Non-professional Phagocytes Research Articles

Stem cells tightly regulate dead cell clearance to maintain tissue fitness

Billions of cells are eliminated daily from our bodies1–4. Although macrophages and dendritic cells are dedicated to migrating and engulfing dying cells and debris, many epithelial and mesenchymal tissue cells can digest nearby apoptotic corpses1–4. How these non-motile, non-professional phagocytes sense and eliminate dying cells while maintaining their normal tissue functions is unclear. Here we explore the mechanisms that underlie their multifunctionality by exploiting the cyclical bouts of tissue regeneration and degeneration during hair cycling. We show that hair follicle stem cells transiently unleash phagocytosis at the correct time and place through local molecular triggers that depend on both lipids released by neighbouring apoptotic corpses and retinoids released by healthy counterparts. We trace the heart of this dual ligand requirement to RARγ–RXRα, whose activation enables tight regulation of apoptotic cell clearance genes and provides an effective, tunable mechanism to offset phagocytic duties against the primary stem cell function of preserving tissue integrity during homeostasis. Finally, we provide functional evidence that hair follicle stem cell-mediated phagocytosis is not simply redundant with professional phagocytes but rather has clear benefits to tissue fitness. Our findings have broad implications for other non-motile tissue stem or progenitor cells that encounter cell death in an immune-privileged niche.

Gene expression differences associated with intrinsic hindfoot muscle loss in the jerboa, Jaculus jaculus.

Vertebrate animals that run or jump across sparsely vegetated habitats, such as horses and jerboas, have reduced the number of distal limb bones, and many have lost most or all distal limb muscle. We previously showed that nascent muscles are present in the jerboa hindfoot at birth and that these myofibers are rapidly and completely lost soon after by a process that shares features with pathological skeletal muscle atrophy. Here, we apply an intra- and interspecies differential RNA-Seq approach, comparing jerboa and mouse muscles, to identify gene expression differences associated with the initiation and progression of jerboa hindfoot muscle loss. We show evidence for reduced hepatocyte growth factor and fibroblast growth factor signaling and an imbalance in nitric oxide signaling; all are pathways that are necessary for skeletal muscle development and regeneration. We also find evidence for phagosome formation, which hints at how myofibers may be removed by autophagy or by nonprofessional phagocytes without evidence for cell death or immune cell activation. Last, we show significant overlap between genes associated with jerboa hindfoot muscle loss and genes that are differentially expressed in a variety of human muscle pathologies and rodent models of muscle loss disorders. All together, these data provide molecular insight into the process of evolutionary and developmental muscle loss in jerboa hindfeet.

Engineering Efferocytosis-Mimicking Nanovesicles to Regulate Joint Anti-Inflammation and Peripheral Immunosuppression for Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation of the synovial joints and the dysfunction of regulatory T cells (Tregs) in the peripheral blood. Therefore, an optimal treatment strategy should aim to eliminate the inflammatory response in the joints and simultaneously restore the immune tolerance of Tregs in peripheral blood. Accordingly, we developed an efferocytosis-mimicking nanovesicle that contains three functional factors for immunomodulating of efferocytosis, including "find me" and "eat me" signals for professional (macrophage) or non-professional phagocytes (T lymphocyte), and "apoptotic metabolite" for metabolite digestion. We showed that efferocytosis-mimicking nanovesicles targeted the inflamed joints and spleen of mice with collagen-induced arthritis, further recruiting and selectively binding to macrophages and T lymphocytes to induce M2 macrophage polarization and Treg differentiation and T helper cell 17 (Th17) recession. Under systemic administration, the efferocytosis-mimicking nanovesicles effectively maintained the pro-inflammatory M1/anti-inflammatory M2 macrophage balance in joints and the Treg/Th17 imbalance in peripheral blood to prevent RA progression. This study demonstrates the potential of efferocytosis-mimicking nanovesicles for RA immunotherapy.

Efferocytosis by macrophages in physiological and pathological conditions: regulatory pathways and molecular mechanisms.

