Study Of Plasmacytiod Dendritic Cells In The Pathogenesis Of Systemic Lupus Erythmatosus

Objective:To study the mechanism of pDC in the pathogenesis of SLEMethods:1.Isolate human pheripheral blood pDC by using anti-BDCA4 beads.The purified pDC were challenged by TLR7 or TLR9 stimulation.The supernatant were harvested for cytokine ELISA including IFNα,TNFα,IL6 and IFNβ.2.Collect the RNA samples and run the miRNA and mRNA profiling.Analysis the profiling data by using bioinformatics tools such as DAVID and IPA to identify the inflammatory related miRNAs.3.In order to further dissect the mechanism of miRNA in pDC activation,the human pDC were transfected with miRNA mimic/inhibitors and the cytokine were measured by ELISA.4.Use Density gradient centrifugation combined with FACS sorting methods to isolate mouse pDC.pDC from different microRNA knockout mice were sorted and simulated with ODN2216.To compare the IFNαlevel after stimulation.Use FACS to test whether kockout mircoRNA would affect pDC number or not.5.To better understand the pDC in SLE pathogenesis,7 lupus prone mice combined their control mice were tested in cluding NZB/W F1,NZB,NZW,NZM2410,MRL-lpr,B6.NZM^Sle/2/3,BXSB/Mp,MRL/Mp,BXSB.B6.Yaa,Balb/c and C57BL/6.To observe the phenotype,total cell of bone marrow from 2 femur and tibias,spleen,thymus and brachial lymph node were counted and the pDC number were calculated by multiply pDC percentage and total cell number.For function study,pDC were sorted and challenged with TLR7 or 9 ligand,the IFNαlevel were compared by using ELISA.Results:1.The gender difference of IFNαlevel produced by human pDC with R837stimulation were observed.But the there were no differences in the level of TNFα,IL6 and IFNβ.The cytokine level did not show difference when challenge pDC with ODN2216.2.After the bioinformatic study,we failed to indentify the miRNA or mRNA which related to the gender difference of IFNαproducing capability with TLR7 ligand stimulation.3.There was a huge species difference between human and mouse regarding to the miRNA relate to pDC activation.What we observed in human could not be equalized to mouse.4.We tested the IFNαproducing abilitity and pDC number among different microRNA knockout strains.pDC number was significantly decreased in miR-21KO mice.miR-150 KO mice exhibit high IFNαproducing ability without number change.The pDC number was decreased miR-155 KO mice but could not reach the statistical difference.5.The FACS results unveiled that BM pDC was the largest pDC population amang all tested strains.NZB/W F1 and NZB mice had higher spleen and BM pDCs than C57BL/6.MRL-lpr,NZM2410 and NZW had higher but less significant spleen pDC number than C57BL/6.NZM2410,NZW and Balb/c had high pDC percentage in LN.The total LN cell was hard to calculate,so whether these 3 strains had higher LN-pDC number was inconclusive.The percentage of pDC in thymus were lower than other lymphoid organs,total pDC number was higher in NZW and lower in NZB and NZM2410 than C57BL/6 mice.The BM pDC number were slightly lower in B6.NZM^Sle1/2/3 strain.BXSB/Mp mice did not show difference compare to C57BL/6.Similar pDC number were observed between MRL/Mp and MRL-lpr as well as BXSB/MP and BXSB.B6-Yaa,therefore both lpr and Yaa mutation could not affect pDC number.6.IFNαproducing ability by pDC from different lymphoid organ were different,BM pDC was extraordinary higher than others.7.The response of pDC to different TLR ligand were different among different strains.Spleen pDC from NZB,NZB/W F1 and NZM2410 could produce higher IFNαthan C57BL/6 mice with ODN2216 stimulation.BM pDC from NZB could produce higher IFNαwhile B6.NZM^Sle1/2/3 was lower than C57BL/6 strain.Conclusion:1.The IFNαproducing ability of human pDC had gender difference when encounter TLR7 ligand.2.Some microRNAs were changed after stimulation but this change did not relate to the gender difference.3.The mircoRNAs identified which related to pDC activation in human could not be verified in mouse.4.Knockout miR-21 resulted in decreasing of pDC number,while knockout miR-150 could enhance the IFNαproducing ability of pDC5.The difference pDC number and function among different lupus prone suggest the role of pDC in different SLE pathogenesis were different.