Foxd3 Suppresses IL-10 Expression In B Cells

IL-10+ regulatory B cells(Bregs)play an important role in restraining excessive inflammatory responses by secreting IL-10.However,some problems limit Bregs development.It contains that Bregs are rare in the body,special surface marker and transcription factors are lacking and the developmental mechanism is unclear.To further study developmental mechanism of Bregs,we firstly focus on a key scientific problem: What transcription factor plays a key role in the expression of IL-10,a pivotal cytokine in Bregs.Objective:This study is designed not only to figure out the influence of Foxd3 on IL-10 producing in IL-10+ regulatory B cells and the underlying mechanism,but also to provide theoretical and experimental basis for the treatment of autoimmune diseases by targeting Foxd3 signaling pathway.Methods:(1)Explore which transcription factor directly binds IL-10 promoter by two predicted softwares.(2)To prove whether Foxd3 directly bound the IL-10 promoter,we used Ch IP-PCR technology.(3)The specific interaction sites of foxd3 and IL-10 promoter were further confirmed by gene mutation technology.(4)For the role of Foxd3 in the activation of the IL-10 promoter,we used IL-10 promoter report system.(5)To explore the role Foxd3 in IL-10 expression in B cells,we developed the in vitro system of IL-10+ regulatory B cell production by lps stimulated and explored foxd3 expression in m RNA and protein levels by q-PCR and western blot.(6)We used Foxd3-specific sh RNA to knock down Foxd3 expression in LPS-activated B cells.Western blot and q PCR analysis demonstrated that Foxd3-specific sh RNA effectively reduced Foxd3 expression in LPS-stimulated B cells.FACS analysis the expression of IL-10.(7)Explore the erpression of foxd3 and IL-10 of lymph nodes and the spleen of lupus-prone mice by western blot,q PCR and FACS.Results:(1)By using two webs of predict unknown transcription factor binding sites,we found that transcription factor Foxd3 had much more binding sites and higher predicted scores on binding the IL-10 promoter.(2)Chip-PCR proved that Foxd3 directly binds the same binding sites about the start codon upstream-1400 bp(that is,600 bp from-2000 bp of the IL-10 promoter)predicted by two webs.(3)By constreucted the truncated IL-10 promoter,we demonstrated that Foxd3 directly binds-1400 bp site of the IL-10 promoter;By constreucted the truncated Foxd3,we demonstrated that Foxd3 binds the IL-10 promoter by N-terminal domain of Foxd3.(4)By performing IL-10 promoter system assay,we demonstrated that Foxd3 directly suppresses the activation of IL-10 promoter.(5)We found that LPS effectively up-regulated Foxd3 m RNA and protein expression.Critically,Foxd3 expression time-dependently increased in LPS-stimulated B cells.(6)B220+B cells were sorted by B220 microbeads.we used Foxd3-specific sh RNA to knock down Foxd3 expression in LPS-activated B cells.Western blot and q PCR analysis demonstrated that knock down of Foxd3 expression up-regulated IL-10+ regulatory B cells by IL-10 expression in LPS-stimulated B cells.(7)The q PCR,FACS and Western blot analysis showed Foxd3 expression was negatively associated with IL-10 expression in B cells from lupus-prone mice.Importantly,the level of foxd3 in B cells from lymph nodes was higher than that from splenic B cells,and the percentages of IL-10+Breg cells from lymph nodes were lower than those from spleen.Conclution:Our data suggest that when activated in vitro and in vivo,B cells up-regulated Foxd3 expression.Foxd3 suppresses the production of IL-10+ Breg cells by limiting IL-10 expression.Together,Foxd3 expression may be up-regulated to suppress IL-10+ Breg cells in lupus-prone MRL/lpr mice.This study may provide a hint for Breg production and its application to the treatment of autoimmune diseases by regulating Foxd3 expression.It not only enriched the basic theory of differentiation and development of IL-10+ Breg cells,but also laid a solid foundation for the application of IL-10+ Breg cells.