Cellular Responses to the Efferocytosis of Apoptotic Cells.

The rapid and efficient phagocytic clearance of apoptotic cells, termed efferocytosis, is a critical mechanism in the maintenance of tissue homeostasis. Removal of apoptotic cells through efferocytosis prevents secondary necrosis and the resultant inflammation caused by the release of intracellular contents. The importance of efferocytosis in homeostasis is underscored by the large number of inflammatory and autoimmune disorders, including atherosclerosis and systemic lupus erythematosus, that are characterized by defective apoptotic cell clearance. Although mechanistically similar to the phagocytic clearance of pathogens, efferocytosis differs from phagocytosis in that it is immunologically silent and induces a tissue repair response. Efferocytes face unique challenges resulting from the internalization of apoptotic cells, including degradation of the apoptotic cell, dealing with the extra metabolic load imposed by the processing of apoptotic cell contents, and the coordination of an anti-inflammatory, pro-tissue repair response. This review will discuss recent advances in our understanding of the cellular response to apoptotic cell uptake, including trafficking of apoptotic cell cargo and antigen presentation, signaling and transcriptional events initiated by efferocytosis, the coordination of an anti-inflammatory response and tissue repair, unique cellular metabolic responses and the role of efferocytosis in host defense. A better understanding of how efferocytic cells respond to apoptotic cell uptake will be critical in unraveling the complex connections between apoptotic cell removal and inflammation resolution and maintenance of tissue homeostasis.

Antiplatelet and Anticoagulant Strategies Following Left Ventricular Assist Device (LVAD) Explantation or Decommissioning: A Scoping Review of the Literature.

Mechanical circulatory support using left ventricular assist devices (LVADs) has transformed management of patients with end-stage heart failure with more patients on LVAD therapy surviving long enough to necessitate either device explantation or decommissioning. Usually, there is foreign material retained following these procedures that requires maintaining antiplatelet and/or anticoagulant therapy. However, there is no consensus on optimal management of antiplatelet and anticoagulant therapy following LVAD explantation or decommissioning. We conducted a scoping review of antiplatelet and anticoagulation strategies, searching EMBASE, PubMed and CENTRAL. A total of 15 case reports and series encompassing 38 patient cases were found that met inclusion criteria. There was a heterogeneity of LVAD types and techniques used for explantation and decommissioning. Most reports identified in our review maintained patients on a vitamin K antagonist for at least 3 months post-explantation or decommissioning with or without concomitant antiplatelet therapy with low-dose aspirin. However, there was no single agreed-upon optimal strategy for antiplatelet and anticoagulant use post-procedure. Factors such as the degree of foreign material retained following device explantation or decommissioning and whether there is another indication for anticoagulation or antiplatelet use must be considered. A lack of overall consensus indicates that more studies are needed in this area to establish definitive guidelines around antiplatelet and anticoagulant therapy following LVAD explantation or decommissioning.

Clinical effectiveness of therapy with continuous-flow left ventricular assist devices in nonischemic versus ischemic cardiomyopathy: a systematic review and meta-analysis.

Background: Clinicians may be less inclined to consider long-term left ventricular assist device (LVAD) therapy in end-stage heart failure (ESHF) as a result of nonischemic cardiomyopathy (NICM) versus ischemic cardiomyopathy (ICM) owing to potentially greater right ventricular involvement in the former; however, it is unknown whether the cause of heart failure has a clinically meaningful effect on outcomes following LVAD implantation. In this systematic review, we aimed to determine whether ischemic versus nonischemic etiology has any impact on patient-relevant outcomes. Methods: We searched MEDLINE, Embase, PubMed and the Cochrane Library for studies published in English between Jan. 1, 2000, and Nov. 22, 2018, that examined survival and transplantation rates following LVAD implantation in patients with NICM or ICM. Randomized clinical trials, cohort studies, case-control studies, cross-sectional studies and case series with a sample size of at least 8 patients were eligible for inclusion. To be included in the meta-analysis, outcomes had to include at least death reported at 30 days or 1 year after LVAD implantation. Quality of included studies was assessed by 2 independent reviewers using the Newcastle-Ottawa Quality Assessment Scale for Cohort Studies. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) quality-assessment tool was used to assess outcomes (30-d survival, 1-yr survival and cardiac transplantation following LVAD therapy) across studies. Results: From a total of 2843 citations identified, 7 studies met all inclusion criteria. Studies were generally of good quality, but reporting of patient demographic characteristics, outcomes and complications was heterogeneous. We found no significant difference in 30-day or 1-year survival or in cardiac transplantation rates after device implantation between the NICM and ICM groups. Patients in the 2 groups had similar outcomes up to 1 year with LVAD therapy. Conclusion: Early outcomes of LVAD therapy do not appear to be affected by heart failure etiology. Ongoing investigation is required to determine the long-term outcomes of LVAD therapy in ICM and NICM. Systematic review registration: PROSPERO register, record ID 76483.

