-
Journal of Cellular Biochemistry Jan 2020Our current understanding of phagocytosis is largely derived from studies of individual receptor-ligand interactions and their downstream signaling pathways. Because...
Our current understanding of phagocytosis is largely derived from studies of individual receptor-ligand interactions and their downstream signaling pathways. Because phagocytes are exposed to a variety of ligands on heterogeneous target particles in vivo, it is important to observe the engagement of multiple receptors simultaneously and the triggered involvement of downstream signaling pathways. Potential crosstalk between the two well-characterized opsonic receptors, FcγR and CR3, was briefly explored in the early 1970s, where macrophages were challenged with dual-opsonized targets. However, subsequent studies on receptor crosstalk were primarily restricted to using single opsonins on different targets, typically at saturating opsonin conditions. Beyond validating these initial explorations on receptor crosstalk, we identify the early signaling mechanisms that underlie the binding and phagocytosis during the simultaneous activation of both opsonic receptors, through the presence of a dual-opsonized target (immunoglobulin G [IgG] and C3bi), compared with single receptor activation. For this purpose, we used signaling protein inhibitor studies as well as live cell brightfield and fluorescent imaging to fully understand the role of tyrosine kinases, F-actin dynamics and internalization kinetics for FcγR and CR3. Importantly, opsonic receptors were studied together and in isolation, in the context of sparsely opsonized targets. We observed enhanced particle binding and a synergistic effect on particle internalization during the simultaneous activation of FcγR and CR3 engaged with sparsely opsonized targets. Inhibition of early signaling and cytoskeletal molecules revealed a differential involvement of Src kinase for FcγR- vs CR3- and dual receptor-mediated phagocytosis. Src activity recruits Syk kinase and we observed intermediate levels of Syk phosphorylation in dual-opsonized particles compared with those opsonized with IgG or C3bi alone. These results likely explain the intermediate levels of F-actin that is recruited to sites of dual-opsonized particle uptake and the notoriously delayed internalization of C3bi-opsonized targets by macrophages.
Topics: Actins; Animals; Biological Transport; Bone Marrow Cells; Complement C3b; Cytoskeleton; Female; Immunoglobulin G; Macrophages; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Opsonin Proteins; Phagocytosis; Phagosomes; Phosphorylation; Protein Binding; RAW 264.7 Cells; Signal Transduction
PubMed: 31172552
DOI: 10.1002/jcb.29043 -
Developmental and Comparative Immunology Apr 2022Zebrafish Fish-egg lectin (zFEL) has been identified and proved to be a maternal factor with antibacterial and opsonic ability in fishes. In this study, we found that...
Zebrafish Fish-egg lectin (zFEL) has been identified and proved to be a maternal factor with antibacterial and opsonic ability in fishes. In this study, we found that zFEL was capable of enhancing the phagocytosis of the bacteria by macrophages of mouse (RAW264.7 and mouse peritoneal macrophages), suggesting a cross-species function of zFEL in higher animals. Further studies showed that zFEL can active the antigen presentation ability by up-regulating the expression of CD80, CD86 and MHC II. Meanwhile, zFEL also promoted the polarization of macrophages to M1-type, which was confirmed by the increase of cytokines TNF-α and IL-6. The expression of p38 gene was up-regulated in macrophages preincubated with zFEL. Taken together, zFEL appears opsonic function in mammal macrophages and has potential application in immunomodulation.
