Johnathan Canton

Scavenger receptors in homeostasis and immunity

Scavenger receptors were originally identified by their ability to recognize and to remove modified lipoproteins; however, it is now appreciated that they carry out a striking range of functions, including pathogen clearance, lipid transport, the transport of cargo within the cell and even functioning as taste receptors. The large repertoire of ligands recognized by scavenger receptors and their broad range of functions are not only due to the wide range of receptors that constitute this family but also to their ability to partner with various co-receptors. The ability of individual scavenger receptors to associate with different co-receptors makes their responsiveness extremely versatile. This Review highlights recent insights into the structural features that determine the function of scavenger receptors and the emerging role that these receptors have in immune responses, notably in macrophage polarization and in the pathogenesis of diseases such as atherosclerosis and Alzheimers disease.

The life cycle of phagosomes: formation, maturation, and resolution

Phagocytosis, the regulated uptake of large particles (>0.5 μm in diameter), is essential for tissue homeostasis and is also an early, critical component of the innate immune response. Phagocytosis can be conceptually divided into three stages: phagosome, formation, maturation, and resolution. Each of these involves multiple reactions that require exquisite spatial and temporal orchestration. The molecular events underlying these stages are being unraveled and the current state of knowledge is briefly summarized in this article.

Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.

Phagosomal pH is regulated in diametrically opposed ways in M1 and M2 macrophages. M2 phagosomes acidify rapidly and monotonically, whereas M1 phagosomes undergo cyclic alkaline oscillations caused by proton consumption upon dismutation of superoxide, followed by activation of HV1 channels.

The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis.

TIM4 is a receptor for phosphatidylserine that mediates engulfment of apoptotic cells. Remarkably, it does not require its cytosolic or transmembrane domains to mediate phagocytosis. TIM4 associates with integrins that serve as signal-transducing coreceptors.

Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages

Macropinocytosis can be induced in several cell types by stimulation with growth factors. In selected cell types, notably macrophages and dendritic cells, macropinocytosis occurs constitutively, supporting the uptake of antigens for subsequent presentation. Despite their different mode of initiation and contrasting physiological roles, it is tacitly assumed that both types of macropinocytosis are mechanistically identical. We report that constitutive macropinocytosis is stringently calcium dependent, while stimulus-induced macropinocytosis is not. Extracellular calcium is sensed by G-protein-coupled calcium-sensing receptors (CaSR) that signal macropinocytosis through Gα-, phosphatidylinositol 3-kinase and phospholipase C. These pathways promote the recruitment of exchange factors that stimulate Rac and/or Cdc42, driving actin-dependent formation of ruffles and macropinosomes. In addition, the heterologous expression of CaSR in HEK293 cells confers on them the ability to perform constitutive macropinocytosis. Finally, we show that CaSR-induced constitutive macropinocytosis facilitates the sentinel function of macrophages, promoting the efficient delivery of ligands to cytosolic pattern-recognition receptors.

Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis

ABSTRACT Aspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report that A. fumigatus escapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3 and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3. Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3 signaling during membrane ruffling and phagocytosis. IMPORTANCE Aspergillus fumigatus is the most frequent cause of human infections in the Aspergillus genus. In immunocompromised populations, invasive aspergillosis (IA) is associated with a mortality rate of up to 90%, and current antifungal therapies have failed to prevent or reverse the infection. Therefore, a deeper understanding of the interactions between A. fumigatus and its host is required. In healthy humans, alveolar macrophages can ingest and eliminate fungal spores, thus limiting their germination into mycotoxin-producing hyphae. Our studies reveal that gliotoxin—the most abundant Aspergillus mycotoxin—undermines the ability of phagocytes to carry out their protective functions. By targeting PtdIns(3,4,5)P3 signaling and downregulating phagocytic immune defenses, the toxin could also exacerbate polymicrobial infections. Notably, we were able to reverse gliotoxin toxicity by addition of diacylglycerol analogues, which may provide the basis for therapeutic interventions. Aspergillus fumigatus is the most frequent cause of human infections in the Aspergillus genus. In immunocompromised populations, invasive aspergillosis (IA) is associated with a mortality rate of up to 90%, and current antifungal therapies have failed to prevent or reverse the infection. Therefore, a deeper understanding of the interactions between A. fumigatus and its host is required. In healthy humans, alveolar macrophages can ingest and eliminate fungal spores, thus limiting their germination into mycotoxin-producing hyphae. Our studies reveal that gliotoxin—the most abundant Aspergillus mycotoxin—undermines the ability of phagocytes to carry out their protective functions. By targeting PtdIns(3,4,5)P3 signaling and downregulating phagocytic immune defenses, the toxin could also exacerbate polymicrobial infections. Notably, we were able to reverse gliotoxin toxicity by addition of diacylglycerol analogues, which may provide the basis for therapeutic interventions.

Measuring lysosomal pH by fluorescence microscopy.

The technique of dual-wavelength ratio fluorescence microscopy provides a powerful tool to measure organellar pH. Unlike single-wavelength measurements, this method is unaffected by changes in focal plane, dye volume, and fluorophore bleaching, providing a quantitative and dynamic readout of the pH of subcellular compartments. This chapter describes the application of dual-wavelength ratio fluorescence microscopy to the measurement of lysosomal pH, highlighting its advantages and limitations. Probe selection, calibration methods, and salient aspects of the required hardware are discussed in detail.

Measuring Phagosomal pH by Fluorescence Microscopy.

Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

Differential ability of proinflammatory and anti-inflammatory macrophages to perform macropinocytosis

Although anti-inflammatory macrophages perform macropinocytosis constitutively, proinflammatory macrophages do not, due to impaired signaling of the calcium-sensing receptor. However, macropinocytosis can be elicited in proinflammatory macrophages by agonists that induce the recruitment of phosphatidylinositol 3-kinase to the plasma membrane.

Mesenchymal stem cells enhance NOX2-dependent reactive oxygen species production and bacterial killing in macrophages during sepsis

Human mesenchymal stem/stromal cells (MSCs) have been reported to produce an M2-like, alternatively activated phenotype in macrophages. In addition, MSCs mediate effective bacterial clearance in pre-clinical sepsis models. Thus, MSCs have a paradoxical antimicrobial and anti-inflammatory response that is not understood. Here, we studied the phenotypic and functional response of monocyte-derived human macrophages to MSC exposure in vitro. MSCs induced two distinct, coexistent phenotypes: M2-like macrophages (generally elongated morphology, CD163+, acute phagosomal acidification, low NOX2 expression and limited phagosomal superoxide production) and M1-like macrophages characterised by high levels of phagosomal superoxide production. Enhanced phagosomal reactive oxygen species production was also observed in alveolar macrophages from a rodent model of pneumonia-induced sepsis. The production of M1-like macrophages was dependent on prostaglandin E2 and phosphatidylinositol 3-kinase. MSCs enhanced human macrophage phagocytosis of unopsonised bacteria and enhanced bacterial killing compared with untreated macrophages. Bacterial killing was significantly reduced by blockade of NOX2 using diphenyleneiodonium, suggesting that M1-like cells are primarily responsible for this effect. MSCs also enhanced phagocytosis and polarisation of M1-like macrophages derived from patients with severe sepsis. The enhanced antimicrobial capacity (M1-like) and inflammation resolving phenotype (M2-like) may account for the paradoxical effect of these cells in sepsis in vivo. Mesenchymal stem cells enhance bacterial killing via production of phagosomal reactive oxygen species in macrophages http://ow.ly/tigl30iGFPG