Effect Of Stem Cells From Human Apical Papilla On Activation And Polarization Of Mononuclear/macrophages

AimHuman stem cells from the apical papilla(SCAPs)is a kind of mesenchymal stem cells derived from papilla tissue of the developing root,and play an important role in root development of young permanent teeth.Mesenchymal stem cells have the immunomodulatory effect,however,it is unclear whether SCAPs have the same function.Mononuclear/macrophages play a major role in immune response.When activated by microbes and various chemokines,mononuclear/macrophages can produce IL-6,TNF-a and other inflammatory factors leading to inflammation reaction.Macrophages can be classified into two contrary polarization states according to phenotype and cytokines secreted,including the M1 macrophages that promote occurrence of inflammation and the M2 macrophages that promote the repairment of tissue.Studies have found that mesenchymal stem cells can inhibit activation of mononuclear/macrophages and promote the polarization of macrophages to M2 type to achieve the immunosuppressive effect.In this study,we investigated the immunomodulatory effect of SCAPs on mononuclear/macrophages activation and polarization for an initiatory research.Methods and results1.Culture of cells(1)Culture of SCAPs:SCAPs were achieved from the undeveloped healthy third molars through enzymatic digestion after separating apical papilla tissue by sterile operation.(2)Mononuclear cell line THP-1 and U937 were cultured in subculturing.(3)Human monocyte-derived macrophages(MDM):peripheral blood mononuclear cells(PBMC)were isolated from peripheral blood of healthy people by lymphocyte separation tube and placed in a culture dish for overnight,then the monocytes would be adherent to macrophages.2.SCAPs promote the polarization of THP-1 to M2-like macrophageIn the experimental group,SCAPs and THP-1 were cocultured in the transwell system for 1d,2d and 3d.In the control group,there was THP-1 only.qPCR results showed that TNF-a and IL-12 were increased in THP-1 in the experimental group after 1d of coculture,but they were decreased at other time points(p<0.5).However,IL-10?TGF-?1?CD 163 and CD206 were increased in the experimental group(p<0.5),and CD206 was decreased after 3d of coculture.The expression of TNF-a was decreased in THP-1 in the experimental group significantly(p<0.5)when detected by flow cytometry after 3d of coculture.The discovery was consistent with the qPCR result.Western blotting was used to detect the expression of stat3/P-stat3?smad3/P-smad3 protein in THP-1 after coculture.The result showed that the relative protein level of P-stat3/stat3 was increased in the experimental group(p<0.5).However,the relative protein level of P-smad3/smad3 was no significant changing(p>0.5).Therefore,SCAPs promotes polarization of THP-1 to M2-like macrophages and the mechanism may be related to the activation of stat3 pathway.3.SCAPs inhibit the expression of TNF-a in PMA-induced macrophagesIn the experimental group,SCAPs were cocultured with mononuclear cell line THP-1 and U937 induced by PMA in the transwell system for Id,2d and 3d.In the control group,there were macrophages only.qPCR was used to detect the expression of inflammatory factors and surface markers in macrophage-like THP-1 after coculture.The result showed that the expression of TNF-a was decreased in the experimental group significantly(p<0.5),but other molecules related macrophages polarization were no changing.IL-10 and CD 163 were increased on the early stage of coculture(p<0.5).mRNA expression of TNF-? was also decreased in macrophage-like U937 in the experimental group(p<0.5).The concentration of TNF-a was decreased in the supernatant of experimental group after detected by ELISA(p<0.5).It was further confirmed that TNF-a secreted by macrophage-like THP-1 or U937 was decreased after cocultured with SCAPs.The results indicated that SCAPs could have no effect on the macrophage-like THP-1 polarization induced by PMA,but their immunosuppressive effect on TNF-a was enhanced in macrophage-like monocyte.4.SCAPs supernatant inhibit the activation of the human monocyte-derived macrophagesThe supernatant was abandoned when mononuclear cells were adherent to macrophages in PBMC,then monocyte-derived macrophages(MDM)were cultured with certain amount of SCAPs conditioned medium for 4d.In the experimental group,MDM were cultured with SCAPs conditioned medium.In the control group,MDM were cultured with same volume of a-MEM medium.qPCR was used to detect the expression of inflammatory factors.It was found that the expression of IL-12?IL-6?TNF-a and IL-1? were decreased in the experimental group(p<0.5).However,the expression of IL-10 and TGF-?1 were no changing in the experimental group(p>0.5).The result indicated that SCAPs supernatant can inhibit the activation of MDM.ConclutionSCAPs can promote the polarization of monocyte cell line THP-1 to M2-like macrophages by coculture,and the mechanism may be related to the activation of stat3 pathway.SCAPs have no obvious effect on the polarization of macrophage-like THP-1 induced by PMA,however they can inhibit the expression of TNF-a in macrophage-like THP-1 and U937 significantly.SCAPs supernatant can inhibit the activation of macrophages derived from human monocyte.