Intra-patient spatial comparison of non-metastatic and metastatic lymph nodes reveals the reduction of CD169+ macrophages by metastatic breast cancers.

BACKGROUND: Breast cancer cells suppress the host immune system to efficiently invade the lymph nodes; however, the underlying mechanism remains incompletely understood. Here, we aimed to comprehensively characterise the effects of breast cancers on immune cells in the lymph nodes. METHODS: We collected non-metastatic and metastatic lymph node samples from 6 patients with breast cancer with lymph node metastasis. We performed bulk transcriptomics, spatial transcriptomics, and imaging mass cytometry to analyse the obtained lymph nodes. Furthermore, we conducted histological analyses against a larger patient cohort (474 slices from 58 patients). FINDINGS: The comparison between paired lymph nodes with and without metastasis from the same patients demonstrated that the number of CD169+ lymph node sinus macrophages, an initiator of anti-cancer immunity, was reduced in metastatic lymph nodes (36.7 ± 21.1 vs 7.3 ± 7.0 cells/mm2, p = 0.0087), whereas the numbers of other major immune cell types were unaltered. We also detected that the infiltration of CD169+ macrophages into metastasised cancer tissues differed by section location within tumours, suggesting that CD169+ macrophages were gradually decreased after anti-cancer reactions. Furthermore, CD169+ macrophage elimination was prevalent in major breast cancer subtypes and correlated with breast cancer staging (p = 0.022). INTERPRETATION: We concluded that lymph nodes with breast cancer metastases have fewer CD169+ macrophages, which may be detrimental to the activity of anti-cancer immunity. FUNDING: JSPS KAKENHI (16H06279, 20H03451, 20H04842, 22H04925, 19K16770, and 21K15530, 24K02236), JSPS Fellows (JP22KJ1822), AMED (JP21ck0106698), JST FOREST (JPMJFR2062), Caravel, Co., Ltd, Japan Foundation for Applied Enzymology, and Sumitomo Pharma Co., Ltd. under SKIPS.

The Role of TIM-1 and CD300a in Zika Virus Infection Investigated with Cell-Based Electrical Impedance.

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.

Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis.

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.

Serum and urine interferon-inducible protein 10, galectin-9, and SIGLEC-1 as biomarkers of disease activity in systemic lupus erythematosus.

Background/aim: In this prospective study, we aimed to investigate the association of serum (s) and urine (u) IP-10, galectin-9, and SIGLEC-1 with disease activity in patients with systemic lupus erythematosus (SLE). Materials and methods: Sixty-three patients with active SLE (31 renal, 32 extrarenal) were included. Thirty patients with inactive SLE (15 renal, 15 extrarenal), 17 with renal active AAV, and 32 healthy volunteers were selected as control groups. Serum and urine IP-10, galectin-9, and SIGLEC-1 were tested using ELISA. Results: Levels of sIP-10 (p = 0.046), uIP-10 (p < 0.001), sGalectin-9 (p = 0.03), and uSIGLEC-1 (p = 0.006) were significantly higher in active SLE group compared to the inactive SLE; however, no differences were detected in the comparison of uGalectin-9 (p = 0.18) and sSIGLEC-1 (p = 0.69) between two groups. None of the biomarkers discriminated patients with active renal SLE from active extrarenal SLE. ROC analyses revealed an AUC of 0.63 (0.52-0.73) for sIP-10, 0.78 (0.68-0.86) for uIP-10, 0.64 (0.53-0.74) for sGalectin-9, and 0.68 (0.57-0.77) for uSIGLEC-1 in discriminating disease activity in SLE, which did not outperform C3 (0.75, 0.64-0.84) and C4 (0.72, 0.61-0.82). sIP-10 (p = 0.001), uIP-10 (p = 0.042), and uGalectin-9 (p = 0.009) were significantly increased in patients with active renal SLE compared to active renal AAV. sGalectin-9 (p < 0.001) and sIP-10 levels (p = 0.06) were decreased after 8 (5-22.5) months of treatment. Conclusion: sIP-10, uIP-10, sGalectin-9, and uSIGLEC-1 reflect global disease activity in SLE but do not outperform C3 and C4. sIP-10 and uIP-10 may be specific for active SLE compared to active AAV. sIP-10 and sGalectin-9 might be valuable in monitoring response after treatment.

