Prevention of LPS- induced Acute Respiratory Distress Syndrome in Sheep by Bone Marrow-Derived Mesenchymal Stem/Stromal Cells

In this study, 10 male Shall sheep were used in two groups and bone marrow samples were collected and BM-MSCs isolated. Then experimental model of ARDS was induced by intrapulmonary injection of LPS to dose of 400 μg/kg. Twenty-four hours after LPS injection, 5×10 7 cells of BM-MSCs were autologous transferred in the group of treatment and 1ml PBS was infused in the group of control as intrapulmonary. Then, the symptoms of clinical, complete blood count, analysis of arterial blood gases and the concentrations of IL6,IL10,TNF-α,total protein, Ig M and albumin BAL were determined before and at times of 3,6,12,24,48,72, and 168 after transplantation/infusion. The results of the investigations 24 hours post-LPS injection(time 0) indicated the occurrence of acute inammation which conrmed ARDS model. These changes included increase in RR, HR and RT, decrease in PO2 and SatO2 and increase in PCO2, WBC, neutrophils, macrophages, total protein, IL6, IL10, TNF-α, Ig M and albumin. But the stem/stromal cells transplantation reduced the severity of clinical signs induced by LPS, caused signicant increase in PO 2 , SatO 2 and IL-10 and signicant decrease in PCO 2 , the total protein, TNF-α, IL-6, Ig M, albumin, WBCs, neutrophils and macrophages at different times of sampling both in compared with before transplantation(time 0) and in compared with the group of control. While in the control group, inammation continued until the end of the study. These results showed that BM-MSCs are able to reduce inammation and have an important role in reconstruction of the damaged lung. the intensity of inammation and increased hemodynamic factors in the experimental ARDS model with LPS of E.coli in sheep 17 . In another study, researchers indicated that the intravenous allogenic transplantation of human BM-MSCs in ARDS sheep decreased the severity of inammation and HR compared to the control group after 24 h, signicantly 15 . These results matched with the results of the present study, which demonstrated signicant decline in HR at 24, 48 and 72 h after transplantation. Increasing blood neutrophils due to their role in processes of chemotaxis, opsonization and phagocytosis 18 at zero time is a characteristic of leukogram inammation and early stages of the disease. The signicant decrease in the amount of inammatory cells (WBC, neutrophils, lymphocytes and monocytes) has been reported following cell therapy in model of lung inammation with endotoxin in mice 19 , which is consistent with the present study. Erythrocytosis, polycythemia and increase of hemoglobin in the present ndings may have occurred following the reduction of blood plasma and dehydration of the animal or to compensate the oxygen deciency of the body due to poor functioning of the heart or lung. studies believe that direct damage to cytokines in inammatory cascades is involved in pulmonary injury Li, In this study, with assessment of cellular content and concentration of cytokines in BAL, the MSC treatment performance evaluated. In the early stages of ARDS, most of the lung cellular secretions are neutrophils 20 The various studies have shown that MSC can reduce the BAL neutrophils and it is clear that MSCs inhibit the activation and proliferation of immune cells by secreting a deterrent agent Many of the current trends in the use of stem cells depend on the effects of soluble agents in these cells. The ability of these cells to secrete different paracrine growth endothelial and epithelial permeability regulators, anti-inammatory cytokines and antimicrobial peptides, can be potentially useful in the treatment of ARDS underlying disorders, including capacity impairment, endothelial permeability change, dysregulated inammation, and infection 6,16 . BAL assessment after autologous transplantation of MSCs in this study displayed decrease of the total inammatory cells, neutrophils and macrophages, proinammatory cytokines (IL-6 and TNF-α) and the amount of total protein, Ig M and albumin and increase of anti-inammatory cytokine (IL-10),these results were consistent with the study’s results of Mei in mice 13 . Mei et al., induced pulmonary inammation with LPS in mice intratracheally and administered MSCs intravenously after 30 minutes. The results of the BAL evaluation showed a signicant decrease in the cell count, neutrophils and proteins 13 . MSCs, through angiopoietin I secretion and keratinocyte growth factor, induce restoration of alveolar vascular endothelial, and prevent leakage of protein-rich uids as well as inammatory cells, which leads to reduced alveolar edema 13 .


