Protective Effect Of Lung Adult Stem Cells Expressing LL37 Against Lung Injury And Infection

BACKGROUNDThe main function of respiratory system is ventilation,which is very important for life activities.The integrity of the respiratory system relies on its own innate immune system that protects against infection and regulates inflammatory responses.After being damaged by external factors,a large number of respiratory epithelial cells would die and induce inflammatory reactions,which leads to massive damage of lung tissue as various acute and chronic lung diseases.For such diseases,due to the inflammation and leakage of lung epithelium layer,relapse or re-infection frequently happens,which leads to the development of irreversible pulmonary fibrosis.In 1986,PJ Cole proposed the famous“vicious cycle”theory to illustrate pathophysiology of bronchiectasis and chronic obstructive pulmonary disease,the damage of epithelial cells caused by external factors are more vulnerable to bacterial infection,recurrent infections can lead to chronic inflammation and tissue damage,this vicious circle will eventually lead to obvious lung function decline,and even death.Respiratory infection has become the leading cause of death in humans and the increasing incidence of antibiotic-resistant cases,enhancing host defense will be a good strategy with great clinical potential.Tissue regeneration has attracted wide attention as an adjuvant therapy for lung injury.Injury or stimulation can activate a special group of lung adult stem cells which repair the damaged lung tissue thro?gh the regeneration process.A special group of endogenous lung stem/progenitor cells has been found to play an important role in maintaining homeostasis and regeneration of lung.Among them,it has been found that distal airway stem cells/progenitor cells(DASCs)expressing of basal cell-restricted transcription factor p63 and keratin-5(KRT5)have been shown to have potent regenerative capacity after lung injury.Influenza-induced lung injury activated DASCs to proliferate and migrate widely to occupy heavily injured areas,whereupon they differentiate toward mature epithelium and the regeneration of injured tissue depending on the extent of injury.The feasibility for large-scale in vitro expansion and remarkable lung engraftment after transplantation make DASC an ideal candidate for cell therapy.Antimicrobial peptides(AMPs)are substances produced by animals,bacteria and plants which are regarded as broad-spectrum antibiotics from nature.As an essential part of innate immunity,AMP possess the ability of killing invading various pathogens including bacteria,fungi,virus and parasites.In addition to its direct antibacterial activity,AMPs can also regulate the innate immune response of the host indirectly to eliminate pathogens.LL37 is the only human cathelicidin identified so far,and is known to involve in multiple host defense processes by directly targeting microbial biofilm as well as stimulating innate immune cell function.In inflamed human lung,LL37 was reported to be highly expressed and had potent antiinfective and anti-inflammatory potential,s?ggesting that the LL37 peptide can be used as alternative of conventional antibiotics to treat pulmonary infection.However,LL37peptide has a short half-life in vivo due to lability to proteases,which limites its application in clinical.Furthermore,the peptide needs to be delivered topically,rather than systemically,to avoid potential toxicity.Therefore,the development of a system to achieve local,long-term LL37 release is highly desirable for pulmonary infection therapy.Damaged lung will be more susceptible to viruses,bacteria and other microorganisms,and the“vicious cycle”will lead to the loss of lung function and even death.Therefore,the new strategy of combining tissue repair(DASC)and anti-microorganism(LL37)has become the optimal choice for complex respiratory diseases.AIMS(1)To observe the protective effect of mice DASCs(mDASCs)on bleomycin(BLM)induced lung injury in mice;(2)To observe the antibacterial effect of antimicrobial peptide LL-37 on pseudomonas aer?ginosa(PAO1)and study its related mechanism;(3)To verify the"vicious cycle"theory thro?gh BLM-induced lung injury combined with lung infection,and then engineer LL37 gene into mDASCs to express LL37,observe the influence of LL37 on proliferation,integration and differentiation of mDASCs,and further observe the antibacterial effect of LL37-mDASCs in vitro and vivo,as well as the influence on the lung function of mice;(4)To observe the antibacterial effect of LL37-hDASCs when they are grafted onto decellularized rat lung scaffold.METHODS(1)The mDASCs were isolated and cultured,and lung was injured by intratracheally instilling with 3 U/kg bleomycin.The mDASCs(1*10~6)were transplanted 7 days after BLM injury,and the distribution and integration of cells in the lung were observed by stereomicroscope 7 and 14 days after transplantation.The proliferation and differentiation of cells in the lung were observed by immunofluorescence.The effect of mDASCs on BLM induced lung injury was observed by HE and Masson staining.The content of pulmonary hydroxyproline acid was determined and the expression of?-SMA was detected by western blot and immunofluorescence.The effect of mDASCs on lung function injuried by BLM was observed by detecting arterial O2 saturation,O2 partial pressure and CO2 partial pressure.The mortality of mice injuried by BLM was observed and recorded.