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Cells Mar 2024Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through...
Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.
Topics: Humans; Disulfiram; Opsonin Proteins; Phagocytosis; Phagocytes; Macrophages
PubMed: 38534379
DOI: 10.3390/cells13060535 -
ImmunoHorizons Mar 2024Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology,...
Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic 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 B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.
Topics: Humans; c-Mer Tyrosine Kinase; Bacillus anthracis; Peptidoglycan; Anthrax; Efferocytosis; Hepatitis A Virus Cellular Receptor 2; Macrophages; Cell Wall
PubMed: 38517345
DOI: 10.4049/immunohorizons.2300109 -
PeerJ 2024Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune...
Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with , , , , and . The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.
Topics: Humans; Animals; Mice; Moths; Apolipoproteins; Larva; Host-Pathogen Interactions; Mammals
PubMed: 38500532
DOI: 10.7717/peerj.17117 -
Brain, Behavior, and Immunity May 2024Complement is dysregulated in the brain in Alzheimer's Disease and in mouse models of Alzheimer's disease. Each of the complement derived effectors, opsonins,...
Complement is dysregulated in the brain in Alzheimer's Disease and in mouse models of Alzheimer's disease. Each of the complement derived effectors, opsonins, anaphylatoxins and membrane attack complex (MAC), have been implicated as drivers of disease but their relative contributions remain unclarified. Here we have focussed on the MAC, a lytic and pro-inflammatory effector, in the App mouse amyloidopathy model. To test the role of MAC, we back-crossed to generate App mice deficient in C7, an essential MAC component. C7 deficiency ablated MAC formation, reduced synapse loss and amyloid load and improved cognition compared to complement-sufficient App mice at 8-10 months age. Adding back C7 caused increased MAC formation in brain and an acute loss of synapses in C7-deficient App mice. To explore whether C7 was a viable therapeutic target, a C7-blocking monoclonal antibody was administered systemically for one month in App mice aged 8-9 months. Treatment reduced brain MAC and amyloid deposition, increased synapse density and improved cognitive performance compared to isotype control-treated App mice. The findings implicate MAC as a driver of pathology and highlight the potential for complement inhibition at the level of MAC as a therapy in Alzheimer's disease.
Topics: Mice; Animals; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Cognitive Dysfunction; Mice, Transgenic; Plaque, Amyloid; Brain; Cognition; Complement Activation; Disease Models, Animal
PubMed: 38485063
DOI: 10.1016/j.bbi.2024.03.017 -
Frontiers in Immunology 2024Single nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on...
INTRODUCTION
Single nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production.
METHODS
We genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants.
RESULTS AND DISCUSSION
In participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.
Topics: Humans; Leishmaniasis, Visceral; Leishmania infantum; Receptors, IgG; Interleukin-12; Tumor Necrosis Factor-alpha; Nucleotides; Protein Isoforms; Genetic Variation; Immunoglobulin G
PubMed: 38455048
DOI: 10.3389/fimmu.2024.1343602 -
The Journal of Biological Chemistry Apr 2024The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to...
The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.
Topics: Humans; Complement Factor H; Recombinant Fusion Proteins; Complement Activation; CD55 Antigens; Hemolysis; Complement Pathway, Alternative; Complement Inactivating Agents; Erythrocytes; Receptors, Complement 3b
PubMed: 38401844
DOI: 10.1016/j.jbc.2024.105784 -
Journal of Immunology (Baltimore, Md. :... Apr 2024FcγR is a significant opsonin receptor located on the surface of immune cells, playing a crucial role in Ab-dependent cell-mediated immunity. Our previous work revealed...
FcγR is a significant opsonin receptor located on the surface of immune cells, playing a crucial role in Ab-dependent cell-mediated immunity. Our previous work revealed opposite expression trends of FcγRII and FcγRIII in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda. This observation suggests that FcγRII and FcγRIII might serve distinct functions in Ig-opsonized immune responses. In this study, we prepared rFcγRIII as well as its corresponding Abs to investigate the potential roles of FcγRII and FcγRIII in the Ab-dependent immune response of IgM+ B cells. Our findings indicate that, unlike FcγRII, FcγRIII does not participate in Ab-dependent cellular phagocytosis. Instead, it is involved in cytokine production and bacterial killing in mIgM+ B lymphocytes. Additionally, we identified platelet-derived ADAM17 as a key factor in regulating FcγRIII shedding and cytokine release in mIgM+ B lymphocytes. These results elucidate the functions of FcγRII and FcγRIII in the innate immunology of mIgM+ B lymphocytes and contribute to an improved understanding of the regulatory roles of FcγRs in the phagocytosis of teleost B lymphocytes.
Topics: Animals; Receptors, IgG; Receptors, Fc; Flounder; Immune System; Cytokines
PubMed: 38380986
DOI: 10.4049/jimmunol.2300429 -
Carbohydrate Polymers Apr 2024Enterococcus faecium, a gram-positive opportunistic pathogen, has become a major concern for nosocomial infections due to its resistance to several antibiotics,...
Enterococcus faecium, a gram-positive opportunistic pathogen, has become a major concern for nosocomial infections due to its resistance to several antibiotics, including vancomycin. Finding novel alternatives for treatment prevention, such as vaccines, is therefore crucial. In this study, we used various techniques to discover a novel capsular polysaccharide. Firstly, we identified an encapsulated E. faecium strain by evaluating the opsonophagocytic activity of fifteen strains with antibodies targeting the well-known lipoteichoic acid antigen. This activity was attributed to an unknown polysaccharide. We then prepared a crude cell wall glycopolymer and fractionated it, guided by immunodot-blot analysis. The most immunoreactive fractions were used for opsonophagocytic inhibition assays. The fraction containing the inhibitory polysaccharide underwent structural characterization using NMR and chemical analyses. The elucidated structure presents a branched repeating unit, with the linear part being: →)-β-d-Gal-(1 → 4)-β-d-Glc-(1 → 4)-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1→, further decorated with a terminal α-d-Glc and a d-phosphoglycerol moiety, attached to O-2 and O-3 of the 4-linked Gal unit, respectively. This polysaccharide was conjugated to BSA and the synthetic glycoprotein used to immunize mice. The resulting sera exhibited good opsonic activity, suggesting its potential as a vaccine antigen. In conclusion, our effector-function-based approach successfully identified an immunogenic capsular polysaccharide with promising applications in immunotherapy.
Topics: Mice; Animals; Antigens, Bacterial; Enterococcus faecium; Opsonin Proteins; Polysaccharides; Antibodies, Bacterial; Vaccine Development
PubMed: 38368077
DOI: 10.1016/j.carbpol.2023.121731 -
ACS Infectious Diseases Mar 2024Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing...
Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, (ATCC 25922) and (ATCC BAA-427), but not of Gram-positive bacteria, (ATCC 6633) and (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria (ATCC 25922) and (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.
Topics: Animals; Mice; Lipopolysaccharides; Escherichia coli; Phagocytosis; Macrophages; Gram-Negative Bacteria
PubMed: 38363869
DOI: 10.1021/acsinfecdis.3c00274 -
Cancer Research Communications Feb 2024In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack...
UNLABELLED
In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403).
SIGNIFICANCE
Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.
Topics: Humans; CD47 Antigen; Receptors, Immunologic; Phagocytosis; Macrophages; Neoplasms; Antibodies, Neoplasm; Opsonin Proteins; Hematologic Neoplasms
PubMed: 38319147
DOI: 10.1158/2767-9764.CRC-23-0634