Efferocytosis is defined as the highly effective phagocytic removal of apoptotic cells (ACs) by professional or non-professional phagocytes. Tissue-resident professional phagocytes ("efferocytes"), such as macrophages, have high phagocytic capacity and are crucial to resolve inflammation and aid in homeostasis. Recently, numerous exciting discoveries have revealed divergent (and even diametrically opposite) findings regarding metabolic immune reprogramming associated with efferocytosis by macrophages. In this review, we highlight the key metabolites involved in the three phases of efferocytosis and immune reprogramming of macrophages under physiological and pathological conditions. The next decade is expected to yield further breakthroughs in the regulatory pathways and molecular mechanisms connecting immunological outcomes to metabolic cues as well as avenues for "personalized" therapeutic intervention.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Stabilin-2 mediated apoptotic cell phagocytosis induces interleukin-10 expression by p38 and Pbx1 signaling

Apoptotic cell death occurs under normal physiological conditions, such as development, tissue remodeling, and inflammation. Appropriate removal of apoptotic cells by phagocytes and the secretion of anti-inflammatory cytokines such as IL-10 are important mechanisms for maintaining tissue homeostasis. Apoptotic cell phagocytosis is mediated by several phosphatidylserine recognition receptors on non-professional or professional phagocytes, such as neighboring epithelial cells or macrophages. Stabilin-2 is reported as a phosphatidylserine recognition receptor for apoptotic cell phagocytosis, and its downstream signaling pathway for cytoskeletal rearrangement for phagocytosis is well known. However, the mechanisms for stabilin-2-mediated IL-10 production has not yet been reported. In this study, we aimed to investigate stabilin-2 receptor-mediated IL-10 transcription regulation signaling pathway.

Brucella infection and Toll-like receptors.

Brucella consists of gram-negative bacteria that have the ability to invade and replicate in professional and non-professional phagocytes, and its prolonged persistence in the host leads to brucellosis, a serious zoonosis. Toll-like receptors (TLRs) are the best-known sensors of microorganisms implicated in the regulation of innate and adaptive immunity. In particular, TLRs are transmembrane proteins with a typical structure of an extracellular leucine-rich repeat (LRR) region and an intracellular Toll/interleukin-1 receptor (TIR) domain. In this review, we discuss Brucella infection and the aspects of host immune responses induced by pathogens. Furthermore, we summarize the roles of TLRs in Brucella infection, with substantial emphasis on the molecular insights into its mechanisms of action.

The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

Professional phagocytes are recruited for the clearance of obsolete nonprofessional phagocytes in the Drosophila ovary.

Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered "immune-privileged" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. Here we analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.

Efferocytosis as One of the Mechanisms for Realizing the Therapeutic Effects of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) stimulate regeneration and exhibit unique immunomodulatory properties, which makes them attractive for use in cell therapies of a wide range of pathologies. The clinical use of MSCs is hampered by the insufficiently clear understanding of their therapeutic action mechanisms. It has been reliably proven that MSCs after transplantation quickly die in the recipient&s body by the mechanism of apoptosis and are cleared by professional, such as macrophages, and non-professional phagocytes, including endothelial cells, hepatocytes, resident stem cells of various tissues, including MSCs. The ingestion and processing of apoptotic cells by the phagocytes was named efferocytosis. Despite rapid elimination of transplanted cells, in most cases MSC transplantation leads to positive therapeutic effects. Clearance of apoptotic MSCs affects phagocytes, changing their phenotype, secretome, and further behavior. This review presents the basic molecular mechanisms of efferocytosis, examines the clearance of apoptotic MSCs and their therapeutic effects in various pathologies in the context of their efferocytosis by various types of phagocytes.

Glial plasticity at nervous system transition zones.

The central and peripheral nervous systems (CNS and PNS, respectively) are two separate yet connected domains characterized by molecularly distinct cellular components that communicate via specialized structures called transition zones to allow information to travel from the CNS to the periphery, and vice versa. Until recently, nervous system transition zones were thought to be selectively permeable only to axons, and the establishment of the territories occupied by glial cells at these complex regions remained poorly described and not well understood. Recent work now demonstrates that transition zones are occupied by dynamic glial cells and are precisely regulated over the course of nervous system development. This review highlights recent work on glial cell migration in and out of the spinal cord, at motor exit point (MEP) and dorsal root entry zone (DREZ) transition zones, in the physiological and diseased nervous systems. These cells include myelinating glia (oligodendrocyte lineage cells, Schwann cells and motor exit point glia), exit glia, perineurial cells that form the perineurium along spinal nerves, as well as professional and non-professional phagocytes (microglia and neural crest cells).

