The Role And Intervention Of Macrophage Differentiation In Tissue Repair After Ventilator-induced Lung Injury

Background:Ventilator-induced lung injury(VILI)is a common complication of invasive ventilation,as well as an important factor for exacerbating and perpetuating lung fibrosis in patients of acute respiratory distress syndrome(ARDS).Studies showed both mechanical and biological factors are involved in the development of VILI,in which some possible cell sources and activation of a few signaling pathways was closely related to mechanical ventilation-associated lung fibrosis.Macrophage,an important party of body immune system,has been widely recognized to be involved in the evolution of repair in a variety of lung injury diseases,with an especially crucial role of macrophage subsets.However,the regulatory pattern and mechanism of macrophage in the evolution following VILI still need to be elucidated.On the other hand,lung protective ventilation that is mainly consist of a lower tidal volume and a higher end-expiratory positive pressure(PEEP)have been proved to reduce the incidence of VILI and improve prognosis.Despite of advanced progress,the protective ventilation could not avoid the development of VILI fibrosis.Traditional Chinese medicine(TCM)is a unique component of Chinese medicine.Nowadays,the research based on effective monomer components of Chinese herb has become one of the hot spots for new drugs exploration.Low-molecular-weight fucoidan(LMWF)is a highly-sulfated heteropolysaccharide extracted from kelp,which have been reported with many biological activities such as anti-inflammation,immune regulation,and anti-fibrogenesis.But to our knowledge,it has not been studied in the treatment of MV-associated lung fibrosis.Based on the above background,this research was divided into the following four parts and aimed to provide novel perspectives and targets for the molecular mechanism and intervention strategies of VILI fibrosis.Part One:The Establishment and Evaluation of Mouse Model of Pulmonary Fibrosis following Ventilator-Induced Lung InjuryObjective:Nowadays,the evolution of repair and fibrosis following VILI still remains to be determined,especially there was a controversy over the animal model of pulmonary fibrosis followed by VILI.In this study,we aimed to establish a successful VILI fibrosis mouse model through high tidal volume ventilation without PEEP,which would help provide a reliable mouse model for further mechanism research.Methods:A total of 111 male C57BL/6 mice were used in the prospective,randomized and controlled animal study.All animals were randomly divided into sham group(n=21)and ventilation group(n=90).4-hour high-tidal volume(VT)ventilation was performed after tracheotomy.The ventilation protocol comprised of the following settings:volume controlled pattern with a high-VT of 20mL/kg,a positive end-expiratory pressure(PEEP)of Ocm H20,a respiratory rate(RR)of 80 breath/min,a inspiratory-expiratory ratio(I:E)of 1:1,and a fraction of inspired O2(FiO2)of 0.4A Samples were collected on Oh and day 1,3,5,7,14 after ventilation,and groups were used only for the survival analysis.Haematoxylin and eosin(H&E),Masson and Sirius staining were performed to evaluate lung histopathological and fibrotic alterations,following lung injury scores,Ashcroft scores,collagen deposition areas and the type of collagen were calculated.The immunohistochemistry were used to observe the expression of a-SMA in lungs.Bicinchoninic acid(BCA)and alkaline hydrolysis were used to measure the levels of total protein and hydroxyproline content in BALF and lung tissue,respectively.Lung wet-to-dry weight ratio was calculated.Moreover,enzyme-linked immunosorbent assay(ELISA)were performed to provide more compelling data on the concentration of TGF-β1 in bronchoalveolar lavage fluid(BALF)and plasma,and TGF-β1,CTGF and Collagen I mRNA level were detected by real time quantitaitive rverse transcription polymerase chain reaction(real time qPCR).Results:VILI induced an early inflammatory alteration with alveolar exudation,inflammatory cells infiltration,following a marked fibroproliferative response that mainly manifested as repaired airway epithelial,severe disordered lung structure and thickened alveolar septum.Collagen deposition can be detected by Masson staining,which were significantly increased on the 7th and 14th day.Sirius staining showed the process of pulmonary fibrosis from type III collagen to type I.a-SMA staining was also most pronounced on the 7th day.Meanwhile,the total protein level in BALF and the lung wet-to-dry weight ratio both peaked in the first day and returned to baseline within 7th day,while lung hydroxyproline content remained increasing to 14d.VILI model also generated a higher TGF-(β1 concentration in BALF and plasma and the upregulated levels of TGF-(β1,CTGF and Collagen I mRNA in lungs with ventilation,which