Toll‐Like Receptor 9 Pathway Inhibited Atheroscierotic Progression By Skewing Of Macrophage Polarization In ApoE^{‐/‐} Mice

ObjectiveAtherosclerosis(AS) is the most common pathological change in cardiovascular disease, its development related to chronic inflammatory response associated with immune reaction. Toll‐like receptors(TLRs) plays an important role in the natural immune response and acquired immune response. The role of TLR9 in atherosclerosis remains controversial. The objective of this study was to investigate the role of TLR9 in atherosclerosis development and macrophage polarization.MethodsRAW264.7 cells were treated for 24 h with agonist(ODN1826) and antagonist(IRS869) of TLR9, cells were divided into for groups: control、ODN、IRS and ODN+ IRS group; Female Apo E-/- mice were randomized into 2 groups, saline and IRS869(40 μg)were administered by intraperitoneal injection for 12 weeks, twice a week. The aorta TLR9 and its downstream molecules including My D88, p‐NF‐κB and IRF7 were determined by western blot analysis; The frequency of M1 and M2 subtype in RAW264.7 cells were evaluated with flow cytometry; The expression of TNF-α in RAW264.7 cells were evaluated with RT‐PCR and ELISA; The expression of M1‐ and M2‐ associated markers in plaques and RAW264.7 cells were detected by RT‐PCR and immune fluorescence; Plaque vulnerability were determined by Immune histochemistry、Sirius Red staining and Oil Red O staining; Oil Red O staining were used to measured the plaque area percentage in aortic sinus serial frozen sections.Results1. The levels of TLR9 in IRS group was markedly down‐regulated compared with thosein the control group in RAW264.7 cells(p<0.05), while The levels of TLR9 in ODN+IRS group was down‐regulated compared with those in ODN group(p<0.05).2. The levels of TLR9,My D88, p‐NF‐κB and IRF7 were markedly down‐regulated by 73%,66%、64% and 58% compared with those in the control littermates in Apo E‐/‐ mice(p<0.05). 3. In RAW264.7 macrophages, ODN induced a remarkably stronger M1 response, While the proportion of M2 cells notably increased when treated with IRS; co-administration of ODN and IRS caused a significant elevation of the ratio of CD206-to-i NOS compared with ODN group. 4. TLR9 inactivation resulted in an increased ratio of CD206-to-i NOS macrophages, and a powerful inhibition of M1 signature m RNA expression such as IL‐1β, i NOS and Ciita(P<0.05),Meanwhile, the expressions o M2 markers Fizz1 and Ym1 were up‐regulated(P<0.05) compared with the control group. 5. ODN1826 induced an obviously increase in the expression of TNF-α in RAW264.7 cell culture medium(P<0.05)compared with the control group, IRS869 reverse the elevated production of TNF-α induced by ODN1826(P<0.05). 6. The plaque burden of IRS869 Apo E‐/‐ decreased by 34.9% in aortic root(P<0.05)and the plaque vulnerability index declined from 3.7 to 2.6 as compared to controls(P<0.05).Conclusions1. IRS869 suppressed the activation of TLR9,p-NF-κB and IRF7. 2. TLR9 inactivation changed the state of macrophage polarization(M2/M1), and induced the increased expression of M2 macrophages. 3. TLR9 inactivation not only caused a quantitative decrease in plaque burden, but also a qualitative change in plaque composition to more stable lesions in Apo E‐/‐ mice.