Phosphoinositides in phagocytosis and macropinocytosis.

Professional phagocytes provide immunoprotection and aid in the maintenance of tissue homeostasis. They perform these tasks by recognizing, engulfing and eliminating pathogens and endogenous cell debris. Here, we examine the paramount role played by phosphoinositides in phagocytosis and macropinocytosis, two major endocytic routes that mediate the uptake of particulate and fluid matter, respectively. We analyze accumulating literature describing the molecular mechanisms whereby phosphoinositides translate environmental cues into the complex, sophisticated responses that underlie the phagocytic and macropinocytic responses. In addition, we exemplify virulence strategies involving modulation of host cell phosphoinositide signaling that are employed by bacteria to undermine immunity. This article is part of a Special Issue entitled Phosphoinositides.

Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes.

Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking.

Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation.

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase ß abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase ß mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.

Vascular endothelial cells evade complement-mediated membrane injury via Weibel-Palade body mobilization.

BACKGROUND: Defective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b-9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. OBJECTIVE/METHODS: We examined the response of human endothelial cells to complement-mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b-9 on their plasma membranes. RESULTS: This triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel-Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement-mediated damage was examined by comparing the effects of C5b-9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement-mediated damage, but became resistant when transfected to express VWF (and thus WPBs). CONCLUSION: We conclude that BOECs exposed to C5b-9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell-specific response may protect the vascular endothelium from complement-mediated damage in vivo.

Screening for Rho GTPase Modulators in Actin-Dependent Processes Exemplified by Phagocytosis.

Rho GTPases, a family of molecular switches, are essential for the assembly and rearrangement of the cellular actin network. Actin remodeling is a central component of many important biological phenomena including chemotaxis, immunological synapse formation, and phagocytosis. Proper execution of these processes requires careful modulation of Rho GTPase activity in space and time. This is accomplished by delicate coordination of Rho GTPase activation and inactivation by Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase-activating proteins (RhoGAPs), respectively. Elucidating the function of these Rho GTPase modulators is complicated by their diversity, varied expression across different tissues, and multiplicity of substrates. To overcome some of these hurdles, we describe here a systematic and unbiased screening approach consisting of three sequential steps: (1) monitoring the subcellular localization of a library of Rho GTPase modulators; (2) assessing endogenous levels of expression of the suitably localized candidates in the cell type of interest; and (3) validating the functional relevance of the identified candidates by siRNA, followed by determining the effects of gene silencing on Rho GTPase activity and actin polymerization. To this end, we describe the expression and visualization of fluorescent Rho GTPase modulators, and the use of genetically encoded biosensors for active Rac/Cdc42 and of fluorescent probes of polymerized actin. Phagocytosis by macrophages is used in this chapter as an experimental paradigm, but the methods described herein can be easily extended to other cells and actin-dependent processes.

Every day I'm rufflin': Calcium sensing and actin dynamics in the growth factor-independent membrane ruffling of professional phagocytes.

Professional phagocytes continuously extend dynamic, actin-driven membrane protrusions. These protrusions, often referred to as membrane ruffles, serve a critical role in the essential phagocyte processes of macropinocytosis and phagocytosis. Small GTPases, such as RAC1/2, spatially and temporally regulate membrane ruffle formation. We have recently shown that extracellular calcium regulates the elaboration of membrane ruffles primarily through the synthesis of phosphatidic acid (PtdOH) at the plasma membrane. RAC1/2 guanine nucleotide exchange factors harbouring polybasic stretches are recruited by PtdOH to sites of ruffle formation. Here we discuss our findings and offer perspectives on how the regulation of dynamic actin structures at the plasma membrane by small GTPases is a critical component of phagocyte function.

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.

UNLABELLED: 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 thatA. fumigatusescapes 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)P3and 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)P3signaling during membrane ruffling and phagocytosis. IMPORTANCE: Aspergillus fumigatusis the most frequent cause of human infections in theAspergillusgenus. 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 betweenA. fumigatusand 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 abundantAspergillusmycotoxin-undermines the ability of phagocytes to carry out their protective functions. By targeting PtdIns(3,4,5)P3signaling 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.

Chemokine Signaling Enhances CD36 Responsiveness toward Oxidized Low-Density Lipoproteins and Accelerates Foam Cell Formation.

Excessive uptake of oxidized low-density lipoproteins (oxLDL) by macrophages is a fundamental characteristic of atherosclerosis. However, signals regulating the engagement of these ligands remain elusive. Using single-molecule imaging, we discovered a mechanism whereby chemokine signaling enhanced binding of oxLDL to the scavenger receptor, CD36. By activating the Rap1-GTPase, chemokines promoted integrin-mediated adhesion of macrophages to the substratum. As a result, cells exhibited pronounced remodeling of the cortical actin cytoskeleton that increased CD36 clustering. Remarkably, CD36 clusters formed predominantly within actin-poor regions of the cortex, and these regions were primed to engage oxLDL. In accordance with enhanced ligand engagement, prolonged exposure of macrophages to chemokines amplified the accumulation of esterified cholesterol, thereby accentuating the foam cell phenotype. These findings imply that the activation of integrins by chemokine signaling exerts feedforward control over receptor clustering and effectively alters the threshold for cells to engage ligands.

Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.

Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large--but not small--phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles.

Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes.

Macrophages and dendritic cells continuously survey their environment in search of foreign particles and soluble antigens. Such surveillance involves the ongoing extension of actin-rich protrusions and the consequent formation of phagosomes and macropinosomes. The signals inducing this constitutive cytoskeletal remodeling have not been defined. We report that, unlike nonphagocytic cells, macrophages and immature dendritic cells have elevated levels of phosphatidic acid (PA) in their plasma membrane. The plasmalemmal PA is synthesized by phosphorylation of diacylglycerol, which is in turn generated by a G protein-stimulated phospholipase C. Inhibition of diacylglycerol kinase activity results in the detachment of T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1)-a Rac guanine exchange factor-from the plasma membrane, thereby depressing Rac activity and abolishing the constitutive ruffling and macropinocytosis that characterize macrophages and immature dendritic cells. Accumulation of PA and binding of TIAM1 to the membrane require the activity of phosphatidylinositol-4,5-bisphosphate 3-kinase. Thus a distinctive, constitutive pathway of PA biosynthesis promotes the actin remodeling required for immune surveillance.