P-selectin Glycoprotein Ligand-1 Research Articles

He effects of NETosis on fibrinolysis in colon cancer patients

Aim. To investigate formation of neutrophil extracellular traps (NETs) and their impact on fibrinolysis in patients with colon cancer.Materials and methods. The study was performed in two groups. The experimental group consisted of patients with stage 2–3 non-metastatic colon cancer (n = 17, average age – 67 years). The control group included healthy volunteers matched by sex and age (n = 30, average age – 68 years). An experimental model was created from the whole blood. It included platelet-poor plasma and an isolated culture of neutrophils, previously induced to NETosis by adding 100 nmol PMA. The samples were incubated for 4 hours, then the test tubes were centrifuged to pellet cells and their remnants, and the plasma was transferred for subsequent examination. The plasma incubated with intact neutrophils was used as a control. The levels of interleukin-8 (IL-8) and P-selectin glycoprotein ligand-1 (PSGL-1) were used to determine the degree of cell activation. NETosis was confirmed by enzyme-linked immunosorbent assay (ELISA) and fluorescent microscopy. Fibrinolysis was assessed using the thrombodynamics test. The results were compared with the levels of fibrinolytic system components measured by flow cytometry.Results. In the control group, NETosis induction contributed to pronounced neutrophil activation that was accompanied by an increase in the IL-8, PSGL-1, and plasminogen levels, a decrease in PAI-1, and enhancement of fibrinolysis, compared with the intact samples. Higher levels of IL-8, PSGL-1, plasminogen, and PAI-1 and intensified fibrinolysis were detected in the intact samples. However, PMA-induced NETosis did not result in an increase in the degree of activation and significant changes in the given parameters.Conclusion. NETosis promotes both formation and lysis of fibrin clots. However, in cancer patients, suicidal NETosis does not contribute to fibrinolysis due to intracellular protease depletion, which may be one of the mechanisms causing hypercoagulation and insufficient fibrinolysis in cancer.

Platelet Phagocytosis via P-selectin Glycoprotein Ligand 1 and Accumulation of Microparticles in Systemic Sclerosis.

It is unclear why activated platelets and platelet-derived microparticles (MPs) accumulate in the blood of patients with systemic sclerosis (SSc). This study was undertaken to investigate whether defective phagocytosis might contribute to MP accumulation in the blood of patients with SSc. Blood samples were obtained from a total of 81 subjects, including 25 patients with SSc and 26 patients with stable coronary artery disease (CAD). Thirty sex- and age-matched healthy volunteers served as controls. Studies were also conducted in NSG mice, in which the tail vein of the mice was injected with MPs, and samples of the lung parenchyma were obtained for analysis of the pulmonary microvasculature. Tissue samples from human subjects and from mice were assessed by flow cytometry and immunochemical analyses for determination of platelet-neutrophil interactions, phagocytosis, levels and distribution of P-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), and HMGB1 on platelets and MPs, and concentration of byproducts of neutrophil extracellular trap (NET) generation/catabolism. Activated P-selectin+ platelets and platelet-derived HMGB1+ MPs accumulated in the blood of SSc patients but not in the blood of healthy controls. Patients with CAD, a vasculopathy independent of systemic inflammation, had fewer P-selectin+ platelets and a negligible number of MPs. The expression of the receptor for P-selectin, PSGL-1, in neutrophils from SSc patients was significantly decreased, raising the possibility that phagocytes in SSc do not recognize activated platelets, leading to a failure of phagocytosis and continued neutrophil release of MPs. As evidence of this process, activated platelets were not detected in the neutrophils from SSc patients, whereas they were consistently present in the neutrophils from patients with CAD. HMGB1+ MPs elicited generation of NETs, which were only detected in the plasma of SSc patients. In mice, P-selectin-PSGL-1 interaction resulted in platelet phagocytosis in vitro and influenced the ability of MPs to elicit NETs, endothelial activation, and migration of leukocytes through the pulmonary microvasculature. The clearance of activated platelets via PSGL-1 limits the undesirable effects of MP-elicited neutrophil activation. This balance is disrupted in patients with SSc. Its reconstitution might curb vascular inflammation and prevent fibrosis.

Abstract 8907: Fatty-Acid-Binding Protein 3 as a Novel Inducer of Adhesion Molecules in Atherosclerosis

