Abstract
TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products. Here, this is demonstrated through the use of classically-activated and alternatively-activated macrophages. We show that, when polarized, human macrophages differentially express and localize TLR4, resulting in biased recognition and subsequent signalling of LPS derived from Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica. Analysis of activation demonstrated that in classically activated macrophages, P. aeruginosa signals from the plasma membrane via TLR4 to p65 dependent on TAK1 and TBK1 signalling. E. coli signals dependent or independent of the endosome, utilizing both TAK1- and TBK1-signalling to induce P65 and IRF3 inducible genes and cytokines. S. enterica however, only induces P65 and IRF3 phosphorylation through signalling via the endosome. This finding outlines clear signalling mechanisms by which innate immune cells, such as macrophages, can distinguish between bacterial species and initiate specialized responses through TLR4.
Highlights
TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products
Differentiated classically‐activated M1 macrophages display internalisation of TLR4. Myeloid lineage cells, such as macrophages are known to express high levels of TLR4 as these innate immune cells can quickly respond to infection through the production of large amounts of chemotactic and inflammatory mediators
This was determined through comparison of surface staining with total staining, via saponin permeabilization. This differential localisation was confirmed through immunofluorescent imaging of the same macrophages clearly showing a punctate distribution of TLR4 in M1 macrophages (Fig. 1Eii), suggesting compartmentalisation, perhaps within the early-endosome, whilst M0 and M2 macrophages revealed an even distribution throughout the cell cytoplasm (Fig. 1Ei,iii)
Summary
TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products. Abbreviations TLR Toll-like receptor MyD88 Myeloid differentiation primary response 88 TRIF TIR-domain-containing adapter-inducing interferon-β NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells IRF3 Interferon regulatory factor 3 LPS Lipopolysaccharide M1 Classically activated macrophage M2 Alternatively activated macrophage TAK1 Mitogen-activated protein kinase kinase kinase 7 TBK1 TANK binding kinase 1 PMA Phorbol 12-myristate 13-acetate IFNγ Interferon gamma IL Interleukin IP-10/CXCL10 Interferon gamma-induced protein 10 CCR7 Chemokine receptor 7 CD206 Mannose receptor C type 1 CD163 Haemoglobin scavenger receptor CCL17 Chemokine (C–C motif) ligand 17 Chronic inflammatory diseases, such as inflammatory bowel disease (IBD), is driven in part by a dysregulated immune response to the host microbiota. Current evidence demonstrates that all TLRs, excluding the endosomal dsRNA receptor TLR3, signal through a myeloid differentiation primary response 88 (MyD88)-dependent pathway
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