牙龈卟啉单胞菌脂多糖对树突状细胞成熟及功能影响的体外研究
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摘要
目的研究牙龈卟啉单胞菌脂多糖(P.gingivalis-LPS)的刺激对大鼠树突状细胞(DCs)成熟及功能的影响,为探索DCs在牙周炎的发生发展中的作用机制提供实验依据。方法采用流式细胞学方法检测P.gingivalis-LPS和大肠杆菌脂多糖(E.coli-LPS)刺激下,CD11c~+MHCⅡ~+、CD11c~+CD80~+、CD11c~+CD86~+和CD11c~+CD40~+DCs的比率;采用ELISA法检测DCs分泌白介素-12(IL-12)、干扰素-γ(IFN-γ)、白介素-10(IL-10)和白介素-13(IL-13)的量。采用CCK8法检测与上述DCs共培养的CD4+T细胞的增殖;采用ELISA法检测T细胞分泌IL-2、IFN-γ、IL-10和IL-13的量。在上述的培养系统中加入Toll样受体4(TLR4)抑制剂(polymyxin B,PmB)或TLR2/TLR4抑制剂(oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine,OxPAPC),观察TLR抑制剂对上述DCs成熟及功能的影响。结果 P.gingivalis-LPS与E.coli-LPS均能刺激DCs成熟。TLR4抑制剂明显抑制E.coli-LPS组DCs成熟和抗原提呈功能,对P.gingivalis-LPS组DCs成熟和抗原提呈功能没有显著抑制。TLR2/TLR4抑制剂对P.gingivalis-LPS组DCs成熟和抗原提呈功能显著抑制。P.gingivalis-LPS组DCs分泌IL-12和IFN-γ的量低于E.coli-LPS组(P<0.05);P.gingivalis-LPS组DCs分泌IL-10和IL-13的量高于E.coli-LPS组(P<0.05)。与P.gingivalis-LPS和E.coli-LPS共培养的DCs均能促进CD4+T细胞增殖。与P.gingivalis-LPS组DCs共培养的T细胞分泌IL-2和IFN-γ的量低于E.coli-LPS组(P<0.05);其分泌IL-10的量高于E.coli-LPS组(P<0.05)。结论 P.gingivalis-LPS能促进DCs的成熟和抗原提呈功能。P.gingivalis-LPS刺激下的DCs促进Th2型免疫应答;E.coli-LPS刺激下的DCs促进Th1型免疫应答。P.gingivalis-LPS通过TLR2通路刺激DCs成熟;E.coli-LPS通过TLR4通路刺激DCs成熟。
Objective dy the roles of P.gingivalis-LPS on the maturation and functions of DCs to provide experimental evidences to explore the possible mechanism of DCs in periodontitis. Methods Flow cytometry was used to detect CD11c,MHCⅡ,CD80,CD86 and CD40 expression on DCs which were stimulated by P.gingivalis-LPS or E. coli-LPS and ELISA was used to detect IL-12,IFN-γ,IL-10 and IL-13 secreted by DCs. CCK8 was used to assay CD4~+T cells proliferation after co-cultured with DCs stimulated by P.gingivalis-LPS or E.coli-LPS and ELISA was used to detect IL-2,IFN-γ,IL-10 and IL-13 secreted by T cells. TLR4 inhibitor( polymyxin B) or TLR2 and TLR4 inhibitor( OxPAPC) was added to P.gingivalis-LPS group and E.coli-LPS group to observe the effects of these two TLR inhibitors on the maturation and antigenpresenting functions of DCs. Results The capacity of P.gingivalisLPS to stimulate DCs maturation was similar to that of E.coliLPS. When TLR4 inhibitor was added to E. coli-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited. When TLR4 inhibitor was added to P. gingivalis-LPS group,maturation and antigen-presenting functions of DCs were not significantly inhibited. When TLR2 and TLR4 inhibitor was added to P.gingivalis-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited.The level of IL-12 and IFN-γ secreted by DCs in P. gingivalis-LPS group was significantly lower than that of E. coli-LPS group( P<0.05),meanwhile,IL-10 and IL-13 secreted by DCs in P.gingivalis-LPS group was significantly higher than that of E.coliLPS group( P<0.05). DCs stimulated by both P.gingivalis-LPS and E.coli-LPS could promote the proliferation of CD4~+T cells. The amount of IL-2 and IFN-γ secreted by T cells stimulated by DCs in P.gingivalis-LPS group was significantly lower than that of E.coliLPS group( P<0.05),meanwhile,IL-10 secreted by T cells stimulated by DCs in P.gingivalis-LPS group was significantly higher than that of E.coli-LPS group( P<0.05). Conclusion P.gingivalis-LPS could promote DCs maturation and antigen-presenting functions.DCs stimulated by P.gingivalis-LPS are prone to induce a stronger Th2 cell responses while DCs stimulated by E.coli-LPS are prone to induce a stronger Th1 cell responses. P. gingivalis-LPS triggers DCs through TLR2 pathway while E. coli-LPS triggers DCs through TLR4 pathway.
