The Distirbution Of Dendritic Cell Within Atherosclerotic Arterial Wall And The Effect Of Dendritic Cells On The Regulation Of Th17Cells In Vitro

Atherosclerosis (atherosclerosis, AS) is a chronic inflammatory autoimmune disease,its main characteristic is that the immune cells are gathered at the lesion site and somespecific cellular immune responses are activated by T lymphocytes. Dendritic cells isthe most powerful professional antigen presenting cells (antigen presenting cell, APC),widely distributed and fewer, but effective antigen capture, processing, and presented toT lymphocytes, through double signal stimulation, stimulate the elementary cellularimmune response. DC plays an important role in the induction and regulation of specificcellular and humoral immune responses. DC has a dual role of immunogenicity andtolerance. It is less reported that DC has involved the formation process of AS.Tolerability of dendritic cells is a kind of cells which could cut the immune responseand maintenance of immune tolerance. Th17cell secretion of IL-17is a powerfulpromoting inflammatory cell factor, which can increase the immune response, topromote inflammation, and is closely related to AS. Several studies have confirmed thatTh17cells is the major pathogenic effect of inflammatory cells. It is not yet clearwhether CD11c+CD11b+tolDC could adjust the function of Th17and inhibitinflammatory reaction in vitro. This study collected arterial specimens which fromnormal and Patients with lower limb atherosclerosis occlusion syndrome. To investigatethe relationship between mature dendritic cell (mDC)、tolerogenic dendritic cell (tolDC)and AS by the observation on the distribution and the content of CD83and CD1apositive reaction mDC and CD11b and DC-SIGN positive reaction tolDC within thearterial wall in AS. The current study was to induce and purify CD11c+CD11b+tolDC in vitro, and to investigate the effect of CD11c+CD11b+tolDC on the regulation ofTh17cells in vitro. Aim:To investigate the distribution of immunogenic and tolerogenic vascular dendriticcell(VDC) within the arterial wall in the lower limb atherosclerosis occlusionsyndrome(ASO), and to discussion the relationship between the immunogenic andtolerogenic VDC and the disease of lower limb ASO. To investigate the effect ofCD11c+CD11b+tolDC on the regulation of Th17cells in vitro.Methods:Forty human arterial specimens were collected from surgical operation and autopsy, andparaffin sections were made and HE stained routinely. Ten of them without obviousmorphosis change were assigned as control group and the other thirty cases of lowerlimb artery lesions clearly were set to lesion group. The distribution of S100positive,immunogenicity CD1a, CD83and CCR7positive and tolerability CD11b, DC-SIGN,TLR-2, IDO positive cells were detected by using immunohistochemistry method onsections. The expression of proteins of immunogenicity CD1a, CD83and tolerabilityCD11b, IDO were detected by the method of western blot. CD11c+CD11b+tolDC wereinduced by rmGM-CSF and rmIL-4in vitro, and were identified by the expressions ofIL-10and TGF-β1. In vitro, the effects of tolDC on T lymphocytes were analyzed bythe cytokine levels IL-17, the percent of Th17, and the mRNA expressions of IL-17. Themethods of real-time PCR (RT-PCR) were utilized to detect the mRNA expressions ofIL-17in the thymus. The levels of IL-10and TGF-β1in cultrue supernatant weredetected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used totest the percentage of the tolDC, mDC and Th17.Results1. The HE staining pathology changes of clinical specimensHE staining of blood vessels in the control group was normal arteries, the HE staining pathology changes of experimental vascular visible vascular endothelial upliftdeformation, intimal thickening, forming fiber atheromatous plaque; a lot of fiberhyperplasia within subendothelial layer of plaques, which scattered a large number offoam cells and smooth muscle fibers; A large number of void shape or needle lipiddeposition in the deep within the plaques.2. The expression of S-100+VDCImmunogenicity S-100positive VDC were heavily found in intima plaque.3．The expression of CD83+、CD1a+、CCR7+VDCImmunogenicity CD1a, CD83and CCR7positive VDC were heavily found in intimaplaque. Western blot results found that they were high expression of CD1a、CD83in theASO group than the control group.4.The expression of CD11b+、DC-SIGN+、TLR-2+and IDO+VDCImmunogenicity CD11b、DC-SIGN、TLR-2and IDO positive VDC were lowerexpression in intima plaque.Western blot results found that they were high expression ofCD11b and IDO in the ASO group than the control group.5. CD11c+CD11b+tolDC derived from bone marrow were induced successfully anddisplayed a tolerogenic characteristic5.1CD11c+CD11b+tolDC were induced successfullyResults showed that the percent of CD11c+CD11b+tolDC in bone marrow cells inducedby rmGM-CSF and IL-4was approximately45%, and the proportion of theCD11c+CD11b+tolDC was99%by further purification. About1×106BMCD11c+CD11b+tolDC could be collected from each mouse.5.2CD11c+CD11b+tolDC were displayed a tolerogenic characteristicThe levels of IL-10and TGF-β1in the CD11c+CD11b+tolDC culture supernatants weresignificantly higher than those in the CD11c+CD83+mDC culture supernatants.CD11c+CD11b+tolDC demonstrate a tolerogenic characteristic. 6. Effects of CD11c+CD11b+tolDC on the t he differentiation of Th17cells in vitro6.1CD11c+CD11b+tolDC inhibited the differentiation of Th17cells in vitroTo investigate the role of tolDC on Th17cells, T cells were re-stimulated with PMA,Ion and BFA in the co-culture system of tolDC and T lymphocytes. Results showed thatcompared with LPS induced mDC, the percentage of CD4+IL-17+Th17cells weresignificantly decreased.6.2tolDC inhibited the expressions of IL-17mRNA in Th17cells in vitroThe expression of IL-17mRNA was analyzed by RT-PCR, and results showed that theexpression of IL-17mRNA in T cells was down-regulated compared with mDC groups.6.3tolDC inhibited co-cultured T cells to express IL-17in vitroCompared with mDC group, the level of IL-17in T cells supernatants was decreased inthe co-culture system.Conclusion:1. tolDC and mDC were assembled in the process of ASO, and the progression of thedisease might be aggravated by DC-maturation.2. CD11c+CD11b+tolDC could be induced successfully with rmGM-CSF and rmIL-4invitro.3. tolDC could suppress Th17functions in vitro.