Podoplanin expressing macrophages and their involvement in tertiary lymphoid structures in mouse models of Sjögren's disease.

Tertiary lymphoid structures (TLSs) are formed in tissues targeted by chronic inflammation processes, such as infection and autoimmunity. In Sjögren's disease, the organization of immune cells into TLS is an important part of disease progression. Here, we investigated the dynamics of tissue resident macrophages in the induction and expansion of salivary gland TLS. We induced Sjögren's disease by cannulation of the submandibular glands of C57BL/6J mice with LucAdV5. In salivary gland tissues from these mice, we analyzed the different macrophage populations prior to cannulation on day 0 and on day 2, 5, 8, 16 and 23 post-infection using multicolored flow cytometry, mRNA gene analysis, and histological evaluation of tissue specific macrophages. The histological localization of macrophages in the LucAdV5 induced inflamed salivary glands was compared to salivary glands of NZBW/F1 lupus prone mice, a spontaneous mouse model of Sjögren's disease. The evaluation of the dynamics and changes in macrophage phenotype revealed that the podoplanin (PDPN) expressing CX3CR1+ macrophage population was increased in the salivary gland tissue during LucAdV5 induced inflammation. This PDPN+ CX3CR1+ macrophage population was, together with PDPN+CD206+ macrophages, observed to be localized in the parenchyma during the acute inflammation phase as well as surrounding the TLS structure in the later stages of inflammation. This suggests a dual role of tissue resident macrophages, contributing to both proinflammatory and anti-inflammatory processes, as well as their possible interactions with other immune cells within the inflamed tissue. These macrophages may be involved with lymphoid neogenesis, which is associated with disease severity and progression. In conclusion, our study substantiates the involvement of proinflammatory and regulatory macrophages in autoimmune pathology and underlines the possible multifaceted functions of macrophages in lymphoid cell organization.

Development of molecular sensors based on fluorescent proteins for polarized macrophages identification.

Macrophages are a type of white blood cells that play key roles in innate immune responses as a part of cellular immunity for host defence and tissue homeostasis. To perform diverse functions, macrophages show high plasticity by transforming to polarized states. They are mainly identified as unpolarized, pro-inflammatory and antiinflammatory states and termed as M0, M1 and M2 macrophages respectively. Discriminating polarized states is important due to strict implication with inflammatory conditions resulting in many diseases as chronic inflammation, neurodegeneration, and cancer etc. Many polarization protein markers have been identified and applied to investigate expression profiles through PCR and other techniques with antibodies. However, they are time and cost consuming and sometimes show insufficient performances. We focused on the mannose receptor (CD206) as representative marker of M2 macrophage recognising terminal mannose. We developed dose dependent mannosylated fluorescent proteins (FPs) by conjugations with mannose derivative for around 20 modifiable sites on FPs surfaces. Maximum modifications did not spoil various features of FPs. We found further sensitive and specific discriminations among M2, M1 and M0 macrophages after treating polarized macrophages with adequately conditioned FPs compared to already established approaches using anti CD206 antibody through flow cytometric analysis. These results might be derived from direct ligand utilizations and increased avidity due to multivalent bindings with abundantly modified multimeric FPs. Our strategy is simple but addresses disadvantages of preceding methods. Moreover, this strategy is applicable to detect other cell surface receptors as FPs can be modified with ligands or recognizable aptamer like molecules.

Decoding the mannose receptor-mAb interaction: the importance of high-mannose N-glycans and glycan-pairing.

During the development process of therapeutic monoclonal antibodies (mAbs), it is crucial to control (critical) quality attributes such as N-glycosylation influencing pharmacokinetics (PK) and Fc effector functions. Previous reports have shown that mAbs containing high-mannose N-glycans are cleared faster from blood circulation, leading to reduced half-lives. The high-mannose N-glycan content of mAbs can be influenced during the cell culture process by factors such as cell lines, process conditions, and media. Furthermore, mAbs have either one high mannose N-glycan (asymmetrical high-mannose glyco-pair) or two high mannose N-glycans (symmetrical high-mannose glyco-pair). The hypothesis that the mannose receptor (MR, CD206) accelerates clearance by facilitating their internalization and subsequent lysosomal degradation is widespread. However, the interaction between MR and mAbs has not been explicitly demonstrated. This study aimed to investigate this interaction, providing the first systematic demonstration of MR binding to the Fc region of mAbs with high-mannose N-glycans. Two novel analytical methods, MR surface plasmon resonance and MR affinity chromatography, were developed and applied to investigate the MR-mAb interaction. The interaction is found to be dependent on high-mannose content, but is independent of the mAb format or sequence. However, different glyco-pairs exhibited varying binding affinities to the MR, with the symmetrical high-mannose glyco-pair showing the strongest binding properties. These findings strengthen the hypothesis for the MR-mediated mAb interaction and contribute to a deeper understanding of the MR-mAb interaction, which could affect the criticality of high-mannose containing mAbs development strategies of IgG-based molecules and improve their PK profiles.

