Macrophage Mannose Receptor Research Articles

Intranasal Administration of Bedaquiline-Loaded Fucosylated Liposomes Provides Anti-Tubercular Activity while Reducing the Potential for Systemic Side Effects.

Liposomal formulations of antibiotics for inhalation offer the potential for the delivery of high drug doses, controlled drug release kinetics in the lung, and an excellent safety profile. In this study, we evaluated the in vivo performance of a liposomal formulation for the poorly soluble, antituberculosis agent, bedaquiline. Bedaquiline was encapsulated within monodisperse liposomes of ∼70 nm at a relatively high drug concentration (∼3.6 mg/mL). Formulations with or without fucose residues, which bind to C-type lectin receptors and mediate a preferential binding to macrophage mannose receptor, were prepared, and efficacy was assessed in an in vivo C3HeB/FeJ mouse model of tuberculosis infection (H37Rv strain). Seven intranasal instillations of 5 mg/kg bedaquiline formulations administered every second day resulted in a significant reduction in lung burden (∼0.4-0.6 Δlog10 CFU), although no differences between fucosylated and nonfucosylated formulations were observed. A pharmacokinetic study in healthy, noninfected Balb/c mice demonstrated that intranasal administration of a single dose of 2.5 mg/kg bedaquiline liposomal formulation (fucosylated) improved the lung bioavailability 6-fold compared to intravenous administration of the same formulation at the same dose. Importantly, intranasal administration reduced systemic concentrations of the primary metabolite, N-desmethyl-bedaquiline (M2), compared with both intravenous and oral administration. This is a clinically relevant finding as the M2 metabolite is associated with a higher risk of QT-prolongation in predisposed patients. The results clearly demonstrate that a bedaquiline liposomal inhalation suspension may show enhanced antitubercular activity in the lung while reducing systemic side effects, thus meriting further nonclinical investigation.

Plasma proteomics of acute tubular injury

The kidney tubules constitute two-thirds of the cells of the kidney and account for the majority of the organ’s metabolic energy expenditure. Acute tubular injury (ATI) is observed across various types of kidney diseases and may significantly contribute to progression to kidney failure. Non-invasive biomarkers of ATI may allow for early detection and drug development. Using the SomaScan proteomics platform on 434 patients with biopsy-confirmed kidney disease, we here identify plasma biomarkers associated with ATI severity. We employ regional transcriptomics and proteomics, single-cell RNA sequencing, and pathway analysis to explore biomarker protein and gene expression and enriched biological pathways. Additionally, we examine ATI biomarker associations with acute kidney injury (AKI) in the Kidney Precision Medicine Project (KPMP) (n = 44), the Atherosclerosis Risk in Communities (ARIC) study (n = 4610), and the COVID-19 Host Response and Clinical Outcomes (CHROME) study (n = 268). Our findings indicate 156 plasma proteins significantly linked to ATI with osteopontin, macrophage mannose receptor 1, and tenascin C showing the strongest associations. Pathway analysis highlight immune regulation and organelle stress responses in ATI pathogenesis.

Macrophages exploit the mannose receptor and JAK-STAT1-MHC-II pathway to drive antigen presentation and the antimycobacterial immune response after BCG vaccination.

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M. tb), remains one of the leading causes of fatal infectious diseases worldwide. The only licensed vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), has variable efficacy against TB in adults. Insufficiency of immune cell function diminishes the protective effects of the BCG vaccine. It is critical to clarify the mechanism underlying the antimycobacterial immune response during BCG vaccination. Macrophage mannose receptor (MR) is important for enhancing the uptake and processing of glycoconjugated antigens from pathogens for presentation to T cells, but the roles of macrophage MR in the BCG-induced immune response against M. tb are not yet clear. Here, we discover that macrophage MR deficiency impairs the antimycobacterial immune response in BCG-vaccinated mice. Mechanistically, macrophage MR triggers JAK-STAT1 signaling, which promotes antigen presentation via upregulated MHC-II and induces IL-12 production by macrophages, contributing to CD4 + T cell activation and IFN-γ production. MR deficiency in macrophages reduces the vaccine efficacy of BCG and increases susceptibility to M. tb H37Ra challenge in mice. Our results suggest that MR is critical for macrophage antigen presentation and the antimycobacterial immune response to BCG vaccination and offer valuable guidance for the preventive strategy of BCG immunization.

