Macrophage Phagocytosis Research Articles

Potential of Pandan anggur (Sararanga Sinuosa Hemsley) as immunomodulator

Background: Pandan Anggur plant is an endemic plant of the Papua archipelago in Indonesia, and the fruit called Pandan Anggur Fruit (PAF) is freshly consumed or prepared as a juice. Based on previous studies on the phytochemical content of PAF, the ethanol and water extract contain flavonoids, which act as immunomodulators, to regulate the response of immunity. Objectives: This study aimed to examine the potential of PAF as an immunomodulator in malnutrition cases, by evaluating six parameters which are phagocytosis capacity and nitric oxide (NO) production of peritoneal macrophage, spleen lymphocyte proliferation, IFN-γ and IL-4 in lymphocyte culture, and intestinal sIgA levels. By proving the potential of PAF as an immunomodulator, this research will lead to significant development of functional food products of Pandan Anggur. Methods: A total of 35 Sprague Dawley rats were used as animal models divided into seven different groups named standard group (healthy standard), malnourished (negative control), malnourished rats given Imboost Force (positive control), as well as PAF ethanol extract doses of I, II, III, and PAF water extract. The treatment lasted for seven weeks in total and the measurement of immunomodulator parameters was carried out at the end of the treatment period. The immunomodulator parameters included phagocytosis capacity and nitric oxide (NO) production of peritoneal macrophage, spleen lymphocyte proliferation, IFN-γ and IL-4 in lymphocyte culture, and intestinal sIgA levels. Result: The results indicated that the administration of PAF ethanol extract at a dose of 3.15 mg/mL provided an optimum immunostimulant effect, by increasing the macrophage phagocytosis capacity, nitric oxide, lymphocyte proliferation, IL-4, and sIgA levels in rat intestine. However, increasing the dose of extract did not produce a better effect, but led to suppression of immunity. Conclusion: PAF extract at a dose of 3.15 mg/mL provides an immunostimulant effect. Keywords: In Vivo, IL-4, IF-γ, sIgA, lymphocyte proliferation, macrophage, Pandan Anggur Fruit

Extraction and molecular characterisation of polysaccharides from Suaeda maritima for their immunomodulatory effects

In the present study, Suaeda maritima polysaccharides (SMPs) were identified, characterised and evaluated for their immune-modulating effects on RAW264.7 macrophages and the underlying biological processes. Two acidic polysaccharides (SMP-F1 and SMP-F2) were obtained via ion-exchange chromatography from the crude polysaccharide (SMP-C). The SMP-C and its fractions mainly contained carbohydrates, proteins and sulphates, in variable proportions. Glucose, mannose, galactose and arabinose were the primary monosaccharide units, accompanied by smaller amounts of rhamnose and xylose. The molecular weight (Mw) of the SMPs ranged from 76.2 × 103 to 245.0 × 103 g/mol. SMPs can significantly induce the production of NO by up-regulating iNOS expression. The results showed that SMPs could increase TNF-α, IL-6, IL-10 and IL-12 expression. In addition, SMP-F2 significantly boosts inflammatory reactions more than SMP-C or SMP-F1. Further study revealed that SMP-F2 was found to stimulate macrophage phagocytosis and the expression of CD11b, CD14, CD40 and TLR4. The major chains of the strongest immunostimulant, SMP-F2, were mainly 1,3-linked Manp, 1,4-linked Glcp and 1,4-linked Galp. The side chain of SMP-F2 was 1,3,6-linked Manp and 1,4,6-linked Galp, with terminals of arabinose, mannose and glucose. These findings show that SMPs, particularly SMP-F2, promote the ability to modulate immunological responses.

An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8+ T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.

Role of MicroRNA-21 in Regulating Intracellular Pathways Associated With Phagocytosis in Human Macrophages: An In Vitro Study.

