术产生的新的治疗性癌症疫苗的原始研究论文,.pdf

Immunobiology 215 2010 53 59 Toll like receptor 2 activation by lipoteichoic acid induces differential production of pro infl ammatory cytokines in human odontoblasts dental pulp fi broblasts and immature dendritic cells Jean Franc ois Kellera b 1 Florence Carrouela 1 Evelyne Colombc Ste phanie H Duranda b Caroline Baudouind Philippe Msikad Franc oise Bleichera Claude Vincentc Marie Jeanne Staqueta Jean Christophe Fargesa b aInstitut de Ge nomique Fonctionnelle de Lyon Universite de Lyon Universite Lyon 1 UMR5242 CNRS INRA Ecole Normale Supe rieure de Lyon Equipe Odontoblastes et Re ge ne ration des Tissus Dentaires Institut Fe de ratif Biosciences Gerland Lyon Sud Faculte d Odontologie Lyon France bHospices Civils de Lyon Service de Consultations et de Traitements Dentaires Lyon France cUniversite de Lyon Universite Lyon 1 EA3732 Hopital E Herriot Lyon France dLaboratoires Expanscience De partement Innovation Recherche et De veloppement Epernon France Received 19 November 2008 received in revised form 8 January 2009 accepted 9 January 2009 Abstract Odontoblasts dental pulp fi broblasts and immature dendritic cells DCs have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process How they regulate the infl ammatory response to Gram positive bacteria remains nevertheless largely unknown In this study we investigated the production of the pro infl ammatory cytokines tumour necrosis factor alpha TNF a interleukin 1beta IL 1b and interleukin 8 CXCL8 in these three cell types upon stimulation with lipoteichoic acid LTA a cell wall component of Gram positive bacteria that activates the pattern recognition molecule Toll like receptor 2 TLR2 We observed that TNF a gene expression was up regulated in all LTA stimulated cell types IL 1b gene expression was not or barely detectable in odontoblast like cells and pulp fi broblasts when stimulated or not but was expressed in immature DCs and increased upon stimulation TNF a and IL 1b proteins were detected in DC culture supernatants but not in odontoblast like cell and pulp fi broblast ones CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation These data indicate that LTA dependent TLR2 activation in odontoblasts and pulp fi broblasts in contrast to immature DCs does not lead to signifi cant TNF a and IL 1b production but that all three cell types infl uence the pulp infl ammatory immune response through CXCL8 synthesis and secretion r 2009 Elsevier GmbH All rights reserved Keywords Tooth Gram positive bacteria pattern recognition molecules immune response pro infl ammatory cytokines chemokines ARTICLE IN PRESS www elsevier de imbio 0171 2985 see front matter r 2009 Elsevier GmbH All rights reserved doi 10 1016 j imbio 2009 01 009 Corresponding author at Laboratoire Odontoblastes et Re ge ne ration des Tissus Dentaires Faculte d Odontologie 11 rue Guillaume Paradin F 69372 Lyon Cedex 08 France E mail address Jean Christophe Farges sante univ lyon1 fr J C Farges 1Both authors contributed equally to this work Introduction Odontoblasts fi broblasts and immature dendritic cells DCs are three major cell populations in the connective tissue localized in the centre of the tooth the so called dental pulp They become exposed to cario genic oral bacteria as these progressively demineralize enamel and dentine and enter the disrupted tissues to gain access to the pulp Due to their peripheral situation odontoblasts are the fi rst cells encountered by pathogens and or their released components and represent in the tooth the fi rst line of defence for the host They thus fulfi ll the role devoted elsewhere to skin keratinocytes andepithelialcellsliningtherespiratory gastro intestinal and uro genital tracts More centrally located pulp fi broblasts and immature DCs are then challenged by bacteria components Love and Jenkinson 2002 Golberg et al 2008 Concomitantly immature DCs accumulateintothe odontoblast layerto capture bacterial components at the peripheral pulp entry site Yoshibaetal 1996 Werecentlyshowedthat odontoblasts stimulated with lipoteichoic acid LTA a cell wall component of Gram positive bacteria that engagesthepatternrecognitionreceptorToll like receptor 2 TLR2 initiate an immune response by producing chemokines and recruiting immature DCs Durand et al 2006 We also demonstrated that odontoblastsdifferfrompulp fi broblastsintheir response to LTA by differential up regulation of pattern recognitionreceptorsandchemokinesandhigher