Introducing Targeting Units or pH-Releasable Immunodrugs into Core-Clickable Nanogels

The development of nano-sized carrier systems plays a fundamental role in immunodrug delivery and the treatment of cancer. Especially functional materials, coupled with a stimuli-responsive drug release, control the selective delivery of small molecular drugs to the target site and avoid systemic side effects. Based on this, we introduce a DBCO core-functionalized nanogel platform for pH-reversible conjugation of highly potent TLR7/8-activating imidazoquinolines and the selective targeting of macrophage mannose receptor (MMR/ CD206) expressed by immunosuppressive macrophages. DBCO-PEG4-amine functionalized polymethacrylates are synthesized by controlled RAFT polymerization and self-assembled into precursor micelles in polar aprotic solvents. Corresponding nanogels are generated via reactive ester chemistry, while conjugated DBCO–units are incorporated into the core, still accessible for click reaction with azide-functionalized structures. Regarding the preparation of targeted nanogels, trimannose equipped with azide moieties can be conjugated to the DBCO nanogels, revealing the efficient targeting of macrophages’ mannose receptor in vitro. Moreover, the broad applicability of the DBCO nanogel is demonstrated by the synthesis of an azide–containing 2-propionic-3-methylmaleic anhydride-based linker sensitive for the pH–reversible conjugation of secondary amine-modified immune modulators, such as IMDQ-Me. Via bioorthogonal DBCO click reaction, the immune modulator can reversibly be conjugated, affording pH-responsive drug-loaded nanogels that conserve the desired immune stimulatory effect in vitro. Overall, these findings highlight the potential of core–functionalized DBCO nanogels, a promising carrier system for pH–sensitive conjugated immunodrugs as well as an attractive platform for controlled targeting of MMR. Altogether, the versatile application of core-functionalized DBCO nanogels may pave the way for enhancing bioorthogonal multifunctionality inside nanocarrier systems that assist in addressing multiple targets in cancer immunotherapy.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ Tcell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Macrophage mannose receptor CD206 targeting of fluoride-18 labeled mannosylated dextran: A validation study in mice

PurposeAluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated mannosylated dextran derivative (Al[18F]F-NOTA-D10CM) is a new tracer for PET imaging. We report here on in vitro and in vivo validation of the tracer’s ability to target the macrophage mannose receptor CD206.MethodsFirst, the uptake of intravenously (i.v.) administered Al[18F]F-NOTA-D10CM was compared between wild-type (WT) and CD206−/− knockout (KO) mice. C57BL/6N mice were injected with complete Freund’s adjuvant (CFA) in the left hind leg and the uptake of Al[18F]F-NOTA-D10CM after i.v. or intradermal (i.d.) injection was studied at 5 and 14 days after CFA induction of inflammation. Healthy C57BL/6N mice were studied as controls. Mice underwent PET/CT on consecutive days with [18F]FDG, i.v. Al[18F]F-NOTA-D10CM, and i.d. Al[18F]F-NOTA-D10CM. After the last imaging, Al[18F]F-NOTA-D10CM was i.v. injected for an ex vivo biodistribution study and autoradiography of inflamed tissues. Blood plasma samples were analyzed using high-performance liquid chromatography. To evaluate the specificity of Al[18F]F-NOTA-D10CM binding, an in vitro competitive displacement study was performed on inflamed tissue sections using autoradiography. CD206 expression was assessed by immunohistochemical staining.ResultsCompared with WT mice, the uptake of Al[18F]F-NOTA-D10CM was significantly lower in several CD206−/− KO mice tissues, including liver (SUV 8.21 ± 2.51 vs. 1.06 ± 0.16, P < 0.001) and bone marrow (SUV 1.63 ± 0.37 vs. 0.22 ± 0.05, P < 0.0001). The uptake of i.v. injected Al[18F]F-NOTA-D10CM was significantly higher in inflamed ankle joint (SUV 0.48 ± 0.13 vs. 0.18 ± 0.05, P < 0.0001) and inflamed foot pad skin (SUV 0.41 ± 0.10 vs. 0.04 ± 0.01, P < 0.0001) than in the corresponding tissues in healthy mice. The i.d.-injected Al[18F]F-NOTA-D10CM revealed differences between CFA-induced lymph node activation and lymph nodes in healthy mice. Ex vivo γ-counting, autoradiography, and immunohistochemistry supported the results, and a decrease of ~ 80% in the binding of Al[18F]F-NOTA-D10CM in the displacement study with excess NOTA-D10CM confirmed that tracer binding was specific. At 60 min after i.v. injection, an average 96.70% of plasma radioactivity was derived from intact Al[18F]F-NOTA-D10CM, indicating good in vivo stability. The uptake of Al[18F]F-NOTA-D10CM into inflamed tissues was positively associated with the area percentage of CD206-positive staining.ConclusionThe uptake of mannosylated dextran derivative Al[18F]F-NOTA-D10CM correlated with CD206 expression and the tracer appears promising for inflammation imaging.Graphical abstract

