Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages

Abstract

Abstract.To study the immune-inflammatory response and signaling mechanism of macrophages to purified Enterococcus faecalis (E. faecalis) lipoteichoic acid (LTA), intact LTA was obtained from an E. faecalis clinical strain P25RC using the butanol method and hydrophobic interaction chromatography purification. The fractions containing LTA were determined using phosphate detection. Contaminations with lipopolysaccharide and proteins were excluded using the Limulus amoebocyte lysate assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. LTA was analyzed using nuclear magnetic resonance. Prior to LTA stimulation assays, THP-1 monocytes were pretreated with phorbol 12-myristate 13-acetate to differentiate into macrophages. Macrophages were treated with LTA in concentration gradients and cells without LTA treatment as the control. Gene expression of TLR2, CD14 and MyD88 were evaluated by quantitative polymerase chain reaction. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 were quantified using ELISA. The activated and total nuclear factor-κB (NF-κB) p65 and three mitogen-activated protein kinases (p38, ERK1/2 and JNK) were assessed using western blot analysis. E. faecalis LTA induced the gene expression of TLR2 and MyD88 whilst it downregulated CD14, suggesting a TLR2-dependent and CD14-independent immune-inflammatory activity. LTA stimulated the expression of pro-inflammatory cytokine TNF-α (P<0.05), but not the anti-inflammatory cytokine IL-10. In conclusion, E. faecalis LTA stimulated the expression of TNF-α in macrophages possibly through the NF-κB and p38 pathways. IntroductionEnterococcus faecalis (E. faecalis), a Gram-positive bacterium, is a predominant cause of hospital-associated infections (1). E. faecalis is also involved in root canal infections (2) and is considered a major pathogen associated with endodontic treatment failure (3). Several virulence attributes that promote bacterial colonization, the invasion of host tissues and the evasion of host defense mechanisms are indicated in persistent infections caused by E. faecalis (4). The virulence factors include lipoteichoic acid (LTA), peptidoglycan, aggregation substances, cytolysin and lytic enzymes. Among them, LTA is the key virulence factor due to its major role in pathogenicity. It has been demonstrated that E. faecalis LTA can stimulate leukocytes to release certain mediators associated with inflammatory response (5) and plays a critical role in biofilm formation (6). E. faecalis LTA is also known to inhibit the repair mechanism of periapical bone by decreasing the proliferation of human osteoblast-like cells and inducing apoptosis (7).E. faecalis LTA belongs to classical (type 1) LTA. Its basic structure is comprised of a glycolipid moiety and 1,3-polyglycerolphosphate substituted at position C-2 with D-alanine and kojibiose residues (8). The alanyl esters with D-configured glycerophosphate influence the antibiotic action, pathogenesis, adhesion, biofilm formation and virulence of 9). The dlt operon consists ...

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