The Signaling Pathways Of Cells Inflammatory Response Induced By Porphyromonas Gingivalis Lipopolysaccharide And Construction And Study Of Gingipains

Chronic periodontitis(CP) is one of the common oral diseases in human beings which is affected by multiple factors.It has been demonstrated that periodontitis is elicited by suppression of periodontal immune defenses,colonization and overgrowth of periodontal bacterial pathogens which produce an array of virulence factors,release of inflammatory cytokines and chemokines by host cells,initiation of cytotoxic or immunopathological events,and subsequently periodontal tissue breakdown.Porphyromonas gingivalis(Pg) is a gram-negative anaerobe that is recognized as an important etiologic agent of human CP and is frequently isolated from the periodontal pockets of patients with chronic periodontal disease.Pg expresses a number of potential virulent factors that enable it to evade innate immmune defense system and destroy host cells,including lipopolysaccharides(LPS),gingipains, fimbriae,collagenase,hemagglutinin and a superoxide dismutase,etc.LPS,a major constituent of outer membrane of Pg,which has a wide biological activities and is a virulent large molecular,stimulates various cell types to release numerous proinflammatory mediators and eventually causes breakdown of periodontal tissue.It is recognized as one of the major virulent factors.Pg-LPS have been shown to differ from Escherichia coli LPS(E-LPS) in structure distinctly,so it is possible that the inflammatory reaction of host cells to Pg-LPS is different from LPS of E-LPS.To date,the signaling pathway of Pg-LPS-induced periodontitis is still unclear and whether it is special from E-LPS needs to be elucidated.Pg cysteine proteinases(gingipains),a group of proteins with similar structure and function,play an important role in the pathgenesis of periodontal disease.It has multiple pathogenic activities,good immunogenicity and can induce host protective immune reaction,recognized as a potential vaccination for periodontitis.Gingpains have double effects on mmunoreactions in complement system(promotion or delay),and may be associated with CP.It is reported that gingipains preferentially hydrolysis human monocyte CD14,the main receptor for LPS,and inhibit a CD14-dependent cell activation,resulting in attenuation of the cellular recognition of bacteria,and as a consequence sustain chronic inflammation.The objective of the present study is:To determine the receptors,the main inflammatory signaling pathways and the ability to induce cytokines in human monocytic cells and neutrophils under inducement of Pg-LPS.To construct a prokaryotic expression systems of ginigpains genes,which lays solid foundation for future study of vaccine of periodontitis,animal experiments and clinical trials. Proteolysis of human monocyte CD14 by Pg gingipains was also studied.The clarification of these aspects would help to further understand the mechanism of Pg in the initiation and progress of CP.The main contents and results are listed below:Part one:The effect of Porphyromonas gingivalis lipopolysaccharide on inducing cytokines in human cells and the associated Toll-like receptors and the intracellular signaling pathwaysIn experiment 1,Pg-LPS was extracted and purified from Pg ATCC 33277 strain according to a modified hot-phenol-water method.The LPS preparations were examined by infrared spectroscopy.The components of saccharides and fatty acids were identified by a Gas Liquid Chromatography system.Tachypleus Amebocyte Lysate (TAL) kit was used to test the bio-activity of purified Pg-LPS.The result showed:The infrared spectrum of Pg-LPS were much similar to that of E-LPS,while the major fatty acids of Pg-LPS were different from that of E-LPS;TAL test indicated the extracted Pg-LPS was biologically active(≥15.0ng/ml),and can be used in this study.In experiment 2,human monocyte THP-1 cell strain and human neutrophil HL-60 cell strain were used as the target cells.Pg-LPS and commercialized lippopolysacchride of Escherichia coli strain O111:B4(E-LPS) was applied to the cells.IL-1β,TNF-αand IL-6 concentrations in the supernatants was detected by commercial enzyme-linked immunosorhent assay kits(ELISAs).The result showed:①When THP-1 cells were stimulated for 0.5,6 and 6 h with 1μg/ml Pg-LPS,and for 6,24 and 24 h stimulated with 1μg/ml E-LPS,the level of TNF-α,IL-1βand IL-6 was obviously(P<0.01) increased respectively.The concentrations of the three cytokines induced by Pg-LPS were higher than those induced by E-LPS(P<0.05).