Transcriptomic Responses to Koi Herpesvirus in Isolated Blood Leukocytes from Infected Common Carp.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.

Mouse Type-I Interferon-Mannosylated Albumin Fusion Protein for the Treatment of Chronic Hepatitis.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-β, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

Cathepsin B-Responsive Programmed Brain Targeted Delivery System for Chemo-Immunotherapy Combination Therapy of Glioblastoma.

Tumor-associated macrophages (TAMs) are closely related to the progression of glioblastoma multiform (GBM) and its development of therapeutic resistance to conventional chemotherapy. TAM-targeted therapy combined with conventional chemotherapy has emerged as a promising strategy to combat GBM. However, the presence of the blood-brain barrier (BBB) severely limits the therapeutic efficacy. Meanwhile, the lack of ability to distinguish different targeted cells also poses a challenge for precise therapy. Herein, we propose a cathepsin B (CTSB)-responsive programmed brain-targeted delivery system (D&R-HM-MCA) for simultaneous TAM-targeted and GBM-targeted delivery. D&R-HM-MCA could cross the BBB via low density lipoprotein receptor-associated protein 1 (LRP1)-mediated transcytosis. Upon reaching the GBM site, the outer angiopep-2 modification could be detached from D&R-HM-MCA via cleavage of the CTSB-responsive peptide, which could circumvent abluminal LRP1-mediated efflux. The exposed p-aminophenyl-α-d-mannopyranoside (MAN) modification could further recognize glucose transporter-1 (GLUT1) on GBM and macrophage mannose receptor (MMR) on TAMs. D&R-HM-MCA could achieve chemotherapeutic killing of GBM and simultaneously induce TAM polarization from anti-inflammatory M2 phenotype to pro-inflammatory M1 phenotype, thus resensitizing the chemotherapeutic response and improving anti-GBM immune response. This CTSB-responsive brain-targeted delivery system not only can improve brain delivery efficiency, but also can enable the combination of chemo-immunotherapy against GBM. The effectiveness of this strategy may provide thinking for designing more functional brain-targeted delivery systems and more effective therapeutic regimens.

Neu1 deficiency and fibrotic lymph node microenvironment lead to imbalance in M1/M2 macrophage polarization.