CD169+ Skin Macrophages Function as a Specialized Subpopulation in Promoting Psoriasis-like Skin Disease in Mice.

Skin macrophages are critical to maintain and restore skin homeostasis. They serve as major producers of cytokines and chemokines in the skin, participating in diverse biological processes such as wound healing and psoriasis. The heterogeneity and functional diversity of macrophage subpopulations endow them with multifaceted roles in psoriasis development. A distinct subpopulation of skin macrophages, characterized by high expression of CD169, has been reported to exist in both mouse and human skin. However, its role in psoriasis remains unknown. Here, we report that CD169+ macrophages exhibit increased abundance in imiquimod (IMQ) induced psoriasis-like skin lesions. Specific depletion of CD169+ macrophages in CD169-ditheria toxin receptor (CD169-DTR) mice inhibits IMQ-induced psoriasis, resulting in milder symptoms, diminished proinflammatory cytokine levels and reduced proportion of Th17 cells within the skin lesions. Furthermore, transcriptomic analysis uncovers enhanced activity in CD169+ macrophages when compared with CD169- macrophages, characterized by upregulated genes that are associated with cell activation and cell metabolism. Mechanistically, CD169+ macrophages isolated from IMQ-induced skin lesions produce more proinflammatory cytokines and exhibit enhanced ability to promote Th17 cell differentiation in vitro. Collectively, our findings highlight the crucial involvement of CD169+ macrophages in psoriasis development and offer novel insights into the heterogeneity of skin macrophages in the context of psoriasis.

Anticancer immune reaction and lymph node sinus macrophages: a review from human and animal studies.

Lymph nodes are secondary lymphoid organs localized throughout the body that typically appear as bean-like nodules. Numerous antigen-presenting cells, including dendritic cells and macrophages, that mediate host defense responses against pathogens, such as bacteria and viruses, reside within lymph nodes. To react to cancer cell-derived antigens in a variety of cancers, antigen-presenting cells induce cytotoxic T lymphocytes (CTLs). In relation to anticancer immune responses, macrophages in the lymph node sinus have been of particular interest because a number of studies involving both human specimens and animal models have reported that lymph node macrophages expressing CD169 play a key role in activating anticancer CTLs. Recent studies have indicated that dysfunction of lymph node macrophages potentially contributes to immune suppression in elderly patients and immunological "cold" tumors. Therefore, in anticancer therapy, the regulation of lymph node macrophages is a potentially promising approach.

Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

Preclinical development of humanized monoclonal antibodies against CD169 as a broad antiviral therapeutic strategy.

New therapies to treat or prevent viral infections are essential, as recently observed during the COVID-19 pandemic. Here, we propose a therapeutic strategy based on monoclonal antibodies that block the specific interaction between the host receptor Siglec-1/CD169 and gangliosides embedded in the viral envelope. Antibodies are an excellent option for treating infectious diseases based on their high specificity, strong targeting affinity, and relatively low toxicity. Through a process of humanization, we optimized monoclonal antibodies to eliminate sequence liabilities and performed biophysical characterization. We demonstrated that they maintain their ability to block viral entry into myeloid cells. These molecular improvements during the discovery stage are key if we are to maximize efforts to develop new therapeutic strategies. Humanized monoclonal antibodies targeting CD169 provide new opportunities in the treatment of infections caused by ganglioside-containing enveloped viruses, which pose a constant threat to human health. In contrast with current neutralizing antibodies that bind antigens on the infectious particle, our antibodies can prevent several types of enveloped viruses interacting with host cells because they target the host CD169 protein, thus becoming a potential pan-antiviral therapy.

Elevated unphosphorylated STAT1 and IRF9 in T and B cells of primary sjögren's syndrome: Novel biomarkers for disease activity and subsets.

OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Identification of Siglec-1-negative alveolar macrophages with proinflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, subpopulations of AMs participating in chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a proinflammatory phenotype and are associated with poor clinical outcomes in patients with COPD. Using flow cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi, and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA sequencing revealed upregulation of multiple proinflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6, and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs displays proinflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.NEW & NOTEWORTHY Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. We find that Siglec-1-negative alveolar macrophages have a wide range of proinflammatory landscapes and a protease-expressing phenotype. Moreover, this subset is associated with the pathogenesis of COPD and responds to viral stimuli.