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Regulators of phagocytosis as pharmacologic targets for stroke treatment.

Stroke, including ischemic and hemorrhagic stroke, causes massive cell death in the brain, which is followed by secondary inflammatory injury initiated by disease-associated molecular patterns released from dead cells. Phagocytosis, a cellular process of engulfment and digestion of dead cells, promotes the resolution of inflammation and repair following stroke. However, professional or non-professional phagocytes also phagocytose stressed but viable cells in the brain or excessively phagocytose myelin sheaths or prune synapses, consequently exacerbating brain injury and impairing repair following stroke. Phagocytosis includes the smell, eating and digestion phases. Notably, efficient phagocytosis critically depends on phagocyte capacity to take up dead cells continually due to the limited number of phagocytes vs. dead cells after injury. Moreover, phenotypic polarization of phagocytes occurring after phagocytosis is also essential to the proresolving and prorepair properties of phagocytosis. Much has been learned about the molecular signals and regulatory mechanisms governing the sense and recognition of dead cells by phagocytes during the smell and eating phase following stroke. However, some key areas remain extremely understudied, including the mechanisms involved in digestion regulation, continual phagocytosis and phagocytosis-induced phenotypic switching following stroke. Here, we summarize new discoveries related to the molecular mechanisms and multifaceted effects of phagocytosis on brain injury and repair following stroke and highlight the knowledge gaps in poststroke phagocytosis. We suggest that advancing the understanding of poststroke phagocytosis will help identify more biological targets for stroke treatment.

Elucidating the role of the host protein, FBXO22, in the infection of macrophages with the zoonotic bacterial pathogen, Brucella.

Abstract Brucella spp. are facultative intracellular bacterial pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes. Successful invasion and chronic intracellular persistence indicate that Brucella manipulates various host cellular processes to create a replication permissive niche. The essential mechanisms and host factors that support the invasion and intracellular replication of Brucella remain poorly understood. RNA interference (RNAi) is a potent molecular tool for understanding gene function by downregulating the target gene expression through mRNA degradation. We used siRNA-based screening to identify the host factors that are involved in Brucella-macrophage interaction. Macrophages were treated with gene specific siRNAs, followed by infection with B. neotomae/B. melitensis and quantification of intracellular Brucella. The screening identified several host factors that are involved in the Brucella infection of macrophages. Subsequently, we performed detailed characterization of one of the identified host proteins, FBXO22. Downregulation of FBXO22 resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted in induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, for degradation through the SCF complex. Funding from Department of Biotechnology (DBT) (BT/PR12301/ADV/90/176/2014)

Natural Isoforms of Listeria monocytogenes Virulence Factor Inlb Differ in c-Met Binding Efficiency and Differently Affect Uptake and Survival Listeria in Macrophage.

Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence.

