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Frontiers in Immunology 2023Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues....
INTRODUCTION
Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid.
METHODS
The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum.
RESULTS
Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9.
CONCLUSION
Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.
Topics: Mice; Animals; Humans; Disease Models, Animal; HEK293 Cells; Plaque, Amyloid; Amyloidosis; Amyloid; Amyloidogenic Proteins; Peptides; Immunoglobulin Light Chains
PubMed: 37854603
DOI: 10.3389/fimmu.2023.1275372 -
BioRxiv : the Preprint Server For... Sep 2023peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including...
peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis that PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163CD206 MΦ to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36 and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.
PubMed: 37066181
DOI: 10.1101/2023.03.30.535001 -
ACS Nano Feb 2022For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and...
For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes' synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or "proteoDNAsome", which maintains its synthetic identity and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles and their synthetic identity.
Topics: Humans; Liposomes; Opsonin Proteins; Polyethylene Glycols; Protein Corona
PubMed: 35040637
DOI: 10.1021/acsnano.1c07687 -
Glycobiology Oct 2018Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains...
Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.
Topics: Animals; Binding Sites; Carbohydrate Conformation; Complement Factor H; Humans; Mice; Mice, Transgenic; N-Acetylneuraminic Acid; Polysaccharides
PubMed: 29982679
DOI: 10.1093/glycob/cwy061 -
Frontiers in Immunology 2020poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial...
poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that spore proteins inhibit complement activation. However, the mechanism through which the capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated strain. Overall, our data have demonstrated that the capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.
Topics: Anthrax; Antigens, Bacterial; Bacillus anthracis; Bacterial Capsules; Complement Activation; Complement C3b; Humans; Immune Evasion; Macrophages; Opsonin Proteins; Phagocytosis; Polyglutamic Acid; Protein Binding; THP-1 Cells; Virulence
PubMed: 32296419
DOI: 10.3389/fimmu.2020.00462 -
Frontiers in Immunology 2017There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific...
There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils using a pH-sensitive fluorescence assay of phagocytosis. In mice, I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.
PubMed: 28928748
DOI: 10.3389/fimmu.2017.01082 -
Frontiers in Immunology 2019Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and...
Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1β, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.
Topics: Animals; Calpain; Cytokines; Inflammation; Macrophages; Mice; NF-kappa B; Phagocytosis; Receptors, Complement
PubMed: 31993058
DOI: 10.3389/fimmu.2019.03049 -
Cell Jun 2018Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with...
Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.
Topics: Animals; Antibodies, Monoclonal; Antigens; Carcinoembryonic Antigen; Gene Editing; Integrins; Leukocyte Common Antigens; Macrophages; Mice; Opsonin Proteins; Phagocytosis; Phosphorylation; RAW 264.7 Cells; Receptors, Fc; Unilamellar Liposomes
PubMed: 29958103
DOI: 10.1016/j.cell.2018.05.059 -
Journal of Neuroinflammation Jun 2016The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with...
BACKGROUND
The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression.
METHODS
We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls.
RESULTS
Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions.
CONCLUSIONS
Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Central Nervous System; Clusterin; Cohort Studies; Complement C1 Inactivator Proteins; Complement C1 Inhibitor Protein; Complement System Proteins; Encephalitis; Female; Gray Matter; HLA-D Antigens; Humans; Male; Microglia; Middle Aged; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Young Adult
PubMed: 27333900
DOI: 10.1186/s12974-016-0611-x -
Infection and Immunity Sep 2014Biofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream...
Biofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream infection and systemic pathogen dissemination. Staphylococcal biofilms are the most common cause of central-line-associated bloodstream infections, and antibiotic resistance makes them difficult to treat. As an alternative to antibiotic intervention, we sought to identify anti-staphylococcal biofilm targets for the development of a vaccine or antibody prophylactic. A screening strategy was devised using a microfluidic system to test antibody-mediated biofilm inhibition under biologically relevant conditions of shear flow. Affinity-purified polyclonal antibodies to target antigen PhnD inhibited both Staphylococcus epidermidis and S. aureus biofilms. PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages, and deletion of phnD inhibited normal biofilm formation. We further adapted our microfluidic biofilm system to monitor the interaction of human neutrophils with staphylococcal biofilms and demonstrated that PhnD-specific antibodies also serve as opsonins to enhance neutrophil binding, motility, and biofilm engulfment. These data support the identification of PhnD as a lead target for biofilm intervention strategies performed either by vaccination or through passive administration of antibodies.
Topics: Antibodies; Antigens, Bacterial; Bacterial Proteins; Biofilms; Humans; Neutrophils; Phagocytosis; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis
PubMed: 24958708
DOI: 10.1128/IAI.02168-14