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Mol Immunol. 2014 Feb;57(2):284-91. doi: 10.1016/j.molimm.2013.10.004. Epub 2013 Nov 12.

Lipoteichoic acid of Streptococcus mutans interacts with Toll-like receptor 2 through the lipid moiety for induction of inflammatory mediators in murine macrophages.

Author information

1
Department of Oral Microbiology and Immunology, DRI, and BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.

Abstract

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA. In this study, we purified LTA from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced TNF-α and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 and TLR2 or CD14 and TLR4, respectively. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. TLR2-dependent induction of NO by Sm.LTA was also confirmed in RAW 264.7 cells using specific antibodies blocking TLR2. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that Sm.LTA lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of other oral bacteria Enterococcus faecalis and Lactobacillus plantarum. In conclusion, our results suggest that the Sm.LTA interacts with TLR2 through the lipid moiety for the induction of inflammatory mediators in macrophages.

KEYWORDS:

CHO; Chinese hamster ovary cell-line; DEAE; ELISA; Ef.LTA; Enterococcus faecalis LTA; IFN-γ; Inflammatory responses; Innate immunity; LTA; Lactobacillus plantarum LTA; Lipoteichoic acid; Lp.LTA; N-hydroxysuccinimide ester sodium salt; NHS; NO; Sm.LTA; Streptococcus mutans; Streptococcus mutans LTA; TLR; TNF-α; Toll-like receptor; diethylaminoethyl; enzyme-linked immunosorbent assay; interferon-gamma; lipoteichoic acid; nitric oxide; tumor necrosis factor-alpha

PMID:
24216318
DOI:
10.1016/j.molimm.2013.10.004
[Indexed for MEDLINE]

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