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Efficient clearance of apoptotic cells by phagocytosis, also known as efferocytosis, is fundamental to developmental biology, organ physiology, and immunology. Macrophages use multiple mechanisms to detect and engulf apoptotic cells, but the signaling pathways that regulate the digestion of the apoptotic cell cargo, such as the dynamic Ca2+ signals, are poorly understood. Using an siRNA screen, we identify TRPM7 as a Ca2+-conducting ion channel essential for phagosome maturation during efferocytosis. Trpm7-targeted macrophages fail to fully acidify or digest their phagosomal cargo in the absence of TRPM7. Through perforated patch electrophysiology, we demonstrate that TRPM7 mediates a pH-activated cationic current necessary to sustain phagosomal acidification. Using mice expressing a genetically-encoded Ca2+ sensor, we observe that phagosome maturation requires peri-phagosomal Ca2+-signals dependent on TRPM7. Overall, we reveal TRPM7 as a central regulator of phagosome maturation during macrophage efferocytosis. Efficient removal of apoptotic cells by phagocytosis underlies tissue development, wound repair, host defense and organ homeostasis. Here, authors identify TRPM7 as a regulator of cargo acidification and Ca2+ signaling during apoptotic cell clearance. [ABSTRACT FROM AUTHOR]

The gene transient receptor potential-melastatin-like 7 (Trpm7) encodes a protein that functions as an ion channel and a kinase. TRPM7 has been proposed to he required for cellular Mg2+ homeostasis in vertebrates. Deletion of mouse Trpm7 revealed that it is essential for embryonic development. Tissue-specific deletion of Trpm7 in the T cell lineage disrupted thymopoiesis, which led to a developmental block of thymocytes at the double-negative stage and a progressive depletion of thymic medullary cells. However, deletion of Trpm7 in T cells did not affect acute uptake of Mg2+ or the maintenance of total cellular Mg2+. Trpm7-deficient thymocytes exhibited dysregulated synthesis of many growth factors that are necessary for the differentiation and maintenance of thymic epithelial cells. The thymic medullary cells test signal transducer and activator of transcription 3 activity, which accounts for their depletion when Trpm7 is disrupted in thymocytes. [ABSTRACT FROM AUTHOR]

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct antiinflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo. [ABSTRACT FROM AUTHOR]

Pannexin 1 (Panx1) forms plasma membrane ion channels that are widely expressed throughout the body. Panx1 activation results in the release of nucleotides such as adenosine triphosphate and uridine triphosphate. Thus, these channels have been implicated in diverse physiological and pathological functions associated with purinergic signaling, such as apoptotic cell clearance, blood pressure regulation, neuropathic pain, and excitotoxicity. In light of this, substantial attention has been directed to understanding the mechanisms that regulate Panx1 channel expression and activation. Here we review accumulated evidence for the various activation mechanisms described for Panx1 channels and, where possible, the unitary channel properties associated with those forms of activation. We also emphasize current limitations in studying Panx1 channel function and propose potential directions to clarify the exciting and expanding roles of Panx1 channels. [ABSTRACT FROM AUTHOR]

Phagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca 2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca 2+ (phase 1), and by concurrently activating the mitochondrial Ca 2+ (mCa 2+ ) uniporter (MCU), they trigger a rapid influx of Ca 2+ into the mitochondria (phase 2). mCa 2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa 2+ signaling, Mcu -/- macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca 2+ signaling is an electrometabolic switch to fuel phagosome killing., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that they have no conflicts of interest to report.

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 2+ -conducting ion channel, mediates the cytosolic Ca 2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca 2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca 2+ 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 2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pannexin 1 (Panx1) forms plasma membrane ion channels that are widely expressed throughout the body. Panx1 activation results in the release of nucleotides such as adenosine triphosphate and uridine triphosphate. Thus, these channels have been implicated in diverse physiological and pathological functions associated with purinergic signaling, such as apoptotic cell clearance, blood pressure regulation, neuropathic pain, and excitotoxicity. In light of this, substantial attention has been directed to understanding the mechanisms that regulate Panx1 channel expression and activation. Here we review accumulated evidence for the various activation mechanisms described for Panx1 channels and, where possible, the unitary channel properties associated with those forms of activation. We also emphasize current limitations in studying Panx1 channel function and propose potential directions to clarify the exciting and expanding roles of Panx1 channels., (© 2018 Chiu et al.)