Soluble forms of Toll-like receptor 4 are present in human saliva and modulate tumour necrosis factor-alpha secretion by macrophage-like cells

Abstract

In health, mucosal inflammation is prevented by tightly regulated responses via Toll-like receptors (TLR) that interact with specific microbe associated molecular patterns. Currently, 13 TLRs have been identified. Based on the specificity of ligand recognition, TLR-2 and TLR-4 can recognize most oral commensal microorganisms. Recent identification of some soluble TLRs (sTLRs) suggests additional regulatory roles for these receptors. We report here the presence of sTLR-4 polypeptides in adult human saliva. Functionally, the salivary sTLR-4 suppressed cytokine secretion by activated macrophages. The sTLR-4 levels were elevated significantly in oral lichen planus (OLP), a chronic inflammatory condition of the oral mucosa characterized by clinical persistence. In contrast, the epithelial cells in the saliva of OLP subjects expressed significantly reduced TLR-2 and TLR-4 mRNA that correlated with fewer bacteria/salivary epithelial cells. Investigating the soluble and cellular components of saliva is useful in identifying potential biomarkers for oral mucosal lesions.

Fig. 1

Detection of soluble Toll-like receptor (sTLR)-4 in human saliva: (a) decreasing concentration of recombinant TLR-4Fc was subjected to immunoblot analysis with goat anti-human TLR-4 polyclonal antibody. (b) Precleaned unstimulated whole saliva (UWS) samples (lanes 3–5) from normal individuals containing 10 µg of proteins were probed with the anti-human TLR-4 polyclonal antibody. Up to four sTLR-4 peptides were detected. Lanes 1 and 2 represent molecular weight (MW) and recombinant TLR-4-Fc respectively. (c) The specificity of the detection was confirmed by performing protein (TLR-4Fc) competition by immunoblotting.

Fig. 3

CD14, Toll-like receptor (sTLR)-2 and TLR-4 are expressed in epithelial cells in unstimulated whole saliva (UWS). (a) Epithelial cells from unstimulated whole saliva (UWS) of patients with oral lichen planus (OLP) (lanes 6–10) or control subjects (lanes 1–5) were assessed for the expression of CD14 [758 base pairs (bp)], TLR-2 (498 bp), TLR-4 (398 bp) mRNA and small proline-rich protein 2a (SPRR2a) (110 bp) mRNA by reverse transcription–polymerase chain reaction using specific primers. (b) Semiquantitative measurement of CD14, TLR-2 and TLR-4 mRNA normalized to the SPRR2a levels. *P < 0·05.

Fig. 2

Soluble Toll-like receptor (sTLR)-2 and sTLR-4 are increased in the unstimulated whole saliva (UWS) of oral lichen planus (OLP) patients: sCD14, sTLR-2 and sTLR-4 were investigated by immunoblot (a, b and c respectively) and measured by enzyme-linked immunosorbent assay (ELISA) (d). The sCD14 was detected as a 55 kDa protein (lane 10). Lanes 1–3 and 4–7 represent UWS samples from control and oral lichen planus (OLP) subjects respectively. (b) sTLR-2 polypeptides present in precleaned UWS (10 µg total protein) by Western blotting. The specificity of the detection was confirmed by performing peptide competition by immunoblotting. The anti-TLR-2 monoclonal antibody (mAb) was preincubated (+) (lanes 1a, 2a) or not (–) (lanes 3a, 4a) with excess of the peptide/TLR-2Fc. Lanes 1–4 and 5–8 represent UWS samples from control and OLP subjects respectively. (Sup) Culture supernatants of human peripharal blood mononuclear cell (PBMC) cultures stimulated with concanavalin A (ConA) (lane 11) or recombinant human TLR-4Fc (lane 12) were loaded as controls. (c) Precleaned UWS samples containing 10 µg of proteins were subjected to immunoblot analysis with anti-human TLR-4 mAb. Recombinant human TLR-4Fc was included as control (lane 14). Lanes 1–7 and 8–13 represent UWS samples from OLP and control subjects respectively. MW, molecular weight. (d) The amount of sCD14, sTLR-2 and sTLR-4/µg of total proteins in the precleaned UWS of normal and OLP patients were determined by ELISA. (e) Quantitative estimation of the cytokines interleukin (IL-8), IL-12 and interferon-γ/µg of total proteins in the precleaned UWS of normal and OLP patients as determined by ELISA. = P < 0·05 and represents statistical significance compared with control saliva.

Fig. 4

Pure populations of epithelial cells were obtained from histologically normal buccal mucosa (lanes 6–10) or tissues that exhibit features of oral lichen planus (OLP) (lanes 1–5) by laser capture microdissection. (a) Expression of CD14 mRNA [758 base pairs (bp)], Toll-like receptor (TLR)-2 mRNA (498 bp) and TLR-4 mRNA (398 bp). The first lane (indicated) in the top three panels represent respective mRNA expression in Thp-1 cells. Expression of SPRR2a (110 bp) abundant in oral epithelium was amplified as control. (b) Quantitation of the band intensity was performed by densitometry. Data are presented as average intensity ± standard error. *P = 0·05.