The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Interaction of human dendritic cell receptor DEC205/CD205 with keratins.

DEC205 (CD205) is one of the major endocytic receptors on dendritic cells and has been widely used as a receptor target in immune therapies. It has been shown that DEC205 can recognize dead cells through keratins in a pH-dependent manner. However, the mechanism underlying the interaction between DEC205 and keratins remains unclear. Here we determine the crystal structures of an N-terminal fragment of human DEC205 (CysR∼CTLD3). The structural data show that DEC205 shares similar overall features with the other mannose receptor family members such as the mannose receptor and Endo180, but the individual domains of DEC205 in the crystal structure exhibit distinct structural features that may lead to specific ligand binding properties of the molecule. Among them, CTLD3 of DEC205 adopts a unique fold of CTLD, which may correlate with the binding of keratins. Furthermore, we examine the interaction of DEC205 with keratins by mutagenesis and biochemical assays based on the structural information and identify an XGGGX motif on keratins that can be recognized by DEC205, thereby providing insights into the interaction between DEC205 and keratins. Overall, these findings not only improve the understanding of the diverse ligand specificities of the mannose receptor family members at the molecular level but may also give clues for the interactions of keratins with their binding partners in the corresponding pathways.

Detection of metastatic lymph node and sentinel lymph node mapping ​using mannose receptor targeting in in vivo mouse footpad tumor models and rabbit uterine cancer models.

BACKGROUND: This study aimed to evaluate the effectiveness of neo-mannosyl human serum albumin-indocyanine green (MSA-ICG) for detecting metastatic lymph node (LN) and mapping sentinel lymph node (SLN) using mouse footpad uterine tumor models. Additionally, the authors assessed the feasibility of MSA-ICG in SLN mapping in rabbit uterine cancer models. MATERIALS AND METHODS: The authors compared the LN targeting ability of MSA-ICG with ICG. Six mouse footpad tumor models and two normal mice were each assigned to MSA-ICG and ICG, respectively. After the assigned tracers were injected, fluorescence images were taken, and the authors compared the signal-to-background ratio (SBR) of the tracers. A SLN biopsy was performed to confirm LN metastasis status and CD206 expression level. Finally, an intraoperative SLN biopsy was performed in rabbit uterine cancer models using MSA-ICG. RESULTS: The authors detected 14 groin LNs out of 16 in the MSA-ICG and ICG groups. The SBR of the MSA-ICG group was significantly higher than that of the ICG group. The metastatic LN subgroup of MSA-ICG showed a significantly higher SBR than that of ICG. CD206 was expressed at a high level in metastatic LN, and the signal intensity difference increased as the CD206 expression level increased. SLN mapping was successfully performed in two of the three rabbit uterine cancer models. CONCLUSIONS: MSA-ICG was able to distinguish metastatic LN for an extended period due to its specific tumor-associated macrophage-targeting property. Therefore, it may be a more distinguishable tracer for identifying metastatic LNs and SLNs during uterine cancer surgery. Further research is needed to confirm these results.

Lipid nanoparticles containing labile PEG-lipids transfect primary human skin cells more efficiently in the presence of apoE.

Nucleic acid-based therapeutics encapsulated into lipid nanoparticles (LNPs) can potentially target the root cause of genetic skin diseases. Although nanoparticles are considered impermeable to skin, research and clinical studies have shown that nanoparticles can penetrate into skin with reduced skin barrier function when administered topically. Studies have shown that epidermal keratinocytes express the low-density lipoprotein receptor (LDLR) that mediates endocytosis of apolipoprotein E (apoE)-associated nanoparticles and that dermal fibroblasts express mannose receptors. Here we prepared LNPs designed to exploit these different endocytic pathways for intracellular mRNA delivery to the two most abundant skin cell types, containing: (i) labile PEG-lipids (DMG-PEG2000) prone to dissociate and facilitate apoE-binding to LNPs, enabling apoE-LDLR mediated uptake in keratinocytes, (ii) non-labile PEG-lipids (DSPE-PEG2000) to impose stealth-like properties to LNPs to enable targeting of distant cells, and (iii) mannose-conjugated PEG-lipids (DSPE-PEG2000-Mannose) to target fibroblasts or potentially immune cells containing mannose receptors. All types of LNPs were prepared by vortex mixing and formed monodisperse (PDI âˆ¼ 0.1) LNP samples with sizes of 130 nm (±25%) and high mRNA encapsulation efficiencies (≥90%). The LNP-mediated transfection potency in keratinocytes and fibroblasts was highest for LNPs containing labile PEG-lipids, with the addition of apoE greatly enhancing transfection via LDLR. Coating LNPs with mannose did not improve transfection, and stealth-like LNPs show limited to no transfection. Taken together, our studies suggest using labile PEG-lipids and co-administration of apoE when exploring LNPs for skin delivery.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Soluble mannose receptor: A potential biomarker in Gaucher disease.