Topics: Animals; B7-1 Antigen; Cytokines; Lectins; Macrophages; Mice; Phagocytosis; Tumor Necrosis Factor-alpha; Zebrafish
PubMed: 34910945
DOI: 10.1016/j.dci.2021.104332 -
Brain Sciences Jun 2017In central nervous system (CNS) demyelinating disorders, such as multiple sclerosis (MS), neuromyelitis optica (NMO) and related NMO-spectrum disorders (NMO-SD), a... (Review)
Review
In central nervous system (CNS) demyelinating disorders, such as multiple sclerosis (MS), neuromyelitis optica (NMO) and related NMO-spectrum disorders (NMO-SD), a pathogenic role for antibodies is primarily projected into enhancing ongoing CNS inflammation by directly binding to target antigens within the CNS. This scenario is supported at least in part, by antibodies in conjunction with complement activation in the majority of MS lesions and by deposition of anti-aquaporin-4 (AQP-4) antibodies in areas of astrocyte loss in patients with classical NMO. A currently emerging subgroup of AQP-4 negative NMO-SD patients expresses antibodies against myelin oligodendrocyte glycoprotein (MOG), again suggestive of their direct binding to CNS myelin. However, both known entities of anti-CNS antibodies, anti-AQP-4- as well as anti-MOG antibodies, are predominantly found in the serum, which raises the questions why and how a humoral response against CNS antigens is raised in the periphery, and in a related manner, what pathogenic role these antibodies may exert outside the CNS. In this regard, recent experimental and clinical evidence suggests that peripheral CNS-specific antibodies may indirectly activate peripheral CNS-autoreactive T cells by opsonization of otherwise unrecognized traces of CNS antigen in peripheral compartments, presumably drained from the CNS by its newly recognized lymphatic system. In this review, we will summarize all currently available data on both possible roles of antibodies in CNS demyelinating disorders, first, directly enhancing damage within the CNS, and second, promoting a peripheral immune response against the CNS. By elaborating on the latter scenario, we will develop the hypothesis that peripheral CNS-recognizing antibodies may have a powerful role in initiating acute flares of CNS demyelinating disease and that these humoral responses may represent a therapeutic target in its own right.
PubMed: 28640199
DOI: 10.3390/brainsci7070070 -
The Journal of Infectious Diseases Apr 2023We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a...
BACKGROUND
We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule.
METHODS
Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73's strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined.
RESULTS
The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined.
CONCLUSIONS
A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.
Topics: Acinetobacter baumannii; Antibodies, Monoclonal; Single-Chain Antibodies; Antibodies, Bispecific
PubMed: 36617220
DOI: 10.1093/infdis/jiac499 -
Bioconjugate Chemistry Oct 2021Translation of intravenously administered nanomaterials to the clinic is limited due to adverse infusion reactions. While these reactions are infrequent, with up to 10%...
Translation of intravenously administered nanomaterials to the clinic is limited due to adverse infusion reactions. While these reactions are infrequent, with up to 10% prone to experiencing infusion reactions, the reactions can be severe and life-threatening. One of the innate immune pathways, the complement activation pathway, plays a significant role in mediating this response. Nanoparticle surface properties are a relevant design feature, as they control the blood proteins the nanoparticles interact with and allow the nanoparticles to evade the immune reaction. PEGylation of nanosurfaces is critical in improving the blood circulation of nanoparticles and reducing opsonization. Our goal was to understand whether modifying the surface architecture by varying the PEG density and architecture can impact the complement response in vitro. We utilized block copolymers of poly(lactic acid)--poly(ethylene glycol) prepared with poly(ethylene glycol) macroinitiators of molecular weights 3400 and 5000 Da. Tracking the complement biomarker C5a, we monitored the impact of changing PEGylation of the nanoparticles. We also investigated how the changing PEG length on the nanoparticle surface impacts further strengthening the stealth properties. Lastly, we determined which cytokines change upon blood incubation with nanoparticles in vitro to understand the extent to which inflammation may occur and the crosstalk between the complement and immune responses. Increasing PEGylation reduced the generation of complement-mediated anaphylatoxin C5a in vitro, with 5000 Da PEG more effectively reducing levels of C5a generated compared to 3400 Da PEG. The insights gathered regarding the impact of PEG density and PEG chain length would be critical in developing stealth nanoparticles that do not lead to infusion reactions upon intravenous administration.