②When HL-60 cells were stimulated for 48,24 and 48 h with 1μg/ml Pg-LPS,and for 6,24 and 24 h stimulated with 1μg/ml E-LPS,the levels of TNF-α,IL-1βand IL-6 were noticeably(P<0.01) promoted respectively.The maximum concentrations of the three cytokines secreted by HL-60 cells were much lower than that by THP-1 cells.The maximum concentrations of TNF-αinduced by E-LPS were similar to those induced by Pg-LPS(P>0.05),but the maximum concentrations of IL-1βand IL-6 were obviously lower than Pg-LPS (P<0.05).In experiment 3,antibodies against TLR2 or TLR4 plus the ELISAs were used to determine the types of Pg-LPS binding Toll-like receptors(TLRs) on the surface of target cells.Before stimulation with 1μg/ml Pg-LPS or E-LPS,cells were incubated with 2μg/ml of either anti-TLR2 or anti-TLR4 antibodies for 1 h.IL-1β,TNF-αand IL-6 levels were detected by ELISA kits.Moreover,reverse transcription polymerase chain reaction(RT-PCR) was used to detect the level of TLR mRNA at the cell surface with or without LPS treatment.The result showed:①The anti-TLR2 antibody had a significant(P<0.05) inhibitory affect on IL-1β,TNF-αand IL-6 production by THP-1 cells stimulated with Pg-LPS.The anti-TLR4 antibody also showed the inhibition on the production of IL-6,while the effect of anti-TLR2 antibody inhibition was more obvious. In contrast,THP-1 cells preincubated with anti-TLR4 antibodies exhibited markedly (P<0.05) reduced levels of the three cytokines upon stimulation with E-LPS.②The activity of Pg-LPS inducing HL-60 cells to secrete the three cytokines could be blocked with anti-TLR2 antibody alone(P<0.05).Anti-TLR4 antibody showed the effects blocking the three cytokine secretion in HL-60 cells under inducement of E-LPS (P<0.05).When 1μg/ml Pg-LPS acted for 12 h in THP-1 cells,TLR2 mRNA level was increased obviously as the band was much stronger than the control,while the level of TLR4 mRNA was not change.In contrast,the level of TLR4 mRNA was increased after treatment with E-LPS.In experiment 4,the specific inhibitors of JNK(SP600125),P38MAPK (SB203580) and NF-κB(SN50) signaling pathways were applied to confirm the associated signaling pathways of Pg-LPS-induced THP-1 and HL-60 cells to secrete cytokines.Phospho-sandwich ELISA kits were used to determine the phosphorylation levels of JNK,P38MAPK and NF-κB after treatment with Pg-LPS or E-LPS.Pg-LPS associated signaling pathways which transfer from cytoplasm to nucleus were analyzed by confocal microscopy.The result showed:①JNK inhibitor(SP600125) and NF-κB inhibitor(SN50) could block Pg-LPS-induced IL-1β,TNF-αor IL-6(P<0.05) secretion in THP-1 cells,and P38MAPK inhibitor(SB203580) and NF-κB inhibitor(SN50) could block E-LPS-induced the three cytokines(P<0.05) secretion in THP-1 cells.②JNK inhibitor and NF-κB inhibitor could block Pg-LPS-induced IL-1β(P<0.05) secretion in HL-60 cells.All of the three signaling pathway inhibitors could block Pg-LPS-induced TNF-α(P<0.05) secretion in HL-60 cells,but JNK inhibitor was the most effective.Only P38MAPK inhibitor and NF-κB inhibitor showed the block all of the E-LPS-induced three cytokines(P<0.05).③In the test of phosphorylation levels of three proteins,although all of the three protein levels showed upregulated,the phosphorylation level of JNK was increased more significantly upon stimulation with Pg-LPS,while the phosphorylation levels of NF-κB and P38MAPK were increased more noticeably induced by E-LPS(P<0.05).④Confocal microscopy analysis showed that both NF-κB and JNK signaling pathways were activated in THP-1 cells after stimulation with Pg-LPS,signaling expression was transferred into nucleus,which confirmed again that Pg-LPS induces cytokines production through JNK and NF-κB. Part two:Construction of a prokaryotic expression system of gingipains R,collection of recombinant proteins,antiserum preparation and hydrolysis of Human Monocyte CD14In experiment 1,The routing Phenol-chloroform method was applied to prepare genomic DNAs of Pg ATCC33277.The DNA preparations was digested with DNase-free RNase and then extracted by Phenol-chloroform method.The DNAs used as templates in PCR were dissolved in TE buffer and the concentration and purification of the two DNA solutions were determined by ultraviolet spectrophotometry.The result showed:The DNA preparations were purity and stored with 100ug/10ul(When OD260=1,the DNA concentration is 50ug/ml,and OD260:OD280≥1.8,the DNA is purity).In experiment 2,oligonucleotide primers were designed to amplify the rgpAc, rgpBc and hagA genes according to the published corresponding nucleotide sequences. The rgpAc,rgpBc and hagA genes from Pg ATCC33277 were amplified by PCR, respectively.The