Introduction: Cardiovascular disease is accompanied by a considerable degree of morbidity and mortality, and the pathogenesis is complex. The fatty-acid-binding protein 3 (FABP-3) is a cytoplasmic protein and can be released from injured myocardium. We had demonstrated that FABP-3 was an independent predictor for cardiovascular outcomes in the patients with stable coronary artery disease (CAD). However, the mechanisms of FABP-3 in atherosclerosis were still unknown. Here we supposed that FABP-3 could lead to atherogenesis via activating the adhesion molecules. Methods and Results: Human coronary endothelial cells (HCAECs) and mononuclear cells (MNCs) cultured from CAD patients and healthy subjects were used for the in vitro study. The ox-LDL and TNF-α were used as stimulators in the in vitro experiments. The expressions of adhesion molecule were evaluated by Western blot analysis. MNCs from CAD patients had higher expressions of FABP-3 than those from healthy subjects. In addition, FABP-3 could be further up-regulated by ox-LDL and TNF-α stimulations in both MNCs from CAD patients and healthy subjects. Exogenous of FABP-3 could directly induce the adhesion molecules including integrin β2, integrin α4, and P-selectin glycoprotein ligand 1 expressions in MNCs from CAD patients and healthy subjects. Nevertheless, direct treatments of FABP-3 did not upregulate the adhesion molecules in HCAECs. Also, the adhesiveness of FABP-3-induced adhesiveness of HCAECs to THP-1 monocytic cells was not enhanced. Conclusions: According to the above results, we suggested that FABP-3 could directly lead to the atherogenesis via the MNC activation instead of endothelial cell activation. The findings of the serial studies may not only elucidate the prognostic role of plasma FABP-3 levels in patients with CAD but define the role of FABP-3 on the pathogenesis of atherosclerosis especially its influence in MNCs. The study might clarify the clinical impact of FABP-3 and as a potential novel therapeutic target for atherosclerosis.

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Direct platelet adhesion potentiates group 2 innate lymphoid cell functions

Platelets are thought to be involved in the pathophysiology of asthma, presumably through direct adhesion to inflammatory cells, including group 2 innate lymphoid cells (ILC2s). Here, we tried to elucidate the effects of platelet adhesion to ILC2s in vitro and in vivo, as well as the mechanisms involved. Alternaria-induced ILC2-dependent airway inflammation models using wild-type and c-mpl-/- mice were evaluated. Both purified CD41+ and CD41- ILC2s were cultured with IL-2 and IL-33 to determine in vitro Type 2 (T2) cytokine production and cell proliferation. RNA-seq data of flow-cytometry-sorted CD41+ and CD41- ILC2s were used to isolate ILC2-specific genes. Flow cytometry was performed to determine the expression of CD41 and adhesion-related molecules on ILC2s in both mouse and human tissues. T2 inflammation and T2 cytokine production from ILC2s were significantly reduced in the c-mpl-/- mice compared to wild-type mice. Platelet-adherent ILC2s underwent significant proliferation and showed enhanced T2 cytokine production when exposed to IL-2 and IL-33. The functions of ILC2-specific genes were related to cell development and function. Upstream regulator analysis identified 15molecules, that are thought to be involved in ILC2 activation. CD41 expression levels were higher in ILC2s from human PBMCs and mouse lung than in those from secondary lymphoid tissues, but they did not correlate with the P-selectin glycoprotein ligand-1 or CD24 expression level. Platelets spontaneously adhere to ILC2s, probably in the peripheral blood and airways, thereby potentiating ILC2s to enhance their responses to IL-33.

Identification of active small-molecule modulators targeting the novel immune checkpoint VISTA

BackgroundCancer immunotherapy has gained increasing popularity as a novel approach to treat cancer. A member of the B7 family, V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint that regulates a broad spectrum of immune responses. VISTA is an acidic pH-selective ligand for P-selectin glycoprotein ligand-1(PSGL-1). CA-170, a first-in-class small-molecule dual antagonist of VISTA/PD-L1, was collaboratively developed by Aurigene Discovery Technologies Limited and Curis, Inc. It is currently in Phase I clinical trial.ResultsIn this study, we develop homology modeling for the VISTA 3D structure and subsequent virtual screening for VISTA small-molecule hit ligands. Visualization of the binding postures of docked ligands with the VISTA protein indicates that some small molecular compounds target VISTA. The ability of antagonist to disrupt immune checkpoint VISTA pathways was investigated though functional studies in vitro.ConclusionsAffinity active molecule for VISTA was obtained through virtual screening, and the antagonist compound activity to VISTA was assayed in cellular level. We reported a small molecule with high VISTA affinity as antagonist, providing ideas for development VISTA-targeted small molecule compound in cancer immunotherapy.

P-Selectin Glycoprotein Ligand 1: A Potential HIV-1 Therapeutic Target.

Antiretroviral therapy (ART), which is a life-long therapeutic option, remains the only currently effective clinical method to treat HIV-1 infection. However, ART may be toxic to vital organs including the liver, brain, heart, and kidneys, and may result in systemic complications. In this context, to consider HIV-1 restriction factors from the innate immune system to explore novel HIV therapeutics is likely to be a promising investigative strategy. In light of this, P-selectin glycoprotein ligand 1 (PSGL-1) has recently become the object of close scrutiny as a recognized cell adhesion molecule, and has become a major focus of academic study, as researchers believe that PSGL-1 may represent a novel area of interest in the research inquiry into the field of immune checkpoint inhibition. In this article, we review PSGL-1’s structure and functions during infection and/or inflammation. We also outline a comprehensive review of its role and potential therapeutic utility during HIV-1 infection as published in contemporary academic literature.

