The Influences Of 3D Collagen Scaffold Microenvironment On Development And Biological Function Of Dendritic Cell

The microenvironment plays a pivotal role for cell survival and functional regulation,and directs the cell fate determination.Dendritic cells?DCs?are the most effective antigen-presenting cells in the mammalian immune system and are versatile regulators for maintaining immune homeostasis.The biological functions of DCs have been extensively investigated to date.However,the influences of the microenvironment on the differentiation of bone marrow cells?BMCs?into dendritic cells?DCs?are not well defined.Here,we established a 3D collagen scaffold microenvironment to investigate whether such3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs.Firstly,we found that cells cultured in 2D presented corona-like-radiating morphology with long and slim dendrites.In comparison,the cells cultured in 3D collagen scaffolds exhibited an irregular shape with short and thick dendrites.Moreover,we analysed the expression of CD11c,CD11b,and MHC-?,as well as co-stimulatory molecules including CD40,CD80,CD86 and CD83,in immature?iDCs?and mature?mDCs?DCs using flow cytometry.We found DCs cultured in 3D collagen scaffolds exhibited low expression of CD11c,co-stimulator?CD40,CD80,CD83,and CD86?and MHC-?molecules compared to those in the DC cultured in 2D.Therefore,on the basis of phenotypic features,the DCs cultured in 3D collagen scaffolds in the current study were regarded as a distinct subset of DCs,CD11b⁺MHC^lo DCs.Secondly,biological function assays showed that iDCs-3D exhibited a lower antigen uptake activity of compared with iDCs-2D.DCs cultured in 3D collagen scaffolds appeared to suppress T-cell proliferation in vitro concomitant with the low expression levels of IL-2 and IFN-?,in accordance with high expression levels of IL-10.In addition,CD11b⁺MHC^lo DCs exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo,with high levels of IL-10and TGF-?1 production.Finally,we performed a global transcriptome analysis using the Affymetrix Mouse 430 2.0expression microarray,revealing a large variation in gene expression profile between iDCs-2D and iDCs-3D.Immature DC-related genes such as Ccl3,Ccl4,Ccr1,Ccr3,and Ccr5 were significantly up-regulated in iDCs-3D compared to iDCs-2D.However,iDCs-3D showed lower expression of T cell activation-related and antigen presentation-related genes such as Cd40,Cd80,Cd83,Cd86,as well as the antigen uptake and processing-related gene Lamp3,but higher levels of immunosuppression-related genes such as Il10,Tgfb1,Socs3,and Ccl24 than iDCs-2D.By quantitative reverse transcription-polymerase chain reaction?RT-PCR?,we further confirmed that the expression levels of Cd40,Cd80,Cd83,Cd86,and Lamp3 were lower in 3D collagen scaffolds than in 2D culture.Then,the results of transwell assays found that soluble factors and cell-matrix contact induced the CD11b⁺MHC^lo DCs production.Moreover,IL-10 and TGF-?1 were neutralized by antibody neutralization assays,we found that the neutralizing antibody of IL-10 and TGF-?1 alone or combination can reduce the CD11b⁺MHC^lo production.In addition,the proportion of cell apoptosis were evaluated in 2D and 3D collagen scaffold culture.We found that DCs differentiated in 3D collagen scaffold induced higher levels of apoptotic cell compared with these cells cultured in 2D.We further confirmed that the expression levels of apoptotic cell-related gene and immunosuppression-related genes by QRT-PCR were higher in 3D collagen scaffolds than in 2D culture.In summary,our results demonstrate that the 3D collagen scaffold microenvironment could modulate the lineage commitment of DCs and provide a suitable niche for certain specialised DC development.3D collagen scaffold culture may therefore represent a promising tool to prepare DCregs for clinical application in autoimmune diseases and for transplantation.