Characteristics and functional analysis of a novel mannose receptor in Penaeus vannamei.

The mannose receptor (MR) plays a key role in the innate immune system as a pattern recognition receptor. Here, a novel type of mannose receptor, named PvMR2, was identified from Penaeus vannamei (P. vannamei). The PvMR2 coding sequence (CDS) obtained was 988 base pairs in length, encoding a protein consisting of 328 amino acids. This protein includes a signal peptide and two classical C-type lectin domains (CTLD). Quantitative real-time PCR showed that PvMR2 was distributed in all detected tissues, with the highest levels in the intestines and stomach. Following a bacterial challenge with Vibrio anguillarum (V. anguillarum), PvMR2 showed significant up-regulation in both the intestines and stomach of shrimp. To validate the function of PvMR2, recombinant proteins were extracted and purified using a His-tag. The resulting rPvMR2 demonstrated binding capability with lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner, affirming its binding affinity. The purified rPvMR2 demonstrated calcium-independent binding activity towards both Gram-positive bacteria (V. anguilliarum and Vibrio parahaemolyticus) and Gram-negative bacteria (Escherichia coli and Aeromonas Veronii). Antibacterial assays confirmed that rPvMR2 inhibits bacterial growth. Intestinal adhesion and adhesion inhibition experiments confirmed that the rPvMR2 can be used to reduce the adhesion capacity of harmful bacteria in the gut. Phagocytosis experiments have shown that rPvMR2 promotes phagocytosis in hemocytes and protects the host from external infection. Treatment with recombinant PvMR2 significantly bolstered bacterial clearance within the hemolymph and markedly augmented shrimp survival post-infection with V. anguillarum. These results suggest that PvMR2 has agglutination, growth inhibition, adhesion inhibition, clearance promotion, and phagocytosis effects on harmful bacteria, and plays a crucial role in the antimicrobial immune response of P. vannamei.

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.

Targeting tumor-associated macrophages with mannosylated nanotherapeutics delivering TLR7/8 agonist enhances cancer immunotherapy.

Tumor-associated macrophages (TAMs) constitute 50-80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic-co-glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from "cold tumor" into "hot tumor", with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy.

Serum monocyte chemotactic protein 1 and soluble mannose receptor aid predictive diagnosis of pediatric sepsis.

BACKGROUND: To investigate the value of serum monocyte chemotactic protein 1 (MCP-1) and soluble mannose receptor (sMR) for predictive diagnosis of pediatric sepsis. METHODS: This study retrospectively analyzed the data of 82 children with acute and severe signs of inflammation. According to the diagnostic criteria of sepsis, these children were divided into a sepsis group (40 cases) and a non-sepsis group (42 cases). In addition, 50 children who received health examinations during the same time period in Cangzhou Central Hospital were selected as a control group. According to the prognosis of the children in the sepsis group, they were further divided into a survival group (33 cases) and a death group (7 cases). The levels of blood indicators, inflammatory markers, liver and kidney function indicators, MCP-1 level, and sMR were collected from the children. The efficacy of using sMR and MCP-1 levels in the predictive diagnosis of sepsis was analyzed by using the area under the ROC curve (AUC). RESULTS: Serum levels of MCP-1 and sMR were (452.32±2.79) µg/ml and (97.23±.15) µg/ml, respectively, in the sepsis group, significantly higher than those in all controls (P<0.001). In the death group, the levels of white blood cells (WBC), C-reactive protein (CRP), procalcitonin (PCT), sMR, and MCP-1 were significantly higher compared to the survival group (P<0.05). The AUC for CRP in predictive diagnosis of sepsis was 0.9075; the AUC for PCT was 0.8759; the AUC for sMR was 0.9244; and the AUC for MCP-1 was 0.9406. CONCLUSIONS: Serum sMR and MCP-1 levels can help predict the diagnosis of pediatric sepsis.

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ T cell-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.

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.