Contexte: Les professionnels de la santé peuvent être moins enclins à envisager un traitement à long terme par dispositif d'assistance ventriculaire gauche (DAVG) en cas d'insuffisance cardiaque terminale résultant d'une myocardiopathie non ischémique plutôt que d'une myocardiopathie ischémique, en raison du risque potentiellement accru d'atteinte du ventricule droit dans le premier cas. Cependant, on ne sait pas si la cause de l'insuffisance cardiaque a un effet clinique significatif sur les issues après l'implantation d'un DAVG. Dans cette revue systématique, nous avons voulu déterminer si l'étiologie ischémique ou non ischémique a une incidence sur les issues pour les patients. Méthodes: Nous avons interrogé MEDLINE, Embase, PubMed et la Bibliothèque Cochrane pour trouver les études publiées en anglais entre le 1er janvier 2000 et le 22 novembre 2018 qui examinaient la survie et le taux de greffe après l'implantation d'un DAVG chez les patients atteints d'une insuffisance cardiaque ischémique ou non ischémique. Les essais cliniques randomisés, les études de cohorte, les études cas­témoins, les études transversales et les séries de cas ayant un échantillon d'au moins 8 patients étaient admissibles pour inclusion. Pour qu'une publication soit incluse dans la méta-analyse, les issues à l'étude devaient comprendre au minimum les décès dans les 30 jours ou dans l'année suivant l'implantation du DAVG. La qualité des études retenues a été évaluée par 2 évaluateurs indépendants au moyen de l'échelle Newcastle­Ottawa pour l'évaluation de la qualité des études de cohorte. L'outil GRADE (Grading of Recommendations Assessment, Development and Evaluation) a servi à évaluer la qualité des données sur les issues (survie après 30 jours, survie après 1 an et greffe cardiaque après le traitement par DAVG) dans l'ensemble des études. Résultats: Sur les 2843 citations recensées, 7 études respectaient tous les critères d'inclusion. Elles étaient généralement de bonne qualité, mais l'indication des caractéristiques démographiques des patients, des issues et des complications était hétérogène. Nous n'avons trouvé aucune différence significative dans la survie après 30 jours ou après 1 an, ni dans le taux de greffe cardiaque après l'implantation du dispositif entre les groupes ischémique et non ischémique. Les patients des 2 groupes avaient des issues similaires jusqu'à 1 an après le traitement par DAVG. Conclusion: Les issues à court terme du traitement par DAVG ne semblent pas influencées par l'étiologie de l'insuffisance cardiaque. Il faudra faire d'autres études pour caractériser les issues à long terme en présence d'insuffisance cardiaque ischémique et non ischémique. Enregistrement de la revue systématique: Registre PROSPERO, numéro 76483.

Correlation between Medication Administration-Related Errors in Patients with Parkinson Disease and Timing of Pharmacy-Led Best Possible Medication Histories.