Macrophages play a pivotal role in tissue homeostasis, pathogen defense, and inflammation resolution. M1 and M2 macrophage phenotypes represent two faces in a spectrum of responses to microenvironmental changes, crucial in both physiological and pathological conditions. Neuraminidase 1 (Neu1), a lysosomal and cell surface sialidase responsible for removing terminal sialic acid residues from glycoconjugates, modulates several macrophage functions, including phagocytosis and Toll-like receptor (TLR) signaling. Current evidence suggests that Neu1 expression influences M1/M2 macrophage phenotype alterations in the context of cardiovascular diseases, indicating a potential role for Neu1 in macrophage polarization. For this reason, we investigated the impact of Neu1 deficiency on macrophage polarization in vitro and in vivo. Using bone marrow-derived macrophages (BMDMs) and peritoneal macrophages from Neu1 knockout (Neu1-/- ) mice and wild-type (WT) littermate controls, we demonstrated that Neu1-deficient macrophages exhibit an aberrant M2-like phenotype, characterized by elevated macrophage mannose receptor 1 (MMR/CD206) expression and reduced responsiveness to M1 stimuli. This M2-like phenotype was also observed in vivo in peritoneal and splenic macrophages. However, lymph node (LN) macrophages from Neu1-/- mice exhibited phenotypic alterations with reduced CD206 expression. Further analysis revealed that peripheral LNs from Neu1-/- mice were highly fibrotic, with overexpression of transforming growth factor-beta 1 (TGF-β1) and hyperactivated TGF-β signaling in LN macrophages. Consistently, TGF-β1 was found to alter M1/M2 macrophage polarization in vitro. Our findings showed that Neu1 deficiency prompts macrophages towards an M2 phenotype and that microenvironmental changes, particularly increased TGF-β1 in fibrotic tissues such as peripheral LNs in Neu1-/- mice, further influence M1/M2 macrophage polarization, highlighting its sensitivity to the local microenvironment. Therapeutic interventions targeting Neu1 or TGF-β signaling pathways may offer the potential to regulate macrophage behavior across different diseases.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Polymer-Based Nanoparticles as Drug Delivery Systems for Purines of Established Importance in Medicine.

Many purine derivatives are active pharmaceutical ingredients of significant importance in the therapy of autoimmune diseases, cancers, and viral infections. In many cases, their medical use is limited due to unfavorable physicochemical and pharmacokinetic properties. These problems can be overcome by the preparation of the prodrugs of purines or by combining these compounds with nanoparticles. Herein, we aim to review the scientific progress and perspectives for polymer-based nanoparticles as drug delivery systems for purines. Polymeric nanoparticles turned out to have the potential to augment antiviral and antiproliferative effects of purine derivatives by specific binding to receptors (ASGR1-liver, macrophage mannose receptor), increase in drug retention (in eye, intestines, and vagina), and permeation (intranasal to brain delivery, PEPT1 transport of acyclovir). The most significant achievements of polymer-based nanoparticles as drug delivery systems for purines were found for tenofovir disoproxil in protection against HIV, for acyclovir against HSV, for 6-mercaptopurine in prolongation of mice ALL model life, as well as for 6-thioguanine for increased efficacy of adoptively transferred T cells. Moreover, nanocarriers were able to diminish the toxic effects of acyclovir, didanosine, cladribine, tenofovir, 6-mercaptopurine, and 6-thioguanine.

Covalent Conjugates of Allylbenzenes and Terpenoids as Antibiotics Enhancers with the Function of Prolonged Action.

