Hyperlipidemia Impaired Innate Response To Porphyromonas Gingivalis And Enhanced Periodontal Disease Development In Apolipoprotein E Knock Out Mice

Hyperlipidemia, a common health problem in adult people especially in theelderly, inflicts more and more adverse effects on general health. Much emphasis hasbeen placed on its effect on general inflammation and cardiovascular disease,especially atherosclerosis; however, its effect on periodontal status has been ignored.Apolipoprotein E knock out (ApoE-/-) mice acquired hyperlipidemia under normaldiet. Previous studies showed that under lipopolysacchride (LPS) and severaldifferent pathogens stimulation in vivo, ApoE-/-mice released more proinflammatorycytokines into blood, which influenced diseases progress. Thus, we hypothesizedthat hyperlipidemia could also influence periodontal disease development byincreased cytokine secretion.ApoE-/-mice and C57BL/6J wild type(C57WT) with the same gene backgroundwas fed until about20wks to observe the effect of long time hyperlipidemia onperiodontal diseases.Firstly we set up a periodontal disease model by ligation of bilateral uppersecond molars and inoculation of P. gingivalis. We found that:1) ApoE-/-mice developed serious hyperlipidemia, however metabolic index suchas body weight and blood glucose was unaffected;2) ApoE-/-mice displayed more serious periodontal destruction compared to C57WT mice by methylene blue staining;3) TNF-α and IL-6in the blood were significantly decreased in ApoE-/-miceduring periodontal disease progress.4) Clearance of P. gingivalis in periodontal tissue was decreased in ApoE-/-mice.In conclusion, hyperlipidemia may exacerbate periodontal destruction byimpaired innate response to P. gingivalis. Secondly, in order to scrutinize the innate response to periodontal pathogen,we injected P. gingivalis into peritoneal cavity, and found that:1) Cytokine antibody array showed that6h after P. gingivalis peritonealinjection, levels of20cytokines such as IL-3, TNF-α and interferon-γ (IFN-γ)was decreased in peritoneal fluid, whereas only5cytokines such asmacrophage inflammatory protein-1-α (MIP-1-α) and leptin were increased inApoE-/-mice;2) At0h,2h,6h,12h and24h after injection, levels of pro-inflammatorycytokine IL-1β,TNF-α,IL-6and monocyte chemotactic protein-1(MCP-1)was decreased and anti-inflammatory cytokine IL-10was not affected inApoE-/-mice;3) Increased levels of IL-6and MCP-1were observed in the blood in ApoE-/-mice.4) Decreased clearance of P. gingivalis in the peritoneal cavity was observed inApoE-/-mice.Thus hyperlipidemia impaired innate immune response to P. gingivalis, andless cytokine releasement in acute phase of bacteria infection has detrimentaleffects on the progress of peritoneal macrophages.In order to discover the mechanism of such impaired innate reponse, weseparated and cultured peritoneal macrophages (PM) of ApoE-/-and C57WTmice, then stimulated with P. gingivalis, whole genome gene expressionmicroarray was utilized to explore global gene expression; real time PCR andELISA was used to confirm the microarray data; chromatinimmunoprecipitation (ChIP) was utilized to investigate binding of nuclearfactor-κB (NF-κB) p65to chromatin, and we found:1)353genes were upregulated and465genes were down-regulated in ApoE-/-mice compared to C57WT mice in blank treated PM; whereas148genesupregulated and236genes down-regulated in P. gingivalis treated PM; 2) gene ontology (GO) analysis showed that after P. gingivalis stimulation,upregulated genes of difference expression in ApoE-/-mice were involved in7biological process, such as nitrogen compound metabolic process,celladhesion and anatomical structure development; whereas upregulated geneswere involved in35biological process, such as leukocyte mediatedcytotoxicity, regulation of cell killing, positive regulation of cell killing,immune system process, leukocyte homeostasis and immune effector process;3) Pathway analysis showed that in P. gingivalis treated macrophages, differenceexpression genes in ApoE-/-mice when compared to C57WT mice showedfollowing features:i. Down-regulated genes belongs to36pathways such as Toll likereceptors (TLR) pathway, Janus kinase/signal transducers and activators oftranscription(JAK-STAT) pathway, cytokine-cytokine receptor pathway;ii. Upregulated genes belong to16pathways such as extracellularmatrix-receptor and focal adhesion;iii. Difference expression genes such as Ccnd2, Csf2, Csf3, Ifnb1, Ifng, Il12a,Il15ra, Il6, Socs1and Stat1that belong to JAK/STAT pathway were alldown-regulated;iv. Genes that belongs to TLR pathway such as Cd86, Cxcl10, Cxcl9, Ifnb1,Il12a, Il6, Irf7and Stat1were all down-regulated.4) Real time PCR confirmed the downregulation of TNF-α,IL-6,IL-12a andMCP-1in ApoE-/-mice PM;5) Lowered level of TNF-α,IL-6and MCP-1was observed in ApoE-/-micePM;6) Hyperlipidemia impaired binding of NF-κB p65to promoter sequence ofTNF-α and IL-10.Thus we concluded that hyperlipidemia affected TLR signal pathway, andimpaired biological process in innate response to P. gingivalis, such as cytokinetranscription and leukocyte mediated cytotoxicity, and such effect might be related to decreased binding of NF-κB p65to chromatin.Expression of receptors on macrophages membrane such as TLR andtriggering receptors expressed on myeloid cells-1(TREM-1) may influencepattern recognition and the following signal transduction in TLR pathway.Lastly we separated and cultured PM, then analyzed alterations in membranereceptors using P. gingivalis as a stimulant, and found that:i. Transcription of more than20receptors such as TLR-1,-2,-4and-6, NOD1,NOD2, CD14and CD36was not affected by hyperlipidemia. Triggeringreceptors expressed on myeloid cells-1(TREM-1) was down-regulated inblank and P. gingivalis treated macrophages in ApoE-/-mice;ii. Flow cytometry showed that TLR-2and TLR-4expression was not affected,whereas TREM-1expression was decreased in ApoE-/-mice;iii. Inducible nitric oxide synthase(iNOS) was affected by hyperlipidemia, andless nitric oxide (NO) was produced, thus intracellular killing bacteriacapability was impaired in ApoE-/-mice;iv. TREM-1could amplify signal transduction of TLR, when blocking TREM-1by LP-17, a synthetic polypeptide with the same amino-sequence, IL-6andTNF-α was significantly decreased in mice RAW264.7macrophages.In conclusion,long term hyperlipidemia impaired innate response to P.gingivalis, decreased release of pro-inflammatory cytokine in acute phase ofbacteria infection, and accelerated periodontal disease progress; hyperlipidemiaimpaired TLR and JAK/STAT pathway during P. gingivalis treatment, anddecreased binding of binding of NF-κB p65to chromatin,leading to inadequateimmune response to periodontal pathogen; impaired innate response to P.gingivalis in ApoE-/-mice might be related to decreased expression of TREM-1,which is an amplifier of TLR pathway, thus leaded to decrease inflammatoryresponse in acute phase of periodontal infection.