ILC2s Activation By Treg Attenuates Atherosclerosis

Background and Objective:Acute cardiovascular events caused by atherosclerosis?AS?have become the number one killer.Regulatory T cells are acquired immune cells that play an important regulatory role in immune homeostasis.It can be seen from previous studies that Treg plays an important role in maintaining vascular immune homeostasis and inhibiting the occurrence and development of atherosclerosis.Innate lymphoid cells?ILCs?are a new type of natural immune cells discovered in recent years,which play an important role in immune response and tissue construction and repair.ILC2s may attenuate the development of atherosclerosis,according to new researches.However,whether Treg may inhibit AS by regulating the activation of ILC2s has not been studied at home and abroad.Therefore,the aim of our study was to investigate the mechanism of role of Treg in atherosclerosis and whether it relieved atherosclerosis by activating ILC2s.Method:1.ApoE-/-mice by high fat diet were adoptively transferred Tregs for 16 weeks.We stained the aortic en face and aortic root by Oil Red O and aortic root plaque by immunohistochemical Masson,CD68,?-SMA.2.To study the effect of Treg on ILC2s,puried CD4⁺Foxp3?GFP?⁺Treg cells and ILC2s?lin^-CD45⁺ICOS⁺CD127⁺CD25⁺?were co-cultured in vitro.IL-5 and IL-13 secretion of ILC2s were measured by ELISA.3.To analysis the effect of Treg on ILC2s in the atherosclerosis model,we adoptively transferred CD4⁺Foxp3?GFP?⁺cells from Foxp3^GFP mice into ApoE-/-mice during HFD.The percentage and number of ILC2s in spleen,bone marrow and PaLN were detected by flow cytometry.4.To analyze the role of ILC2s in atherosclerosis,we treated ApoE-/-Rag1-/-mice with IL-2/Jes6-1 complex,anti-CD90.2 antibody,or PBS during HFD.We stained the aortic en face and aortic root by Oil Red O and aortic root plaque by immunohistochemical Masson,CD68,?-SMA.5.To analysis the effect of Treg on ILC2s in the atherosclerosis model,ApoE-/-Rag1-/-mice were maintainded on HFD for 16 weeks.The mice received injections of Tregs for the duration of the experiment.To analyze Treg activated ILC2s then Treg reduced atherosclerosis,we treated PBS,activated Treg,or activated Treg meanwhile anti-CD90.2 into ApoE-/-Rag1-/-mice during HFD.We stained the aortic en face and aortic root by Oil Red O and aortic root plaque by immunohistochemical Masson,CD68,?-SMA.6.We co-cultured Treg cells and ILC2s with neutralizing IL-10 and or TGF-?antibody,and we cultured both types of cells in transwell stimulation assay.IL-5 and IL-13 secretion of ILC2s were measured by ELISA to study the mechanism of how Treg stimulated ILC2s.Result:1.The Oil Red O positive lesion area of en face aorta and aortic sirus was significantly reduced in the recipients of CD4⁺Foxp3?GFP?⁺Treg.A significant deacrese of CD68 immunostained macrophages was observed.Interestingly,increases were revealed for collagen immune-stained area on the surface of the plaques.2.ILC2s secretion of IL-5 and IL-13 was detected by ELISA,and Treg could promote ILC2s secretion of IL-5 and IL-13 in vitro.3.ApoE-/-mice received injections of Treg and were fed with high fat diet for 16 weeks,which could increase the proportion and number of ILC2s.4.En face aorta and aortic roots were stained by Oil O Red and plaque lesions were analyzed.The mice treated with IL-2/Jes6-1 complex had reduced lesion size of en face aorta and aortic roots.Lesional area positive for macrophages was also reduced in IL-2/Jes6-1 complex treated mice.However we did not observe collagen and muscle smooth differences.There was no significant effect on lesion size or composition in anti-CD90.2 treated mice compared to PBS control.5.ApoE-/-Rag1-/-mice received injections of Treg and were fed with high fat diet for 16 weeks,which could increase the proportion and number of ILC2s.The Oil Red O positive lesion area of en face aorta and aortic sirus was significantly reduced in the recipients of Treg.A significant deacrese of CD68 immunostained macrophages was observed.Interestingly,increases were revealed for collagen immune-stained area on the surface of the plaques.The quantification of atherosclerosis in aorta and aortic root from activated Treg mice displayed significantly less plaque area,compared to activated Treg and anti-CD90.2 mice.The quantification of atherosclerosis in aorta and aortic root from activated Treg mice displayed significantly less plaque area,compared to activated Treg and anti-CD90.2 mice.There were no difference observed in collagen and muscle smooth content per plaque area between two groups.6.We co-cultured ILC2s and Tregs with neutralizing antibodies against IL-10 and TGF-?.In these cultures,presence of neutralizing antibody for IL-10 with or without TGF-?abrogated the Treg-enhancement of IL-5 and IL-13 production by ILC2.And we cultured both types of cells in transwell stimulation assay showed that cell-to-cell contact was required for activation of ILC2s by Treg.Conclusion:1.Tregs reduces atherosclerosis.2.Tregs induce production of type 2 cytokines by mouse ILC2s in vitro and vivo.3.IL-2/Jes6-1 complex treatment reduces atherosclerosis.4.Tregs treatment protected atherosclerosis,by which induced the expansion of ILC2s?5.Treg cells activated ILC2s by cell-to-cell contact and by the secretion of IL-10 and TGF–?...