Antitumor immunostimulatory activity of the traditional Chinese medicine polysaccharide on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignancy, often associated with compromised immune function in affected patients. This can be attributed to the secretion of specific factors by liver cancer cells, which hinder the immune response and lead to a state of immune suppression. Polysaccharides derived from traditional Chinese medicine (TCM) are valuable constituents known for their immunomodulatory properties. This review aims to look into the immunomodulatory effects of TCM polysaccharides on HCC. The immunomodulatory effects of TCM polysaccharides are primarily manifested through the activation of effector T lymphocytes, dendritic cells, NK cells, and macrophages against hepatocellular carcinoma (HCC) both in vivo and in vitro settings. Furthermore, TCM polysaccharides have demonstrated remarkable adjuvant antitumor immunomodulatory effects on HCC in clinical settings. Therefore, the utilization of TCM polysaccharides holds promising potential for the development of novel therapeutic agents or adjuvants with advantageous immunomodulatory properties for HCC.

IL-6 signaling drives self-renewal and alternative activation of adipose tissue macrophages.

Introduction: Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that neutralization of interleukin (IL)-6 in obese AT organ cultures inhibits proliferation of ATMs, which occurs preferentially in alternatively activated macrophage phenotype. Methods: In this study, we investigated AT biology and the metabolic phenotype of mice with myeloid cell-specific IL-6Rα deficiency (Il6ra Δmyel) after normal chow and 20 weeks of high-fat diet focusing on AT inflammation, ATM polarization and proliferation. Using organotypical AT culture and bone marrow derived macrophages (BMDMs) of IL-4Rα knockout mice (Il4ra -/-) we studied IL-6 signaling. Results: Obese Il6ra Δmyel mice exhibited no differences in insulin sensitivity or histological markers of AT inflammation. Notably, we found a reduction of ATMs expressing the mannose receptor 1 (CD206), as well as a decrease of the proliferation marker Ki67 in ATMs of Il6ra Δmyel mice. Importantly, organotypical AT culture and BMDM data of Il4ra -/- mice revealed that IL-6 mediates a shift towards the M2 phenotype independent from the IL-6/IL-4Rα axis. Discussion: Our results demonstrate IL-4Rα-independent anti-inflammatory effects of IL-6 on macrophages and the ability of IL-6 to maintain proliferation rates in obese AT.

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

PURPOSE: Aluminum 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. METHODS: First, 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. RESULTS: Compared 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. CONCLUSION: The uptake of mannosylated dextran derivative Al[18F]F-NOTA-D10CM correlated with CD206 expression and the tracer appears promising for inflammation imaging.

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.

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.

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-ß, 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.

Genetic insights into carbohydrate sulfotransferase 8 and its impact on the immunotherapy efficacy of cancer.

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p < 5 × 10-8) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress T cell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction.

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.

The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish's (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion.

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1ß, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

Estudos de síntese de dendrons dirigidos de NFOH com potencial antichagásico e modelagem molecular do receptor de manose de macrófagos / Studying the synthesis of NFOH targeted dendrons, potentially active in Chagas disease, and the molecular modeling of manose receptor from macrophages

