Paul Verkuijlen

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors

Background Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. Methodology/Principal Findings We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. Conclusions/Significance SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.

Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components

Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content.

PKCδ is dispensible for oxLDL uptake and foam cell formation by human and murine macrophages.

AIMS Uptake of oxidized lipoprotein particles (oxLDL) and foam cell formation by macrophages is one of the first steps in the development of atherosclerosis. Recently, protein kinase C δ (PKCδ) has been implicated as a regulator of oxLDL uptake and foam cell formation via down-regulation of PKCβ and scavenger receptors CD36 and SR-A expression. Here, we describe studies in which we have re-evaluated the role of PKCδ in oxLDL uptake and foam cell formation. METHODS AND RESULTS PKCδ expression was silenced in the human monocytic cell lines and also in primary human monocytes to analyse oxLDL uptake and CD36 expression. Additionally, bone marrow-derived macrophages of PKCδ knockout mice and macrophages cultured from patients with rare null mutations in the PRKCD gene were tested for uptake of oxLDL and foam cell formation. Expression of scavenger receptor CD36 was determined and levels of PKCβ isoforms were quantified. Neither a reduction in PKCδ levels nor its complete absence resulted in a detectable effect on the uptake of oxLDL and the formation of foam cells. CONCLUSION PKCδ is dispensible for oxLDL uptake and foam cell formation by monocytes and macrophages.

Impaired microbial killing by neutrophils from patients with protein kinase C delta deficiency

Health, University of Genoa, Genoa, Italy; the Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Md; ‘‘Angelo Nocivelli’’ Institute for Molecular Medicine, University of Brescia, Brescia, Italy; the Pediatric Hematology Oncology Unit, Spedali Civili, Brescia, Italy; the Division of Allergy and Clinical Immunology, Rebagliati Martins National Hospital, Lima, Peru; the Division of Pediatric Hematology, Children’s Hospital Orange County, University of California at Irvine, Irvine, Calif; the Department of Immunology, ‘‘Aghia Sophia’’ Children’s Hospital, Athens, Greece; the Division of Pediatric Immunology, Hospital Luis CalvoMackenna, Santiago, Chile; the Clinic of Pediatric Hematology-Oncology, Department for Woman and Child Health, University Hospital, Padua, Italy; and the Department of Pediatrics and Adolescent Medicine, American University of Beirut, Beirut, Lebanon. E-mail: luigi. notarangelo@childrens.harvard.edu. *These authors contributed equally to this work. Supported by a grant from the National Heart, Lung, and Blood Institute/National Institutes of Health (grant 5P01HL059561-13 to L.D.N.); an educational grant (5T32AI007512) from the National Institute of Allergy and Infectious Diseases (to E.C. [Dr Raif S. Geha, principal investigator]); an educational grant from the National Heart, Lung and Blood Institute/National Institutes of Health (grant 5T32HL00757433 to J.C.); and a grant from the UNIL-CHUV (CGRB 29583 to F.C.). Disclosure of potential conflict of interest: F. Candotti has received a grant from University of Lausanne-Centre hospitalier universitaire vaudois and is employed by Centre hospitalier universitaire vaudois. J. Chu has received a grant from the National Institutes of Health (NIH). J. Chou is employed by Boston Children’s Hospital and has received grants from the NIH and the JeffreyModell Foundation. F. Porta has received payment for lectures from Pfizer. S.-Y. Pai has received a grant from Translational Investigator Service, is employed by Boston Children’s Hospital, and has a grant pending from the National Heart, Lung, and Blood Institute. L. D. Notarangelo has received grants from the NIH and the March of Dimes; is an Associate Editor for the Journal of Allergy and Clinical Immunology and the Journal of Clinical Immunology; has consultant arrangements with Novimmune and Sigma-Tau; is employed by Children’s Hospital Pediatric Associates; and has received royalties from UpToDate. The rest of the authors declare that they have no relevant conflicts of interest.

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody‐dependent cellular cytotoxicity towards cancer cells

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti‐cancer antibodies act through different mechanisms, including antibody‐dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47‐SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab‐coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa‐131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa‐131R. Furthermore, ADCC was consistently enhanced by targeting CD47‐SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47‐SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47‐SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.

Altered Intracellular Localization and Mobility of SBDS Protein upon Mutation in Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.