The drug resistance of pathogenic bacteria is often due efflux pumps-specific proteins that remove foreign compounds from bacterial cells. To overcome drug resistance, adjuvants are often used that can inhibit efflux pumps or other systems that ensure the resistance of bacteria to the action of antibiotics. We assumed that a new level of effectiveness with the use of an antibiotic + an adjuvant pair could be achieved by their joint delivery into the pathogen. To test this hypothesis, we constructed a series of molecular carriers based on poly-(olygo-, dendry)mers based on cyclodextrin-grafted PEI or mannan, as well as glycol chitosan, covalently bound to antibiotic, adjuvant, and the oligosaccharide ligand to the macrophage mannose receptor (CD206), which we studied earlier and showed high efficiency and selectivity of delivery of a therapeutic "cargo" to macrophages. Moxifloxacin was used as an antibiotic, and terpenoid and allylbenzene compounds were used as adjuvants, for which we previously discovered the ability to inhibit bacterial efflux pumps. We show that: (a) the resulting structures were stable in vitro for a long time (up to 10 days); (b) they were adsorbed on bacterial cells, providing a local increase in the concentration of the antibiotic and adjuvant in pathogen cells; (c) they were internalized by bacterial cells, ensuring the accumulation of both antibiotic and adjuvant inside bacterial cells; (d) the adjuvant, after entering the bacterial cell, provided inhibition of the efflux pumps; (e) due to this action of the adjuvant, combined with the targeted delivery by the carrier, the antibiotic's half-life in rats increased by more than 2 times, the effective concentration of the drug in the blood plasma (AUC) increased up to 8-10 times; (f) a significant increase in the effectiveness of the antibacterial action against Gram+ and Gram- cells was achieved (up to 3 times). Potentially, such an approach would significantly increase the effectiveness of therapies for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing bacterial resistance. Moreover, the use of a polymer carrier with covalently bound organic molecules of different structures will avoid problems linked to different (suboptimal) solubility and bio-distribution of the administered molecules, which would be almost inevitable when using the same compounds separately. It would be very difficult to find antibiotic/adjuvant pairs that simultaneously achieve optimal concentrations in the same target cells. In our case, terpenoids and alkylbenzenes used as adjuvants are practically insoluble as individual compounds, and their unacceptable pharmacological properties would not allow them to be used as efflux pump inhibitors.

Exploring the conformational dynamics of the CRD4: a model for receptor lysosomal activity shifts in search of 'sweet’ and ‘sour’ states

The macrophage mannose receptor (RMM) is a crucial component of the immune system involved in immune responses, inflammation resolution, and tissue remodeling. When RMM is activated by a specific ligand, it undergoes internalization, forming an endosome that matures into a lysosome. Within the lysosome, structural changes in RMM facilitate the dissociation of ligands for further processing. However, the precise details of these structural changes are not well understood. In this study, we used molecular dynamics simulations to investigate the conformational dynamics of a specific region called CRD4 in RMM. Our simulations explored different conditions, including pH variations and the presence of Ca2+ ions. By analyzing the simulation data, we found that conformational changes primarily occur in loop regions, while the secondary structure remains stable. The binding site of CRD4, essential for ligand interaction, is located on the protein surface between two specific loop regions. Ligand binding is stabilized by three important amino acids. Interestingly, the interaction patterns differ between monosaccharide and disaccharide ligands. These findings improve our understanding of CRD4's dynamics and how it recognizes ligands. They provide insights into the structure of CRD4 and its role in ligand dissociation within lysosomes. The study also highlights the significance of loop regions in functional dynamics and interactions. Further research is needed to fully uncover the complete structure of CRD4, understand ligand binding modes, and explore the influence of environmental factors. This study lays the foundation for future investigations targeting carbohydrate-protein interactions and the development of therapeutics based on RMM's unique properties. Communicated by Ramaswamy H. Sarma

Competitive ELISA for the identification of 35-55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan.

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG35-55 ) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG35-55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG35-55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG35-55 peptide may undergo when it is bound to mannan during production and stability studies.

Bio-inspired Glycosylated Nano-hydroxyapatites Enhance Endogenous Bone Regeneration by Modulating Macrophage M2 Polarization.