Introduction The efficient clearance of bacteria by macrophages is crucial for the timely resolution of inflammation. In this study, we investigated the role of microRNA-21 (miR-21)-induced phagocytosis and its intracellular signaling pathways in human macrophages in vitro. Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from 15 healthy volunteers. Differentiated human macrophages were incubated with lipopolysaccharide (LPS) to determine the time course of changes in phagocytic activity and miR-21 expression. The expression of candidate genes targeted by miR-21 and its downstream effectors was quantitatively assessed. The effects of miR-21 modulation were also examined via transfection with miR-21 mimics and inhibitors. Results Incubation of human macrophages with LPS upregulated both phagocytosis and miR-21 expression. Notably, changing miR-21 expression levels using miR-21 mimics or inhibitors led to significant and opposite changes in the expression of its downstream effectors. miR-21 induction in macrophages downregulated PDCD4 and PTEN, promoted the phosphorylation of Akt and the production of the anti-inflammatory cytokine IL-10, and facilitated phagocytosis. Conclusion This study directly confirms that LPS upregulates macrophage phagocytosis and miR-21 expression. Elevated miR-21 levels in macrophages enhanced phagocytosis, contributing to an anti-inflammatory phenotype. These findings underscore the importance of miR-21 in resolving inflammation.

Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni.

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire's Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.

Abnormal HCK/glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis.

The presence of extensive infiltrated macrophages with impaired phagocytosis is widely recognised as a significant regulator for the development of endometriosis (EMs). Nevertheless, the metabolic characteristics and the fundamental mechanism of impaired macrophage phagocytosis are yet to be clarified. Here, we observe that there is the decreased expression of haematopoietic cellular kinase (HCK) in macrophage of peritoneal fluid from EMs patients, which might be attributed to high oestrogen and hypoxia condition. Of note, HCK deficiency resulted in impaired macrophage phagocytosis, and increased number and weight of ectopic lesions in vitro and in vivo. Mechanistically, this process was mediated via regulation of glutamine metabolism, and further upregulation of macrophage autophagy in a c-FOS/c-JUN dependent manner. Additionally, macrophages of EMs patients displayed insufficient HCK, excessive autophagy and phagocytosis dysfunction. In therapeutic studies, supplementation with glutamine-pre-treated macrophage or Bafilomycin A1 (an autophagy inhibitor)-pre-treated macrophage leads to the induction of macrophage phagocytosis and suppression of EMs development. This observation reveals that the aberrant HCK-glutamine-autophagy axis results in phagocytosis obstacle of macrophage and further increase the development risk of Ems. Additionally, it offers potential therapeutic approaches to prevent EMs, especially patients with insufficient HCK and macrophage phagocytosis dysfunction.

GSDME-mediated pyroptosis promotes anti-tumor immunity of neoadjuvant chemotherapy in breast cancer

Paclitaxel and anthracycline-based chemotherapy is one of the standard treatment options for breast cancer. However, only about 6–30% of breast cancer patients achieved a pathological complete response (pCR), and the mechanism responsible for the difference is still unclear. In this study, random forest algorithm was used to screen feature genes, and artificial neural network (ANN) algorithm was used to construct an ANN model for predicting the efficacy of neoadjuvant chemotherapy for breast cancer. Furthermore, digital pathology, cytology, and molecular biology experiments were used to verify the relationship between the efficacy of neoadjuvant chemotherapy and immune ecology. It was found that paclitaxel and doxorubicin, an anthracycline, could induce typical pyroptosis and bubbling in breast cancer cells, accompanied by gasdermin E (GSDME) cleavage. Paclitaxel with LDH release and Annexin V/PI doubule positive cell populations, and accompanied by the increased release of damage-associated molecular patterns, HMGB1 and ATP. Cell coculture experiments also demonstrated enhanced phagocytosis of macrophages and increased the levels of IFN-γ and IL-2 secretion after paclitaxel treatment. Mechanistically, GSDME may mediate paclitaxel and doxorubicin-induced pyroptosis in breast cancer cells through the caspase-9/caspase-3 pathway, activate anti-tumor immunity, and promote the efficacy of paclitaxel and anthracycline-based neoadjuvant chemotherapy. This study has practical guiding significance for the precision treatment of breast cancer, and can also provide ideas for understanding molecular mechanisms related to the chemotherapy sensitivity.

