Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

Abstract

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca-conducting ion channel, mediates the cytosolic Ca elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1β and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model

“…Our results also suggested that Mg could reduce endothelial cell leakage induced by LPS in vitro through suppressing NF-κB signaling pathway. It was reported that Ca ions influx was necessary for NF-κB activation and translocation to nuclear in both macrophages [65] and endothelial cells [66]. Influx of extracellular Ca 2+ via TRPV4/ 5 (Transient receptor potential cation channel subfamily V members) channels in cell membrane was also necessary in RANKL induced osteoclast differentiation [67,68].…”

Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry

“…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.…”

“…Characteristic TLR4 and CD14 endocytosis measured over time in bone-marrow derived macrophages. Data was modified from its original presentation in Schappe et al ., 2018 with author permission.…”

“…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.…”

Biophysical regulation of macrophages in health and disease

“…TRPV4 appears to negatively regulate LPS‐induced inflammation, but enhances both LPS‐mediated phagocytosis and ox‐LDL uptake . In contrast, a recent study found that TRPM7 positively regulates LPS‐induced inflammatory activation by enhancing endocytosis of the LPS‐TLR4 complex, and its depletion has been shown to protect mice from LPS‐induced peritonitis . Finally, manipulation of the expression and activity of TRPV2 and TRPC6 reveals that these channels are required for phagocytosis in macrophages .…”