A macrophage-associated immune response is vital in bone regeneration. Mannose receptor (MR), a macrophage pattern-recognition receptor, is crucial for the maintenance of immune homeostasis. Here, we designed MR-targeted glycosylated nano-hydroxyapatites (GHANPs) to reprogram macrophages into polarized M2s, promoting bone regeneration by improving the osteoimmune microenvironment. The prepared GHANPs induced macrophage M2 polarization, which then promoted osteoblastic differentiation of stem cells. Further, the mechanistic study showed that GHANPs might influence macrophage polarization by modulating cell metabolism, including enhancing mitochondrial oxidative phosphorylation and activating autophagy. Finally, a rat cranial defect model was used to verify the effect of GHANPs on endogenous bone regeneration in vivo, revealing that GHANPs promoted bone regeneration within the defect and increased the ratio of M2/M1 macrophages in early bone repair. Our results indicate that the MR-targeted macrophage M2 polarization strategy is promising in endogenous bone regeneration. STATEMENT OF SIGNIFICANCE: Macrophage is a pivotal immunity component for bone regeneration. A switch to M2 macrophage has been considered to contribute to osteogenesis. For inducing macrophage M2 polarization, an effective strategy to overcome off-target effects and insufficient specificity is a critical challenge. The mannose receptor on the surface of macrophages has been involved in regulating macrophage directional polarization. The glucomannan presented on the nano-hydroxyapatite rods acts as ligands targeting macrophage mannose receptors to promote their M2 polarization, improving the immunomicroenvironment and achieving bone regeneration. This approach has the advantage of easy preparation, specific regulation, and safety.

Abstract 2385: Targeting mannose receptor expression on macrophages for sentinel lymph node mapping in endometrial cancer: murine model

Abstract Background: Sentinel lymph node (SLN) mapping has been used in various cancer surgery including endometrial cancer as a targeted assessment tool for identifying lymph node metastases, an alternative for complete lymphadenectomy to minimize surgical morbidity. Neomannosyl human serum albumin (MSA) targets the macrophage mannose receptor CD206 which is highly expressed in metastatic lymph node. The aim of this study was to evaluate the effectiveness of indocyanine green-neomannosyl human serum albumin (MSA:ICG) which targets macrophage mannose receptor CD206 for SLN mapping. Methods: LN targeting ability was examined on mouse footpad models of uterine cancer using MSA:ICG and ICG for comparison. Seven mice with uterine cancer and 2 mice without cancer as a control group were each assigned to MSA:ICG and ICG groups. After injection of tracers, fluorescence images were taken and SLN biopsy was done to confirm LN metastasis status. We compared the signal-to-background ratio (SBR) according to time. Results: In both MSA:ICG and ICG group, 16 lymph nodes out of 18 were detected using fluorescence imaging (detection rate: 88.9%). Four lymph nodes in MSA:ICG group and 5 lymph nodes in ICG group were metastatic among 16 lymph node each group. Excluding lymph nodes that were not detected, the SBR after the injection of MSA:ICG group was significantly higher than ICG group in metastatic LN (MSA:ICG 4.36 vs ICG 3.07, p&amp;lt;0.001), non-metastatic lymph nodes (MSA:ICG 3.11 vs ICG 1.93, p=0.001), and normal control lymph nodes (MSA:ICG 1.38 vs ICG 0.54, p&amp;lt;0.001). Comparing signal intensity according to CD206 expression, there was no difference in SBR values between the two groups in patients without CD206 expression, but the MSA group had a statistically significantly higher SBR value than the ICG group (p&amp;lt;0.001) in patients with CD206 moderate or high expression. Conclusions: This study showed that MSA:ICG may be an effective tracer for sentinel lymph node mapping. In addition, the metastatic lymph nodes expressing CD206 showed higher signal than normal lymph nodes in MSA:ICG group, unlike in ICG group. Further studies using larger animal models and clinical trials are needed to confirm this result. Citation Format: Hyun-Woong Cho, OK Hwa Jeon, Sanghoon Lee, Hyun Koo Kim, Jae Kwan Lee, Jin Hwa Hong. Targeting mannose receptor expression on macrophages for sentinel lymph node mapping in endometrial cancer: murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2385.

Targeted O-glycoproteomics for the development of diagnostic markers for advanced colorectal cancer.