capacity to attract immature DCs Staquet et al 2008 To date how odontoblasts pulp fi broblasts and immature DCs each intervene and interact to regulate infl ammatory events that accompany the pulp immune response to pathogens remains largely unknown Datahavedemonstratedthat pro infl ammatory cytokines are rapidly produced by human cells upon contact with pathogens sometimes within tenths of minutes and are able to trigger very potent infl amma tory responses Among these cytokines tumour necrosis factor alpha TNF a and interleukin 1beta IL 1b have a critical role in the regulation of the many outputs of infl ammation Tincani et al 2007 In particular both cytokines induce angiogenesis and synthesis of pro infl ammatory chemokines including CXCL8 Kooloos et al 2007 TNF a IL 1b and CXCL8 are normally absent or produced at an extremely low level in healthy tissues due to their potentially deleterious effects on tissue integrity They are not or barely expressed in healthy human pulps but are up regulated in infl amed pulps under caries lesions D Souza et al 1989 Huang et al 1999 Levin et al 1999 Nakanishi et al 2001 Pezelj Ribaric et al 2002 Zehnder et al 2003 Kokkas et al 2007 Pa a kko nen et al 2007 Veerayutthwilai et al 2007 Farges et al 2008 In infl amed pulp cell origin of TNF a is unknown but IL 1b was shown to be produced by macrophages and CXCL8 by macro phages lymphocytes endothelial cells and odontoblasts D Souza et al 1989 Huang et al 1999 Nakanishi et al 2001 To date the relative capabilities of odontoblasts pulp fi broblasts and DCs to produce TNF a IL 1b and CXCL8 are unknown The aim of this study was to compare time dependent TNF a IL 1b and CXCL8 gene and protein expression by human odontoblast likecells dentalpulp fi broblastsand immature DCs in response to TLR2 activation by LTA Materials and methods Dental pulp cell culture Three healthy non erupted human third molars from three different donors were collected with informed consent of the patients in accordance with the French Public Health Code and following a protocol approved by the local ethics committee Odontoblast like cells and fi broblasts were obtained by culturing dental pulp explants as detailed Couble et al 2000 After 4 weeks cells were stimulated with 0 1 1 or 10mg mL highly purifi ed Staphylococcus aureus LTA Invivogen San Diego CA USA for 0 5 1 2 4 8 or 16h We previously showed that these conditions functionally activated TLR2 and led to chemokine production by odontoblast like cells and dental pulp fi broblasts Dur and et al 2006 Staquet et al 2008 Generation of immature DCs Immature DCs were generated from human CD34 hematopoietic progenitors isolated from human umbilical cord blood mononuclear fraction by immunomagnetic selection with mini MACS Miltenyi Biotec Bergisch Gladbach Germany Purifi edprogenitors 90 98 purity were cultured in RPMI 1640 supplemented with 5 heat inactivated fetal bovine serum Myoclone super plus 10mMHEPES 2mMl glutamine 100U ml penicillin 100mg ml streptomycin all from Invitrogen 5 10 5M b mercaptoethanol Sigma 200U ml recom binant human GM CSF Immunotools Friesoythe Ger many and50U mlrecombinanthumanTNF a Genzyme Corporation Boston MA USA for 7 days Under these conditions cells remain poorly differentiated and display features of immature DCs Noirey et al 2003 Cells were then cultured without TNF a for 24h and stimulated with 10mg mL LTA as above Real time PCR RNA extraction from cultured cells reverse transcrip tion and real time PCR were performed essentially as ARTICLE IN PRESS J F Keller et al Immunobiology 215 2010 53 5954 described Durand et al 2006 Briefl y total RNA was extracted from LTA stimulated and control cultures with a Nucleospin RNA II kit Macherey Nagel Du ren Germany according to the manufacturer s instructions RNA samples 1mg were then converted to fi rst strand cDNAs using 500ng oligo dT 15primers Roche Diag nostics Meylan France and SuperScript III Reverse Transcriptase Invitrogen Life Technologies Grand Island NY USA Real time PCR was performed in a LightCycler instrument Roche with the FastStart DNA Master SYBR Green I kit according to the manufacturer s specifi cations Cyclophilin A housekeep ing gene was used for sample normalization Primer sets and annealing temperatures are listed in Table 1 Standard curves for cyclophilin A and target genes were constructed with known quantities of PCR products generated by conventional RT PCR Analysis of melting curves confi rmed