IL-37 Inhibits The Maturation Of Dendritic Cells Through The IL-1R8-TLR4-NF-κB Pathway

Part IThe effect of IL-37 on the progression of atherosclerosis in Apo E^–/–miceObjective:At present,IL-37 is considered as an anti-inflammatory substance that can down-regulate a variety of pro-inflammatory factors and is involved in the occurrence and development of a variety of chronic diseases.Atherosclerosis is one of the most common chronic diseases,but it lacks effective prevention and treatment.This study is to explore the protective effect of IL-37 on the progression of atherosclerosis and its immunoregulatory effect in the progression of atherosclerotic disease and to search for potential new therapeutic targets for atherosclerosis.Methods:The experimental group was divided into two groups:Apo E^–/–and IL-37tg Apo E^–/–mice,with about 15～20 mice in each group.The western diet was started at 8 weeks of age,and the mice were sacrificed,weighed after 12 weeks.After collecting the mice,we isolated the serum from the blood and then detected the level of total cholesterol,triglyceride,glucose.We used ELISA to test the levels of various inflammatory factors,Th1-related cytokines,such as TNF-α,IL-6,IL-1β,IL-12βand IFN-α,Th2 related cytokines,such as IL-4 and IL-10 in the serum.After that,the whole aorta was dissected and the heart was embedded for aortic valve annular plaque section,and the plaque was stained with Oil Red O to analyze the size of the plaque.To further evaluate the immunomodulatory role of IL-37in atherosclerotic plaques,we performed immunohistochemical staining of CD4,CD68,CD31,andα-SMA in aortic valve ring plaques in two groups of mice.Results:First of all,we constructed IL-37tg mice to enable the stable expression of IL-37 in mice.IL-37 had no significant effect on body weight,cholesterol,triglyceride and glucose in mice.Compared with ordinary Apo E^–/–mice,the total aortic plaque area and aortic root plaque area of IL-37tg Apo E^–/–mice were significantly decreased.Moreover,IL-37 changed the expression levels of various inflammatory cytokines in the serum of Apo E^–/–mice,including some Th1-related cytokines significantly decreased in the peripheral blood of IL-37tg Apo E^–/–mice,while Th2-related cytokines significantly increased.Further immunohistochemical staining of aortic root plaques showed that CD4-positive T lymphocytes and CD68-positive macrophages were decreased in the IL-37tg Apo E^–/–group,while endothelial cells were not significantly changed,and smooth muscle cells were increased.Conclusion:IL-37 can significantly reduce the progression of atherosclerosis,inhibit the level of Th1 related cytokines in the whole body,promote the secretion of Th2 related cytokines,and reduce the infiltration of CD4 positive T cells and macrophages in plaques.Part II The regulation of IL-37 on dendritic cells infiltrating in plaques Objective: At present,IL-37 and its precursors have been confirmed to be produced and secreted by a variety of immune and non-immune cells,and dendritic cells are one of the important sources of IL-37.Mature dendritic cells can promote the progression of atherosclerosis.The purpose of this study was to explore the regulatory role of IL-37 on dendritic cells and its influence on the progression of atherosclerotic disease.Methods: After being fed a Western diet for 12 weeks,the hearts of Apo E–/– and IL-37 tg Apo E–/– mice were collected and sectioned from the aortic valve annulus at the aortic root.In order to detect the infiltration location and number of dendritic cells in the plaque,CD11 c staining was performed on the plaque in the valve annulus.In addition,the spleen cells of the two groups were detected by flow cytometry to test the expression levels of CD86 and MHCII in the spleens of mice.We then cultured bone-marrow-isolated and differentiated dendritic cells to further test the effect of IL-37 on dendritic cells in vitro.After pretreatment with 100 ng/m L IL-37,we stimulated the dendritic cells with ox LDL.After 24 hours,the expression of MHCII and CD86 was detected by flow cytometry and RT-PCR.At the same time,we also detected the expression level of cytokines in DCs by RT-PCR.To further explore the antiinflammatory properties of IL-37,we treated dendritic cells with different concentrations of IL-37,and measured the m RNA levels of CD80,CD86,and TH cell-related cytokines.In addition,the serum levels of IL-37 in IL-37 tg mice and IL-37 tg Apo E–/– mice were detected by ELISA.Results: CD11 c positive DCs mainly infiltrated on the surface of the plaque closer to the lumen.Compared with Apo E–/– mice,CD11 c positive dendritic cells were significantly reduced in the plaques of IL-37 tg Apo E–/– mice.In addition,flow cytometry analysis of spleen cells in mice showed that IL-37 tg overexpression reduced the maturity of dendritic cells in spleen.The results of in vitro experiments showed that 10 ng/m L IL-37 could most effectively inhibit the maturation of DCs and the expression of Th1-related cytokines.When the concentration of IL-37 was lower than 100 ng/m L,the anti-inflammatory ability of IL-37 was slightly weakened with the increase of IL-37 concentration.When pretreated with 200 ng/m L IL-37,m RNA levels of inflammatory cytokines did not follow the previous trend,IL-1β,IL-6,and