Topics: Lactates; Nanoparticles; Opsonization; Polyesters; Polyethylene Glycols
PubMed: 34499487
DOI: 10.1021/acs.bioconjchem.1c00339 -
IET Nanobiotechnology Jun 2019A brain tumour is amongst most devastating and challenging condition to overcome with suitable treatment as the drug has to cross the blood-brain barrier (BBB) with... (Review)
Review
A brain tumour is amongst most devastating and challenging condition to overcome with suitable treatment as the drug has to cross the blood-brain barrier (BBB) with several physiological barriers like opsonisation by the reticuloendothelial system. Presently various techniques such as surgical, chemotherapeutic agents, and radiotherapy techniques have performed to extend the lifespan of patients diagnosed with glioblastoma, which did not maximise the overall survival of patients with a tumour. Nanotechnology is relied upon to diminish the requirement for intrusive methods for conveyance of therapeutics to the central nervous system. Colloidal nanocarriers sizing range 1-1000 nm have been utilised to cross BBB delivers the drug at cell levels with enhanced bioavailability and reduced toxicity. However, solid lipid nanoparticles (SLNs) are considered a highly flexible carrier for more successful remedially in brain tumour. The treatment of a brain tumour via SLNs is gaining greater potency due to its inimitable size and lipidic nature. This review focuses and represents the current strategies of SLNs in the brain tumour treatment with appropriate techniques adopted are highlighted. Based on this review, the authors concluded that SLNs embrace exclusive promising lipidic nanocarrier that could be utilised to target a brain tumour effectively.
Topics: Animals; Antineoplastic Agents; Brain; Brain Neoplasms; Drug Delivery Systems; Humans; Lipids; Liposomes; Mice; Micelles; Nanomedicine; Nanoparticles
PubMed: 31171738
DOI: 10.1049/iet-nbt.2018.5322 -
Cells Dec 2022In phagocytes, cytoskeletal and membrane remodeling is finely regulated at the phagocytic cup. Various smaFll G proteins, including those of the Arf family, control...
In phagocytes, cytoskeletal and membrane remodeling is finely regulated at the phagocytic cup. Various smaFll G proteins, including those of the Arf family, control these dynamic processes. Human neutrophils express AGAP2, an Arf GTPase activating protein (ArfGAP) that regulates endosomal trafficking and focal adhesion remodeling. We first examined the impact of AGAP2 on phagocytosis in CHO cells stably expressing the FcγRIIA receptor (CHO-IIA). In unstimulated CHO-IIA cells, AGAP2 only partially co-localized with cytoskeletal elements and intracellular compartments. In CHO-IIA cells, AGAP2 transiently accumulated at actin-rich phagocytic cups and increased Fcγ receptor-mediated phagocytosis. Enhanced phagocytosis was not dependent on the N-terminal GTP-binding protein-like (GLD) domain of AGAP2. AGAP2 deleted of its GTPase-activating protein (GAP) domain was not recruited to phagocytic cups and did not enhance the engulfment of IgG-opsonized beads. However, the GAP-deficient [R618K]AGAP2 transiently localized at the phagocytic cups and enhanced phagocytosis. In PLB-985 cells differentiated towards a neutrophil-like phenotype, silencing of AGAP2 reduced phagocytosis of opsonized zymosan. In human neutrophils, opsonized zymosan or monosodium urate crystals induced AGAP2 phosphorylation. The data indicate that particulate agonists induce AGAP2 phosphorylation in neutrophils. This study highlights the role of AGAP2 and its GAP domain but not GAP activity in FcγR-dependent uptake of opsonized particles.
Topics: Animals; Cricetinae; Humans; Cricetulus; GTPase-Activating Proteins; Phagocytosis; Receptors, IgG; Signal Transduction; Zymosan; GTP-Binding Proteins
PubMed: 36611866
DOI: 10.3390/cells12010072 -
European Journal of Pharmaceutics and... Dec 2023The complement system is a multicomponent and multifunctional arm of the innate immune system. Complement contributes to non-specific host defence and maintains...