amplicon specifi city PCR amplifi ca tion was also verifi ed for expected products mispairing and primer dimer formation on 3 agarose gel For each target gene mRNA levels were normalized to cyclophilin A and relative gene expression was deter mined using the RelQuant software Roche Results wereexpressedasfoldchangevaluesrelativeto unstimulated control samples ELISA TNF a IL 1b and CXCL8 concentrations in cell culture supernatants were determined using commer cially available human specifi c ELISA kits Instant ELISA Bender MedSystems Vienna Austria Assay sensitivities were 1 65pg mL for TNF a 0 7pg mL for IL 1b and 1 3pg mL for CXCL8 Data are presented as picograms of cytokines per 105viable cells Statistical analysis Results were expressed as mean values 7 standard deviation SD obtained from three different donors Statistical analysis was determined with Student s t test Results Effects of LTA on pro infl ammatory cytokine gene expression by odontoblast like cells and pulp fi broblasts TNF a IL 1bandCXCL8geneexpressionwas differentially up regulated by LTA in human odonto blast like cells and dental pulp fi broblasts TNF a gene expression was not augmented in odontoblast like cells and pulp fi broblasts upon stimulation with 0 1mg mL LTA but was increased in a dose dependent manner in cells stimulated with 1 and 10mg mL LTA after 1h in odontoblast like cells and after 2h in pulp fi broblasts Fig 1 Expression was maximal after 2h of stimulation in both cell types and rapidly decreased to the baseline level after 4h in 1mg mL LTA stimulated cultures and after 16h in 10mg mL LTA stimulated samples IL 1b gene expression was not or barely detectable in unstimu lated odontoblast like cells and pulp fi broblasts and remained unchanged upon stimulation not shown CXCL8 gene expression was augmented only in cells stimulated with 1 and 10mg mL LTA after 1h in odontoblast like cells and after 2h in pulp fi broblasts Expression was maximal after 2 4h of stimulation in both cell types and slowly decreased between 4 and 16h Fig 1 Effects of LTA on pro infl ammatory cytokine gene expression by immature DCs Immature DCs were then stimulated with 10mg mL LTA as this concentration was clearly effective in inducing cytokine gene expression in odontoblast like cells and pulp fi broblasts In these conditions TNF a gene expression rapidly increased in immature DCs upon LTA stimulation peaked at 30min and then progressively decreased thereafter Up regulation of IL 1a and CXCL8 genes was observed after 30min of stimulation peaked at 2h and progressively decreased thereafter Fig 2 Production of TNF a and IL 1b in LTA stimulated odontoblast like cells pulp fi broblasts and immature DCs At protein level TNF a and IL 1b were not detected by ELISA in any supernatant of odontoblast like cell or ARTICLE IN PRESS Table 1 Primers used for real time PCR analysis GeneForward primer 50 30 Reverse primer 50 30 Annealing temperature 1C TNF aGGCGTGGAGCTGAGAGATAACGGTGTGGGTGAGGAGCACAT64 IL 1bAGCACCTCTCAAGCAGAAAACATTTGCATGGTGAAGTCAGTTATATCC62 CXCL8CTGGCCGTGGCTCTCTTGCCTTGGCAAAACTGCACCTT68 Cyclophilin AATGGCACTGGTGGCAAGTCCTTGCCATTCCTGGACCCAAA58 J F Keller et al Immunobiology 215 2010 53 5955 fi broblast cultures stimulated or not with LTA not shown They were produced by non stimulated im mature DCs at low level 7 10pg 105cells and 3 5pg 105cells respectively at all culture times tested DC production was augmented for each molecule about two fold after 1h of LTA stimulation and was sustained until 8 16h Fig 3 Effects of LTA on CXCL8 production by odontoblast like cells pulp fi broblasts and immature DCs CXCL8 was present in all supernatants of unstimu lated cultures It was sharply up regulated by LTA after 4h of stimulation in odontoblast like cells and pulp fi broblasts It remained high in odontoblast like cells while slightly decreasing in pulp fi broblasts after 16h Fig 4 Augmentationwasmoreprogressivein immature DCs and CXCL8 production was maximal after 16h CXCL8 production was lower in stimulated DCs than in odontoblast like cells and pulp fi broblasts Of note great variations were observed in the intensity of the response to LTA between dental cells from the three different donors Nevertheless when observed increase of gene and protein expression was found in each of the primary cultures tested Discussion IntradentinalprogressionofGram positiveoral bacteria during the caries process induces immune and infl ammatory events in the human