Glucomannan is a promising isoniazid's enhancer that inducing macrophage phagocytosis.

Isoniazid (INH) is a frontline antituberculosis agent effective against Mycobacterium tuberculosis (Mtb), but the increasing challenge of avoiding multidrug-resistant tuberculosis, including INH resistance, necessitates innovative approaches. This study focused on enhancing macrophage phagocytosis to overcome INH resistance. Glucomannan, an immunomodulatory polysaccharide, emerged as a potential macrophage activator. Our objective was to characterize the glucomannan-INH mixture and assess its impact on INH efficacy and macrophage activity. Detailed examination of the glucomannan from Amorphophallus muelleri (0.05%-0.2%) was performed in several methods. INH sensitivity tests were carried out with the Mtb strain H37RV on Löwenstein-Jensen medium. Murine macrophage (RAW264.7) viability and activity were evaluated through MTT and latex bead phagocytosis assays. Ultraviolet-wavelength spectrophotometry was used to analyze chemical structure changes. Glucomannan (0.05%-0.2%) significantly enhanced murine macrophage viability and activity. When glucomannan was combined with INH, the IC50 value was greater compared to INH only. Phagocytosis assays revealed heightened macrophage activity in the presence of 0.05% and 0.1% glucomannan. Importantly, glucomannan did not compromise INH efficacy or alter its chemical structure. This study underscores the potential of glucomannan, particularly with a lower molecular weight, as a promising enhancer of INH, boosting macrophage phagocytosis against INH-resistant Mtb. These findings challenge the assumptions about the impact of glucomannan on drug absorption and prompt potential reevaluation. While specific receptors for glucomannan in macrophage phagocytosis require further exploration, the complement receptors are proposed to be potential mediators.

Microwave‐Responsive Nanoplatform with Robust ROS Generation and Inhibiting Effects of Two‐Component System and Quorum Sensing for the Treatment of MRSA‐Induced Osteomyelitis

AbstractThe hypoxic environment of deep tissue and the weak energy of microwave (MW) are still the major limitations of MW catalytic therapy. In this work, an MW‐responsive nano‐platform consisting of an Mn‐doped porphyrin metal–organic framework loaded with calcium peroxide (Mn0.1PCC) is designed to regulate the microenvironment of the injection site and enhance the generation of reactive oxygen species. The excellent MW absorbing properties of Mn0.1PCC improve the Fenton reaction between hydrogen peroxide formed by the hydrolysis of calcium peroxide and released Mn2+ generating more hydroxyl radicals under MW. This process does not consume the oxygen substrate and even produces oxygen to improve the hypoxic environment. Therefore, at a low and safe concentration (125 µg mL−1), the antibacterial rate of Mn0.1PCC against 4 × 108 CFU mL−1 of methicillin‐resistant Staphylococcus aureus (MRSA) after MW irradiation for 15 min is 99.71% ± 0.18%. Mn0.1PCC, upon exposure to MW, prevents the recurrence of bacterial infections and overcomes drug resistance by inhibiting quorum sensing and the two‐component system of MRSA. It can also induce macrophage M1 polarization, up‐regulate the pro‐inflammatory factor tumor necrosis factor‐α, and enhance the phagocytosis of macrophage to bacteria. This strategy provides insights into the development of high‐performance MW‐responsive nanocomposites for the treatment of infections in deep tissues.

Highly Drug-Loaded Nanoaggregate Microparticles for Pulmonary Delivery of Cyclosporin A.