Introduction
The BM-MSCs at passage three were harvested and labeled with PE-conjugated antibodies against CD45 (Biolegend, Inc), CD29, CD31 and CD44 (Abcam, Inc.), and then the cells were analyzed using ow cytometry (BD Bioscience, USA) and the Flowjo program version 7.6.1. In addition, to check multi-lineage differentiation ability of BM-MSCs, they were separately treated with osteogenic differentiation medium for 21 days and adipogenic differentiation medium for 14 days. Then the cells were xed with Paraformaldehyde 4% for 20 min at room temperature and were respectively stained with Alizarin Red (Bioidea-Iran) for 10 min and Oil Red O (Bioidea-Iran) for 15 min at room temperature. After that, cells were washed with distilled water to remove excess stain and were observed using the Olympus IX71 inverted microscope.

ARDS modeling in sheep
The dose calculation of E. coli LPS and ARDS establishment First, one vial of LPS from E. coli O55: B5, 10 mg (Sigma-Aldrich) was dissolved in 10 ml of sterile PBS and divided into sterilized microtubes, and were stored in freezer at -80 C. After this, ve Shall sheep weighing 30-35 kg were selected and anesthetized and intubated, and doses of 50, 100, 200, 400 and 800 μg/kg diluted in PBS heated to 37 °C were intrapulmonary prescribed. Then, the sheep were clinically and paraclinically investigated and nally, according to the ndings, the dose of 400 μg/kg was con rmed for in ammation in this study.
In the next stage, all sheep both in the treatment group and in the control group, were anesthetized and intubated and ARDS experimental model was induced by LPS at 400 µg/kg. The ndings of clinical signs, radiography, blood and BAL pro les and blood gases analysis were recorded before and after administration of LPS.

Autologous transplantation of BM-MSCs
In the treatment group, 24 h after induction of ARDS, rst, viability and number of BM-MSCs were assessed by trypan blue staining. Then, under general anesthesia, sheep were sternaly placed, trachea was cannulated. Then, autologous transplantation of 5×10 7 BM-MSCs were intrapulmonary done. Also, in the control group, 1 ml PBS was intrapulmonary infused only, one day after induction of ARDS.

Clinical and laboratory investigations
The

Clinical examination
The clinical symptoms of RR, HR, RT, breathing sound, cough, nasal discharge, mucosal membrane, appetite and physical condition were determined and documented based on clinical scores for each sheep. The scoring is based on examination of clinical benchmark according to scienti c texts and articles 11,12 that were individually speci ed for each sheep (Table 1).

Sampling and assessment of blood and BAL samples
Blood samples were collected from the ear artery and jugular vein into syringes containing the anticoagulation. Arterial samples containing heparin were used for blood gases analyses by Blood Gas Analyzers (OPTI CCA-TS) and the parameters included pH, PO2, PCO2, HCO3, TCO2, SatO2 and anion gap and electrolytes such as Na + , K +, and Clwere measured.
Venous samples containing EDTA were distributed in two microtubes. One microtube was immediately used for haematologic parameters analysis, and the other was centrifuged at 800 g at 4 °C for 20 min. Then, plasma was separated and frozen at -80 °C for measurement of cytokines. The pro and antiin ammatory cytokines concentrations including TNF-α, IL-6, and IL-10 were measured with commercially available ELISA kits (Eastbiopharm-USA) following the manufacturer's protocols.
Also, after general anaesthetizing with ketamine and xylazine, sheep were sternaly positioned and lung wash was performed with PBS via endotracheal tube.
10 ml sterile normal saline (room temperature) was instilled into lung and immediately aspirated and samples of BAL were collected. Then, samples were centrifuged at 400 g at 4 °C for 10 min and the supernatants were placed at reezer at -80 °C for measurement of cytokines and supernatants from pellets were used for complete cell counts . The pro and anti-in ammatory cytokines concentrations including TNF-α, IL-6, and IL-10 and concentrations of Ig M,  albumin and total protein to evaluate the pulmonary vascular permeability were measured with commercially available ELISA kits (Eastbiopharm-USA) following the manufacturer's protocols. In addition, smears were prepared from pellets and stained by Giemsa and total cell count, neutrophils and macrophages were counted.

Statistical analysis
The results were analyzed statistically by SPSS software (version 24). Data were assessed by the independent samples t-test, repeated measure, Mann-Whitney U and Friedman tests at the signi cance level p<0.05.