BACKGROUND: Poor prescribing and incomplete medication administration have been linked to increased lengths of hospitalization for patients with Parkinson disease. The Institute for Safe Medication Practices (ISMP) has recommended that patients with Parkinson disease receive a pharmacy consultation within 2 h of admission to hospital. OBJECTIVES: To examine whether the time for a pharmacy team member to obtain a best possible medication history (BPMH) was associated with administration-related medication errors. The primary outcome was the proportion of doses with a medication error during a patient's admission in relation to the time to completion of the initial BPMH by a registered pharmacist (RPh) or registered pharmacy technician (RPhT). The secondary objective was to compare the proportion of doses with a medication error in relation to whether the BPMH was completed by an RPh or an RPhT. METHODS: This retrospective chart review involved patients with Parkinson disease who were admitted to the medicine services at London Health Sciences Centre from September 30, 2014, to September 30, 2018. Patients were included if they had Parkinson disease and a medication regimen that included levodopa-carbidopa. For all patients, an RPhT or RPh conducted the initial BPMH or updated the BPMH. Pearson correlation analysis was used to determine whether a correlation existed between administration-related errors and completion of the BPMH by a pharmacy staff member. RESULTS: A total of 84 patients with 104 admissions were included. There was no significant correlation between the time to completion of the initial BPMH by a pharmacy team member and the proportion of doses with medication errors (p = 0.32). Although RPhTs completed the BPMHs more quickly than RPhs (p < 0.001), there was no significant difference between pharmacy team members in terms of the proportion of doses with medication errors (p = 0.86). CONCLUSIONS: Completing a BPMH within 2 h of a patient's admission, as per the ISMP recommendation, is unlikely to affect administration-related medication errors, given that no correlation was identified. Expediting BPMH without addressing other factors is insufficient, and initiatives are required to improve the medication administration process.

CONTEXTE: La mauvaise prescription et l'administration incomplète de médicaments ont été liées à une augmentation de la durée du séjour à l'hôpital des patients atteints de la maladie de Parkinson. L'Institute for Safe Medication Practices (ISMP) a recommandé que les patients atteints de la maladie de Parkinson obtiennent une consultation en pharmacie dans les 2 heures après leur admission à l'hôpital. OBJECTIFS: Vérifier si le temps d'attente pour l'obtention, par un membre de l'équipe de la pharmacie, du meilleur schéma thérapeutique possible (MSTP) était associé ou non à des erreurs de médication liées à l'administration. Le résultat principal portait sur la proportion des doses comportant une erreur de médication lors de l'admission d'un patient par rapport au temps nécessaire à un pharmacien ou à un technicien en pharmacie autorisés pour réaliser le MSTP initial. L'objectif secondaire visait à comparer la proportion des doses comportant une erreur de médication entre un MSTP réalisé par un pharmacien autorisé et un MSTP réalisé par un technicien en pharmacie autorisé. MÉTHODES: Cet examen rétrospectif des dossiers impliquait des patients atteints de la maladie de Parkinson ayant été admis aux services de médecine au London Health Sciences Centre entre le 30 septembre 2014 et le 30 septembre 2018. Les patients pouvaient participer à l'étude s'ils avaient la maladie de Parkinson et qu'ils suivaient un traitement médicamenteux comprenant du lévodopa-carbidopa. Un pharmacien autorisé ou un technicien en pharmacie autorisé réalisait ou actualisait le MSTP initial de tous les patients. La corrélation de Pearson a servi à déterminer s'il existait une corrélation entre les erreurs liées à l'administration et la réalisation du MSTP par un membre du personnel de la pharmacie. RÉSULTATS: Au total, 84 patients correspondant à 104 admissions ont été inclus dans l'étude. Il n'y avait aucune corrélation importante entre le moment de la réalisation du MSTP initial par un membre du personnel de la pharmacie et la proportion des doses comportant des erreurs de médication (p = 0,32). Bien que les techniciens en pharmacie autorisés aient terminé plus rapidement leur MSTP que les pharmaciens autorisés (p < 0,001), aucune différence importante n'a été notée entre les membres du personnel de la pharmacie en termes de proportion des doses et d'erreur de médication (p = 0,86). CONCLUSIONS: Il est peu probable que la réalisation d'un MSTP dans les 2 heures après l'admission d'un patient, conformément à la recommandation de l'ISMP, ait une influence sur les erreurs de médication liées à l'administration, vu qu'aucune corrélation n'a été décelée. Précipiter la réalisation du MSTP sans aborder les autres facteurs ne suffit pas et des actions sont nécessaires pour améliorer le processus d'administration des médicaments.

Rab GTPases in the differential processing of phagocytosed pathogens versus efferocytosed apoptotic cells.