showed no changes in sham group.Conclusion:4-hour high-VT and 0 cmH2O PEEP could generate a successful VILI model,and it will develop into a well-established VILI fibrosis mouse model on the 7th day after ventilation.Part Two:Landscape of Transcription and Long Non-Coding RNAs Reveals New Insights into the Inflammatory and Fibrotic Response Following Ventilator-Induced Lung InjuryObjective:Mechanical ventilation can cause ventilator-induced lung injury(VILI)and lung fibrosis;however,the underlying mechanisms are still not fully understood.RNA sequencing(RNA-seq)is a powerful means for detecting vitally important protein-coding transcripts and long non-coding RNAs(IncRNAs)on a genome-wide scale,which may be helpful for reducing this knowledge gap.Methods:To compare sham-operated lungs,and 0-and 7-day post-ventilated lungs,nine male C57BL/6 mice(n=3)were involved in the RNA-seq to elucidate the expression patterns,biological processes.and functional pathways involved in inflammation and fibrosis.Differentially Expressed Quantification and Analysis were used to identify total differentially expressed transcriptions(DETs)and the important expression patterns.Gene Ontology(GO)and Kyoko Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed to determine the most important biological processes and functional pathways involved in the inflammatory and fibrosis stages.Additionally,the target genes of IncRNAs predicted by the methods of co-location and co-expression were also identified by GO and KEGG analysis,which may help provide more potential molecular mechanism for the lung pathophysiologiy following VILI.Results:From pairwise comparisons among the sham and VILI groups on day 0 and day 7,we obtained a total of 1,297 differentially expressed transcripts(DETs).In the early stage of VILI,expression patterns were represented as a significant inflammatory response with the activation of damage associated molecular patterns(DAMPs)and considerable chemokines.After one week,numerous fibrogenic DETs were obviously up-regulated,including activities for ECM production,deposition,as well as degradation.Additionally,genes indicated macrophages activation and epithelial cell biological characteristics were also identified.GO analysis determined that the stimulus response and the immune response were the most important processes involved in inflammation and fibrosis,respectively.KEGG analysis revealed that mTOR,JAK-STAT,cAMP signaling,and TGF-β,HIF-1,Toll-like receptor and NF-kappB signaling pathways were respectively implicated in inflammation and fibrosis of VILI.Additionally,332 DE IncRNAs were identified that were enriched in the processes of cellular and biological regulation,and they may potentially regulate fibrosis through pathways,such as Wnt,HIF-1 and Toll-like receptor signaling pathways.Conclusion:This is the first transcriptomic study that reveals the potential role of macrophage differentiation and epithelial cells proliferation,migration and differentiation in the development of VILI fibrosis.It also suggested that signaling pathways such as TGF-β,HIF-1,TLR,Wnt may be associated with the fibrogenesis following VILI.Part Three:The Role of Macrophages differentiation in the Evolution of Repair Following Ventilator-Induced Lung Injury and the Potential MechanismObjective:VILI is closely related to the lung repair and prognosis in ARDS.It is well-known that macrophage played an important regulatory role during the acute phase of VILI for activating and secreting a large number of cytokines and growth factors.However,it is not yet clear about how the macrophages subsets are characterized during the process of repair and fibrosis following VILI,as well as the potential mechanism.Therefore,this study aimed to explore the role of macrophages differentiation in the evolution of lung repair following VILI and the potential mechanism.Methods:A total of 100 male C57BL/6 mice were involved in the prospective,randomized and controlled animal study.All animals were randomized either to spontaneous breathing(n =10)or to high tidal volume ventilation(n=90).After 4 hours,mice were allocated to undergo a period of recovery for 0,1,3,5,7,14 days.Macrophage subsets were determined by using doule immunofluorescence staining and Western Bolt.Then,Immunohistochemistry,Western Bolt were used for observing the epithelial-mesenchymal transition(EMT)phenotype.The expression levels of TGF-β1,Smad2/3 and phosphorylated Smad2/3 were analyzed by using Western Bolt.In vitro experiment,the primary macrophages were derived from bone marrow of male,health C57BL/6 mice,and differentiated to M1 and M2 phenotype by co-cultured with LPS(100ng/ml),IFN-γ20ng/ml and IL-4(20ng/ml),IL-13(10ng/ml),respectively.The MLE-12 cells then co-cultured with three subsets of macrophages.At 48 hour,the morphological alterations,the proliferation,migration abilities and apoptosis