SREC-I inactivation reduced LPS-TLR4 mediated proinflammatory cytokine release in BMDM.

A, TLR4 KO and SREC-I KD in macrophages led to reduced IL-6 release compared with WT cells. Primary macrophages from WT and TLR4 KO mice were transfected with siRNA-SREC-I or control hairpins for 72 hours and then incubated with LPS for 12 hours with or without SR-A blocking antibody. IL-6 release by cells was measured in the collected medium by ELISA. B, TLR4KO and SREC-I KD in BMDMs led to reduced TNF-α compared to WT cells. Primary macrophages from WT and TLR4 KO mice were transfected with siRNA-SREC-I or a control hairpin for 72 hours and then incubated with LPS for 12 hours with or without SR-A blocking antibody. TNF-α release was measured in medium by ELISA. C, SREC-I KO and TLR4KD in BMDM led to reduced IL-6 release compared with WT cells. Primary macrophages from WT and SREC-I KO mice were transfected with or without siRNA-TLR4 for 72 hours. IL-6 release into the medium was measured according to manufacturer's instructions. F, SREC-I KO and TLR4 KD BMDMs led to reduced release of TNF-α compared to WT cells. Primary macrophages from WT and SREC-I KO mice were transfected with or without siRNA-TLR4 for 72 hours. TNF-α release by was then assayed as above. E,F, TLR4 KO and SREC-I KO BMDMs led to reduced release of IFN-β compared to WT cells. Primary macrophages from WT and TLR4 KO (E), SREC-I KO were transfected or not with SREC-I siRNA (E) and TLR4 siRNA (F) for 72 hours. IFN-β release in media was assayed as above. G, H, I, Primary BMDM cells (WT, TLR4/SREC-I KO mice, TLR4/SREC-I KO + SREC-I siRNA/TLR4 siRNA) were trypsinized and cell lysates were collected. Equal amount of protein was subjected to SDS-PAGE and Western blotting. All experiments were performedreproducibly 3 times. Error bars in graph show S.D. between three replicate experiments. ***P<0.0001 when compared to the control, and #P<0.0001 when compared to the WT. Values were generated by ANOVA using the Bonferroni post-test.