PURPOSE: Soluble mannose receptor (sMR) relates to mannose receptor expression on macrophages, and is elevated in inflammatory disorders. Gaucher disease (GD) has altered macrophage function and utilises mannose receptors for enzyme replacement therapy (ERT) endocytosis. sMR has not previously been studied in GD. METHODS: sMR was measured by ELISA and correlated with GD clinical features including spleen and liver volume, haemoglobin and platelet count, bone marrow burden (BMB) scores and immunoglobulin levels. sMR was compared with biomarkers of GD: chitotriosidase, lyso-GL1, PARC, CCL3, CCL4, osteoactivin, serum ACE and ferritin. RESULTS: Median sMR in untreated GD patients was 303.0 ng/mL compared to post-treatment 190.9 ng/mL (p = .02) and healthy controls 202 ng/mL. Median sMR correlated with median spleen volume 455 mL (r = .70, p = .04), liver volume 2025 mL (r = .64, p = .04), BMB 7 (r = .8, p = .03), IgA 1.9 g/L (r = .54, p = .036), IgG 9.2 g/L (r = .57, p = .027), IgM 1.45 g/L (r = .86, p < .0001), with inverse correlation to median platelet count of 125 × 109/L (r = -.47, p = .08) and haemoglobin of 137 g/L (r = -.77, p = .0008). sMR correlated with established biomarkers: osteoactivin 107.8 ng/mL (r = .58, p = .0006), chitotriosidase 3042 nmol/mL/h (r = .52, p = .0006), PARC 800 ng/mL (r = .67, p = .0068), ferritin 547 µg/L (r = .72, p = .002) and CCL3 50 pg/mL (r = .67, p = .007). CONCLUSIONS: sMR correlates with clinical features and biomarkers of GD and reduces following therapy.

Transcriptional regulation of the HIV-1 inhibitory factor human mannose receptor 1 by the myeloid-specific transcription factor PU.1.

HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.

[Albumin-based Drug Delivery System Targeting Mannose Receptors and Its Application to Medical Treatments].

The onset and progression of liver diseases and cancer have shown to be affected by over-active macrophages and fibroblasts. Therefore, developing methods to suppress the activation of these cells has become an urgent task. Prior to this study, a mannosylated-albumin (Man-HSA) that targets mannose receptors expressed in hepatic macrophages (Kupffer cells) or fibroblasts was created. Here, we report on the development of medical treatments based on Man-HSA. To target the reactive oxygen species or inflammation derived from Kupffer cells, we developed a nano-antioxidant, i.e., polythiolated (SH)-Man-HSA, by introducing thiol groups into Man-HSA, or a nano-anti-inflammatory drug, i.e., Man-HSA-IFNα2b, by fusing Man-HSA and IFNα2b. SH-Man-HSA or Man-HSA-IFNα2b attenuated Kupffer cell-derived oxidative stress or inflammation, respectively, resulting in the suppression of liver damage and overall improvement of the survival rate in mice with acute and chronic liver injuries. Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF), both of which are present in the stroma of intractable cancers, also express mannose receptors. Thus, mono-polyethylene glycol modified Man-HSA (monoPEG-Man-HSA) was synthesized as a novel drug delivery carrier targeting TAM/CAF. A complex of monoPEG-Man-HSA with paclitaxel suppressed tumor growth by decreasing the number of TAM/CAF and the stroma area. For the present study, we focused on the mannose receptors expressed in macrophages and fibroblasts, and developed drug delivery carriers that target these cells. Considering the excellent drug-carrying capacity and high biocompatibility of HSA, it is expected that this research will pave the way for innovative pharmacotherapy to treat unmet medical needs, i.e., intractable liver diseases and cancer.

Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer.

Chromosome instability (CIN) contributes to resistance to therapies and tumor evolution. Although natural killer (NK) cells can eliminate cells with complex karyotypes, high-CIN human tumors have an immunosuppressive phenotype. To understand which CIN-associated molecular features alter immune recognition during tumor evolution, we overexpress Polo-like kinase 1 (Plk1) in a Her2+ breast cancer model. These high-CIN tumors activate a senescence-associated secretory phenotype (SASP), upregulate PD-L1 and CD206, and induce non-cell-autonomous nuclear factor κB (NF-κß) signaling, facilitating immune evasion. Single-cell RNA sequencing from pre-neoplastic mammary glands unveiled the presence of Arg1+ macrophages, NK cells with reduced effector functions, and increased resting regulatory T cell infiltration. We further show that high PLK1-expressing human breast tumors display gene expression patterns associated with SASP, NF-κß signaling, and immune suppression. These findings underscore the need to understand the immune landscape in CIN tumors to identify more effective therapies, potentially combining immune checkpoint or NF-κß inhibitors with current treatments.

