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.

Highlights

  • Glucocorticoids (GCs) represent the most effective antiinflammatory drugs in the therapy of inflammatory lung diseases

  • Further analysis showed that TLR2 upregulation was already detectable 4 h after treatment (Figure 1B), and TLR2 protein production was maximal after 16 h, as shown by Western blot analysis (Figures 1C,D)

  • We evaluated whether Dex binding to the GR is necessary for TLR2 induction

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Summary

Introduction

Glucocorticoids (GCs) represent the most effective antiinflammatory drugs in the therapy of inflammatory lung diseases. Alveolar macrophages (AMs) are the tissue-resident macrophages in the lung alveolar space They represent the first line of defense against pathogens in the lower airspace and recognize microbial ligands via pattern recognition receptors [4, 5]. After recognition and binding of a specific PAMP, TLRs induce an intracellular signaling cascade that culminates in the activation of the activator protein (AP), nuclear factor (NF)-κB, and interferon regulatory factors (IRFs). These signaling cascades result in the secretion of proinflammatory factors that protect the host from microbial infection [6, 9]

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