A Preliminary Study On The Mechanisms Of Differentiation And Maturation Of Dendritic Cells Regulated By SOCS3 Ablation Against C.Albicans Infection

Part 1:Preparation of siRNA mediated SOCS3 silencing BMDC Objective:To establish an effective method of amplify and culture mouse bone marrow derived dendritic cells in vitro and prepare siRNA mediated SOCS3 silencing dendritic cells.Methods:(1)Dendritic cells were generated from C57BL/6 mice by culturing bone marrow precursors with recombinant mouse granulocyte-marophage colony-stimulating factor(rmGM-CSF)and interlukin-4(rmIL-4),and were identified from the cell morphology and surface molecular markers.(2)Three siRNA trgeting SOCS3 were designed and synthesized,and non-target siRNA was used as negative control.These siRNAs were inserted into the recombinant plasmid GV248 and transfected into BMDC with lipofectin.Incubated 24 hours after transfection,the alteration of SOCS3 in gene and protein levels were detected by qRT-PCR and western blot analysis respectively to identify the most effective siRNA sequence.(3)Cell apoptosis after transfection was analyzed by flow cytometry and trypan blue.Results:(1)We collected DCs for the experiments at day 8.Typical morphology with dendrites could be observed under microscope.Flow cytometric analysis confirmed that>90%cells in the culture plate were CD11c+ cells.The positive rates of MHCII,CD40,CD86 were low,indicating that they were imDCs.(2)qRT-PCR and western blot analysis indicated that the expression of SOCS3 in DCs was downregulated effectively by the three siRNA sequences compared with the control group,of which siRNA SOCS3#2 exhibited the highest silencing effectiveness.(3)Flow cytometric analysis and trypan blue exclusion test showed low apoptosis rates,with no statistic difference among control DCs?siRNA SOCS3#2 transfected DCs and non-target siRNA DCs.Conclusion:A great quantity of immature DC with high purity was obtained by the method in vitro and siRNA mediated SOCS3 silencing dendritic cells were successfully prepared.Part 2:The influence of SOCS3 ablation on DC against C.albicans infectionObjective:To explore the bionomics and functions of SOCS3 silenced DC against C.albicans infection.Methods:(1)We used laser scanning confocal microscope(LSCM)to observe the antigen uptake in all DCs groups.We use flow cytometry to examine the phagocytosis of fungal cells from 15 to 75 min both in the siSOCS3-treated DCs and control DCs.(2)The number of colony forming units was counted to determine the killing rate in all DC groups.(3)The DC surface markers MHC-?,CD86 and CD40 were examined by flow cytometry analysis.(4)We measured the migration of DCs in an in vitro transwell system and the CCR7 expression on DCs were detected by flow cytometry.Results:(1)The phagocytosis in all DC groups reached a peak at 60 min of co-incubation and declined as time extended,of which the siSOCS3-treated DCs had a higher phagocytotic activity.(2)The killing rate is higher in the siSOCS3-treated DCs than control DCs and non-target siRNA treated DC either at the rate of DC:yeast =1:1 or 1:2.(3)All groups of DCs without C.albicans showed a typical immature phenotype at the beginning of the experiments with a low level of MHC-?,CD86 and CD40.The expression of MHC ?,CD86,CD40 in all DC groups co-incubated with C.albicans were increased compared to those without C.albicans stimulation,and the siSOCS3 treated-DCs had higher levels of those markers than control DCs in the presence of C.albicans.