Phagocytosis is an important feature of innate immunity in which invading microorganisms are engulfed, killed and degraded - and in some immune cells, their antigens presented to adaptive immune system. A closely related process, efferocytosis, removes apoptotic cells, and is essential for the maintenance of homeostasis. Both phagocytosis and efferocytosis are tightly regulated processes that involve target recognition and uptake through specific receptors, followed by endolysosomal trafficking and processing of the internalized target. Central to the uptake and trafficking of these targets are the Rab family of small GTPases, which coordinate the engulfment and trafficking of both phagocytosed and efferocytosed materials through the endolysosomal system. Because of this regulatory function, Rab GTPases are often targeted by pathogens to escape phagocytosis. In this review, we will discuss the shared and differential roles of Rab GTPases in phagocytosis and efferocytosis.

International training considerations of Canadian Clinician-Scientist Trainees - A national survey.

PURPOSE: Canadian clinician-scientist trainees enrolled in dual degree programs often pursue an extended training route following completion of MD and MSc or PhD degrees. However, the proportion, plans and reasoning of trainees who intend to pursue training internationally following dual degree completion has not been investigated. In this study, we assessed the international training considerations of current clinician-scientist trainees. METHODS: We designed an 11-question survey, which was sent out by program directors to all current MDPhD program and Clinician Investigator Program (CIP) trainees. Responses were collected from July 8, 2019 to August 8, 2019. RESULTS: We received a total of 191 responses, with representation from every Canadian medical school and both MD-PhD program and CIP trainees. The majority of trainees are considering completing additional training outside Canada, most commonly post-doctoral and/or clinical fellowships. The most common reasons for considering international training include those related to quality and prestige of training programs. In contrast, the most common reasons for considering staying in Canada for additional training are related to personal and ethical reasons. Irrespective of intentions to pursue international training, the majority of trainees ultimately intend to establish a career in Canada. CONCLUSION: While most trainees are considering additional training outside of Canada due to prestige and quality of training, the majority of trainees intend to pursue a career as a clinician-scientist back in Canada. Trainees would likely benefit from improved guidance and mentorship on the value of international training, as well as enhanced support in facilitating cross-border mobility.

Efferocytic Defects in Early Atherosclerosis Are Driven by GATA2 Overexpression in Macrophages.

The loss of efferocytosis-the phagocytic clearance of apoptotic cells-is an initiating event in atherosclerotic plaque formation. While the loss of macrophage efferocytosis is a prerequisite for advanced plaque formation, the transcriptional and cellular events in the pre-lesion site that drive these defects are poorly defined. Transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions identified a 50-fold increase in the expression of GATA2, a transcription factor whose expression is normally restricted to the hematopoietic compartment. GATA2 overexpression in vitro recapitulated many of the functional defects reported in patient macrophages, including deficits at multiple stages in the efferocytic process. These findings included defects in the uptake of apoptotic cells, efferosome maturation, and in phagolysosome function. These efferocytic defects were a product of GATA2-driven alterations in the expression of key regulatory proteins, including Src-family kinases, Rab7 and components of both the vacuolar ATPase and NADPH oxidase complexes. In summary, these data identify a mechanism by which efferocytic capacity is lost in the early stages of plaque formation, thus setting the stage for the accumulation of uncleared apoptotic cells that comprise the bulk of atherosclerotic plaques.

Advancing the understanding of research during medical education through collaborative learning: the Collaboration of Practitioners and Researchers Seminar Series.

BACKGROUND: The Collaboration of Practitioners and Researchers Seminar Series is student-led program comprised of seminars delivered jointly by medical and graduate students on a topic in medicine of mutual interest to an audience of both medical and graduate students. METHODS: Following its inaugural year in 2016-2017, we evaluated changes in attendees' perceived understanding of translational research through an electronic survey and semi-structured interviews with attendees. RESULTS: Study participants rated their understanding of translational research and comfort with interacting with students from the other program higher following attending seminars. Participants believed that the seminars helped in breaking barriers between medical and graduate students. CONCLUSIONS: We conclude that this seminar series positively impacted attendees' understanding of translational research and attitudes towards collaboration between medical and graduate students. We believe that similar initiatives may be of value in fostering new opportunities for collaboration between medical and graduate students at other institutions.