Human Mannose Receptor 1 Attenuates HIV-1 Infectivity in a Virus Isolate-Specific Manner.

Human mannose receptor 1 (hMRC1) is a transmembrane glycoprotein that belongs to the C-type lectin family and is expressed on the surface of most tissue macrophages. hMRC1 contributes to the binding and transmission of HIV-1 and is involved in the endocytic uptake of HIV-1 for subsequent antigen presentation. We previously reported that hMRC1 functions as an antiviral factor by inhibiting virus release through a BST-2-like mechanism. The inhibition of virus release was not virus isolate-specific and, surprisingly, was not Env-dependent. We now report on another hMRC1 antiviral function that affects the infectivity of viral particles. Unlike its effect on virus release, the inhibition of viral infectivity by hMRC1 was virus isolate-specific. An analysis of chimeric Env revealed that the Env V3 region was a critical determinant for the inhibitory effect of hMRC1. Of note, exogenously expressed hMRC1 was packaged into viral particles in an Env-independent manner. Co-immunoprecipitation studies revealed a strong interaction of the hMRC1-sensitive NL43 Env with hMRC1, while the hMRC1-insensitive Envs of AD8 and 49.5 isolates interacted poorly if at all with hMRC1. An analysis of a panel of Transmitted/Founder (T/F) viruses revealed that all of them were R5-tropic, and more than half of them were inhibited by hMRC1. The detailed mechanism of how hMRC1 inhibits viral infectivity remains to be investigated. However, the high-affinity binding of hMRC1 to Env may cause a conformational change around the Env V3 region or obstruct the Env V3 region and may make it inaccessible for subsequent interaction with the coreceptor during virus entry.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation. Binding and uptake capacities of naked and targeted LPR increase with the percent of cationic lipid, but the latter are 2-fold more up taken by the cells. Cationic LPR bearing 5 % and 10 % TM were localized in acidic compartments in contrast to naked LPR and 2.5 % TM-LPR. The drawback is the dramatic decrease of TE as the number of TM-units increases. Cationic LPR bearing 5 % and 10 % TM strongly induced NF-κB and PKR phosphorylation at 6 h. Conversely, mTOR is less activated in line with their low TE. Those side effects are overcome by using 5-methoxyuridine mRNA resulting in an improved TE due to non-phosphorylation of NF-κB and PKR and mTOR activation. Our results point out that targeting DC via mannose receptor triggers a higher uptake of cationic LPRs and fast routing to acidic compartments, and that efficient TE requires low number of TM units use or modified mRNA to escape RNA sensors activation to enhance the translation.

Intra-Operative Definition of Glioma Infiltrative Margins by Visualizing Immunosuppressive Tumor-Associated Macrophages.

Accurate delineation of glioma infiltrative margins remains a challenge due to the low density of cancer cells in these regions. Here, a hierarchical imaging strategy to define glioma margins by locating the immunosuppressive tumor-associated macrophages (TAMs) is proposed. A pH ratiometric fluorescent probe CP2-M that targets immunosuppressive TAMs by binding to mannose receptor (CD206) is developed, and it subsequently senses the acidic phagosomal lumen, resulting in a remarkable fluorescence enhancement. With assistance of CP2-M, glioma xenografts in mouse models with a tumor-to-background ratio exceeding 3.0 for up to 6 h are successfully visualized. Furthermore, by intra-operatively mapping the pH distribution of exposed tissue after craniotomy, the glioma allograft in rat models is precisely excised. The overall survival of rat models significantly surpasses that achieved using clinically employed fluorescent probes. This work presents a novel strategy for locating glioma margins, thereby improving surgical outcomes for tumors with infiltrative characteristics.

Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

A Mannose Receptor from Litopenaeus vannamei Involved in Innate Immunity by Pathogen Recognition and Inflammation Regulation.

Mannose receptor, as a member of the C-type lectin superfamily, is a non-canonical pattern recognition receptor that can internalize pathogen-associated ligands and activate intracellular signaling. Here, a mannose receptor gene, LvMR, was identified from the Pacific white shrimp Litopenaeus vannamei. LvMR encoded a signal peptide, a fibronectin type II (FN II) domain, and two carbohydrate-recognition domains (CRDs) with special EPS and FND motifs. LvMR transcripts were mainly detected in the hepatopancreas, and presented a time-dependent response after pathogen challenge. The recombinant LvMR (rLvMR) could bind to various PAMPs and agglutinate microorganisms in a Ca2+-dependent manner with strong binding ability to D-mannose and N-acetyl sugars. The knockdown of LvMR enhanced the expression of most NF-κB pathway genes, inflammation and redox genes, while it had no obvious effect on the transcription of most phagocytosis genes. Moreover, the knockdown of LvMR caused an increase in reactive oxygen species (ROS) content and inducible nitric oxide synthase (iNOS) activity in the hepatopancreas after Vibrio parahaemolyticus infection. All these results indicate that LvMR might perform as a PRR in immune recognition and a negative regulator of inflammation during bacterial infection.