A novel subpopulation of monocytes with a strong interferon signature indicated by SIGLEC-1 is present in patients with in recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is a T cell-mediated autoimmune disease characterised by pancreatic beta cell destruction. In this study, we explored the pathogenic immune responses in initiation of type 1 diabetes and new immunological targets for type 1 diabetes prevention and treatment. METHODS: We obtained peripheral blood samples from four individuals with newly diagnosed latent autoimmune diabetes in adults (LADA) and from four healthy control participants. Single-cell RNA-sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells to uncover transcriptomic profiles of early LADA. Validation was performed through flow cytometry in a cohort comprising 54 LADA, 17 adult-onset type 2 diabetes, and 26 healthy adults, matched using propensity score matching (PSM) based on age and sex. A similar PSM method matched 15 paediatric type 1 diabetes patients with 15 healthy children. Further flow cytometry analysis was performed in both peripheral blood and pancreatic tissues of non-obese diabetic (NOD) mice. Additionally, cell adoptive transfer and clearance assays were performed in NOD mice to explore the role of this monocyte subset in islet inflammation and onset of type 1 diabetes. RESULTS: The scRNA-seq data showed that upregulated genes in peripheral T cells and monocytes from early-onset LADA patients were primarily enriched in the IFN signalling pathway. A new cluster of classical monocytes (cluster 4) was identified, and the proportion of this cluster was significantly increased in individuals with LADA compared with healthy control individuals (11.93% vs 5.93%, p=0.017) and that exhibited a strong IFN signature marked by SIGLEC-1 (encoding sialoadhesin). These SIGLEC-1+ monocytes expressed high levels of genes encoding C-C chemokine receptors 1 or 2, as well as genes for chemoattractants for T cells and natural killer cells. They also showed relatively low levels of genes for co-stimulatory and HLA molecules. Flow cytometry analysis verified the elevated levels of SIGLEC-1+ monocytes in the peripheral blood of participants with LADA and paediatric type 1 diabetes compared with healthy control participants and those with type 2 diabetes. Interestingly, the proportion of SIGLEC-1+ monocytes positively correlated with disease activity and negatively with disease duration in the LADA patients. In NOD mice, the proportion of SIGLEC-1+ monocytes in the peripheral blood was highest at the age of 6 weeks (16.88%), while the peak occurred at 12 weeks in pancreatic tissues (23.65%). Adoptive transfer experiments revealed a significant acceleration in diabetes onset in the SIGLEC-1+ group compared with the SIGLEC-1- or saline control group. CONCLUSIONS/INTERPRETATION: Our study identified a novel group of SIGLEC-1+ monocytes that may serve as an important indicator for early diagnosis, activity assessment and monitoring of therapeutic efficacy in type 1 diabetes, and may also be a novel target for preventing and treating type 1 diabetes. DATA AVAILABILITY: RNA-seq data have been deposited in the GSA human database ( https://ngdc.cncb.ac.cn/gsa-human/ ) under accession number HRA003649.

The selection of animal models influences the assessment of anti-tumor efficacy: promising sialic acid-conjugate modified liposomes demonstrate remarkable therapeutic effects in diverse mouse strains.

Mice as a crucial tool for preclinical assessment of antineoplastic agents. The impact of physiological differences among mouse strains on the in vivo efficacy of antitumor drugs, however, has been significantly overlooked. Mononuclear phagocyte system (MPS) is the major player in clearance in vivo, and differences in MPS among different strains may potentially impact the effectiveness of antitumor preparations. Therefore, in this study, we employed conventional liposomes (CL-EPI) and SA-ODA modified liposomes (SAL-EPI) as model preparations to investigate the comprehensive tumor therapeutic effects of CL-EPI and SAL-EPI in KM, BALB/c, and C57BL/6 tumor-bearing mice. The results demonstrated significant variability in the efficacy of CL-EPI for tumor treatment across different mouse strains. Therefore, we should pay attention to the selection of animal models in the study of antitumor agents. SAL-EPI effectively targeted tumor sites by binding to Siglec-1 on the surface of peripheral blood monocytes (PBMs), and achieved good therapeutic effect in different mouse strains with little difference in treatment. The SA modified preparation is therefore expected to achieve a favorable therapeutic effect in tumor patients with different immune states through PBMs delivery (Siglec-1 was expressed in both mice and humans), thereby possessing clinical translational value and promising development prospects.