(2)Different concentrations of synthetic LL37(80?g/ml,40?g/ml,20?g/ml,10?g/ml,50?g/ml and 2.5?g/ml)and PAO1(1*10~5 CFUs/ml)were co-cultured for 12h,or 10?g/ml LL37 and 1*10~5 CFUs/ml and 1*10~6 CFUs/ml PAO1 were co-cultured for4h,4h,12h and 24h,respectively,and the OD values were measured at the wavelength of 595nm to observe its antibacterial effect.Rat alveolar macrophages NR8383 cells were stimulated by LPS.The Control group was cultured in normal medium for 12h,the LPS group was cultured in medium containing 1?g/ml LPS for 12h,the LL37group was cultured in medium containing 10?g/ml LL37 for 12h,and the LPS+LL37group was cultured in medium containing 1?g/ml LPS and 10?g/ml LL37 for 12h.The LPS+LL37+anti-LL37 group added 10?g/ml of LL37 neutralizing antibody on the basis of the previous group.Cell viability was detected by Cell Counting Kit-8 assay,cell apoptosis was detected by flow cytometry,The levels of inflammatory factors TNF-?and IL-6 in the cell supernatant were detected by ELISA,The protein expression levels of P-NF-??p65 were detected by Western blotting.(3)To express LL37 in mDASC,LL37 was cloned from human genome into plasmid vector and then pHIV-LL37-EGFP was used to generate lentiviral particles which was transduced into mDASC.The effect of LL37 on cells was observed by cell proliferation experiment.The integration of mDASCs and LL37-mDASCs in lung was compared by stereoscopic fluorescence microscopy.The proliferation and differentiation of mDASCs and LL37-mDASCs in the lung were observed and compared by immunofluorescence.The expression of LL37 was observed by PCR,western blot and immunofluorescence.To observe the antibacterial effect of LL37-mDASCs,the cells(1*10~6)or or their cellular supernatant were co-cultured with PAO1 or E.coli(1*104CFUs)for 16h,then optical density(OD at?=595 nm)was measured and bacterial CFUs was counted.7 days after BLM(3U/g)injury,30ul PAO1or E.coli(5*10~6CFUs)and mDASCs or LL37-mDASCs(1*106)labeled with GFP were instilled into the lung.Two days post infection,mice were sacrificed,and the lung samples and BALFs were collected and the bacterial CFUs in alveolar lavage(BALF)and lung tissue homogenate was counted.The effect of LL37-mDASCs on BLM induced injury and infection was observed by HE staining.The effect of LL37-mDASCs on lung immune cells after BLM injury and lung infection were observed by immunohistochemical staining of CD68 molecule.The effect of LL37-mDASCs lung function after injury and infection were observed by detecting arterial O2 saturation,O2 partial pressure and CO2 partial pressure.(4)The hDASCs were made to overexpress LL37 by lentivirus transfection.The expression of LL37 was observed by PCR,western blot and immunofluorescence.The decellularized rat lung scaffolds were prepared,then hDASCs and LL37-hDASCs were transplanted into the lungs,and the transplantation was observed under the fluorescence microscope.Lung lobes with stem cells were cultured with PAO1 or E.coli(2*10~4CFUs)in 24-well plates(500?l)for 16h,then optical density(OD at?=595 nm)was measured to observe the antibacterial effect.RESULT(1)7 days after administration of BLM,Endogenous mDASCs were found in bronchioles and they had proliferation activity.Substantial incorporation of mDASCs into mouse lung was detected along the days,cells could differentiated into alveolar-like structure in later days and express AECI markers AQP5,PDPN and HOPX.Transplantation of mDASC could significantly protect lung tissue injury thro?gh histopathological changes.In addition,mDASCs could reduce collagen deposition in lung,inhibit fibroblasts formation,improve lung function and reduce mortality.(2)LL37 could directly inhibit the proliferation of PAO1,and its antimicrobial activity was concentration-and time-dependent.LL37 could suppress the decrease of viability and apoptosis of NR8383 cells caused by LPS.LL37 could effectively inhibit the secretion of pro-inflammatory cytokines induced by LPS and decreased the protein expression of P-NF-??p65.(3)The lung clearance ability to bacterial decreased and the injury was more serious after BLM injury.LL37 transfection has no effect on cell proliferation,colonization and differentiation.In vitro experiments,mDASC expressing LL37 has inhibitory effect on the proliferation of PAO1 or E.coli,and this antibacterial effect can be inhibited by neutralizing antibodies of LL37.In vivo experiments,LL37-mDASCs could reduce the CFUs of bacterial in BALF and lung tissue homogenate,protect lung injury,significantly reduce the infiltration of immune cells in lung tissue,and improve the lung function of mice after injury.(4)LL37-hDASCs could graft onto decellularized rat lung scaffold and expressed LL37.In vitro experiments,hDASC overexpression of LL37 could inhibit the proliferation of PAO1 and E.coli.CONCLUSION(1)The mDASCs have protective effect on lung injury induced by BLM in mice.(2)LL-37 have direct antibacterial effect on PAO1.It can protect NR8383 cells injuried by LPS which may be related to the down-regulation of NF-??signaling pathway,thereby inhibiting the production of inflammatory factors.(3)The clearance ability of mouse lung to bacterial infection is decreased and the injury is more serious after BLM injury,LL37-mDASCs have inhibitory effect on PAO1 or E.coli,which can improve lung injury,alleviate lung infection and then improve lung function.(4)LL37-hDASCs can inhibit the proliferation of PAO1 and E.coli.