Impaired clearance of <i>P. gingivalis</i> by inhibiting iNOS induction in ApoE ^−/− mice.

<p>A and B, in vitro studies: peritoneal macrophages from wild type (WT) and ApoE ^−/− mice were infected with <i>P. gingivalis</i> (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 <i>P. gingivalis</i> (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 <i>P. gingivalis</i> (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 <i>P. gingivalis</i>, 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 (<i>n</i> = 3) and were confirmed in repeated experiments. *, <i>P</i><0.05.</p

qPCR validation for selected genes affected by hyperlipidemia.

<p>qPCR validation for selected genes affected by hyperlipidemia.</p

Different pattern recognition receptors (PRRs) expression between wild type (WT) and ApoE^−/− mice macrophages.

<p>Mice peritoneal macrophages were infected with <i>P. gingivalis</i> (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 <i>P. gingivali</i>s infection in peritoneal macrophages. (n = 3). *, <i>P</i><0.05.</p

Inhibited cytokine production in ApoE^−/− mice macrophages.

<p>Macrophages from wild type (WT) and ApoE^−/− mice were exposed to live <i>P. gingivalis</i> (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). *, <i>P</i><0.05.</p

Biological process affected by hyperlipidemia in <i>P. gingivalis</i> infected macrophages.

1<p>Total count of difference expression genes.</p>2<p>The proportion of total count versus the number of genes in the biological process.</p>3<p>Only the biological process with p-value <0.05 were shown.</p><p>Only biological processes that are related to innate immune response to bacterial infection were shown<b>.</b></p

Inhibited cytokine production in ApoE^−/− mice in vivo.

<p>Wild type (WT) and ApoE^−/− mice were infected i.p. with <i>P. gingivalis</i> (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 <i>P</i><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). *, <i>P</i><0.05.</p

Proposed mechanism for impaired host innate immune response to viable <i>P. gingivalis</i> infection by hyperlipidemia in ApoE^−/− mice.

<p>Increased blood lipid would be incorporated into cell membrane and intracellular components of macrophages, leading to the decreased TREM-1 expression upon <i>P. gingivalis</i> 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.</p

Inhibited recruitment of NF-κB to cytokine promoters in ApoE^−/− mice macrophages.

<p>Peritoneal macrophages from wild type (WT) and ApoE^−/− mice were treated with live <i>P. gingivalis</i> (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).</p

The list of pathway category (Kegg) in difference expression genes in <i>P. gingivalis</i> infected macrophages.

<p>Only pathways correlated to innate response to bacterial infection were shown.</p