The complement system is a multicomponent and multifunctional arm of the innate immune system. Complement contributes to non-specific host defence and maintains homeostasis through multifaceted processes and pathways, including crosstalk with the adaptive immune system, the contact (coagulation) and the kinin systems, and alarmin high-mobility group box 1. Complement is also present intracellularly, orchestrating a wide range of housekeeping and physiological processes in both immune and nonimmune cells, thus showing its more sophisticated roles beyond innate immunity, but its roles are still controversial. Particulate drug carriers and nanopharmaceuticals typically present architectures and surface patterns that trigger complement system in different ways, resulting in both beneficial and adverse responses depending on the extent of complement activation and regulation as well as pathophysiological circumstances. Here we consider the role of complement system and complement regulations in host defence and evaluate the mechanisms by which nanoparticles trigger and modulate complement responses. Effective strategies for the prevention of nanoparticle-mediated complement activation are introduced and discussed.
Topics: Complement System Proteins; Immunity, Innate; Complement Activation; Drug Carriers; Nanoparticles
PubMed: 37949325
DOI: 10.1016/j.ejpb.2023.11.006 -
Molecular Immunology Nov 2015Mohamed Daha and I share a common interest in innate immunity. Working in institutes only 25 miles away from each other, that meant ample opportunity and relevance for... (Review)
Review
Mohamed Daha and I share a common interest in innate immunity. Working in institutes only 25 miles away from each other, that meant ample opportunity and relevance for collaboration. And so we did. Moreover, we have both been members of boards and councils of Dutch national organizations, and we have also become good friends. In this short recollection, I look back on 40 years of common activities in complement research and friendship.
Topics: Allergy and Immunology; Antibodies; Biomedical Research; Complement Activation; Complement System Proteins; Cooperative Behavior; History, 20th Century; History, 21st Century; Humans; Immunity, Innate; Inflammation; Macrophages; Neutrophils; Opsonin Proteins; Phagocytosis
PubMed: 26597203
DOI: 10.1016/j.molimm.2015.05.001 -
Critical Reviews in Therapeutic Drug... 2016The mononuclear phagocyte system (MPS) defends the body against the invasion of microorganisms by phagocytosis. In the presence of opsonins, the invading matter is... (Review)
Review
The mononuclear phagocyte system (MPS) defends the body against the invasion of microorganisms by phagocytosis. In the presence of opsonins, the invading matter is readily recognized by phagocytes because of the interaction between receptors on the phagocytic cell surfaces and the modified conformation of opsonins. The particulate carriers, which are otherwise capable of optimizing drug delivery, are subjected to opsonization and phagocytosis by the MPS immediately following intravenous administration. These drug carriers should remain in the bloodstream in order to spatially locate the drug to the target site and temporally control the drug's release from there on; however, they are devastated by opsonization by serum proteins. Therefore, to restrict opsonization, which is critical for recognition of particulate carriers by the MPS, stealth devices have been developed by engineering the carriers' surface characteristics. Physicochemical properties that influence protein immunogenicity include particle size, surface charge, and surface hydrophobicity. Steric stabilization using polyethylene glycol (PEG) and polyethylene oxide (PEO) chains attached to the particle surface is principally effective in preventing the adsorption of serum opsonins. This article reviews the literature on the MPS and its development and functions, as well as approaches for designing long-circulating carrier particles. It also comprehensively reviews parameters affecting the steric characteristics of drug carriers, such as particle size, shape, surface charge, and surface affinity, including PEGylation of carriers.
Topics: Drug Carriers; Humans; Hydrophobic and Hydrophilic Interactions; Opsonin Proteins; Particle Size; Phagocytes; Phagocytosis; Polyethylene Glycols; Static Electricity; Surface Properties
PubMed: 27992308
DOI: 10.1615/CritRevTherDrugCarrierSyst.2016012303