Macropinocytosis: New Insights Into Its Underappreciated Role in Innate Immune Cell Surveillance

Macropinocytosis has received increasing attention in recent years for its various roles in nutrient acquisition, immune surveillance, and virus and cancer pathologies. In most cases macropinocytosis is initiated by the sudden increase in an external stimulus such as a growth factor. This “induced” form of macropinocytosis has been the subject of much of the work addressing its mechanism and function over the years. An alternative, “constitutive” form of macropinocytosis restricted to primary innate immune cells also exists, although its mechanism has remained severely understudied. This mini-review focuses on the very recent advances that have shed new light on the initiation, formation and functional relevance of constitutive macropinocytosis in primary innate immune cells. An emphasis is placed on how this new understanding of constitutive macropinocytosis is helping to define the sentinel function of innate immune cells including polarized macrophages and dendritic cells

Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus

Disruption of the fusion of Leishmania parasitophorous vacuoles with ER vesicles results in the control of the infection

Parasitophorous vacuoles (PV) that harbour Leishmania parasites acquire some characteristics from fusion with host cell vesicles. Recent studies have shown that PVs acquire and display resident endoplasmic reticulum (ER) molecules. We investigated the importance of ER molecules to PV biology by assessing the consequence of blocking the fusion of PVs with vesicles that originate from the early secretory pathway. This was achieved by targeting the N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that mediate the fusion of early secretory vesicles. In the presence of dominant negative variants of sec22b or some of its known cognate partners, D12 and syntaxin 18, PVs failed to distend and harboured fewer parasites. These observations were confirmed in studies in which each of the SNAREs listed above including the intermediate compartment ER/Golgi SNARE, syntaxin 5, was knocked down. The knock-down of these SNARES had little or no measurable effect on the morphology of the ER or on activated secretion even though they resulted in a more significant reduction of PV size. Moreover, the knock-down of the ER/Golgi SNAREs resulted in significant reduction in parasite replication. Taken together, these studies provide further evidence that PVs acquire ER components by fusing with vesicles derived from the early secretory pathway; disruption of this interaction results in inhibition of the development of PVs as well as the limitation of parasite replication within infected cells

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

The original article is available via the journal's website http://www.molbiolcell.org/content/early/2014/08/25/mbc.E14-05-0967.full.pdfMacrophages respond to changes in environmental stimuli by assuming distinct functional phenotypes, a phenomenon referred to as macrophage polarization. We generated classically (M1) and alternatively (M2) polarized macrophages--two extremes of the polarization spectrum--to compare the properties of their phagosomes. Specifically, we analyzed the regulation of the luminal pH after particle engulfment. The phagosomes of M1 macrophages had a similar buffering power and proton (equivalent) leakage permeability but significantly reduced proton-pumping activity compared with M2 phagosomes. As a result, only the latter underwent a rapid and profound acidification. By contrast, M1 phagosomes displayed alkaline pH oscillations, which were caused by proton consumption upon dismutation of superoxide, followed by activation of a voltage- and Zn(2+)-sensitive permeation pathway, likely HV1 channels. The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes. The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify. Thus, in M1 cells, elimination of pathogens through deployment of the microbicidal NADPH oxidase is given priority at the expense of delayed acidification. By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.J.C. is supported by a Cystic Fibrosis Canada post-doctoral fellowship. This work was supported by grants MOP7075, MOP93634 and TBO-122068 from the Canadian Institutes of Health Research awarded to S.G. and M.G

The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis

The original article is available via the journal's website http://www.molbiolcell.org/content/25/9/1511.full.pdfT-cell immunoglobulin mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear, and conflicting data have emerged. To define the mode of action of TIM4, we used two distinct but complementary approaches: 1) we compared bone marrow-derived macrophages from wild-type and TIM4(-/-) mice, and 2) we heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier by others, TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Of interest, TIM4-driven phagocytosis depends on the activation of integrins and involves stimulation of Src-family kinases and focal adhesion kinase, as well as the localized accumulation of phosphatidylinositol 3,4,5-trisphosphate. These mediators promote recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1, and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model in which TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.This work was supported by Grants MOP7075, MOP93634, and TBO-122068 from the Canadian Institutes of Health Research (to S.G. and M.G.). R.S.F. was supported by a Restracomp Fellowship from the Hospital for Sick Children Research Training Center. TIM4−/ bones were kindly provided by John Brumell (Hospital for Sick Children, Toronto, Canada)

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.</p

EveryWear, a Human Research and Health Mobile Assistant for Exploration

EveryWear (EVW) is a software solution developed by CNES integrating joint CNES and ESA objectives improving the monitoring of Astronaut's health through recording of physiological and science data during their mission on-board the International Space Station (ISS). EVW is made of a fully custom tablet (iPad) application, an onboard server part for Space to Ground data synchronisation and ground services components for data dissemination and exchange. EVW provides data recording, pre-processing and synchronization during human spaceflights in support of crew medical and human research objectives. EVW was introduced onboard the International Space Station during Increment 49 (2016) and since then has been extensively used and continuously improved with very positive feedback received from astronauts enjoying the enhanced usability offered by mobile application ergonomics. EveryWear medical functionalities, represented through different modules, include nutritional assessment medication intake tracking and as well as private messaging between crew and medical teams on ground. EveryWear science functionalities allow interfacing with connected devices such as a Commercial Off The Shelf (COTS) glucometer or custom dosimeter. It also offers dynamic questionnaires, and private messaging with operation support teams. Multiple support teams and crewmembers are using EveryWear simultaneously. The system ensures end-to-end encryption between crew and designated recipient for each type of data and for each crewmember. After several years of joint European efforts, ESA with CNES decided to jointly develop EveryWear with NASA. This will be an evolution of the existing EveryWear application, integrating existing functions like NASA's Food Intake Tracker (FIT) and Data Collection Tool (DCT) as well as new requirements from ESA and NASA into a single centralized modular system. The joint development is expected to represent the minimum total cost path for all involved parties and is composed of three incremental phases. Phase 1 and Phase 2 are dedicated to ISS operational use cases while Phase 3 will include enhanced ISS functions considering Exploration scenarios

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