. After samples collection, rats were euthanized following the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.

For umbilical cord blood collection, we obtained approval from IACUC and consent from subjects with a consent form.

Conflict of Interest Statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Funding Sources

The research was supported by the National Natural Science Foundation of China (NSFC 81701900 and NSFC 81571894).

Author contribution

XHH and JKC conceived and designed study; LYL, YW, YNL and ZYL performed experiments; XHH, LYL and YW interpreted results of experiments; LYL prepared figures; XHH and YW drafted manuscript; XHH and YHY edited and revised manuscript; All authors read and approved the final version of manuscript.

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Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Acute Lung Injury Caused by Severe Burn via Secreting TSG-6 and Inhibiting Inflammatory Response

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Abstract

Figures

Introduction

Materials And Methods

hUC-MSCs Preparation

Ex vivo evaluation of TSG-6 release from hUC-MSCs by serum of burn animals

hUC-MSCs were transfected with siRNA for TSG-6

Animals

Experimental Groups

Animal Treatment

Specimen collection and detection

Pulmonary function test

Arterial blood gas analysis

Histological analyses

Western blotting

Enzyme-linked immunosorbent assay (ELISA)

Micro-CT Scan detection

Statistical analysis

Results

TSG-6 levels in supernatant/lung tissues/serum and TSG-6 knockdown efficiency

The effect of hUC-MSCs and TSG-6 knockdown on lung function/pulmonary metabolism function of rats after severe burn

The effect of hUC-MSCs and TSG-6 knockdown on lung imageological changes induced by severe burn

The effect of hUC-MSCs and TSG-6 knockdown on the structural damage and total inflammatory cells infiltrations in lung tissues after severe burn

The effect of hUC-MSCs and TSG-6 knockdown on neutrophil infiltrations in lung tissues after severe burn

The effect of hUC-MSCs and TSG-6 knockdown on macrophage infiltrations in lung tissues after severe burn

The effect of hUC-MSCs and TSG-6 knockdown on inflammatory cytokines in lung tissues after severe burn

Discussion

Abbreviations

Declarations

References

Table

Supplementary Files

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