Metalloproteinase-dependent TLR2 ectodomain shedding is involved in soluble toll-like receptor 2 (sTLR2) production

Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14+ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders

Production of sTLR2 involves a post-translation mechanism.

<p>(<b>A</b>) THP-1 cells were pretreated or not with cycloheximide (100 µg/mL) for 30 min and stimulated with Pam₃CSK₄ (1 µg/mL) for 5 h and the amount of sTLR2 was quantified in the cell culture supernatants by ELISA. Student t test, * p<0.05. (<b>B</b>) Detection of sTLR2 in 18 h culture supernatant (SN) of THP-1 cells by Western blotting using a N-terminal anti-TLR2 antibody. One representative of three experiments is shown. SN: cell culture supernatant; rhTLR2: recombinant human TLR2 protein. (<b>C</b>) Cell surface TLR2 levels evaluated by flow cytometry after stimulation with Pam₃CSK₄ (1, 10 or 20 µg/mL) for 2 h. ***, p<0.0001. (<b>D</b>) Cell surface TLR2 levels evaluated by flow cytometry after treatment with cycloheximide and then stimulation for 2 h with Pam₃CSK₄. MFI =  mean fluorescence intensity. *, p<0.05; **, p = 0.009.</p

Metalloproteinase activity regulates sTLR2 release and TLR2 surface content from THP-1 cells.

<p>(<b>A</b>) Cells were treated with APMA (10 µM), vehicle (DMSO) or left untreated (-) for 5 h and the concentration of sTLR2 in the supernatants was determined by ELISA. Student t-test, **, p = 0.003. (<b>B</b>) Cell surface TLR2 was examined on these cells after 2 h addition of APMA (10 µM), vehicle (DMSO) or left untreated (-) using anti-TLR2-FITC conjugated antibody and analysis by flow cytometry. ***, p<0.0001. MFI =  mean fluorescence intensity. A representative histogram is shown using cells stained with isotype control antibody (filled histogram), untreated cells (dot line) and APMA-treated cells (black line). (<b>C</b>) Cells were treated for 18 h with EDTA (2 mM), TAPI-1 (25 µM or 75 µM), GI254023X (5 µM) (GI) or left untreated (-) and the sTLR2 concentration in the cell supernatants was determined by ELISA. *, p<0.05; **, p = 0.003. Westernblot (10% SDS-PAGE reducing gel) shows sTLR2 release in cell supernatant of TAPI-1 or left untreated (-) treated THP-1 cells using N-terminal anti-TLR2 antibody. One representative of three experiments is shown. (<b>D</b>) Cells treated with TAPI-1 (25 µM or 75 µM), GI254023X (GI) (5 µM) or left untreated (-) were added 30 min prior to cell stimulation with Pam₃CSK₄ (1 µg/mL) for 18 h and the concentration of sTLR2 in cell supernatants was assayed by ELISA. Data represent the mean ± SE of three independent determinations. Student t test **, p = 0.001; ***, p = 0.0001.</p

TLR2 shedding is involved in sTLR2 generation in human peripheral monocytes.

<p>(<b>A</b>) Isolated human peripheral CD14⁺ cells were treated with APMA (10 µM), vehicle (DMSO) or left untreated (-) for 5 h. *, p<0.05. (<b>B</b>) Cells were incubated for 18 h with TAPI-1 (25 µM or 75 µM), GM6001 (10 µM) or left untreated (-).*, p<0.05.(<b>C</b>) Cells treated with TAPI-1 (25 µM or 75 µM), GI254023X (GI) (5 µM) or left untreated (-) were added 30 min prior to cell stimulation with Pam₃CSK₄ (1 µg/mL) for 18 h. Supernatants were harvested and soluble receptor was measured by ELISA. *, p<0.05; ** p<0.01. Data are express as percentage of maximal release ± SE of two independent determinations using four different healthy donors.</p

ADAM10 and ADAM17 are involved in TLR2 shedding.

<p>MEF cells were transfected with an expression plasmid encoding human TLR2 or with an empty vector; sTLR2 content in cell supernatants was analyzed by ELISA. sTLR2 content (pg/mL) was normalized to total TLR2 cell levels (ng/mg total protein). Data represent the mean ± SE of three independent determinations. *, p<0.05.</p