IL-12β did not decrease,and IL-10 did not increase when compared with DCs pretreated with 100 ng/m L IL-37.The maturation of dendritic cells is no longer inhibited.ELISA results showed that the level of IL-37 in the serum of normal IL-37 tg mice was low,but significantly increased in the serum of atherosclerotic IL-37 tg mice（less than 150 ng/m L）.Conclusion: IL-37 can reduce the number of DCs infiltration in plaque and the maturity of DCs in vivo.Ox LDL-induced dendritic cell maturation was inhibited in vitro by lowconcentration IL-37 treatment,but this inhibition was not concentration-dependent.Part III The molecular mechanism of IL-37 in regulating the maturation of dendritic cells in plaques Objective: IL-37 has been confirmed to be able to exert its effect by binding to IL-1R8.In previous studies,IL-37 was found to inhibit the maturation of dendritic cells in plaques,but the exact mechanism was unclear.This part aims to explore the specific molecular mechanism of IL-37 inhibiting the progression of atherosclerosis through dendritic cells.Methods: First,the plaques of Apo E–/– mice and IL-37 tg Apo E–/– mice were immunohistochemical stained with IL-1R8,TLR4,and p65,and the protein levels of IL-1R8,TLR4,and p65 in the spleen and thymus were detected by Western blot.In addition,in order to investigate whether IL-1R8-TLR4-NFκB pathway exists in plaque-infiltrated DCs,we used CD11 c to co-stain the cells with IL-1R8,TLR4 and p65 by immunofluorescence.Finally,in order to explore the effects of different concentrations of IL-37 on the IL-1R8,TLR4 and NF-κB pathways of DCs in vitro,we pretreated the bone marrow isolated and induced DCs with different concentrations of IL-37 and detected the changes in the levels of each molecule in the pathway.Results: Immunohistochemical staining results showed that the expression of IL-1R8 in the plaques of IL-37 tg Apo E–/– group was increased,TLR4 and p65 were significantly decreased,and IL-1R8 was mainly expressed in the cells near the lumen on the plaque surface.Western blot results of the spleen showed that IL-1R8 was slightly increased in Apo E–/– mice and significantly increased in IL-37 tg Apo E–/– mice compared with WT mice.TLR4 was significantly increased in Apo E–/– mice but decreased in IL-37 tg Apo E–/– mice.The p65 changes of NF-κB pathway were similar to those of TLR4,but the difference was small.The results of immunofluorescence co-staining showed that IL-1R8,TLR4 and p65 were highly expressed in DCs.The expression of IL-1R8 in DCs of IL-37 tg Apo E–/– mouse plaques was increased,and the expression of TLR4 and p65 was decreased.In vitro experiment results showed that when DCs was pretreated with high concentration of IL-37,although IL-1R8 continued to be produced and increased,TLR4 expression was not inhibited accordingly.Conclusion: IL-37 regulates the changes of IL-1R8,TLR4 and NF-κB in plaque and the whole body of Apo E–/– mice,and the activation of this molecular pathway also explains the non-concentration-dependent phenomenon of IL-37.Part IVIL-1R8 and TLR4 are important molecular pathways for IL-37 to inhibit the maturation of dendritic cells Objective: In previous studies,we found that IL-1R8 and TLR4 were closely related to the regulation of IL-37,but whether IL-37 inhibited dendritic cell maturation through IL-1R8 and TLR4 has not been proved.This part of the study aims to explore whether IL-1R8 and TLR4 are important pathways for IL-37 to inhibit the maturation of dendritic cells.Methods: In order to investigate whether IL-1R8 is the key molecule of IL-37’s protective effect,we attempted to interfere with IL-1R8 and monitor the protective effect of IL-37.First,we isolated bone marrow cells from IL-1R8–/– mice and induced them to differentiate into DCs.DCs were pretreated with 100 ng/m L IL-37.After the same stimulation with ox LDL,CD86 and CD80 expression on the surface of DCs were detected by flow cytometry,and m RNA levels of TLR4 and p65 in BMDCs were detected subsequently.We cultured TLR4–/– murine-derived BMDCs to confirm the effect of TLR4 on the maturation process of DCs.After stimulation with 50 μg/m L ox LDL,the proportion of CD86 and MHCII positive cells in TLR4–/– murine-derived BMDCs and C57 murine-derived BMDCs was determined by flow cytometry Results: The results showed that IL-37 effectively reduced the expression of MHCII and CD86 in BMDCs isolated from C57 mice after stimulation with ox LDL.However,these effects were not observed on BMDCs isolated from IL-1R8–/– mice,suggesting that IL-1R8 plays an indispensable role in the process of IL-37 inhibiting BMDCs maturation and inhibiting the secretion of pro-inflammatory factors.In IL-1R8–/– BMDCs,IL-37 no longer inhibited the expression of IL-1β,IL-12β and IL-6,and the expression of TLR4 was significantly increased,while the m RNA levels of p65 and i KBe increased slightly.The presence of IL-1R8 can inhibit the expression of TLR4,which in turn affects the activities of p65 and i KBe in the downstream NF-κB pathway.The proportion of CD86 and MHCII positive cells in TLR4–/– mouse BMDCs was significantly reduced.Conclusion: IL-37 inhibited the maturation of dendritic cells through IL-1R8-TLR4-NF-κB pathway,and significantly alleviated the progression of atherosclerosis in Apo E–/– mice.