| BackgroundUPR is a protective program for maintaining endoplasmic reticulum steady-state under adverse environment and preventing endoplasmic reticulum apoptosis in cell.In previous studies,UPR related molecular expression in periodontitis tissue is higher than normal periodontal tissue,and periodontitis patients PDLSC(P-PDLSC)UPR activation level is higher than health PDLSC(H-PDLSC).But there is no report about UPR in the periodontal ligament fibroblasts from periodontitis patients.In the periodontal ligament,the periodontal ligament fibroblasts is one of the most important cells,and its main function is to participate in the production and metabolism of extracellular matrix.PDLF also has the function of many similar innate immune cells such as expression of pattern recognition receptors,recognition patterns of the pathogen associated molecular,producing proinflammatory cytokines and chemokines,promoting the gather and activation of innate immune and acquired immune cells in tissue of inflammation.There is no report about what dose UPR impact on immune function of the periodontal ligament fibroblasts.M1/M2 of macrophage polarization plays an important role in the removal of the pathogens and tissue repair in the development of periodontal inflammation.M1 macrophages secrete a large number of cytokines which could promote the development of inflammation,promote osteoclast differentiation and promote the extracellular matrix degradation.M2 macrophages could promote tissue repair and regeneration by secreting anti-inflammatory cytokines and growth factors.M1,M2 cell proportion and function were different in the different stage of inflammation,and the prognosis of inflammation was influenced by excessive polarization and abnormal transformation of phenotype.There were many previous reports about HPDLF for their regulation to macrophages,but the effects of UPR state on HPDLF regulatory fuctions have not been reported.LAIR-1 called CD305 is an immune inhibitory receptor which expressed on most surface of the immune cells.In vitro studies,CD305 transmit strong inhibitory signals after crosslinking activation with monoclonal antibody(m Ab).But the relationship between LAIR-1 and monocytes-macrophages polarization has not been reported.LAIR-1 is closely related with Rheumatoid Arthritis(Rheumatoid Arthritis,RA)and inhibit osteoclast differentiation.The pathogenesis of periodontitis and RA were similar.But there were no reports about the relationship between periodontal disease and LAIR-1.This study compares the UPR status between the inflammatory and normal periodontal ligament fibroblasts after vitro cultivation.In order to study the impact of the UPR on HPDLF's immune response,the HPDLF,which were preconditioning with UPR inducers,were stimulated by LPS and then the production of cytokine,the expression of TLR,the activation of NF-?B were detected.This research mainly divided into three parts: Part one: The expression of UPR in inflammatory human periodontal ligament fibroblastsObjective 1.To compare the UPR related gene expression in P-HPDLF and P-HPDLF;2.To observe the expression of UPR in H-PDLSCs after treatment with LPS,TNF-??IL-1? respectively.Methods q RT-PCR was used to analyze the expression of UPR related genes in P-HPDLF and H-HPDLF.GRP78?PERK?CHOP were detected by q RT-PCR and western blot in HPDLF which were treated with TNF-?(10ng/ml),IL-1?(0.1ng/ml),LPS(100ng/ml).Results The expression of GRP78?CHOP?PERK?XBP-1s m RNA was higher in P-HPDLF than H-HPDLF.The m RNA expression of GRP78?CHOP?PERK?XBP-1s and the protein expression of GRP78?PERK?CHOP were increased,after stimulated by LPS,TNF-? or IL-1?.Conclusion UPR was activated in vitro cultivation P-HPDLF and the expression of PERK pathways related molecules increased obviously.UPR can be induced in H-HPDLF by LPS,TNF-?,IL-1?.Part two: The effects of UPR on HPDLF immune fuctionsObjective 1.To observe whether HPDLF activated by LPS could induce the M1 polarization of macrophages;2.To observe whether the M1 polarization of macrophage were affected by the UPR state of HPDLF;3.To observe whether the secretion of TNF-?,IL-1?,IL-6,IL-8 was affected in HPDLF in UPR state;4.To observe whether the expression of pattern recognition receptors TLR4/CD14 were affected in HPDLF in UPR state;5.To observe whether the activation of NF-?B pathway was affected in HPDLF in UPR state.Methods 1.FCM was used to detect the CCR7,CD86,CD163,CD206,IL-10 and