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- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemToll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide(Lausanne : Frontiers Media, 2019) Hoppstädter, Jessica; Dembek, Anna; Linnenberger, Rebecca; Dahlem, Charlotte; Barghash, Ahmad; Fecher-Trost, Claudia; Fuhrmann, Gregor; Koch, Marcus; Kraegeloh, Annette; Huwer, Hanno; Kiemer, Alexandra K.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.