Objective dy the roles of P.gingivalis-LPS on the maturation and functions of DCs to provide experimental evidences to explore the possible mechanism of DCs in periodontitis. Methods Flow cytometry was used to detect CD11c,MHCⅡ,CD80,CD86 and CD40 expression on DCs which were stimulated by P.gingivalis-LPS or E. coli-LPS and ELISA was used to detect IL-12,IFN-γ,IL-10 and IL-13 secreted by DCs. CCK8 was used to assay CD4~+T cells proliferation after co-cultured with DCs stimulated by P.gingivalis-LPS or E.coli-LPS and ELISA was used to detect IL-2,IFN-γ,IL-10 and IL-13 secreted by T cells. TLR4 inhibitor( polymyxin B) or TLR2 and TLR4 inhibitor( OxPAPC) was added to P.gingivalis-LPS group and E.coli-LPS group to observe the effects of these two TLR inhibitors on the maturation and antigenpresenting functions of DCs. Results The capacity of P.gingivalisLPS to stimulate DCs maturation was similar to that of E.coliLPS. When TLR4 inhibitor was added to E. coli-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited. When TLR4 inhibitor was added to P. gingivalis-LPS group,maturation and antigen-presenting functions of DCs were not significantly inhibited. When TLR2 and TLR4 inhibitor was added to P.gingivalis-LPS group,maturation and antigen-presenting functions of DCs were significantly inhibited.The level of IL-12 and IFN-γ secreted by DCs in P. gingivalis-LPS group was significantly lower than that of E. coli-LPS group( P<0.05),meanwhile,IL-10 and IL-13 secreted by DCs in P.gingivalis-LPS group was significantly higher than that of E.coliLPS group( P<0.05). DCs stimulated by both P.gingivalis-LPS and E.coli-LPS could promote the proliferation of CD4~+T cells. The amount of IL-2 and IFN-γ secreted by T cells stimulated by DCs in P.gingivalis-LPS group was significantly lower than that of E.coliLPS group( P<0.05),meanwhile,IL-10 secreted by T cells stimulated by DCs in P.gingivalis-LPS group was significantly higher than that of E.coli-LPS group( P<0.05). Conclusion P.gingivalis-LPS could promote DCs maturation and antigen-presenting functions.DCs stimulated by P.gingivalis-LPS are prone to induce a stronger Th2 cell responses while DCs stimulated by E.coli-LPS are prone to induce a stronger Th1 cell responses. P. gingivalis-LPS triggers DCs through TLR2 pathway while E. coli-LPS triggers DCs through TLR4 pathway.
引文
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[3]陈哲,胡明道,田大广,等.重组腺病毒介导人类白细胞抗原-G基因转染恒河猴树突状细胞对T细胞的增殖作用[J].医学研究生学报,2017,30(1):5-9.
[4]周建光,杨梅,曹海涛,等.淋巴细胞亚群的检测在临床的应用[J].东南国防医药,2015,17(3):298-300,321.
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[6]Kocgozlu L,Elkaim R,Tenenbaum H,et al.Variable Cell Responses to P-gingivalis Lipopolysaccharide[J].J Dent Res,2009,88(8):741-745.
