Christiane Rondeau

Endoplasmic Reticulum-Mediated Phagocytosis Is a Mechanism of Entry into Macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.

Trafficking of Leishmania donovani promastigotes in non-lytic compartments in neutrophils enables the subsequent transfer of parasites to macrophages.

Inoculation of Leishmania (L.) spp. promastigotes in the dermis of mammals by blood‐feeding sand flies can be accompanied by the rapid recruitment of neutrophils, inflammatory monocytes and dendritic cells. Despite the presence of these lytic leucocytes, parasitism is efficiently established. We show here that Leishmania donovani promastigotes are targeted to two different compartments in neutrophils. The compartments harbouring either damaged or non‐damaged parasites were characterized at the electron microscopy (EM) level using the glucose 6‐phosphatase cytochemistry and endosome–phagosome fusion assays. One involves the contribution of lysosomes leading to the formation of highly lytic compartments where parasites are rapidly degraded. The other is lysosome‐independent and involves the contribution of a compartment displaying some features of the endoplasmic reticulum (ER) where parasites are protected from degradation. Using genetically modified parasites, we show that the promastigote surface lipophosphoglycan (LPG) is required to inhibit lysosome fusion and maintain parasites in neutrophil compartments displaying ER features. L. donovani‐harbouring neutrophils that eventually enter apoptosis can be phagocytosed by macrophages enabling the stealth entry of parasites into their final replicative host cells. Thus, the ability of L. donovani to avoid trafficking into lysosomes‐derived compartments in short‐lived neutrophils constitutes a key process for the subsequent establishment of long‐term parasitism.

A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase‐dependent manner by serum‐starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho‐associated, coiled‐coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ∼20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.

Subcellular Localization of Iron and Heme Metabolism Related Proteins at Early Stages of Erythrophagocytosis

Background Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined. Methodology/Principal Findings We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes. Conclusions/Significance Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection

Exosome-like vesicles containing an active 20S proteasome core contribute to autoimmunity and vascular allograft inflammation. Friendly fire from organ failure Despite advances in organ transplantation, rejection still poses a substantial risk. Autoantibodies contribute to rejection, but how these autoantibodies are generated remains unknown. Dieudé et al. found that injection of apoptotic exosome-like vesicles apoExo stimulated autoantibody production in mice, which led to increased graft rejection after transplant. The apoExo contained active 20S proteasome core complexes, and inhibition of proteasome activity decreased the immunogenicity of apoExo and graft rejection in transplanted mice. Circulating apoExo and increased anti-autoantibody titers were also observed in mouse models of ischemia-reperfusion injury, suggesting that the same organ failure that necessitates the transplant might increase the risk of rejection. Therefore, proteasome inhibitors could provide a new therapeutic avenue for graft rejection. Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Interleukin-4 boosts the capacity of dendritic cells to present endogenous antigens on MHC II and to resist bacterial infection through a mechanism shown to be partially dependent on RUFY4 expression.

Inhibition of the Host Translation Shutoff Response by Herpes Simplex Virus 1 Triggers Nuclear Envelope-Derived Autophagy

ABSTRACT Macroautophagy is a cellular pathway that degrades intracellular pathogens and contributes to antigen presentation. Herpes simplex virus 1 (HSV-1) infection triggers both macroautophagy and an additional form of autophagy that uses the nuclear envelope as a source of membrane. The present study constitutes the first in-depth analysis of nuclear envelope-derived autophagy (NEDA). We established LC3a as a marker that allowed us to distinguish between NEDA and macroautophagy in both immunofluorescence and flow cytometry. NEDA was observed in many different cell types, indicating that it is a general response to HSV-1 infection. This autophagic pathway is known to depend on the viral protein γ34.5, which can inhibit macroautophagy via binding to beclin-1. Using mutant viruses, we were able to show that binding of beclin-1 by γ34.5 had no effect on NEDA, demonstrating that NEDA is regulated differently than macroautophagy. Instead, NEDA was triggered in response to γ34.5 binding to protein phosphatase 1α, an interaction used by the virus to prevent host cells from shutting off protein translation. NEDA was not triggered when late viral protein production was inhibited with acyclovir or hippuristanol, indicating that the accumulation of these proteins might stress infected cells. Interestingly, expression of the late viral protein gH was sufficient to rescue NEDA in the context of infection with a virus that otherwise does not support strong late viral protein expression. We argue that NEDA is a cellular stress response triggered late during HSV-1 infection and might compensate for the viral alteration of the macroautophagic response.