Abstract
After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.
Keywords: Toll-like receptor, TLR, TLR4, CD14, Endocytosis, Macrophage, BMDM, Innate immunity, LPS
Background
Innate immune cells, including macrophages and dendritic cells, employ a variety of pattern recognition receptors (PRRs) to survey their environments for danger- and pathogen-associated molecular patterns. Trafficking and signaling of PRRs from various subcellular compartments enables wider immune surveillance and has emerged as an important design principle of innate immunity (Brubaker et al., 2015). The detection of the bacterial endotoxin LPS is highly dependent on TLR4 and its co-receptor CD14. The endocytosis of the TLR4 complex requires CD14 and is essential for LPS-induced macrophage activation (Zanoni et al., 2011; Tan et al., 2015). Endocytosis of TLR4 is essential to activate secondary signaling complexes at the newly-formed ‘signaling endosome,’ which promotes interferon regulatory factor 3-dependent transcription through the signaling adaptor TIR-domain containing adapter-inducing interferon-β (TRIF) (Kagan et al., 2008). TLR4 endocytosis has been observed in macrophages, dendritic cells, and epithelial cells (Roy et al., 2014). Understanding the underlying mechanisms of this critical step in macrophage activation requires a robust and quantitative method to measure LPS-triggered TLR4 endocytosis. Here, we describe a version of a flow cytometry-based method that was initially reported by Kagan and colleagues (Kagan et al., 2008), and used by others, to monitor TLR4 endocytosis. We have used the method recently to study the role of transient receptor potential melastatin-like 7 (TRPM7), an ion channel, in TLR4 endocytosis (Schappe et al., 2018). The experimental logic of this method relies on measuring the loss of TLR4 and CD14 staining at the cell surface after LPS treatment. We stain LPS-treated cells with an anti-TLR4 (or anti-CD14) fluorophore-conjugated antibody without permeabilization. The fluorescence intensity acquired using flow cytometry reports the relative quantity of receptor resident in the plasma membrane (Figure 1). Although specific for TLR4 and CD14, the assay can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.Figure 1. Schematic of TLR4 and CD14 endocytosis protocol. Experimental workflow described in protocol “Procedure”.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
We would like to thank Dr. Jonathan Kagan (Boston Children’s Hospital) and colleagues whose protocol (cited herein) we adapted for our research. We also would like to thank members of the Desai laboratory for their assistance in editing this manuscript. We also thank the UVA Flow Cytometry Core and Carter Immunology Center Flow Cytometry Core. We appreciate our funding that supported this work: grants GM108989 (BND) and 5T32GM007055-41 (MSS) from the National Institutes of Health. The authors declare that they have no conflicts of interest to report.
References
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