Rab17 mediates intermixing of phagocytosed apoptotic cells with recycling endosomes.

Efferocytosis-the phagocytic removal of apoptotic cells-is required for preventing the presentation of apoptotic cell-derived antigens. This process is regulated by Rab17-dependent sorting of efferocytosed cargos from the phagolysosome to recycling endosomes. In this study we demonstrate that Rab17 is rapidly recruited to efferosomes, followed by migration of the efferosome to the cell center where it intermixes with lysosomes and undergoes Rab17-dependent vesiculation. These efferosome-derived vesicles then traffic in a Rab17-dependent manner to the cell periphery, where they transfer cargo to recycling endosomes. Combined, our observations support a model wherein efferosomes migrate to the cell center to acquire degradative enzymes, followed by peripheral migration to prevent further phagolysosome maturation and to enable cargo transfer to recycling endosomes.

Fourth Annual Clinician Scientist Trainee Symposium at the Schulich School of Medicine and Dentistry.

The Annual Clinician Scientist Trainee Symposium (CSTS) gathers the local medical research community at the Schulich School of Medicine and Dentistry, including research trainees at the medical student, resident and fellow levels. Trainees showcase their current and upcoming work, and leaders in the community impart their perspectives on the importance and future of research in medicine. At the 4th Annual CSTS, perseverance and cautious optimism emerged as the characteristics that trainees should foster through their future careers as clinician scientists. This was echoed by the challenges and encouraging findings presented by trainees, who conducted their work as part of research projects within the MD, MD/PhD and clinical investigator programs. The four oral presentations and 10 three-minute thesis presentations covered the full breadth of medical disciplines across the spectrum of translational medicine, from fundamental sciences through knowledge translation. The CSTS concluded with the keynote presentation by Michael Strong, current president of the Canadian Institutes of Health Research, who gave a glimpse into his decades-long path of overcoming and overturning dogmatic views in the world of amyotrophic lateral sclerosis research, demonstrating how roadblocks in research can become an impulse for stronger science.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

The 2nd Annual Clinical Scientist Trainee Symposium, August 22, 2017, London, Canada.

Clinician scientists play a critical role in bridging research and clinical practice. Unfortunately, the neglect of research training in medical schools has created clinicians who are unable to translate evidence from literature to practice. Furthermore, the erosion of research training in medical education has resulted in clinicians who lack the skills required for successful scientific investigation. To counteract this, the Schulich School of Medicine & Dentistry has made an effort to engage trainees, at all levels, in the research process. The 2nd Annual Clinician Scientist Trainee Symposium was held in London, Ontario, Canada on August 22, 2017. Organized each year since 2016 by the Schulich Research Office, the symposium features research being conducted by trainees in Schulich's Clinical Research Training Program. The focus this year was on the current state of clinician-scientist training in Canada and visions for the path ahead.

Human-Specific Mutations and Positively Selected Sites in MARCO Confer Functional Changes.

Macrophage Receptor with COllagenous structure (MARCO) is a class A scavenger receptor that binds, phagocytoses, and modifies inflammatory responses to bacterial pathogens. Multiple candidate gene approach studies have shown that polymorphisms in MARCO are associated with susceptibility or resistance to Mycobacterium tuberculosis infection, but how these variants alter function is not known. To complement candidate gene approach studies, we previously used phylogenetic analyses to identify a residue, glutamine 452 (Q452), within the ligand-binding Scavenger Receptor Cysteine Rich domain as undergoing positive selection in humans. Herein, we show that Q452 is found in Denisovans, Neanderthals, and extant humans, but all other nonprimate, terrestrial, and aquatic mammals possess an aspartic acid (D452) residue. Further analysis of hominoid sequences of MARCO identified an additional human-specific mutation, phenylalanine 282 (F282), within the collagenous domain. We show that residue 282 is polymorphic in humans, but only 17% of individuals (rs6761637) possess the ancestral serine residue at position 282. We show that rs6761637 is in linkage disequilibrium with MARCO polymorphisms that have been previously linked to susceptibility to pulmonary tuberculosis. To assess the functional importance of sites Q452 and F282 in humans, we cloned the ancestral residues and loss-of-function mutations and investigated the role of these residues in binding and internalizing polystyrene microspheres and Escherichia coli. Herein, we show that the residues at sites 452 and 282 enhance receptor function.