Nanoparticles have the advantages of improving the solubility of poorly water-soluble drugs, facilitating the drug across biological barriers, and reducing macrophage phagocytosis in pulmonary drug delivery. However, nanoparticles have a small aerodynamic particle size, which makes it difficult to achieve optimal deposition when delivered directly to the lungs. Therefore, delivering nanoparticles to the lungs effectively has become a popular research topic. Nanoaggregate microparticles were used as a pulmonary drug delivery strategy for the improvement of the bioavailability of cyclosporine A (CsA). The nanoaggregate microparticles were prepared with polyvinyl pyrrolidone (PVP) as the excipient by combining the anti-solvent method and spray drying process. The physicochemical properties, aerodynamic properties, in vivo pharmacokinetics and inhalation toxicity of nanoaggregate microparticles were systematically evaluated. The optimal nanoparticles exhibited mainly spherical shapes with the particle size and zeta potential of 180.52 nm and -19.8 mV. The nanoaggregate microparticles exhibited irregular shapes with the particle sizes of less than 1.6 µm and drug loading (DL) values higher than 70%. Formulation NM-2 as the optimal nanoaggregate microparticles was suitable for pulmonary drug delivery and probably deposited in the bronchiole and alveolar region, with FPF and MMAD values of 89.62% and 1.74 μm. In addition, inhaled NM-2 had C max and AUC0-∞ values approximately 1.7-fold and 1.8-fold higher than oral cyclosporine soft capsules (Neoral®). The inhalation toxicity study suggested that pulmonary delivery of NM-2 did not result in lung function damage, inflammatory responses, or tissue lesions. The novel nanoaggregate microparticles for pulmonary drug delivery could effectively enhance the relative bioavailability of CsA and had great potential for clinical application.

Investigation of Factors Influencing the Effectiveness of Deformable Nanovesicles for Insulin Nebulization Inhalation.

Nebulized inhalation offers a noninvasive method for delivering drugs to treat both local respiratory and systemic diseases. In this study, insulin was used as a model drug to design a series of deformable nanovesicles (DNVs) with key quality attributes, including particle size, deformability, and drug load capacity. We investigated the effects of these properties on aerosol generation, macrophage phagocytosis, and bloodstream penetration. The results showed that deformability improved nebulization performance and reduced macrophage phagocytosis, benefiting local and systemic delivery. However, the advantage of DNVs for transmembrane penetration was not evident in the alveolar epithelium. Within the size range of 80-490 nm, the smaller the particle size of IPC-DNVs, the easier it is to evade clearance by macrophages and the more effective the in vivo hypoglycemic efficacy will be. In the drug load range of 3-5 mg/mL, a lower drug load resulted in better hypoglycemic efficacy. The area above the blood glucose decline curve with time (AAC) of nebulized DNVs was 2.32 times higher than that of the insulin solution, demonstrating the feasibility and advantages of DNVs in the pulmonary delivery of biomacromolecule drugs. This study provides insights into the construction and formulation optimization of pulmonary delivery carriers.

Single-cell topographical profiling of the immune synapse reveals a biomechanical signature of cytotoxicity

Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force.

Radiation-induced eCIRP impairs macrophage bacterial phagocytosis.

Macrophages are essential immune cells for host defense against bacterial pathogens after radiation injury. However, the role of macrophage phagocytosis in infection following radiation injury remains poorly examined. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that dysregulates host immune system responses such as phagocytosis. We hypothesized that radiation-induced eCIRP release impairs macrophage phagocytosis of bacteria. Adult healthy mice were exposed to 6.5-Gy total body irradiation (TBI). Primary peritoneal macrophages isolated from adult healthy mice were exposed to 6.5-Gy radiation. eCIRP-neutralizing monoclonal antibody (mAb) was added to the cell culture prior to irradiation. Bacterial phagocytosis by peritoneal macrophages was assessed using pHrodo Green-labeled E. coli 7 days after irradiation ex vivo and in vitro. Bacterial phagocytosis was also assessed after treatment with recombinant murine CIRP (rmCIRP). Rac1 and ARP2 protein expression in cell lysates and eCIRP levels in the peritoneal lavage were assessed by Western blotting. Bacterial phagocytosis by peritoneal macrophages was significantly decreased after irradiation compared to controls ex vivo and in vitro. Rac1 and ARP2 expression in the peritoneal macrophages were downregulated after TBI. TBI significantly increased eCIRP levels in the peritoneal cavity. rmCIRP significantly decreased bacterial phagocytosis in a dose-dependent manner. eCIRP mAb restored bacterial phagocytosis by peritoneal macrophages after irradiation. Ionizing radiation exposure impairs bacterial phagocytosis by macrophages after irradiation. Neutralization of eCIRP restores the phagocytic ability of macrophages after irradiation. Our findings elucidate a novel mechanism of immune dysfunction and provide a potential new therapeutic approach for limiting infection after radiation injury.