Sialic acid-decorated liposomes enhance the anti-cancer efficacy of docetaxel in tumor-associated macrophages.

Tumor-associated macrophages (TAMs) in the tumor microenvironment potentially enhance tumor growth and invasion through various mechanisms and are thus an essential factor in tumor immunity. The highly expressed siglec-1 receptors on the surfaces of TAMs are potential targets for cancer drug delivery systems. Sialic acid (SA) is a specific ligand for siglec-1. In this study, the sialic acid-polyethylene glycol conjugate (DSPE-PEG2000-SA) was synthesized to modify the surface of liposomes and target TAMs by interacting with the siglec-1 receptor. Three docetaxel (DTX)-loaded liposomes, conventional (DTX-CL), DSPE-PEG2000-coated (DTX-PL), and DSPE-PEG2000-SA-coated (DTX-SAPL) liposomes, were prepared, with a particle size of <100 nm, uniform polydispersity index (PDI) values, negative zeta potential, and % encapsulation efficiency (EE) exceeding 95 %. Liposomes showed high stability after 3 months of storage at 4 °C without significant changes in particle size, PDI, zeta potential, or % EE. DTX was released from liposomes according to the Weibull model, and DTX-SAPL exhibited more rapid drug release than other liposomes. In vitro studies demonstrated that DTX-SAPL liposome exhibited a higher uptake and cytotoxicity on RAW 264.7 cells (TAM model) and lower toxicity on NIH3T3 cells (normal cell model) than other formulations. The high cell uptake ability was demonstrated by the role of the SA-SA receptor. Biodistribution studies indicated a high tumor accumulation of surface-modified liposomal formulations, particularly SA-modified liposomes, showing high signal accumulation at the tumor periphery, where TAMs were highly concentrated. Ex vivo imaging showed a significantly higher accumulation of SA-modified liposomes in the tumor, kidney, and heart than conventional liposomes. In the anti-cancer efficacy study, DTX-SAPL liposomes showed effective inhibition of tumor growth and relatively low systemic toxicity, as evidenced by the tumor volume, tumor weight, body weight values, and histopathological analysis. Therefore, DSPE-PEG2000-SA-coated liposomes could be promising carriers for DTX delivery targeting TAMs in cancer therapy.

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

Sialoadhesin (CD169/Siglec-1) is an extended molecule that escapes inhibitory cis-interactions and synergizes with other macrophage receptors to promote phagocytosis.

Sialoadhesin (CD169/Siglec-1, Sn) is a macrophage receptor that interacts with sialic acids on both host cells and pathogens. It is a type 1 membrane protein with an unusually large number of 17 extracellular immunoglobulin (Ig)-like domains, made up of an N-terminal V-set domain that binds sialic acid and 16 adjacent C2-set domains. The potential importance of 17 Ig domains in Sn for mediating cellular interactions has not been investigated experimentally. In the present study, Chinese Hamster Ovary (CHO) cells were stably transfected with full-length or truncated forms of Sn. Using human red blood cells (RBC) as a model system, CHO cells expressing truncated forms of Sn with 4 or less Ig domains were unable to bind RBC in comparison to the full-length protein. Immunoelectron microscopy of the CHO cells indicated that full-length Sn extends ~ 33 nm from the plasma membrane compared with ~ 14 nm for a truncated form with 6 N-terminal Ig domains. Co-expresssion of Sn-expressing CHO cells with heavily glycosylated membrane proteins of differing predicted lengths resulted in selective modulation of Sn-dependent binding to RBC and supported the hypothesis that Sn has evolved 17 Ig domains to escape inhibitory cis-interactions. The functional significance of the extended length of Sn was demonstrated in experiments with macrophages showing that Sn synergizes with phagocytic receptors FcR and TIM-4 to strongly promote uptake of IgG-opsonized and eryptotic RBC respectively.

Sialylation-dependent interaction between PD-L1 and CD169 promotes monocyte adhesion to endothelial cells.