A doença de Chagas, considerada doença extremamente negligenciada, acomete mais de 6 milhões de pessoas ao redor do mundo e mais de 75 milhões de pessoas vivem sob risco da doença. Considerada endêmica em 21 países da América Latina. No Brasil, grassa, sobretudo, na região Norte, especialmente, na região amazônica. Apesar de se constituir em risco global, a doença de Chagas conta com apenas com dois fármacos, o benznidazol e o nifurtimox, que, além de tóxicos, não apresentam eficácia significativa na fase crônica da parasitose. Assim sendo, torna-se imperativa a busca por quimioterápicos mais eficazes, mormente na fase crônica da doença. A introdução de novos fármacos da terapêutica várias fases, consumindo tempo e recursos. No entanto, há processos que permitem a otimização de fármacos já existentes e de compostos bioativos, com vistas à busca de candidatos a fármacos, que, uma vez bem-sucedidos nos ensaios clínicos, são aprovados para uso terapêutico. Entre esses processos, destaca-se a latenciação, forma de aprimoramento de propriedades farmacêuticas, farmacocinéticas e, indiretamente, farmacodinâmicas, que utiliza, em geral, transportadores para a resolução de problemas dessas naturezas. Os transportadores variam de acordo com o problema a ser resolvido e, entre eles, os dendrons e dendrímeros podem ser ressaltados pela sua natureza química, que permite a ligação de várias moléculas de fármacos/compostos bioativos e, também, de grupos diretores para certos compartimentos ou células. Dessa forma, podem-se obter fármacos dirigidos, que se constituem em formas latentes de alta seletividade. Face ao exposto e, estimulados pela busca de novas alternativas terapêuticas para a doença de Chagas, o objetivo deste trabalho foi a obtenção de dendrons dirigidos, por meio de manose, derivados de hidroximetilnitrofural (NFOH). Esse composto foi mostrou-se altamente ativo contra T. cruzi, também na fase crônica NFOH e menos tóxico que o protótipo e o benznidazol. Efetuaram-se estudos para a síntese desses compostos derivados de dendron triazólico, sintetizado através de click chemistry, tendo a manose como grupo diretor para os macrófagos, onde, também, são encontrados os amastigotas de Trypanosoma cruzi. Obtiveram-se alguns intermediários, que foram caracterizados por RMN 1H e 13C. A rota sintética proposta não pôde ser finalizada. Por outro lado, efetuaram-se estudos de modelagem molecular, utilizando-se dinâmica molecular, com o intuito de conhecer como se dá a interação da manose e de polimanosídeos com seu respectivo receptor e como se realiza a liberação do composto bioativo da ligação com o dendron. Anteriormente, procedeu-se à caracterização da biologia estrutural do receptor de manose e de suas estruturas primárias, secundárias e terciárias, com ênfase para o domínio CRD4 o papel do cálcio principal na interação com o monossacarídeo. A movimentação do domínio foi muito pouco diferente nos meios simulados (neutro, ácido, contendo ligantes e contendo o cálcio auxiliar), evidenciado pelo RMSF e estudo de PCA desses sistemas. Foi possível concluir que este domínio não apresenta nenhuma alteração conformacional responsável pela liberação de ligantes em meio lisossômico, e que o cálcio auxiliar e os ligantes não causam impactos na estabilidade conformacional do CRD4. Há necessidade de mais estudos para o conhecimento dos requisitos estruturais envolvidos na da formação do complexo receptor-composto bioativo

Chagas disease, considered an extremely neglected one, affects more than 6 million people all over de world, with more than 75 million people living under its risk, while endemics in 21 countries in Latin America. In Brazil, it propagates, mainly in North region, especially in Amazon region. Although being a global risk, only two drugs, benznidazole and nifurtimox, are currently available for Chagas disease. These drugs are toxic and not significantly efficient against the chronic phase of the disease. Therefore, the search for more active chemotherapeutic agents, mainly against the chronic phase of the parasitosis, is imperative. The introduction of new drugs in the therapeutics involves many phases, consuming time, and money. Notwithstanding, there are processes that allow either drugs or bioactive compounds to be optimized, towards drug candidates. These derivatives, once well-succeeded in the clinical trials, can be approved for therapeutic uses. Among those processes, prodrug design stands out. It is a way to improve the pharmaceutics, pharmacokinetics and, indirectly, pharmacodynamics, properties of drugs/bioactive compounds, which requires adequate carriers, in general, for these problems´ solution. The carriers vary according to the problem to be solved, and, among them, dendrons and dendrimers can be emphasized due to their chemical nature, which allows the link of many molecules/bioactive compounds and of directing groups to specific compartments or cells. Thus, targeted drugs, which are latent forms of drugs/bioactive compounds with high selectivity. In this connection and stimulated by the search for new therapeutic alternatives for Chagas disease, the objective of this work was obtaining hydroxymethylnitrofurazone (NFOH) targeted dendrons, by means of mannose, as directing groups. NFOH is highly active against T. cruzi, even in chronic phase of the disease, and less toxic than the prototype and benznidazole. Studies have been developed to synthesize these compounds with a triazole dendron, planned to be obtained by click chemistry. Mannose was designed to be the directing groups to macrophages, where the T. cruzi amastigotes can also be found. Some intermediaries have been obtained and structurally characterized by 1H and 13C NMR, but the proposed synthetic route could not be finished. On the other hand, molecular modeling studies have been developed, using molecular dynamics, with the aim to know how the interaction of mannose, and also of polymannoside, occur with the specific receptor, and how NFOH is released from its linkage to the dendron. The structural biology characterization, as well as of primary, secondary and tertiary structures of the mannose receptor was previously performed, with emphasis onCRD4 and main calcium role in the interaction of the mannoside. All systems simulated (neutral medium, acid medium, complexes with ligands and auxiliary calcium) showed little movement differences when analyzed by RMSF and PCA calculations. It was possible to conclude that this domain shows no conformational changes involved in ligand releasing in lysosomal environment and its conformation is not altered when in presence of ligands or the auxiliary calcium. Much more studies are needed to the knowledge of the structural requirements to the complex receptor-drug-compound bioactive to the receptor