Armed for destruction: formation, function and trafficking of neutrophil granules.

Neutrophils respond nearly instantly to infection, rapidly deploying a potent enzymatic and chemical arsenal immediately upon entering an infected site. This capacity for rapid and potent responses is endowed by stores of antimicrobial proteins contained in readily mobilizable granules. These granules contain the proteins necessary to mediate the recruitment, chemotaxis, antimicrobial function and NET formation of neutrophils. Four granule types exist, and are sequentially deployed as neutrophils enter infected sites. Secretory vesicles are released first, enabling recruitment of neutrophils out of the blood. Next, specific and gelatinase granules are released to enable neutrophil migration and begin the formation of an antimicrobial environment. Finally, azurophilic granules release potent antimicrobial proteins at the site of infection and into phagosomes. The step-wise mobilization of these granules is regulated by calcium signaling, while specific trafficking regulators and membrane fusion complexes ensure the delivery of granules to the correct subcellular site. In this review, we describe neutrophil granules from their formation through to their deployment at the site of infection, focusing on recent developments in our understanding of the signaling pathways and vesicular trafficking mechanisms which mediate neutrophil degranulation.

Training the next generation of Canadian Clinician-Scientists: charting a path to success.

Clinician-scientists are physicians with training in both clinical medicine and research that enables them to occupy a unique niche as specialists in basic and translational biomedical research. While there is widespread acknowledgement of the importance of clinician-scientists in today's landscape of evidence-based medical practice, training of clinician-scientists in Canada has been on the decline, with fewer opportunities to obtain funding. With the increasing length of training and lower financial compensation, fewer medical graduates are choosing to pursue such a career. MD-PhD programs, in which trainees receive both medical and research training, have the potential to be an important tool in training the next generation of clinician-scientists; however, MD-PhD trainees in Canada face barriers that include an increase in medical school tuition and a decrease in the amount of financial support. We examined the available data on MD-PhD training in Canada and identified a lack of oversight, a lack of funding and poor mentorship as barriers experienced by MD-PhD trainees. Specific recommendations are provided to begin the process of addressing these challenges, starting with the establishment of an overseeing national body that would track long-term outcome data for MD-PhD trainees. This national body could then function to implement best practices from individual programs across the country and to provide further mentorship and support for early-career physician-scientists. MD-PhD programs have the potential to address Canada's growing shortage of clinician-scientists, and strengthening MD-PhD programs will help to effect positive change.

Quantitative Efferocytosis Assays.

Efferocytosis, the phagocytic removal of apoptotic cells, is a dynamic process requiring recruitment of numerous regulatory proteins to forming efferosomes in a tightly regulated manner. Herein we describe microscopy-based methods for the enumeration of efferocytic events and characterization of the spatiotemporal dynamics of signaling molecule recruitment to efferosomes, using genetically encoded probes and immunofluorescent labeling. While these methods are illustrated using macrophages, they are applicable to any efferocytic cell type.

Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis.

Macrophages engulf and destroy pathogens (phagocytosis) and apoptotic cells (efferocytosis), and can subsequently initiate adaptive immune responses by presenting antigens derived from engulfed materials. Both phagocytosis and efferocytosis share a common degradative pathway in which the target is engulfed into a membrane-bound vesicle, respectively, termed the phagosome and efferosome, where they are degraded by sequential fusion with endosomes and lysosomes. Despite this shared maturation pathway, macrophages are immunogenic following phagocytosis but not efferocytosis, indicating that differential processing or trafficking of antigens must occur. Mass spectrometry and immunofluorescence microscopy of efferosomes and phagosomes in macrophages demonstrated that efferosomes lacked the proteins required for antigen presentation and instead recruited the recycling regulator Rab17. As a result, degraded materials from efferosomes bypassed the MHC class II loading compartment via the recycling endosome - a process not observed in phagosomes. Combined, these results indicate that macrophages prevent presentation of apoptotic cell-derived antigens by preferentially trafficking efferocytosed, but not phagocytosed, materials away from the MHC class II loading compartment via the recycling endosome pathway.

A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function.

Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.