IL-6 Secreted By Endothelial Cells Under High Shear Stress Regulates Macrophage Polarization To M2 Through STAT3/KLF4 Pathway

Objective: The purpose of this study was to establish a co-culture system of vascular endothelial cells and macrophages under shear stress by using shear force devices,in order to investigate the effect of shear stress on the polarization phenotype and function of macrophages in the co-culture system and the mechanism of action.Method:1.Effect of shear stress on macrophage polarization in co-culture system: a co-culture system of vascular endothelial cells C166 and macrophages Raw264.7 were established and co-cultured in shear force devices for 24 hours with shear stress stimulating,the macrophages and culture medium supernatant were collected respectively.The cell viability was detected by Calcein/PI staining,Western blot for i NOS,Arg1 protein expression,RT-q PCR for TNF-α,IL-1β,IL-6,i NOS,Mrc1,Arg1,IL-10 m RNA expression,ELISA for TNF-α,IL-1β,IL-6,IL-10 protein expression,and cellular immunofluorescence for the protein expression of CD86/ CD163.2.Effect of shear stress on the expression of inflammatory factors in endothelial cells: C166 and Raw264.7 co-culture systems were co-cultured in shear stress devices for24 hours with shear stress stimulating,the vascular endothelial cells and culture medium supernatants were collected respectively.RT-q PCR and ELISA were used to detect the expression of TNF-α,IL-1β,IL-6,IL-10 m RNA and protein.3.Exploration of the effect of IL-6 secreted by endothelial cells on macrophage polarization:(1)C166 were pretreated with LMT-28,and then co-cultured with Raw264.7in shear force device;(2)Raw264.7 were transfected with si-IL6 R,and then co-cultured with C166 in shear devices;with shear stress stimulating for 24 hours,and the macrophages and medium supernatant were collected.Western blot for i NOS,Arg1,STAT3,p-STAT3,and KLF4 protein expression,RT-q PCR for STAT3,KLF4 m RNA expression,ELISA for TNF-α,IL-1β,IL-6,IL-10 protein expression,and cellular immunofluorescence for the protein expression of CD86/ CD163.4.Exploration of the mechanism of STAT3/KLF4 pathway on macrophage polarization:(1)Raw264.7 were pretreated with Stattic;(2)Raw264.7 were transfected with si-KLF4,and then co-cultured with C166 in shear devices for 24 hours with shear stress stimulating,and macrophages and medium supernatants were collected.The cell viability was detected by Calcein/PI staining,Western blot for i NOS,Arg1,STAT3,p-STAT3,and KLF4 protein expression,RT-q PCR for STAT3,KLF4 m RNA expression,ELISA for TNF-α,IL-1β,IL-6,IL-10 protein expression,and cellular immunofluorescence for the protein expression of CD86/ CD163.Result:1.Calcein/PI staining showed that there was no difference in cell survival rate(cell survival rate=living cells/(living cells+dead cells))between the static and shear stress groups.Western blot showed when compared with static group,the expression of Arg1 protein in shear stress group was increased,and there was no difference in i NOS between this two groups.RT-q PCR showed that the m RNA expression of Mrc1,Arg1 and IL-10 were increased in the shear stress group compared with the static group,but i NOS,TNF-α,IL-1β and IL-6 were not difference between this two groups.ELISA showed that the protein expression of IL-10 was increased in the shear stress group,but TNF-α,IL-1βand IL-6 were not difference between this two groups.Cellular immunofluorescence showed the fluorescence signal of CD163 was increased in the shear stress group when compared with static group,while CD86 was not difference between this two groups.2.RT-q PCR showed that the m RNA expression of IL-6 and IL-10 were increased in the shear stress group,while TNF-α and IL-1β were not difference between this two groups.ELISA showed that the protein expression of IL-6 was increased in the shear stress group compared with the static group,but TNF-α,IL-1β and IL-10 were not difference between this two groups.3.