Abstract 74: Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis

Abstract Increased cellular apoptosis is commonly seen in myelodysplastic syndrome (MDS), but how clearance of apoptotic cells, especially neutrophils, impacts disease progression is unknown. Moreover, while dysfunction of bone marrow (BM) stromal cell populations has been described in patients with MDS, the mechanisms inducing stromal defects are not well understood. Our laboratory found that BM macrophages are defective in a murine model of MDS (Vav-Nup98 HoxD13, aka NHD13), with decreased efferocytotic rates and increased inflammatory mediators. The increased apoptotic burden and defective macrophages would be expected to recruit non-professional phagocytes (osteoblasts) in the bone and bone marrow. Based on these data, we hypothesize that OBs (osteoblasts) participate in the clearance of apoptotic cells in the bone marrow microenvironment (BMME) to maintain BM homeostasis. However, the increased osteoblastic efferocytosis burden in MDS contributes to OBs’ dysfunction as a mechanism of MDS-dependent BMME disruption that may lead to MDS progression. We isolated from wild-type (B6) mice bone-associated cells (BAC), which are enriched with OBs. In vitro co-culture with labeled human end-stage neutrophils at 1:1 and 1:10 (OB: PMN) increased OB apoptosis, while senescence was increased only in high-dose PMN co-culture. We found that these changes were cell-autonomous. In vivo injection of neutrophils into WT mice showed around 5-10% efferocytosis rate in OBs. In efferocytotic OBs, apoptosis rates were significantly increased. Together, these data show that OBs participate in efferocytosis and that efferocytosis disrupts OBs, causing apoptosis and senescence. To mimic the MDS microenvironment, we used a genetic model where one of the Isocitrate dehydrogenase 2 (IDH2) mutations found in MDS (R140Q) is targeted to hematopoietic cells via the Vav promoter (IDH2R140Q mice) as well as NHD13 mice, representing early and late MDS BMME respectively. We observed that 6-to-8 months old NHD13 mice experienced more end-stage neutrophils[CL1] in the BMME (4.6% rate of end-stage neutrophils in WT compared to 9.5% rate in NHD13 mice), where OB apoptosis was also increased in vivo. In IDH2R140Q mice at 3 months of age, we confirmed the presence of mutated IDH2 in bone marrow cells by increased intracellular levels of the oncometabolite 2-hydroxyglutarate. At this early time point, IDH2R140Q mice did not have increased rates of apoptosis in CD45+ cells, and we found no increase in OB apoptosis. Analysis of IDH2R140Q mice at later time points is ongoing. Our data identify novel mechanisms to explain the relationship between defective myeloid cells and OB apoptosis in MDS that may contribute to the inflammatory MDS microenvironment, impact disease progression, and serve as future therapeutic approaches for MDS patients. Citation Format: Chunmo Chen, Emily R. Quarato, Yuko Kawano, Noah A. Salama, Hiroki Kawano, Michael W. Becker, Jeevisha Bajaj, Laura Calvi. Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 74.

SCAV-3 affects apoptotic cell degradation in Caenorhabditis elegans.

As the final step in apoptosis, apoptotic cells (ACs) are swiftly removed by specialized phagocytes, such as macrophages, or nonprofessional phagocytes, such as epidermal cells. Genetic studies of model organisms such as Caenorhabditis elegans have helped to elucidate the mechanisms of AC clearance and the underlying causes of disorders related to the dysregulation of these pathways. C. elegans possesses six class B scavenger receptor homologs, but whether they affect apoptosis is unknown. Here, we show that only the loss of function of scav-3, the C. elegans homolog of human lysosomal integral membrane protein-2, resulted in a considerable accumulation of cell corpses, which was caused by a failure in degradation rather than engulfment. SCAV-3 was found to be widely distributed and localized in lysosomes to maintain the integrity of the lysosomal membrane. Further study revealed that loss of scav-3 had no effect on phagosome maturation or the recruitment of lysosomes to phagosomes carrying cell corpses. Moreover, we discovered that the hydrolytic enzymes contained in the lysosomes were reduced in phagosomes in scav-3 mutants. Thus, hydrolases may leak from the damaged lysosome during phagolysosome formation due to the loss of scav-3 function, which reduces lysosome digestion activity and thus directly contributes to the elimination of ACs.

Protein Disulfide Isomerase and Extracellular Adherence Protein Cooperatively Potentiate Staphylococcal Invasion into Endothelial Cells.

Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5β1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated β1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5β1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.

Unrevealing the Mystery of Latent Leishmaniasis: What Cells Can Host Leishmania?

Leishmania spp. (Kinetoplastida) are unicellular parasites causing leishmaniases, neglected tropical diseases of medical and veterinary importance. In the vertebrate host, Leishmania parasites multiply intracellularly in professional phagocytes, such as monocytes and macrophages. However, their close relative with intracellular development-Trypanosoma cruzi-can unlock even non-professional phagocytes. Since Leishmania and T. cruzi have similar organelle equipment, is it possible that Leishmania can invade and even proliferate in cells other than the professional phagocytes? Additionally, could these cells play a role in the long-term persistence of Leishmania in the host, even in cured individuals? In this review, we provide (i) an overview of non-canonical Leishmania host cells and (ii) an insight into the strategies that Leishmania may use to enter them. Many studies point to fibroblasts as already established host cells that are important in latent leishmaniasis and disease epidemiology, as they support Leishmania transformation into amastigotes and even their multiplication. To invade them, Leishmania causes damage to their plasma membrane and exploits the subsequent repair mechanism via lysosome-triggered endocytosis. Unrevealing the interactions between Leishmania and its non-canonical host cells may shed light on the persistence of these parasites in vertebrate hosts, a way to control latent leishmaniasis.