rates of MLE-12 cells were evaluated and the EMT phenotype and protein expression levels of TGF-β1,p-Smad2/3,t-Smad2/3 were detected by using immunofluorescence and Western Bolt.Results:High-VT MV caused a dramatically macrophage phenotype alteration in the progression of VILI fibrosis.The results of Immunofluorescence,flow cytometry and real time qPCR showed that the M1 macrophages dominated at day 0-3 after ventilation,while the M2 macrophages was gradually increased on the 5th day and would lasted high expression levels for two weeks(P<0.05).In the comparisons between the sham and VILI group on day 0 and 7,the EMT phenotype was significantly observed in lungs with high-VT MV on the 7th day.It could observed that a decreased expression level of epithelial marker of E-cadherin and increased expression levels of interstitial markers of α-SMA,fibronectin and collagen I in mice with high-VT MV on day 7.The Western Bolt also suggested that the expression levels of TGF-β1 and p-Smad2/3 protein were higher in VILI group on day 7 compared with that in the sham and VILI group on day Od.In vitro experiment,MLE-12 cells that cocultured with mouse primary M2 macrophages were transformed from cobblestone to more spindle and fibroblast like appearance.It also had increased abilities in cell proliferation and migration compared to that co-culture with M0,M1 macrophages.The cells apoptosis rates were also decreased after coculturing with M2 macrophages.Immunofluorescence and Western Bolt indicated that the expression level of E-cadherin in MLE-12 was reduced,and the expression levels of α-SMA,fibronectin were increased after coculturing with M2 macrophages.Additionally,the expression levels of TGF-β1 and p-Smad2/3 were both increased in MLE-12 cells that co-cultured with M2 macrophages.Conclusion:Collectively,our findings showed the M2 macrophages was over activated after high-VT ventilation,which could peak at 5th day and remain high expression level for two weeks.Also,we found M2 macrophages were associated with the epithelial cell proliferation,migration and EMT during the lung evolution following VILI,which may eventually contributed to the abnormal lung repair and pulmonary fibrosis.Part Four:The Role of Low Molecular Weight Fucoidan in Attenuating the Fibrotic Response Following Ventilator-Induced Lung injury in Healthy Lungs and the Potential MechanismObjective:Low molecular weight fucoidan(LMWF)is a highly-sulfated heteropolysaccharide extracted from seaweed kelp.Current studies have demonstrated that this monomer could play important roles in renal fibrogensis and M2 macrophages differentiation.This part aimed to investigate the role of LMWF in the treatment of VILI fibrosis and potential mechanism,which may provide novel insights into the treatment of lung fibrotic disease.Methods:Sixty adult,healthy male C57BL/6 mice were randomly divided into sham-operated groups(Sham),and model groups(VILI),LMWF treatment group(VILI+LMWF)and dexamethasone treatment group(VILI+DXM).The control mice were intubated without ventilation,and the other three groups received continuous high tidal volume ventilation for 4 hours.Among them,the two treatment groups were begun to intervene on the first day after ventilation.The LMWF and DXM were intraperitoneal injection with a respective dose of 100 mg/kg and 1 mg/kg.7 days later,all animals were sacrificed and evaluated by using lung histopathology,hydroxyproline concent,lung wet-to-dry weight ratio and the expression levels of TGF-β1 in BALF and plasma.Immunofluorescence,flow cytometry were used to determine the macrophage subsets.Immunohistochemistry,Immunofluorescence,and Western Bolt were used to determine the EMT phenotypic proteins,and the expression levels of TGF-β1,p-Smad2/3 and t-Smad2/3.Results:Lung histopathological staining showed that LMWF could partially alleviate the lung architecture,interstitial cells proliferation,thickened alveolar septum,as well as decrease the collagen deposition areas.Compared to the model group,LMWF-treated mice had decreased concentrations of lung hydroxyproline,TGF-β1,although it was still higher than that in controls.Except for the model animals,there was no significant difference of wet-to-dry weight ratio among other three groups.Immunofluorescence,flow cytometry showed M2 macrophages were reduced in BALF and lungs after the treatment of LMWF.Additionally,Immunohistochemistry,Immunofluorescence,and Western Bolt demonstrated that LMWF could effectively attenuate EMT phenotype and inhibit the expression of TGF-β1 and p-Smad2/3 in lung homogenates compared with that in model group,and DXM administration could also improve these alterations.Conclusion:In health lungs,LMWF could effectively alleviate VILI induced lung EMT and fibrosis,which may be associated with the inhibited role of LMWF in M2 macrophage-mediated epithelial TGF-β1/Smad signaling pathway.