Recrultment Of MSCs To The Lung Of Mice With Allerglc Asthma And The Mechanism

Objective: To determine whether exogenous mesenchyma l stem cells (MSCs)were involved in airwa y-lung infla mma tion of allergic asthmatic mice , and toelucida te the mecha nism of MSCs recruitment to airwa y-lung.Method:1.Role of exogenous MSCs in the lung of allergic asthmatic mice①Recruitment of exogenous MSCs to the lung of mice with allergicasthmaFifteen C57BL/6 mice were randomly divided into three groups: asthmagroup(O:M:O), non-asthma group (P:M:P) and control group (P:P:P). Asthmaticmice were sensitized with OVA on day 1 and day 8, then were challenged withOVA injected intratrachea lly on day 15 to day 17. MSCs were labeled withCFSE and were administered into tail vein of mice both asthmatic mice and nonasthmaticmice on day 14. Control mice were treated with PBS. Twenty-fourhours after the fina l challenge (day 18), mice were sacrificed and lungs wereremoved for single-cell analysis by flow-cytometry.②Reduced allergic airway infla mmation by exogenous MSCsC57BL/6 mice were randomly divided into three groups: asthma group(O:P:O), MSCs treatment group (O:M:O) and control group (P:P:P). Asthmaticand MSCs treated mice were sensitized with OVA on day 1 and day 8, then were challenged with OVA injected intratrachea lly on day 15 to day 17. On day 14,exogenous MSCs were administered into tail vein of mice of MSCs treatmentgroup. Control mice were treated with PBS. All mice were sacrificed on day 18,lung tissue and BALF were collected. Histopa thologica l analysis was carried outby hematoxylin and eosin staining, Cell number and eosinophilic cell in BALFwere direct counted and mast cell degra nulation was estimated byβ-hexosa minidase detection in BALF.2. Role of CXCR4/SDF-1 axis in the migration of exogenous MSCs①Expression of CRCX4 on MSCs in vitro and vivoExogenous MSCs were labeled with CFSE and anti-CXCR4-PE[?]. MSCs-CFSE were analyzed by flow cytometry . The frequency of each cell subset wascalculated. Five C57BL/6 mice were sensitized with OVA on day 1 and day 8.Then, MSCs-CFSE were administered into tail vein of mice on day 14. Micewere challenged with OVA injected intratrachea lly on day 15 to day17. Twentyfourhours after the final challenge (day 18), all mice were sacrificed and lungswere removed for single-cell analysis by flow-cytometry.The frequency of eachcell subset was calculated. CXCR4 expression on MSCs in vitro and vivo weredetected also.②Expression of SDF-1 in mice lungTen C57BL/6 mice were randomly divided into two groups: asthma groupand control group. Asthmatic mice were sensitized with OVAon day 1 and day 8.Then, mice were challenged with OVA injeted intratrachea lly on day 15 to day17.Control mice were treated with PBS. Twenty-four hours after the finalchallenge (day 18), all mice were sacrificed . The expression of SDF-1, a liga ndof CXCR4, in lung tissues was detected by RT-PCR and Western-blot.③CXCR4 block out and recruitment of exogenous MSCs to lung of allergic asthmatic miceTen C57BL/6 mice were sensitized with OVA on day 1 and day 8. Then,MSCs-CFSE, treated with AMD3100 or not, were administered into tail vein ofmice on day14, Mice were challenged with OVA injected intratrachea lly on day15 to day 17. Twenty-four hours after the final challenge (day 18), all mice weresacrificed and lungs were removed for single-cell analysis by flow-cytometry.ResultResults1.Role of exogenous MSCs in lung of allergic asthmatic mice:①Distribution of exogenous MSCs in lung tissues of asthmatic miceIn OVA-induced asthmatic mice, the propotion and the number of CFSEpositive cells (0.1520±0.0302 and 1.6400±0.1435) were highThe data in controlmice were the lowest among the three groups.②Airway infla mma tion were significa ntly reduced by exogenous MSCsCell number and eosinophilic cell (1.9990±0.5257and 0.7330±0.1546) inBALF of MSCs treated mice were found lower than those in asthmatic mice(3.940±0.7133 and 1.3200±0.1657)(P＜0.05). However,the data were stillhigher than those in control mice (1.0880±0.2287 and 0.1110±0.0198) (P＜0.01).In asthmatic mice , airwa y challenge leads to a dense peribronchiolarinfla mma tory infiltrate of lymphocytes, mono cytes and polymorphonuclear cellswith epithelia l shedding and extended columnar cells. In MSCs treatment group,however, tissue infla mma tion was greatly alleviated with less peribronchial andperivascular cellular infiltration.In addition, the allergen-induced increase ofβ-hexosa minidase in BALF ,indica ting the release of mast cell med iator, was attenuated in MSCs treatedmice(0.2112±0.0166), compared with asthmatic mice (0.2875±0.0234)(P＜0.05). In control group, the data(0.1043±0.0124)were the lowest among the three groups.2.Role of CXCR4/SDF-1 axis in the migration of exogenous MSCs①The proportion of CXCR4+ cells in total CFSE+ cells significa ntlyincreased in the lungs of MSCs-treated asthmatic mice compared with culturedMSCs in vitro(P<0.01).②The mRNA and protein expression of SDF-1 significa ntly increased inOVA-induced asthmatic mice than those in control mice .③AMD3100 is the antagonist of CXCR4 . MSCs were treated withAMD3100 in vitro prior to impla ntation. The proportion and the number ofCFSE+ cells significa ntly decreased in AMD3100 treated cells compared with noAMD3100 treated mice (P<0.05).ConclusionThis study showed that exogenous MSCs can migrate to sites ofinfla mma tion in asthmatic mice through SDF-1/CXCR4 dependent mecha nism.Administration of exogenous MSCs significa ntly reduced infla mma tory cellularinfiltration in airwa y. Immunotherapy based on MSCs ma y be feasible, efficientfor asthma therapy.