results of PCR were observed after electrophoresis in 15 g/L agarose pre-stained with ethidium bromide.The target DNA amplification fragments were recovered with 3S PCR Product Purification Kit V2.0(SNBC) and ligated to pUCm-T vectors,respectively.The three recombinant pUCm-T vectors for sequencing were respectively named as pUCm-T-rgpAc,pUCm-T-rgpBc and pUCm-T-hagA,and transfer into E.coli DH5α.The obtained positive colonies by blue-white screening were amplified and then the plasmids in the bacteria were respectively extracted by alkaline-denature method.The plasmids were identified with restriction endonucleases and then the inserted target fragments were sequenced by dideoxynucleotide chain termination method.Homologies of nucleotide sequences of the inserted fragments were compared with the corresponding sequences from GeneBank.Then the target fragments and pET-42a were recovered for ligation.The recombinant expression vector pET-42a-rgpAc,ET-42a-rgpBc and pET-42a-hagA were transformed into E.coli BL21DE3,which was named as pET-42a-rgpAc BL21DE3,pET-42a-rgpBc BL21DE3 and pET-42a-hagA BL21DE3.The target gene fragments inserted into pET-42a plasmid was sequenced again.The result showed:①The sizes of target fragments amplified from Pg ATCC 33277 rgpAc,rgpBc and hagA genes were approximate 1479bp,1305 bp and 2961bp,respectively.②The target genes inserted into pUCm-T vectors were confirmed by the results of the digestion with two restriction endonucleases and sequence analysis.③In comparison with the corresponding sequences from GeneBank,homologies of the nucleotide sequences of the cloned rgpAc,rgpBc and hagA genes were >97%.In experiment 3,0.5mmol/L IPTG was used to induce the expression of recombinant proteins(rRgpAc,rRgpBc and rHagA) in the prokaryotic expression systems.The molecular weight of rRgpAc,rRgpBc and rHagA were measured by SDS-PAGE.Ni-NTA affinity chroma-tography was applied to extract and purify the recombinant proteins.Rabbits were immunized with purity recombinant proteins to prepare the antiserum and Western blot was applied again to determine the antigenicity of rRgpAc,rRgpBc and rHagA.The result showed:①The results of SDS-PAGE demonstrated that IPTG can induce the expression of rRgpAc,rRgpBc and rHagA. Purified rRgpAc,rRgpBc and rHagA protein showed only one band in SDS-PAGE. Western blots showed rRgpAc,rRgpBc and rHagA could combine with the rabbit antiserum against whole cell of Pg and induce rabbits to produce specific antibodies, respectively,which indicated rRgpAc,rRgpBc and rHagA have good antigenicity and immunoreactivity.In experiment 4,rRgpAc and rRgpBc were applied to human monocyte THP-1 cell strain,Flow cytometric analyses were performed to detect the expression of CD14 on the cell surface.The result showed:differentiated THP-1 cells could expression a certain amount CD14.When 0.5μM rRgpAc or rRgpBc was treated for 30 min with the THP-1 cells,the expression of CD14 was reduced obviously.After 120 min treatment,CD14 on the THP-1 surface was almost completely abolished,which indicated that Rgp might delay the inflammation induced by LPS.Conclusion:1.The structure of Pg-LPS were generally similar to that of E-LPS,while the major fatty acids of Pg-LPS were different from that of E-LPS,Which may be the reason why they activate different signaling pathways.2.Pg-LPS showed ability to induce THP-1 cells and HL-60 cells to secrete cytokines. The maximum concentrations of the three cytokines secreted by HL-60 cells were much lower than that by THP-1 cells,indicating that the response of different cell types induced by LPS can be different.3.Differ to E-LPS stimulate cells via TLR4 to secrete cytokines,TLR2 acts as the Pg-LPS main receptor,Pg-LPS could also utilize TLR4.TLR2 or TLR2/TLR4 acted as the Pg-LPS receptors is dependent on cell types.4.The Signaling pathways of Pg-LPS in the same type of target cells or in different target cells to regulate different cytokines production remarkably differ to that of E-LPS.JNK pathway was the major signaling pathway for Pg-LPS inducing the secretion of cytokines and NF-κB participates as well,whereas P38MAPK and NF-κB pathways were the major signaling pathway for E-LPS inducing the secretion of cytokines.5.Prokaryotic expression systems of RgpAc,RgpBc and HagA genes was successfully established in this study,which lays solid foundation for future study of vaccine of periodontitis and immunological mechanism.6.rRgpAc and rRgpBc could hydrolysis monocyte CD14,the main receptor for LPS. The cross-reaction between gingipains and LPS might be the mechanism of CP induced by Pg.