Arginase-1 expression on d THP-1 after co-culture with HPDLF pretreated by LPS,and q RT-PCR was used to detect the expression of m RNA of polarization related molecules;2.FCM was used to detect M1/M2 polarization related molecules expression on the THP-1 and CD14+ macrophage,which co-culture with HPDLF pretreated with TM/TG for 6h and stimulation with LPS for 12h;3.HPDLF were pretreated with TM/TG,followed by stimulation with LPS.The levels of TNF-?,IL-1?,IL-6 and IL-8 were then analyzed by ELISA and q RT-PCR;4.HPDLF were pretreated with TM/TG,followed by stimulation with LPS.The expression of CD14 and TLR4 were then analyzed by FCM;5.HPDLF were pretreated with TM/TG,followed by stimulation with LPS.The expression of p-NF-?B p65,NF-?B p65,p-I?B and I?B were then analyzed by Western Blot;Results 1.After cocultivation with HPDLF pretreated with LPS,the protein expression of CCR7,CD86 was significantly increased on d THP-1.The m RNA of CCR7,CD86,IL-1?,TNF-? and IL-12 were also increased.The expression of IL-10 and Arginase-1 in d THP-1 were decreased;2.After 6 h TM or TG pretreatment,HPDLF is restrained in the function of promoting macrophage M1 polarization.The expression of CCR7 and CD86 were significantly decreased in THP-1 or CD14+ macrophage compared with the control group;3.The m RNA levels of IL-1?,IL-6,TNF-? and IL-8 were decreased in the HPDLF which were pretreated by TM or TG compared with that of cells which were not pretreated.Meanwhile,in cell culture supernatant of the HPDLF at UPR state,the IL-1?,IL-6,IL-8 and TNF-? expression levels were significantly decreased;4.FCM results showed that preconditioning with UPR decreased the expression of TLR4 induced by LPS in HPDLF;5.Western Blot results show that TM itself do not affect the expression and phosphorylation of NF-?B p65 or I-?B,but can reduce the phosphorylation of NF-?B p65 induced by LPS.TG itself increased the expression and phosphorylation of NF-?B p65(P < 0.01).TG can inhibit the increasing the expression of NF-?B p65 and phosphorylation of NF-?B p65 and I-?B induced by LPS.Conclusion In UPR state,the reactions of HPDLF to LPS were alleviated.UPR in HPDLF could reduce the pattern recognition receptors,decrease the secretion of cytokines and the activation of NF-?B pathways.UPR activation plays a reducer role in HPDLF promoting M1 polarization functions.Part three: The effects of UPR state of HPDLF on the expression of inhibitory receptor LAIR-1 on the macrophageObjective 1.To observe the expression of LAIR-1 on the surface of monocytes-macrophages in chronic inflammatoty microenvironment of periodontitis;2.To detect the monocytes-macrophages polarization when LAIR-1 was activated by monoclonal antibody 91C3.Methods 1.FCM used to detect the expression of LAIR-1 on d THP-1 which were treated with TNF-?(10ng/ml),IL-1?(0.1ng/ml),E.coli LPS(100ng/ml)and P.g LPS(100ng/ml);2.FCM used to detect the expression of LAIR-1 on macrophages co-culture with HPDLF,which were pretreated with TM 6 h or not;3.Using flow cytometry and q RT-PCR to detect whether LAIR-1 activated by 91C3 have an effect on polarization of the M1 and M2.Results 1.Flow cytometry test shows: the expression of LAIR-1 on macrophages surface were significantly decreased,after treated with TNF-?,IL-1?,E.coli LPS and P.g LPS.Co-culture with HPDLF pretreated with E.coli LPS could reduce the the expression of LAIR-1 on macrophages surface.Decreasing of LAIR-1 from macrophages co-culture with HPDLF alleviated when HPDLF were pretreated with UPR inducer;2.LAIR-1 on monocytes-macrophages can be crosslinked activated by monoclonal antibody 91C3.Flow cytometry results showed that increasing expression of CCR7,CD86 induced by IFN-? were inhibited by activation of LAIR-1.q RT-PCR results showed that the expression of CCR7,CD86,IL-1? and IL-6 m RNA were significantly inhibited by activation of LAIR-1.Flow cytometry results showed that activation of LAIR-1 does not influence CD163,CD206 induced by IL-4.But the q RT-PCR results showed that CD163 and CD206 m RNA expression were increased by activation of LAIR-1.Activation of LAIR-1 has no effect on the m RNA expression of IL-10 and Arginase-1.Conclusion We proved that activation of LAIR-1 can significantly inhibit the macrophages M1 polarization.Expression of LAIR-1 decreased under the stimulation of pathogenic factors or cytokines.UPR preconditioning has a positive significance for maintaining this surface molecular. |
PDF Full Text Request