Figure 1.
Metabolic characteristics of experimental animals.
Six-week-old wild type (WT) and ApoE−/− C57BL/6 mice were fed a normal chow diet for 14 weeks. Body mass (A), blood glucose (B) and serum lipid levels (C) were measured (n = 5). *, P<0.05.
Figure 2.
Impaired clearance of P. gingivalis by inhibiting iNOS induction in ApoE −/− mice.
A and B, in vitro studies: peritoneal macrophages from wild type (WT) and ApoE −/− mice were infected with P. gingivalis (MOI = 25∶1) and CFU of internalized bacteria were determined at 1.5 and 15 h after infection. C, in vivo studies: mice were infected i.p. with P. gingivalis (5×107). Serial dilutions of peritoneal fluid were plated for anaerobic growth and enumeration of recovered peritoneal CFU. D, peritoneal macrophages were incubated with FITC-labeled P. gingivalis (MOI = 25∶1) for 30 min. Association (i.e., representing both adherence and phagocytosis) or phagocytic indices were determined by flow cytometry, as described in Materials & Methods, using the following formula: (% positive cells for FITC-P. gingivalis×MFI)/100. E, 24 h after peritoneal infection, the induction of iNOS was determined by Western blot using the cellular components of the peritoneal fluid after centrifugation, data were normalized to GAPDH. F, mouse macrophages were infected with P. gingivalis, and iNOS production was determined by Western blot 24 h post infection (i), and iNOS mRNA was determined by qPCR 2 h post infection (ii). Results were means ±SD (n = 3) and were confirmed in repeated experiments. *, P<0.05.
Figure 3.
Inhibited cytokine production in ApoE−/− mice in vivo.
Wild type (WT) and ApoE−/− mice were infected i.p. with P. gingivalis (5×107). Peritoneal fluid of 6 h post infection was subjected to cytokine antibody array. Each cytokine is represented by duplicate spots (A). The cytokine array image represented results of three independent experiments (B). Cytokines with fold change >2.0 and P<0.05 was shown (C). TNF-α, IL-6, IL-1β, MCP-1, IL-12 and IL-10 levels at 0 h∼24 h was determined by ELISA (D-I) (mean ± SD; n = 3; representative of duplicate independent tests). *, P<0.05.
Figure 4.
Inhibited cytokine production in ApoE−/− mice macrophages.
Macrophages from wild type (WT) and ApoE−/− mice were exposed to live P. gingivalis (MOI = 25∶1). TNF-α, IL-6, IL-1β, MCP-1, IL-12 and IL-10 in culture supernatants were analyzed by ELISA (mean ± SD; n = 3). *, P<0.05.
Figure 5.
Disrupted gene profile in ApoE−/− mice macrophages.
Macrophages from wild type (WT) and ApoE−/− mice were exposed to live P. gingivalis (MOI = 25∶1) for 2 h. Heat map of hierarchical clustering was utilized to reveal gene profiles; shown were qualified genes with fold change of >5.0 (P<0.05) between any groups (A). Red indicated up-regulation, whereas green indicated down-regulation, and black indicated no change. Differentially expressed genes in control and bacteria infected macrophages were categorized as up-regulated and down-regulated (B), then a Venn diagram was utilized to explore the logical relation between the difference expression genes(C), showing 130 genes were affected by hyperlipidemia in both the untreated and infected macrophages. Further analysis demonstrated 101 genes were down-regulated in the 130 genes (D).
Table 1.
Biological process affected by hyperlipidemia in P. gingivalis infected macrophages.
Table 2.
The list of pathway category (Kegg) in difference expression genes in P. gingivalis infected macrophages.
Table 3.
qPCR validation for selected genes affected by hyperlipidemia.
Figure 6.
Different pattern recognition receptors (PRRs) expression between wild type (WT) and ApoE−/− mice macrophages.
Mice peritoneal macrophages were infected with P. gingivalis (MOI = 25∶1) for 2 h, then mRNA of PRRs related to TLR and NLR pathway were determined by qPCR (A). TLR-2 and TREM-1 expression were confirmed by flow cytometry (B and C), showing significantly inhibited expression of TREM-1 in ApoE−/− mice macrophages. Blockage of TREM-1 with LP-17 peptide pretreatment decreased TNF-α and IL-6 production significantly at 6 h after P. gingivalis infection in peritoneal macrophages. (n = 3). *, P<0.05.
Figure 7.
Inhibited recruitment of NF-κB to cytokine promoters in ApoE−/− mice macrophages.
Peritoneal macrophages from wild type (WT) and ApoE−/− mice were treated with live P. gingivalis (MOI = 100∶1) for 15 min and 30 min. NF-κB p65 polyclonal antibody precipitated DNA was quantified by real time PCR, and the results were expressed as percentage of ChiP DNA to input DNA. *, P<0.05. (n = 3).
Figure 8.
Proposed mechanism for impaired host innate immune response to viable P. gingivalis infection by hyperlipidemia in ApoE−/− mice.
Increased blood lipid would be incorporated into cell membrane and intracellular components of macrophages, leading to the decreased TREM-1 expression upon P. gingivalis infection. Reduced TREM-1 activation resulted in inadequate signal transduction, less p65 nuclear translocation and binding to gene promoters region at DNA; therefore, innate immune responses including iNOS production and release of cytokines, such as TNF-α, IL-6 and MCP-1, were disrupted in the hyperlipidemic host. LDL, low density lipoprotein; TREM-1, triggering receptors expressed on myeloid cells-1; PRRs, pattern recognition receptors, TLR, Toll-like receptors; NLR, NOD-like receptors; NF-κB, nuclear factor κB; IκB, inhibitory κB; IKK, IκB kinase complex; iNOS, inducible nitric oxide synthase; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein 1; TNF-α, tumor necrosis factor α.