Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide

Abstract

Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam₃CSK₄. In addition, the presence of EVs reduced inflammatory responses in Pam₃CSK₄-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.

Keywords: corticosteroid; exosome; innate immunity; microvesicle; pulmonary macrophage.

Figure 1

Dexamethasone induces TLR2 in AMs. (A–E) AMs were incubated with solvent control (0.1% DMSO, Co) or dexamethasone (Dex, 1 μM) for up to 24 h (A–D) or at the indicated concentrations for 4 h (E). (E–G) AMs were preincubated with the GR inhibitor RU486 (10 μM) or solvent control (0.1% EtOH) and treated with Dex (1 μM) for 24 h. Data from at least three independent experiments performed in duplicate with cells from different donors are presented as means ± SEM. TLR expression was measured by or qPCR (A,B,E,F) or Western blot (C,D,G,H). (A) TLR expression upon Dex treatment was normalized to the TLR expression values for the respective vehicle-treated control (indicated by the dotted line). (B–H) TLR2 expression in solvent-treated cells were set as 1. (C,G) Representative blots. (D,H) Densitometric analysis. TLR2 signal intensities were quantified and normalized to the loading control tubulin. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^###p < 0.001 vs. vehicle-treated cells. p-values were generated by ANOVA with Bonferroni's post-hoc test or Mann–Whitney U-test.

Figure 2

TLR2 is overexpressed during the immunosuppressive phases of SIRS and sepsis. (A,B) Dataset GSE4607 was obtained from Gene Expression Omnibus (GEO) and normalized using log2-RMA. The dataset included transcriptional profiles of human whole blood samples of 15 healthy controls, 27 patients with non-infectious SIRS and 12 samples from patients with resolved non-infectious SIRS. Patients were classified as SIRS, or SIRS resolved (no longer meeting criteria for SIRS) on d3 after ICU admittance. (C,D) Dataset GSE8121 was retrieved from GEO and normalized using log2-RMA. The dataset included transcriptional profiles of human whole blood samples of 15 healthy controls and 30 patients with sepsis. Samples were obtained at d1 and d3 after admittance to the ICU. The statistical significance was determined by the Kolmogorov–Smirnov test.

Figure 3

Long-Term LPS exposure upregulates TLR2 in AMs. (A–D) AMs were incubated with LPS (100 ng/mL) for 24 h (A) or the indicated time points (B–D). TLR expression was measured by qPCR (A,B) or Western blot (C,D). (A) TLR expression was normalized to the TLR expression values for the respective vehicle-treated control which was set as 1 (indicated by the dotted line). (E–F) AMs were treated with LPS (100 ng/mL), Pam₃CSK₄ (Pam3, 100 ng/mL) or Poly(I:C) (PIC, 1 μg/mL) for 24 h. TLR2 expression was analyzed by qPCR (E) and Western blot (F). (G,H) Long-Term LPS exposure results in LPS tolerance. AMs were pretreated with LPS (100 ng/mL) for 24 h and restimulated with LPS (1 μg/mL) for 2 h. TNF, CXCL10, IL10, MMP9, FPR2, and TLR2 mRNA expression levels were determined by qPCR. NT/NT, not treated; NT/LPS, LPS stimulation without pretreatment; LPS/NT, LPS pretreatment only; LPS/LPS, LPS pretreatment followed by LPS stimulation. Data from at least three independent experiments performed in duplicate with cells from different donors are shown and are presented as x-fold of solvent-treated cells ± SEM. ^#p < 0.05, ^###p < 0.001 vs. untreated cells, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 as indicated, ^§p < 0.05 vs. Pam3-treated cells. p-values were generated with ANOVA and Bonferroni's post-hoc test.

Figure 4

Synergistic upregulation of TLR2 by dexamethasone and LPS. AMs were preincubated with the GR inhibitor RU486 (10 μM) or solvent control (0.1% EtOH) and treated with LPS (100 ng/mL), Dex (1 μM) or both for 24 h. TLR2 expression was measured by qPCR (A) or Western blot (B,C). (B) Representative blot. (C) Densitometric analysis. TLR2 signal intensities were quantified, normalized to tubulin values, and expressed as x-fold of untreated cells. (A,C) Data from at least three independent experiments performed in duplicate with cells from different donors are presented as means + SEM. ^*p < 0.05, ^**p < 0.01. p-values were generated by ANOVA with Bonferroni's post-hoc test.