The monocyte adhesion to endothelial cells is an early step in chronic inflammation. Interferon-γ (IFN-γ) is regarded as a master regulator of inflammation development. However, the significance and mechanisms of IFN-γ in the monocyte adhesion to endothelial cells remains largely unknown. IFN-γ up-regulates PD-L1 on various types of cells. Here, we performed flow cytometry to examine the contribution of IFN-γ-induced PD-L1 expression on monocyte adhesion to endothelial cells. Up-regulation of PD-L1 by IFN-γ enhanced the adhesion of monocytes to endothelial cells. By immunoprecipitation and lectin blot, PD-L1 in endothelial cells interacted with CD169/Siglec 1 in monocytes depending on the α2,3-sialylation of PD-L1. ST3Gal family (ST3ß-galactoside α-2,3-sialyltransferase) was the major glycosyltransferase responsible for the α2,3-sialylation of membrane proteins. Down-regulation of ST3Gal4 by RNAinterference partially reduced the α2,3-sialylation of PD-L1 and the PD-L1-CD169 interaction. Finally, purified PD-L1 protein with α2,3-sialylation, but not PD-L1 protein without α2,3-sialylation, partially reduced IFN-γ-induced monocyte adhesion to endothelial cells. These findings provide evidence that the interaction between PD-L1 and CD169 promoted monocyte adhesion to endothelial cells and might elucidate a new mechanism of monocyte adhesion to endothelial cells.

The role of sialic acid-binding immunoglobulin-like-lectin-1 (siglec-1) in immunology and infectious disease.

Siglec-1, also known as Sialoadhesin (Sn) and CD169 is highly conserved among vertebrates and with 17 immunoglobulin-like domains is Siglec-1 the largest member of the Siglec family. Expression of Siglec-1 is found primarily on dendritic cells (DCs), macrophages and interferon induced monocyte. The structure of Siglec-1 is unique among siglecs and its function as a receptor is also different compared to other receptors in this class as it contains the most extracellular domains out of all the siglecs. However, the ability of Siglec-1 to internalize antigens and to pass them on to lymphocytes by allowing dendritic cells and macrophages to act as antigen presenting cells, is the main reason that has granted Siglec-1's key role in multiple human disease states including atherosclerosis, coronary artery disease, autoimmune diseases, cell-cell signaling, immunology, and more importantly bacterial and viral infections. Enveloped viruses for example have been shown to manipulate Siglec-1 to increase their virulence by binding to sialic acids present on the virus glycoproteins allowing them to spread or evade immune response. Siglec-1 mediates dissemination of HIV-1 in activated tissues enhancing viral spread via infection of DC/T-cell synapses. Overall, the ability of Siglec-1 to bind a variety of target cells within the immune system such as erythrocytes, B-cells, CD8+ granulocytes and NK cells, highlights that Siglec-1 is a unique player in these essential processes.

An Application of Tumor-Associated Macrophages as Immunotherapy Targets: Sialic Acid-Modified EPI-Loaded Liposomes Inhibit Breast Cancer Metastasis.

Breast cancer metastasis is an important cause of death in patients with breast cancer and is closely related to circulating tumor cells (CTCs) and the metastatic microenvironment. As the most infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs), which highly express sialic acid (SA) receptor (Siglec-1), are closely linked to tumor progression and metastasis. Furthermore, the surface of CTCs also highly expressed receptor (Selectin) for SA. A targeting ligand (SA-CH), composed of SA and cholesterol, was synthesized and modified on the surface of epirubicin (EPI)-loaded liposomes (EPI-SL) as an effective targeting delivery system. Liposomes were evaluated for characteristics, stability, in vitro release, cytotoxicity, cellular uptake, pharmacokinetics, tumor targeting, and pharmacodynamics. In vivo and in vitro experiments showed that EPI-SL enhanced EPI uptake by TAMs. In addition, cellular experiments showed that EPI-SL could also enhance the uptake of EPI by 4T1 cells, resulting in cytotoxicity second only to that of EPI solution. Pharmacodynamic experiments have shown that EPI-SL has optimal tumor inhibition with minimal toxicity, which can be ascribed to the fact that EPI-SL can deliver drugs to tumor based on TAMs and regulate TME through the depletion of TAMs. Our study demonstrated the significant potential of SA-modified liposomes in antitumor metastasis. Schematic diagram of the role of SA-CH modified EPI-loaded liposomes in the model of breast cancer metastasis.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.