| Objective: To confirm the capsular polysaccharide of Mycoplasma penumoniae（M.pneumoniae） as pathogen-associated molecular patterns (PAMP) of dendriticcell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN/CD209) andstudy the effect of M.pneumoniae capsular polysaccharide on the cytokine secretion andmaturation of the human PBMCs derived dendritic cells by binding to DC-SIGN. Providetheoretical basis for further studying of Mp immune evasion mechanisms.Methods: Recovered and cultivated M.pneumoniae M129strain. Mp precipitate wascollected by centrifugation. Extract and purify capsular polysaccharide (CPS) and lipidassociated membrane proteins (LAMPs). Phenol-sulfuric acid method and BCA wererespectively performed for CPS and LAMPs concentration determination. Human Peripheralblood mononuclear cells (PBMCs) with density gradient centrifugation was separated fromfresh peripheral blood of healthy volunteers and induced to dendritic cells.Observe cells wecultivated directly or through Giemsa’s staining under the microscope firstly and then flowcytometry(FCM) was performed to identify whether these cells were dendritic cells.Detectethe binding of CPS and DC-SIGN with indirect immunofluorescence assay (IFA). IL10andIL12expression levels was detected by Enzyme-linked immunosorbent assay(ELISA) in thesupernatant of DC culture medium treated with CPS and(or) LAMPs. Immature DC werestimulated by CPS and LAMPs, FITC-dextran Phagocytosis and surface markersCD80/CD80/CD86/HLA-DR expression levels of DC treated by CPS and (or) LAMPs weredetected by FCM and then analyze their effect on DC maturitResults:1. CPS concentration was0.751mg/mL and LAMPs concentration was0.217mg/mL.2.Cells were observed directly or through Giemsa’s staining under the microscope.Some of PBMCs were adherent to the plastic surface of the cells culture dish and could be induced toimmature dendritic cells3～5days later. The morphology of PBMCs tended to be colonygroups and the number of colony groups increased with the growth time.After incubatedwith LPS for18h, some of the DC became mature.FCM detection showed that theirsurface markers expression levels increased as follows: HLA-DR were incresded from(29.525±8.19)%to (77.47±19.11)%, CD80were incresded from (48.50±0.59)%to(86.85±11.24)%and CD86were incresded from (32.71±12.70)%to (84.95±1.81)%.3. CPS and DC-SIGN interaction was analyzed by IFA. After staining with Homologousfluorescent antibody, CPS was stained to be green fluorescence, DC-SIGN was stained withfluorescent antibody to be red fluorescence.Nucleolus were stained to be blue fluorescenceafter DAPI staining. Results showed that red fluorescence was observed on the surface ofDC while it could disappear after cells treated by anti-DC-SIGN-specific antibody. Thissuggested that this antibody had blocked DC-SIGN receptors. When CPS co-incubated withDC, green fluorescence was observed on the surface of DC. The green fluorescence and thered fluorescence merged to be yellow fluorescence. Red and green fluorescence disappearedwhen DC-SIGN receptors blocked by anti-DC-SIGN-specific antibody.4. Immature DC were stimulated18h by CPS and (or) LAMPs. We detected IL-10and IL-12expression levels in cell culture supernatant. The results showed that: compared withuntreated group, IL-10expression level in LAMPs treatment cells culture supernatantremained unchaged (P>0.05). IL-10expression level in CPS stimulating as well asco-stimulating (CPS and LAMPs) cell culture supernatant increased considerably(P<0.001).Co-stimulating (CPS and LAMPs) group,compared with either CPS or LAMPstreatment group, IL-10expression was significantly higher(P<0.05). IL-10level was highestwhen CPS stimulating concentration was2.8μg/mL and LAMPs is7.2μg/mL. There is nosignificant difference exists（P>0.05）in IL-12expression level between each treatmentgroups.5. FITC-dextran Phagocytosis of iDC was detected by FCM after iDC stimulated by CPSand LAMPs for18h. Results showed that:compared with untreated group, DC uptookFITC-Dextran fluorescence mean of CPS treated group increased significantly(P<0.01), while co-stimulating (CPS and LAMPs) group’s remained unchanged (P＞0.05).6. After stimulated with CPS and (or) LAMPs,DC surface markers (CD80,CD86HLA-DRCD83)expression levels detected by FCM.Results showed that:Compared with untreatediDC group,CPS stimulation could considerably down-regulate the expression ofCD80,CD86,CD83and HLA-DR.In contrast, LAMPs could considerably up-regulate theexpression of CD80,CD86,CD83and HLA-DR（P＜0.05）. Expression of CD80CD86andHLA-DR（P＜0.001）was up-regulated by CPS and LAMPs co-stimulation as LAMPstreated group. CD83was down-regulated（P＜0.001） by CPS and LAMPs co-stimulatedgroups but Compared with CPS stimulation group, the down-regulate effect was notsignificant（P＜0.05）.Conclusion:1. M.pneumoniae CPS could recognize and bind to DC-SIGN.2. M.pneumoniae CPS could enhance IL-10express.3. Mp CPS could promote the phagocytic function of DC and inhibite iDC maturationwhile Mp LAMPs could stimulate iDC maturation. |
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