Results
Isolation, culture and characterizations of BM-MSCs in sheep Cells that are isolated from the BM were spherical early. Because of the MSCs adhesion, gradually with changing the cell medium and passage, the purity of the MSCs increased and other cells were removed. BM-MSCs were initially appendages, and then had a broblastic-like appearance and spindle shape. After some days, 80-90% of the culture ask was covered with spindle cells. In the present study, the cells had broad and stretched morphology and vortex patterns after three consecutive passages.
Differentiation of BM-MSCs to lineages of adipogenic and osteogenic revealed, MSCs preserved their ability to form adipocytes and osteoblasts in medium of differentiation and proved potential multipotent MSCs. Also, the ndings Flow cytometry showed that BM-MSCs expressed cell surface markers of CD29 and CD44, 91% and 89%, respectively, but did not express markers of CD31 (marker of endothelial cells) and CD45 (marker of hematopoietic cells) ( Figure 1). These results con rmed cultured cells were MSCs.
The lethal dose of E. coli LPS and ARDS model con rmation Doses of 50, 100 and 200 μg/kg did not cause signi cant pulmonary in ammation but the 400 μg/kg dose caused acute pulmonary in ammation, and the dose of 800 μg/kg caused death in sheep. Thus, the 400 μg/kg dose was used for establishment of ARDS experimental model. It should also be mentioned that individual sensitivities in sheep were not observed in the 400 μg/kg dose.
The results of the investigations one day after injection of LPS compared to the normal state indicated the occurrence of acute in ammation. Clinical changes included respiratory abnormal sounds (crackle and wheeze), di culty breathing, cough, increase in RR (p=0.018), HR (p= .014), RT (p=0.001), mucosal hyperemia, abnormal discharge from the nose, decreased appetite and abnormal physical condition. In addition, hematological changes included WBC (p=0.015) and segmented neutrophils (p=0.03), PO2 (p=0.019), PCO2 (p=0.04) and measurement of in ammatory factors in BAL demonstrated changes in total protein (p=0.045), IL6/total protein (p=0.02), IL10/total protein (p=0.02), TNF-α/total protein (p=0.002), Ig M/total protein (p=0.000) and albumin/total protein (p=0.031). All these results con rmed the acute pulmonary involvement and ARDS model. RT decline was begun after stem cells transplantation and at the end of the study, it returned to the base level, so that was signi cant at 12, 24, 48, 72 and 168 times compared with time 0 (p value was 0.030, 0.016, 0.040, 0.018 and 0.002, respectively). In addition, a signi cant difference seen after the cell therapy in comparison with the control group at hours of 24, 48, 72 and 168 (p value was 0.044, 0.019, 0.011 and 0.021, respectively) ( Table 2).  It should be noted that in the control group, clinical symptoms were abnormal until the end of the study and never returned to the state before in ammation   Figure   3). Survey of the number of monocytes showed that, in the control group there is monocytosis while in the treatment group the absolute number of monocytes remained constant during the study. Comparison the treatment group with the control group revealed the signi cant difference at 72 (p=0.040) and 168 hours (p=0.034).
In addition, there was a signi cant difference in RBC at 24 h (p=0.044), HGB at times of 24 (p=0.034) and 48 (p=0.026) and HCT at 24 hour (p=0.044) in the cell receiver group in comparison with the PBS receiver group. But, there was no signi cant difference in the PLT count in or between the groups during the study (Figure 4).
The BAL results displayed that the cell count, macrophages and neutrophils were signi cantly increased in time of in ammation ( Figure 5) Our results were consistent with Mauricio et al.'s, study. They showed that the allogenic transplantation of human BM-MSCs administrated intratracheally reduced the intensity of in ammation and increased hemodynamic factors in the experimental ARDS model with LPS of E.coli in sheep 17 . In another study, researchers indicated that the intravenous allogenic transplantation of human BM-MSCs in ARDS sheep decreased the severity of in ammation and HR compared to the control group after 24 h, signi cantly 15 . These results matched with the results of the present study, which demonstrated signi cant decline in HR at 24, 48 and 72 h after transplantation. Increasing blood neutrophils due to their role in processes of chemotaxis, opsonization and phagocytosis 18 at zero time is a characteristic of leukogram in ammation and early stages of the disease. The signi cant decrease in the amount of in ammatory cells (WBC, neutrophils, lymphocytes and monocytes) has been reported following cell therapy in model of lung in ammation with endotoxin in mice 19 , which is consistent with the present study. Erythrocytosis, polycythemia and increase of hemoglobin in the present ndings may have occurred following the reduction of blood plasma and dehydration of the animal or to compensate the oxygen de ciency of the body due to poor functioning of the heart or lung.