Figure 5

Impaired response toward TLR2 ligands in LPS- and/or Dex-pretreated AMs. (A) AMs were preincubated with LPS (100 ng/mL), Dex (1 μM), or both for 24 h and treated with Pam₃CSK₄ (Pam3, 1 μg/mL, 4 h). TNF secretion was assessed by TNF bioassay. (B,C) Primary human AMs were incubated with LPS (100 ng/mL, 24 h) before restimulation with TLR2 ligands for 2 h. LTA: lipoteichoic acid (5 μg/mL), HKSA: heat-killed S. aureus (10⁸ cells/mL), Pam2: Pam₂CSK₄ (1 μg/mL), Pam3: Pam₃CSK₄ (1 μg/mL). TNF (B) and TLR2 (C) mRNA levels were determined by qPCR. Data from at least three independent experiments performed in duplicate with cells from different donors are presented as means + SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. p-values were generated by ANOVA with Bonferroni‘s post-hoc test.

Figure 6

TLR2 in AM supernatants. (A,B) AMs were incubated with solvent control (0.1% DMSO), LPS (100 ng/mL), Dex (1 μM), or LPS+Dex for 24 h. 4-aminophenylmercuric acetate (APMA, 10 μM) was added to the indicated samples 5 h before supernatants were harvested. Soluble TLR2 (sTLR2) and full-length TLR2 (flTLR2) were detected in the supernatants by Western blot. (A) representative blot. (B) Relative sTLR2/TLR2 signal intensities are presented as means + SEM (n = 5). ^*p < 0.05 (Student's t-test). (C,D) Cells were either left untreated (Co) or treated with LPS (100 ng/mL) + Dex (1 μM) for 3 days, and EVs were isolated by sequential centrifugation. (C) Representative cryo-TEM images of EVs from untreated (Co) and LPS+Dex-treated cells. (D) Representative Western blot analysis for TLR2 in AM supernatants before and after ultracentrifugation (UC) and in EVs is shown (n = 5).

Figure 7

Characterization of THP-1 vesicles. Cells were incubated with medium only (Co), LPS (100 ng/mL), Dex (1 μM), or LPS+Dex for 72 h and EVs were isolated by sequential centrifugation. (A) Vesicles were visualized by cryo-TEM. Scale bar = 500 nm. (B,C) Average EV size (B) and concentration (C) were determined by nanoparticle tracking analysis. Data are presented as means + SEM (n = 7). ^*p < 0.05, ^**p < 0.01. p-values were generated by Mann–Whitney U-test. (D,E) EVs originating from differentially treated THP-1 cells were subjected to proteomics analysis (n = 3). (D) Overlap of identified proteins among treatment groups. (E) TLR2 and EV marker distribution. Log2 values of unique spectrum counts are shown for all three independent preparations per treatment. (F) Volcano plot of p-value vs. fold change in expression level in EV_LPS+Dex vs. EV_Co. Proteins that were upregulated at least 4-fold in EV_LPS+Dex vs. EV_Co with a p < 0.01 are highlighted. (G) Representative Western blot result for TLR2 detection in THP-1 supernatant before and after ultracentrifugation (UC) and in EV fractions (n = 3).

Figure 8

TLR2-containing vesicles act as decoy receptors. Differentiated THP-1 cells were incubated with medium only (Co) or LPS (100 ng/mL) + Dex (1 μM) for 72 h and EVs (EV_Co and EV_LPS+Dex, respectively) were isolated by sequential centrifugation. (A–F) Bead-bound EVs from untreated or LPS+Dex-treated THP-1 cells were analyzed by flow cytometry. Unloaded latex beads served as controls. Histograms show bead counts vs. log fluorescence intensity. (A,B) EV loading was confirmed by staining for vesicle markers CD9 (A) and CD63 (B). Representative histograms are shown (n = 3). (C–F) TLR2 staining and binding of rhodamine-labeled Pam₃CSK₄ (Pam3). (C,D) Representative histograms. (E,F) Mean fluorescence intensities were expressed as x-fold of EV_Co values + SEM (n = 3, duplicates). (G) Pam3-induced gene expression in HUVECs was measured by qRT-PCR. Pam3 was preincubated with the specified vesicles for 30 min at 37°C (2 × 10¹⁰ EVs/μg Pam3), and HUVECs were treated with the Pam3/EV mix (2 × 10¹⁰ EVs and 1 μg Pam3/mL) for 3 h. Data from 3 independent THP-1 vesicle preparations and HUVEC donors are presented as a percentage of EV_Co-treated cells + SEM. H: Pam3-induced CXCL8 promoter-dependent luciferase activity was quantified in hTLR2 HEK-Dual cells. Cells were either treated with Pam3 only (1 ng/mL, Co) or co-treated with Pam3 (1 ng/mL) and the specified vesicles (5 × 10⁹ EVs/ml) for 24 h. The Pam3/vesicle mix was preincubated for 30 min at 37°C before it was added to the cells. Data are expressed as percentage of Co-values + SEM (n = 4, duplicates). ^*p < 0.05, ^**p < 0.01, n.s.: not significant. P-values were generated by ANOVA with Bonferroni‘s post-hoc test or Student's t-test.