Bioengineer mesenchymal stem cell for treatment of glioma by IL‐12 mediated microenvironment reprogramming and nCD47‐SLAMF7 mediated phagocytosis regulation of macrophages

AbstractHigh expression of cellular self‐activated immunosuppressive molecules and extensive infiltration of suppressive immune cells in the tumor microenvironment are the main factors contributing to glioma's resistance to immunotherapy. Nonetheless, technology to modify the expression of glioma cellular self‐molecules through gene editing requires further development. This project advances cell therapy strategies to reverse the immunosuppressive microenvironment of glioma (TIME). Bone marrow‐derived mesenchymal stem cells (MSCs) are engineered to express bioactive proteins and demonstrate tumor‐homing characteristics upon activation by TGF‐β. These MSCs are designed to secrete the anti‐tumor immune cytokine IL‐12 and the nCD47‐SLAMF7 fusion protein, which regulates T‐cell activity and macrophage phagocytosis. The engineered MSCs are then injected in situ into the glioma site, circumventing the blood‐brain barrier to deliver high local concentrations of bioactive proteins. This approach aims to enhance the M1 polarization of infiltrating macrophages, stimulate macrophage‐mediated tumor cell phagocytosis, activate antigen‐presenting cells, and promote effector CD8+ T cell infiltration, effectively controlling glioma. Additionally, the engineered MSCs may serve as a universal treatment for other tumors that express TGF‐β at high levels. This study proposes a novel treatment strategy for the clinical management of glioma patients.

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging

Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Retrieval analysis of PEEK rods pedicle screw system: three cases analysis

PurposeTo analyze the characteristics of PEEK rods retrieved in vivo, specifically their wear and deformation, biodegradability, histocompatibility, and mechanical properties.MethodSix PEEK rods were retrieved from revision surgeries along with periprosthetic tissue. The retrieved PEEK rods were evaluated for surface damage and internal changes using Micro-CT, while light and electron microscopy were utilized to determine any histological changes in periprosthetic tissues. Patient history was gathered from medical records. Two intact and retrieved PEEK rods were used for fatigue testing analysis by sinusoidal load to the spinal construct.ResultsAll implants showed evidence of plastic deformation around the screw-rod interface, while the inner structure of PEEK rods appeared unchanged with no visible voids or cracks. Examining images captured through light and electron microscopy indicated that phagocytosis of macrophages around PEEK rods was less severe in comparison to the screw-rod interface. The results of an energy spectrum analysis suggested that the distribution of tissue elements around PEEK rods did not differ significantly from normal tissue. During fatigue testing, it was found that the retrieved PEEK rods cracked after 1.36 million tests, whereas the intact PEEK rods completed 5 million fatigue tests without any failure.ConclusionPEEK rods demonstrate satisfactory biocompatibility, corrosion resistance, chemical stability, and mechanical properties. Nevertheless, it is observed that the indentation at the junction between the nut and the rod exhibits relatively weak strength, making it susceptible to breakage. As a precautionary measure, it is recommended to secure the nut with a counter wrench, applying the preset torque to prevent overtightening.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

Abstract PO-038: Unbiased discovery of novel antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma

Abstract Monoclonal antibodies are among the most effective treatments for patients with blood cancers, and they primarily function by marking cells for destruction by the innate immune system. Macrophages are critical innate effectors of antibody therapies for lymphoma, but their anti-tumor capacity is limited by the CD47/SIRPa interaction. CD47 acts a “don’t eat me” signal that is highly expressed on the surface of many lymphomas, and it prevents phagocytosis by binding to the inhibitory receptor SIRPa expressed on macrophages. In multiple clinical trials, CD47-blocking therapies have demonstrated encouraging signs of efficacy for B-cell lymphomas, particularly when used in combination with rituximab, an opsonizing anti-CD20 antibody. However, the best antibodies and combination strategies to activate macrophages remains unknown. Here, we sought to define the repertoire of cell surface antigens that can be targeted to stimulate macrophage-mediated destruction of B-cell lymphomas. To achieve this goal, we developed a high-throughput functional screening platform to measure the ability of primary macrophages to attack B-cell lymphoma cells. We successfully applied this system to screen monoclonal antibody libraries targeting hundreds of distinct cell surface antigens across both mouse and human systems. We conducted screens using each antibody as a single agent, in combination with anti-CD20, or in combination with anti-CD47. From these efforts, we identified CD24, CD38, CXCR4, CD71, and multiple other novel and unique antigens that could be targeted alone or in combination to exert maximal macrophage-mediated destruction of B-cell lymphoma. Of note, some of the identified targets are predominantly expressed by the lymphoma cells, whereas others are expressed by the macrophages and act as unappreciated immune checkpoints. In validation studies, we defined a multitude of new antibody combinations that robustly stimulate macrophages to attack and eliminate lymphoma cells. Since some anti-CD47-antibodies have been limited by on-target hematologic toxicity, we also used the identified targets to engineer a collection of novel bispecific antibodies that induce macrophage phagocytosis without causing hematologic toxicity. We generated 156 bispecific antibodies, produced them recombinantly, and demonstrated they can maximize macrophage-mediated cytotoxicity of human B-cell lymphoma cells while minimizing binding to healthy blood cells. In mouse xenograft models, these bispecific antibodies exhibited significant single-agent activity in a model of aggressive B-cell lymphoma. Thus, our study has led to the development of a multitude of novel therapeutic candidates and combination strategies that can be developed further to maximize anti-tumor function and benefit patients with lymphoma. Furthermore, our approach can be rapidly applied to other hematologic malignancies to create innovative bispecific agents that maximize anti-tumor responses by macrophages or other innate immune cells. Citation Format: Juliano Ribeiro, Carlota Pages Geli, José Velarde, Anna Meglan, Jasmine Blandin, Kyle Vaccaro, Marta Crespo, Kipp Weiskopf. Unbiased discovery of novel antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-038.

Engineering nanoparticles-enabled tumor-associated macrophages repolarization and phagocytosis restoration for enhanced cancer immunotherapy

Tumor-associated macrophages (TAMs) are pivotal within the immunosuppressive tumor microenvironment (TME), and recently, have attracted intensive attention for cancer treatment. However, concurrently to promote TAMs repolarization and phagocytosis of cancer cells remains challenging. Here, a TAMs-targeted albumin nanoparticles-based delivery system (M@SINPs) was constructed for the co-delivery of photosensitizer IR820 and SHP2 inhibitor SHP099 to potentiate macrophage-mediated cancer immunotherapy. M@SINPs under laser irradiation can generate the intracellular reactive oxygen species (ROS) and facilitate M2-TAMs to an M1 phenotype. Meanwhile, inhibition of SHP2 could block the CD47-SIRPa pathway to restore M1 macrophage phagocytic activity. M@SINPs-mediated TAMs remodeling resulted in the immunostimulatory TME by repolarizing TAMs to an M1 phenotype, restoring its phagocytic function and facilitating intratumoral CTLs infiltration, which significantly inhibited tumor growth. Furthermore, M@SINPs in combination with anti-PD−1 antibody could also improve the treatment outcomes of PD−1 blockade and exert the synergistic anticancer effects. Thus, the macrophage repolarization/phagocytosis restoration combination through M@SINPs holds promise as a strategy to concurrently remodel TAMs in TME for improving the antitumor efficiency of immune checkpoint block and conventional therapy.

AI-2 quorum sensing-induced galactose metabolism activation in Streptococcus suis enhances capsular polysaccharide-associated virulence

Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.