(4)Compared to DCs group without C.albicans,the migration of DCs pre-incubated with C.albicans is slightly increased in control DCs or non-target siRNA-treated DCs,but significantly enhanced in SOCS3 siRNA-treated DCs.The basal levels of CCR7 expression among control DCs,non-target siRNA-treated DCs and SOCS3 siRNA-treated DCs are barely detectable without C.albicans stimulation.However,after inoculation with C.albicans,the CCR7 expressions in all DC groups are greatly increased with its highest in SOCS3-silenced DCs.Conclusion:Silencing SOCS3 not only increased fungal phagocytosis and killing capacity of DCs,but also promoted DC maturation and migration in response to C.albicans.Part 3:SOCS3 ablation enhances DC-derived Th17 immune response against C.albicans via activating IL-6/STAT3Objective:To investigate the effects of SOCS3 silenced DC on the proliferation and differentiation of T cells.Methods:(1)The protein and mRNA levels of IL-6 and IL-12 in the DCs were examined by ELISA?qRT-PCR and WB.(2)We evaluated the STAT3 pathway activation through measuring p-STAT3 and total STAT3 proteins by western blot.(3)CD4+ T lymphocytes were separated from spleen of C57 mice using immunomagnetic beads.To analyze the functions of SOCS3 in the priming of CD4+ T cells,DCs were co-cultivated with CD4+ T cells prior to being challenged with or without C.albicans and the proliferation of CD4+T cells were determined using flow cytometry.(4)We examined the percentage distribution of CD4+ T cells that produce IFN-y,IL-4 or IL-17 by intracellular cytokine staining.(5)The gene markers of T-bet for the Thl,GATA3 for Th2 and RORyt for Th17 are quantified by qRT-PCR.(6)We added anti-IL-6 mAbs to the co-culture experiments and used western blot to determine the protein levels of IL-17A?IL-23R and RORyt.Results:(1)The basal levels of IL-6 and IL-12p70/IL-12p35 expression in DCs without C.albicans are very low regardless of transinfection of SOCS3 siRNA.However,the gene and protein expression levels of IL-6 and IL-12p70/IL-12p3 5 in DCs are significantly increased upon C.albicans stimulation.Moreover,with C.albicans stimulation,protein level of IL-6 in SOCS3 siRNA-treated DCs is higher than non-target siRNA and control DCs.There are no significant differences in IL-12p35 mRNA and IL-12p70 protein among control DCs,non-target siRNA-treated DCs and siSOCS3-treated DCs upon C.albicans stimulation.(2)There are no significant differences in protein expression of p-STAT3 and total STAT3 among control DCs,non-target siRNA-treated DCs and SOCS3 siRNA-treated DCs without C.albicans stimulation.However,the total STAT3 and p-STAT3 protein levels in each of the DC groups are significantly increased with C.albicans stimulation compared to their corresponding non-stimulated DC.s.In presence of C.albicans,the siSOCS3-treated DCs have greater expression levels of p-STAT3 and total STAT3 protein in comparison with control DCs and non-target siRNA treated DCs.(3)Without C.albican stimulation,there is no significant difference in CD4+ T lymphocyte proliferation among all DC groups.However,DCs stimulated with C.albicans can enhance the proliferation of CD4+T cells compared with DCs without C.albicans stimulation.In response to C.albicans,SOCS3 silenced DCs further increase the proliferation of T cells at a DC-to-T cell ratio of 1:50 as well at a ratio of 1:200 when compared to control DCs and non-target siRNA-treated DCs.(4)The protein level of IFN-y,IL-4 or IL-17 produced by CD4+T cells after being co-cultured with DCs is notably enhanced when stimulating with C.albicans,but there is no marked difference among control DCs,non-target treated DCs and siSOCS3-treated DCs.However,compared with control DCs.the siSOCS3-treated DCs when co-cultured with C.albicans generate a greater population of IL-17 producing CD4+ T cells.(5)In the presence of C.albicans,T-bet,GATA3 and RORyt are all significantly increased in DCs in comparison with DCs without C.albicans.However,the increased RORyt expression is greater in siSOCS3 treated DCs than control DCs and non-target siRNA treated DCs in presence of Calbicans.(5)Exogenous anti-IL-6 mAbs partly reduces the protein levels of IL-17A?IL-23R and RORyt in siSOCS3-treated DCs.Conclusions:SOCS3 silencing promotes IL-6-induced tyrosine phosphorylation of STAT3 in DCs,which favors the differentiation of naive CD4+T cells to the Th 17 subset and protects against C.albicans.