Aryl Hydrocarbon Receptor Regulates MSC In The Airway Inflammation Of Neutrophilic Asthma

Objective: As a special phenotype of asthma,neutrophilic asthma is characterized by poor clinical symptom control,frequent acute exacerbations and glucocorticoid resistance.Mesenchymal stem cell(MSC)has been shown to play a therapeutic role in the eosinophilic asthma,while the effect exerting on the neutrophilic asthma needs to be confirmed by more studies.Aryl hydrocarbon receptor(Ah R)is a transcription factor which is activated by some kind of ligands.It is widely expressed in lots of cells and involves in many important biological processes.We found a downregulation of Ah R in mice with neutrophilic asthma.Thus,present study has been performed to investigate the following issues:(1)the isolation,culture and identification of MSC and the effect of Ah R activation on the MSC;(2)to explore the role of Ah R-activated MSC in the alleviation of airway inflammation of neutrophilic asthma;(3)the mechanism of Ah R-activated MSC in regulating the proliferation of splenocytes;(4)and the role of Ah R-activated MSC in the Th17 differentiation.Methods:(1)MSCs were isolated from the murine bone marrow and cultured in the dishes.MSCs were identified by flow cytometry and differentiation capacity experiment and the Ah R-activated MSCs were also identified in the same way.(2)The mouse model of neutrophilic asthma was established with the use of ovalbumin(OVA)and lipopolysaccharide(LPS).MSCs or Ah R-activated MSCs were administrated intranasally and bronchoalveolar lavage fluid(BALF),hematoxylin-eosin(HE)and periodic acid Schiff(PAS)staining,enzyme-linked immunosorbent assay(ELISA) and flow cytometry were performed to assess the alleviation of the airway inflammation.(3)Splenocytes were isolated in a way of density gradient centrifugation.MSCs were co-cultured with splenocytes in different proportions in the cell-cell contact way and transwell system to detect the regulation of the Ah R activation of MSC for the splenocytes proliferation.Real-Time PCR was conducted to explore the expression of inducible nitric oxide synthase(i NOS)with the activation of Ah R of MSCs and the nitric oxide(NO)concentration was also measured using a NO assay kit.i NOS inhibitor N-nitro-L-arginine methyl ester(L-NAME),NO scavenger2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide(PTIO)and NO donor S-nitroso-N-acetylpenicillamine(SNAP)were also given to detect the mechanism of the inhibitory effect of Ah R-activated MSC on the proliferation of splenocytes.(4)Na(?)ve CD4+ T cells from WT mouse were primed in vitro under Th17 polarizing conditions.The cells were co-cultured with MSCs or Ah R-activated MSCs in the cell-cell contact way and Th17 percentage was detected using flow cytometry.NO concentration was measured using a NO assay kit.i NOS inhibitor L-NAME,NO scavenger PTIO and NO donor SNAP were given to detect the mechanism of the inhibitory effect of Ah R-activated MSC on the differentiation of Th17.Results:(1)MSCs were plastic adherent and demonstrated fibroblast-like shape.The flow cytomycin analysis showed that MSCs were positive for CD29,Sca-1,CD105 and CD90 and negative for CD45,CD11 b,CD34 and CD80.MSCs were capable of differentiating into osteocytes,adipocytes,and chondrocytes.Ah R activation did not influence the inherent characters of MSCs.(2)Ah R activation enhanced the function of MSCs in alleviating the airway inflammation of neutrophilic asthma through the inhibition of Th17 differentiation and antigen presenting function of neutrophils.(3)Ah R activation of MSCs upregulated the i NOS expression which promoted the production of NO,thus enhancing the inhibitory effect on splenocytes proliferation.NO donor SNAP could also inhibit the splenocytes proliferation and i NOS inhibitor L-NAME and NO scavenger PTIO partly reversed the immunosuppressive function.(4)Ah R activation primed the suppressive function of MSCs against Th17 by increasing the NO production.NO donor SNAP could also inhibit the Th17 differentiation and i NOS inhibitor L-NAME and NO scavenger PTIO partly reversed the phenomenon.Conclusion: Ah R activation did not affect the morphology,surface molecule expression and differentiation capacity of MSCs.Ah R activation enhanced the function of MSCs in alleviating the airway inflammation of neutrophilic asthma through the inhibition of Th17 differentiation and antigen presenting function of neutrophils.Ah R activation of MSCs reinforced the inhibition of splenocytes proliferation and Th17 differentiation by up-regulating the i NOS expression with more NO production.