Most studies believe that direct damage to cytokines in in ammatory cascades is involved in pulmonary injury (Y. Li, et al., 2016). In this study, with assessment of cellular content and concentration of cytokines in BAL, the MSC treatment performance was evaluated. In the early stages of ARDS, most of the lung cellular secretions are neutrophils 20  The study of the effect of human MSCs on the reduction of acute pulmonary damage induced by intravenous olycic acid in the pig showed that increasing IL8 in ARDS acts as a chemokine for neutrophil and has a close relationship with the intensity and duration of ARDS, and a signi cant relationship between neutrophils and concentration of IL8 was found but there was a signi cant reduction in NF-κB in ammatory factor transcription 16 . Also, the effect of human MSCs transplantation as intrapulmonary in sheep model of ARDS with intravenous endotoxin revealed the number of blood neutrophils and plasma level of IL-8 returned to before in ammation level while in the control group they did not return to normal until the end of the study. Total cells count cell, neutrophils and lymphocytes in BAL were xed during the study, however, pulmonary edema was signi cantly eliminated after transplantation of MSCs 17 . Asmussen et al., did not observe signi cant change in amount of BAL neutrophils after MSCs intravenous therapy in model of acute lung in ammation in sheep but pulmonary edema was reduced.
Mokhber Dezfouli et al., demonstrated BM-MSCs intrapulmonary transplantation in ARDS rabbit model with E. coli LPS causes signi cant decline in total cell count, neutrophils, macrophages and pro-in ammation cytokines (IL-6 and TNF-α) and a signi cant increase in cytokines in ammation (IL-10) in plasma and BAL and the rate of all of them returned to before in ammation levels. But in the control group these did not return to normal until the end of the study.
The results of the analysis of blood gases have an important role in the diagnosis and management of pulmonary capacity, the status of oxygenation and the balance of acid and base. Low levels of oxygen and high levels of carbon dioxide in the blood can occur due to decreasing gas exchange, which results from severe in ammation and obstruction of airway 28 . This condition occurred after the onset of in ammation in the present study. Cell therapy was able to improve arterial blood oxygen level by mediators and reducing in ammation. Stem cells transplantation induced a similar effect to bronchodilator drugs and made a signi cant increase in PO2 and SatO2 and a signi cant decrease in PCO2. These results matched with our previous study in ARDS rabbits model 7 .
Also, researchers demonstrated human MSCs in ARDS of sheep cause increase in PO2 and decrease in PCO2 15,17 . In cell therapy of in uenza-induced pulmonary in ammation in mice, blood gas analysis has shown improvement in hypoxemia, which is consistent with the current study data 6 . Zhou et al., investigated the effects of mesenchymal cells isolated from bone marrow in pulmonary injury caused by aspiration of mouse gut contents. In this study, GFPpositive cells were transplanted by tail vein. Increasing partial pressure of arterial oxygen, decreasing protein levels and the total cells and neutrophils in BAL, decreasing TNF and cytokines caused by neutrophil activity and reduction of alveolar edema and the lung in ammation in histopathology are consistent with the current study 25 . In this study, there was no change in the acidity with metabolic origin. Hypoventilation, as it happened, at the time of zero to 6 h after in ammation resulted in accumulation of CO 2 in the blood and reduction of pH (respiratory acidosis). Following cell transplantation, deep and fast breathing (hyperventilation) increased the removal of CO 2 and resulted in an increase in pH.

Conclusions
In       The sheep BAL cytokines/total protein amount (mean ± SD) in the treatment group (ARDS+BM-MSCs) and the control group (ARDS+PBS) in the period of study. (A) IL-6/ total protein concentration, (B) TNF-α/total protein concentration, (c) IL-10/total protein concentration. Note: In the tables and gures, information are as mean ± SD (n=5 sheep/group). *p < 0.05; The changes that are signi cantly in comparison with time 0 in the same group. #p < 0.05; The changes that are signi cantly in comparison to the group of control (ARDS+PBS) at the same time.