[7]Kirikae T,Nitta T,Kirikae F,et al.Lipopolysaccharides(LPS)of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/He J macrophages in a way different from that of Salmonella LPS[J].Infect Immun,1999,67(4):1736-1742.
[8]Jones KJ,Ekhlassi S,Montufar-Solis D,et al.Differential cytokine patterns in mouse macrophages and gingival fibroblasts after stimulation with porphyromonas gingivalis or Escherichia coli lipopolysaccharide[J].J Periodontol,2010,81(12):1850-1857.
[9]Barksby HE,Nile CJ,Jaedicke KM,et al.Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide[J].Clin Exp Immunol,2009,156(3):479-487.
[10]Jotwani R,Moonga BS,Gupta S,et al.Nuclear factor-kappa B p50 subunits in chronic periodontitis and Porphyromonas gingivalis lipopolysaccharide-pulsed dendritic cells[J].Ann N Y Acad Sci,2010,1192:278-285.
[11]Diya Z,Lili C,Shenglai L,et al.Lipopolysaccharide(LPS)of Porphyromonas gingivalis induces IL-1beta,TNF-alpha and IL-6production by THP-1 cells in a way different from that of Escherichia coli LPS[J].Innate Immun,2008,14(2):99-107.
[12]Sun Y,Li H,Sun MJ,et al.Endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli:alternations in Toll-like receptor 2 and 4 signaling pathway[J].Inflammation,2014,37(1):268-276.
[13]Andrukhov O,Ertlschweiger S,Moritz A,et al.Different effects of P.gingivalis LPS and E.coli LPS on the expression of interleukin-6 in human gingival fibroblasts[J].Acta Odontol Scand,2014,72(5):337-345.
[14]Cutler CW,Jotwani R.Dendritic cells at the oral mucosal interface[J].J Dent Res,2006,85(8):678-689.
[15]Cury PR,Furuse C,Rodrigues AE,et al.Interstitial and Langerhans'dendritic cells in chronic periodontitis and gingivitis[J].Braz Oral Res,2008,22(3):258-263.
[16]Kowsar R,Hambruch N,Marey MA,et al.Evidence for a novel,local acute-phase response in the bovine oviduct:progesterone and lipopolysaccharide up-regulate alpha 1-acid-glycoprotein expression in epithelial cells in vitro[J].Mol Reprod Dev,2014,81(9):861-870.
[17]Padial-Molina M,Volk SL,Rios HF.Periostin increases migration and proliferation of human periodontal ligament fibroblasts challenged by tumor necrosis factor-alpha and Porphyromonas gingivalis lipopolysaccharides[J].J Periodontal Res,2014,49(3):405-414.
[18]Knobloch J,Feldmann M,Wahl C,et al.Endothelin receptor antagonists attenuate the inflammatory response of human pulmonary vascular smooth muscle cells to bacterial endotoxin[J].J Pharmacol Exp Ther,2013,346(2):290-299.
[19]Dadaglio G,Fayolle C,Zhang X,et al.Antigen Targeting to CD11b(+)Dendritic Cells in Association with TLR4/TRIF Signaling Promotes Strong CD8(+)T Cell Responses[J].J Immunol,2014,193(4):1787-1798.
[20]von Schlieffen E,Oskolkova OV,Schabbauer G,et al.Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids[J].Arterioscler Thromb Vasc Biol,2009,29(3):356-362.
[21]Erridge C,Kennedy S,Spickett CM,et al.Oxidized phospholipid inhibition of Toll-like receptor(TLR)signaling is restricted to TLR2 and TLR4-Roles for CD14,LPS-binding protein,and MD2as targets for specificity of inhibition[J].J Biol Chem,2008,283(36):24748-24759.
[2]Vroman H,van den Blink B,Kool M.Mode of dendritic cell activation:the decisive hand in Th2/Th17 cell differentiation.Implications in asthma severity?[J]Immunobiology,2015,220(2):254-261.
[3]陈哲,胡明道,田大广,等.重组腺病毒介导人类白细胞抗原-G基因转染恒河猴树突状细胞对T细胞的增殖作用[J].医学研究生学报,2017,30(1):5-9.
