Bacterial Lipoprotein TLR2 Agonists Broadly Modulate Endothelial Function and Coagulation Pathways In Vitro and In Vivo

Abstract

Toll-like receptor 2 (TLR2) activation induces cellular and organ inflammation, and affects lung function. Since deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells (EC) to produce IL-6 and IL-8, and to express E-selectin on their surface, including human umbilical vein EC (HUVEC), human lung microvascular EC, and human coronary artery EC. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen-activator inhibitor 1 (PAI-1) and tissue factor, and decreased production of tissue plasminogen activator (tPA) and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. PAL induced IL-6 production by EC from wild-type, but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged intravenously with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

N-Arachidonoyl Dopamine Modulates Acute Systemic Inflammation via Nonhematopoietic TRPV1

“…Primary peritoneal cells were isolated from adult mice as described (42). Primary lung endothelial cells and microglia were prepared from 6-7-d-old mouse pups immediately after decapitation, as described (43)(44)(45). The purity of the cell populations was verified by flow cytometry, as described below.…”

Peptidoglycan-associated lipoprotein ofAggregatibacter actinomycetemcomitansinduces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7in vitro

“…However, studies have shown contradictory results for the apoptotic ability of other bacterial lipoproteins. Whereas most studies suggest that lipoproteins do induce apoptosis [34–36], others report that lipoproteins in fact have anti-apoptotic properties [37]. The observed anti-apoptotic effects could be due to the lipoprotein-stimulated production of TNF-α, as indicated by the finding that LPS-induced long-term survival of macrophages depended on TNF-α autocrine signalling [38].…”

“…However, Hemophilus influenzae PAL (P6) stimulation of human macrophages induced high levels of the neutrophil-attracting chemokine IL-8 [39]. Moreover, E. coli PAL upregulates the MCP-1 expression of mouse lung cells in vivo [36]. …”

“…The PAL of E. coli has also been shown to induce IL-6 and TNF-α production by mouse macrophages via TLR2 in vivo , as demonstrated by using a TLR2 knockout mouse model [22]. The TLR2 dependency is not restricted to macrophages because TLR2 also mediates the response of mouse lung cells to PAL, because TLR2 knockout mice are unable to upregulate the leukocyte adhesion molecules E-selectin and P-selectin as well as MCP-1 when exposed to E. coli PAL in vivo [36]. The results indicating that the synthetic lipopeptide Pam 3 CSK 4 , a well-known TLR2 agonist, did not induce the production of IL-6, TNF-α and MCP-1 through TLR2 were puzzling.…”

“…Bacterial lipoproteins are established bacterial virulence factors [11–13,24,26,36] that function as inflammatory stimulants in infection and disease progression. Our study is the first to show pro-inflammatory effects of AaPAL, an outer membrane lipoprotein of a major periodontal pathogen, A. actinomycetemcomitans .…”