Targeting the Mannose Receptor with Functionalized Fucoidan/Chitosan Nanoparticles Triggers the Classical Activation of Macrophages.

Understanding how nanoparticles' properties influence their cellular interactions is a bottleneck for improving the design of carriers. Macrophage polarization governs their active role in solving infections or tissue repair. To unravel the effect of carbohydrate-targeting mannose receptors on the macrophage surface, drug-free fucoidan/chitosan nanoparticles were functionalized using mannose (M) and mannan (Mn). Polyelectrolyte complex nanoparticles were obtained upon chitosan self-assembly using fucoidan. The functionalized nanoparticles were characterized in terms of their physicochemical characteristics, chemical profile, and carbohydrate orientation. The nanoparticles varied in size from 200 to 400 nm, were monodisperse, and had a stable negative zeta potential with a low aggregation tendency. The nonfunctionalized and functionalized nanoparticles retained their properties for up to 12 weeks. Cell viability and internalization studies were performed for all the designed nanoparticles in the THP-1 monocytes and THP-1-differentiated macrophages. The expression of the mannose receptor was verified in both immune cells. The carbohydrate-functionalized nanoparticles led to their activation and the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumour necrosis factor (TNF)-α. Both M- and Mn-coated nanoparticles modulate macrophages toward an M1-polarized state. These findings demonstrate the tailoring of these nanoplatforms to interact and alter the macrophage phenotype in vitro and represent their therapeutic potential either alone or in combination with a loaded drug for future studies.

A Meaningful Attempt: Applying Dielectric Barrier Discharge Plasma to Induce Polarization of Macrophages.

Macrophage polarization plays an important role in many macrophage-related diseases. This study was designed to preliminarily explore the effects of dielectric barrier discharge (DBD) plasma on the polarization direction and cell activity of macrophages with different phenotypes (ie, M0, M1, and M2). The M1 macrophage marker inducible nitric oxide synthase (iNOS) and M2 macrophage marker cluster of differentiation 206 (CD206) were detected by western blot (WB). The effects of DBD plasma on macrophage viability were analyzed by using a cell counting kit-8 detection kit. M0, M1, and M2 macrophages exhibited a decrease in iNOS expression and an increase in CD206 expression after the DBD plasma intervention. Additionally, the decrease in macrophage viability remained non-significant after initiating the intervention. DBD plasma can promote the transformation of M0 and M1 macrophages to M2 macrophages, and can further enhance the expression of the M2 macrophage phenotype marker CD206. Our study not only demonstrates the potential therapeutic value of DBD plasma for macrophage-related diseases, but it also provides a new direction for research to improve the treatment of macrophage-related diseases. © 2023 Bioelectromagnetics Society.

Evaluation of a CD206-Targeted Peptide for PET Imaging of Macrophages in Syngeneic Mouse Models of Cancer.

Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 µM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.

Aluminum Fluoride-18 Labeled Mannosylated Dextran: Radiosynthesis and Initial Preclinical Positron Emission Tomography Studies.

PURPOSE: In addition to being expressed on liver sinusoidal endothelial cells, mannose receptors are also found on antigen-presenting cells, including macrophages, which are mainly involved in the inflammation process. Dextran derivatives of various sizes containing cysteine and mannose moieties have previously been labeled with 99mTc and used for single-photon emission computed tomography imaging of sentinel lymph nodes. In this study, we radiolabeled 21.3-kDa D10CM with positron-emitting 18F for initial positron emission tomography (PET) studies in rats. PROCEDURES: D10CM was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and radiolabeled with the aluminum fluoride-18 method. The whole-body distribution kinetics and stability of the intravenously administered tracer were studied in healthy male Sprague-Dawley rats by in vivo PET/CT imaging, ex vivo gamma counting, and high-performance liquid chromatography analysis. RESULTS: Al[18F]F-NOTA-D10CM was obtained with a radiochemical purity of >99% and molar activity of 9.9 GBq/µmol. At 60 minutes after injection, an average of 84% of the intact tracer was found in the blood, indicating excellent in vivo stability. The highest radioactivity concentration was seen in the liver, spleen, and bone marrow, in which mannose receptors are highly expressed under physiological conditions. The uptake specificity was confirmed with in vivo blocking experiments. CONCLUSIONS: Our results imply that Al[18F]F-NOTA-D10CM is a suitable tracer for PET imaging. Further studies in disease models with mannose receptor CD206-positive macrophages are warranted to clarify the tracer's potential for imaging of inflammation.

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.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.