dental pulp the molecular effectors of which remain largely unknown Golbergetal 2008 Werecentlyshowedthat odontoblasts and pulp fi broblasts initiate an immune response to Gram positive bacteria upon TLR2 engage ment by LTA Durand et al 2006 Staquet et al 2008 Farges et al 2008 In the present study we sought to determine whether these cells play a role in the early infl ammatory events that accompany this response by analyzing their production of pro infl ammatory cyto kines TNF a IL 1b and CXCL8 We compared this response with that of immature DCs another key component in the early pulp response to cariogenic bacteria Yoshiba et al 1996 Jontell et al 1998 We failed to detect any IL 1b gene or protein expression in odontoblast like cells and pulp fi broblasts when stimulated or not TNF a gene expression was found sharply augmented in these cells upon LTA stimulation for 2h and then rapidly decreased TNF a protein was not detected in culture supernatants by the use of a highly sensitive ELISA kit This absence was confi rmed by antibody array analysis fl ow cytometry and immunohistochemistry not shown This indicates ARTICLE IN PRESS Fold control TNF 0 20 40 60 80 100 120 Fold control Fib 200 100 80 60 40 20 0 Od 40 30 20 10 0 Fib CXCL8 Control 0 1 g mLLTA 1 g mLLTA 10 g mLLTA Od 40 30 20 10 0 hh 0 5124816 h 0 5124816 h 1248160 5 0 5124816 Fig 1 LTA treatment induces increase of TNF a and CXCL8 gene expression in odontoblast like cells and pulp fi broblasts Cells were stimulated with 0 1 1 or 10mg mL highly purifi ed Staphylococcus aureus LTA for 0 5 1 2 4 8 or 16h At indicated time gene expression was quantifi ed by real time PCR analysis Results were normalized to cyclophilin A and are expressed as fold change values relative to control unstimulated cells Data represent the mean7SD obtained from three different donors pp0 05 versus control cells Od odontoblast like cells Fib pulp fi broblasts J F Keller et al Immunobiology 215 2010 53 5956 that TNF a is not produced at detectable level by odontoblast like cells and pulp fi broblasts in response to LTAandsuggestsanegativepost transcriptional regulatory process that remains to be identifi ed Never theless it appears that TLR2 engagement by LTA does not trigger pulp infl ammatory events by inducing a signifi cant production of TNF a and IL 1b in odonto blasts and underlying fi broblasts This hypothesis is in accordance with the absence of TNF a increase in pulp fi broblastsco culturedwithStreptococcusmutans Gram positive bacteria Engels Deutsch et al 2003 Immature DC accumulation in the odontoblast layer is an early event of dental pulp response to cariogenic bacteria Yoshiba et al 1996 Jontell et al 1998 Our previous studies suggest that Gram positive bacteria recognition and chemokine production by odontoblasts trigger this accumulation Durand et al 2006 In the odontoblast layer DC sensing of bacterial by products which gain access to the dental pulp requires the immature state of DCs As IL 1b and TNF a promote DC maturation Banchereau and Steinman 1998 Ueno et al 2007 it is conceivable that odontoblasts do not express molecules that could induce a maturation of DCs before these have migrated to the pulp dentine ARTICLE IN PRESS Fold control IL 1 30 20 10 0 Fold control CXCL8 40 30 20 10 0 Fold control Control 10 g mL LTA TNF 0 5 10 5 0 124816 h 0 5124816 h 0 5124816 h Fig 2 LTA treatment induces increase of TNF a Il 1b and CXCL8geneexpressioninimmatureDCs Cellswere incubated with 10mg mL LTA for 0 5 16h Gene expression was quantifi ed by real time PCR analysis Results were normalized to cyclophilin A and are expressed as fold change values relative to control unstimulated cells Data represent the mean7SD obtained from three different donors pp0 05 versus control cells Control 10 g mL LTA TNF pg 105 cells 50 40 30 20 10 0 1 30 20 10 0 IL 1 pg 105 cells 24816 h 124816 h Fig 3 LTA treatment induces increase of TNF a and Il 1b protein release in immature DCs Cells were incubated with 10mg mL LTA for 1 16h Protein amount was assessed in culture supernatants by ELISA Results are expressed as picograms of cytokine per 105cultured cells Data represent the mean7SD obtained from three different donors pp0 05 versus control cells J F Keller et al Immunobiology 215 2010 53 5957 interface to capture bacterial components Immature DCs express several TLRs including TLR2 whose activation induces DC maturation and cytokine produc tion Ueno et al 2007 Our results confi