Aberrant glycosylation is a prominent feature of cancer, that can be used as targets to improve the existing cancer biomarkers, and help to assess metastasis risks, and therapeutic effects. We developed a targeted O-glycoproteomics method using serum specimens, and evaluated its utility in identifying advanced colorectal cancer (CRC) markers. To this end, we combined consecutive lectin affinity purification using Maclura pomifera lectin (MPL), jacalin, and Sambucus nigra lectin, which have affinities for the following O-glycans, that have received attention as cancer-related antigens, Tn (GalNAc-Ser/Thr), Sialyl Tn (Siaα2-6GalNAc-Ser/Thr), T (Galβ1-3GalNAc-Ser/Thr), Sialyl T (Siaα2-3Galβ1-GalNAc-Ser/Thr), and di-Sialyl T (Siaα2-3Galβ1-3[Siaα2-6] GalNAc-Ser/Thr), with a unique O-glycoproteomics approach. A total of 2,068 O-glycoforms derived from 265 proteins were identified in healthy individuals and patients with advanced CRC, of which 44 CRC-specific O-glycoforms were extracted. Particularly, five glycoproteins with T, Sialyl T, and di-Sialyl T antigens in specific peptide regions were evaluated quantitatively and statistically. We found that fibulin-2 (FBLN2) (aa330-349)/T antigen (area under the curve [AUC] = 0.92); macrophage colony-stimulating factor 1 (CSF1) (aa370-395)/(T + di-Sialyl T) (AUC = 0.94); macrophage mannose receptor 1 (MRC1) (aa1083-1101 and aa1215-1229)/T (AUC = 0.96 and 0.99); fibrinogen alpha chain (FGA) (aa354-367, aa511-527 and aa559-573)/Sialyl T (AUC = 0.98, 0.90 and 0.94); and complement component C7 (C7) (aa692-701)/di-Sialyl T (AUC = 1.00), can have high diagnostic efficacy to strategically predict advanced CRC groups. Hence, they could be promising markers for detection of advanced CRC, and provide new clinical test indicators along with lectins, such as MPL and jacalin. Our O-glycoproteomics platform provides a novel tool and resource, for researchers and clinicians seeking to better understand and treat advanced CRC.

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP)

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.

The Long Noncoding RNA Gm9866/Nuclear Factor-κB Axis Promotes Macrophage Polarization.

Macrophages are a type of immune cells with high levels of plasticity and heterogeneity. They can polarize into M1 or M2 functional phenotypes. These two phenotypes exhibit a dynamic balance during polarization-related diseases and play opposing roles. Long noncoding RNAs (lncRNAs) play an important role in biological processes such as cell proliferation, death, and differentiation; however, how long noncoding RNAs affect the cellular functionality of macrophages remains to be studied. Long noncoding RNA Gm9866 was found to be closely related to macrophage polarization through bioinformatics analysis. In this study, by conducting real-time polymerase chain reaction analysis, it was observed that long noncoding RNA Gm9866 expression significantly increased after treatment with interleukin-4 but significantly decreased after treatment with lipopolysaccharide. Fluorescence in situ hybridization revealed that long noncoding RNA Gm9866 was expressed mainly in the nucleus. Real-time polymerase chain reaction analysis showed that overexpression of long noncoding RNA Gm9866 in RAW264.7 cells further promoted the expression of M2 markers MRC1 (macrophage mannose receptor 1) and MRC2 (macrophage mannose receptor 2). Western blotting analysis demonstrated inhibition of nuclear factor-κB (NF-κB) expression. EdU (5-ethynyl-2'-deoxyuridine) and TUNEL (TdT-mediated dUTP nick-end labeling) staining assays revealed that overexpression of long noncoding RNA Gm9866 promoted cell proliferation and inhibited apoptosis. These findings thus indicated that long noncoding RNA Gm9866 promoted macrophage polarization and inhibited the nuclear factor-κB signaling pathway. Thus, long noncoding RNA Gm9866 may serve as a potential diagnostic and therapeutic target for polarization-related diseases such as infectious diseases, inflammatory diseases, liver fibrosis, and tumors.