(1)Inhibition of IL-6 protein function:Western blot showed that the protein expression of Arg-1,KLF4,STAT3,p-STAT3,p-STAT3/STAT3 were increased in the shear stress+DMSO group when compared with static+DMSO,whereas decreased in the shear stress+LMT-28 group while compared with shear stress+DMSO,the i NOS was not difference between this groups.RT-q PCR showed that the m RNA expression of STAT3 and KLF4 were increased in the shear stress+DMSO group when compared with static+DMSO group,decreased when compared with the shear stress+LMT-28 group,were not difference between the static+DMSO and static+LMT-28 groups.ELISA showed that the protein expression of IL-10 was increased in the shear stress+DMSO group when compared with static+DMSO,decreased when compared with the shear stress+LMT-28 group,while TNF-α,IL-1β,and IL-6 were not difference among this groups.Cellular immunofluorescence showed that the fluorescence signal of CD163 was increased in the shear stress+DMSO group when compared with static+DMSO group,while decreased when compared with the shear stress+LMT-28 group,whereas CD86 were not difference among this groups.(2)Knockdown of IL-6R gene in macrophages,Western blot showed that the protein expression of Arg1,STAT3,p-STAT3,KLF4,p-STAT3/STAT3 were decreased in the shear stress+si-IL6 R group compared with the shear stress+si-NC group,while i NOS was not difference between this two groups.RT-q PCR showed that the m RNA expression of STAT3 and KLF4 were reduced in the shear stress+si-IL6 R when compared with the shear stress+si-NC group.ELISA showed that the expression of IL-10 in the shear stress+si-IL6 R group was decreased when compared with the shear stress+si-NC group,while TNF-α,IL-1β and IL-6 were not difference between this two groups.Cellular immunofluorescence showed that the fluorescence signal of CD163 was diminished in the si-IL6 R group when compared with the si-NC group,while the CD86 was not difference between the two groups.4.(1)Macrophages were treated with Stattic: Calcein/PI cell staining showed there was no difference in cell survival between the two groups.Western blot showed that the protein expression of Arg1 and KLF4 were decreased in the shear stress+stattic group when compared with the shear stress+DMSO group,while i NOS was not difference between the two groups.RT-q PCR showed that the m RNA expression of KLF4 was decreased in the shear stress+stattic group when compared with the shear stress+DMSO group.ELISA showed that the protein expression of IL-10 was decreased in the shear stress+stattic group when compared with the shear stress+DMSO group,while TNF-α,IL-1β and IL-6 were not difference between the two groups.Cellular immunofluorescence showed that the fluorescence signal of CD163 was diminished in the shear stress+stattic group when compared with shear stress+DMSO group,while CD86 was not difference between the two groups.(2)Knockdown of macrophage KLF4gene: Western blot showed that the protein expression of Arg1 and KLF4 were reduced in the shear stress+si-KLF4 group when compared with the shear stress+si-NC group,while i NOS,STAT3,p-STAT3,p-STAT3/STAT3 were not significantly difference between this two groups.RT-q PCR showed that the m RNA expression of STAT3 was no significant difference in the two groups.ELISA showed that the protein expression of IL-10 was decreased in the shear stress+si-KLF4 group when compared with the shear stress+si-NC group,while TNF-α,IL-1β and IL-6 were not significantly difference between the two groups.Cellular immunofluorescence showed that the fluorescent signal of CD163 was diminished in the shear stress+si-KLF4 group when compared with the shear stress+si-NC group,while CD86 was not difference between the two groups.Conclusion:High shear stress promotes macrophage polarization toward M2;IL-6secreted by endothelial cells under high shear stress;IL-6 secreted by endothelial cells under high shear stress regulates macrophage polarization to M2 through STAT3/KLF4 pathway.Regulation the gene expression or functions of IL-6,IL-6R,STAT3 and KLF4,it was further confirmed that IL-6 secreted by endothelial cells under high shear stress regulates macrophage polarization to M2 through STAT3/KLF4 pathway.