[4]周建光,杨梅,曹海涛,等.淋巴细胞亚群的检测在临床的应用[J].东南国防医药,2015,17(3):298-300,321.
[5]Hajishengallis G,Tapping RI,Harokopakis E,et al.Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus[J].Cellular Microbiol,2006,8(10):1557-1570.
[6]Kocgozlu L,Elkaim R,Tenenbaum H,et al.Variable Cell Responses to P-gingivalis Lipopolysaccharide[J].J Dent Res,2009,88(8):741-745.
[7]Kirikae T,Nitta T,Kirikae F,et al.Lipopolysaccharides(LPS)of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/He J macrophages in a way different from that of Salmonella LPS[J].Infect Immun,1999,67(4):1736-1742.
[8]Jones KJ,Ekhlassi S,Montufar-Solis D,et al.Differential cytokine patterns in mouse macrophages and gingival fibroblasts after stimulation with porphyromonas gingivalis or Escherichia coli lipopolysaccharide[J].J Periodontol,2010,81(12):1850-1857.
[9]Barksby HE,Nile CJ,Jaedicke KM,et al.Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide[J].Clin Exp Immunol,2009,156(3):479-487.
[10]Jotwani R,Moonga BS,Gupta S,et al.Nuclear factor-kappa B p50 subunits in chronic periodontitis and Porphyromonas gingivalis lipopolysaccharide-pulsed dendritic cells[J].Ann N Y Acad Sci,2010,1192:278-285.
[11]Diya Z,Lili C,Shenglai L,et al.Lipopolysaccharide(LPS)of Porphyromonas gingivalis induces IL-1beta,TNF-alpha and IL-6production by THP-1 cells in a way different from that of Escherichia coli LPS[J].Innate Immun,2008,14(2):99-107.
[12]Sun Y,Li H,Sun MJ,et al.Endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli:alternations in Toll-like receptor 2 and 4 signaling pathway[J].Inflammation,2014,37(1):268-276.
[13]Andrukhov O,Ertlschweiger S,Moritz A,et al.Different effects of P.gingivalis LPS and E.coli LPS on the expression of interleukin-6 in human gingival fibroblasts[J].Acta Odontol Scand,2014,72(5):337-345.
[14]Cutler CW,Jotwani R.Dendritic cells at the oral mucosal interface[J].J Dent Res,2006,85(8):678-689.
[15]Cury PR,Furuse C,Rodrigues AE,et al.Interstitial and Langerhans'dendritic cells in chronic periodontitis and gingivitis[J].Braz Oral Res,2008,22(3):258-263.
[16]Kowsar R,Hambruch N,Marey MA,et al.Evidence for a novel,local acute-phase response in the bovine oviduct:progesterone and lipopolysaccharide up-regulate alpha 1-acid-glycoprotein expression in epithelial cells in vitro[J].Mol Reprod Dev,2014,81(9):861-870.
[17]Padial-Molina M,Volk SL,Rios HF.Periostin increases migration and proliferation of human periodontal ligament fibroblasts challenged by tumor necrosis factor-alpha and Porphyromonas gingivalis lipopolysaccharides[J].J Periodontal Res,2014,49(3):405-414.
[18]Knobloch J,Feldmann M,Wahl C,et al.Endothelin receptor antagonists attenuate the inflammatory response of human pulmonary vascular smooth muscle cells to bacterial endotoxin[J].J Pharmacol Exp Ther,2013,346(2):290-299.
[19]Dadaglio G,Fayolle C,Zhang X,et al.Antigen Targeting to CD11b(+)Dendritic Cells in Association with TLR4/TRIF Signaling Promotes Strong CD8(+)T Cell Responses[J].J Immunol,2014,193(4):1787-1798.
[20]von Schlieffen E,Oskolkova OV,Schabbauer G,et al.Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids[J].Arterioscler Thromb Vasc Biol,2009,29(3):356-362.
[21]Erridge C,Kennedy S,Spickett CM,et al.Oxidized phospholipid inhibition of Toll-like receptor(TLR)signaling is restricted to TLR2 and TLR4-Roles for CD14,LPS-binding protein,and MD2as targets for specificity of inhibition[J].J Biol Chem,2008,283(36):24748-24759.