Neutrophils aid cellular therapeutics by enhancing glycoengineered stem cell recruitment and retention at sites of inflammation

The efficacy of cell-based therapies relies on targeted payload delivery and enhanced cell retention. In vitro and in vivo studies suggest that the glycoengineering of mesenchymal and cardiosphere-derived cells (CDCs) may enhance such recruitment at sites of injury. We evaluated the role of blood cells in amplifying this recruitment. Thus, the human α(1,3)fucosyltransferase FUT7 was stably expressed in CDCs, sometimes with P-selectin glycoprotein ligand-1 (PSGL-1/CD162). Such FUT7 over-expression resulted in cell-surface sialyl Lewis-X (sLeX) expression, at levels comparable to blood neutrophils. Whereas FUT7 was sufficient for CDC recruitment on substrates bearing E-selectin under flow, PSGL-1 co-expression was necessary for P-/L-selectin binding. In both cone-plate viscometer and flow chamber studies, chemokine driven neutrophil activation promoted the adhesion of glycoengineered-CDCs to blood cells. Here, blood neutrophils activated upon contact with IL-1β stimulated endothelial cells, amplified glycoengineered-CDC recruitment. In vivo, local inflammation in a mouse ear elicited both glycoengineered-CDC and peripheral blood neutrophil homing to the inflamed site. Glycoengineering CDCs also resulted in enhanced (~16%) cell retention at 24 h in a murine myocardial infarction model, with CDCs often co-localized with blood neutrophils. Overall, peripheral blood neutrophils, activated at sites of injury, may enhance recruitment of glycoengineered cellular therapeutics via secondary capture mechanisms.

Binding kinetics of liposome conjugated E-selectin and P-selectin glycoprotein ligand-1 measured with atomic force microscopy

Various ligand-functionalized liposomes have been developed for targeted therapies. Typically, the binding properties of the ligands and targeted proteins are measured with surface plasmon resonance (SPR), where the proteins are immobilized on a rigid surface. However, the difference of protein-ligand binding kinetics between liposome-conjugated protein and rigid surface-conjugated protein is not fully understood. In this work, the binding kinetics of P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin conjugated on liposome and on rigid surfaces are investigated with Atomic Force Microscopy (AFM). The results suggest that protein orientation and diffusion on liposomal membrane can alter the binding kinetics of the protein-ligand interaction. Specifically, the association and dissociation rate constant of AFM probe-conjugated E-selectin and glass-conjugated PSGL-1 are measured as 9.32 × 104 M−1s−1 and 1.54 s−1, respectively. While for the liposome-conjugated E-selectin and glass-conjugated PSGL-1, the kinetic constants are measured as 5.00 × 107 M−1s-1 and 2.76 s−1, respectively. Thus, there is an order’s magnitude increase of binding affinity (from kd = 16.51 μM to kd = 0.06 μM) when protein is attached to liposome compared to attached to a rigid surface. The results might provide better understanding and pave the way for the future design of the ligand-targeted liposomes.

Screening of Atherosclerotic Druggable Targets from the Proteome Network of Differentially Expressed Genes.

Differently expressed genes of atherosclerotic sample analysis are helpful to sort the prominent genes that influence the plaque formation and progression. Scientific evidence-based protein-protein interaction network (PPIN) studies were used to find hub proteins in complex disease conditions. Druggable capacity is one of the important parameters to confirm as a successful drug target. Construction of protein interaction network and principal node analysis (PNA) on atherosclerotic data sets lead to screen the hub proteins. Furthermore, druggable property of protein pocket confirms the targetability of susceptible target candidates and for target selection. Differentially expressed genes are identified through GEO2R analyzer on data sets of various atherosclerotic samples. STRING database and Cytoscape are employed to construct PPIN. Targets were identified by PNA such as centrality measures and clustering algorithm. Gene Ontology enrichment also used as one of the screening parameters to filter the candidates related to atherosclerotic terms. Topological evaluation of target protein was successfully done by ITASSER and GROMACS, respectively. Grid-based principle of DoGSiteScorer is utilized for druggability analysis. Six proteins such as integrin alpha L (ITGAL), metallothionein 1F (MT1F), metallothionein 1X (MT1X), P-selectin glycoprotein ligand-1 (SELPLG), solute carrier family 30 A, zinc transporter protein (SLC30A1), and TNFSF13B are screened as potential biomarkers through network-based analysis. Among the six, ITGAL, SELPLG, SLC30A1, and TNSF13B are identified as better prioritized atherosclerotic targets through druggability efficiency.

Constitutive PSGL-1 Correlates with CD30 and TCR Pathways and Represents a Potential Target for Immunotherapy in Anaplastic Large T-Cell Lymphoma.

Simple SummaryP-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is the major ligand of selectins and plays a pivotal role in tethering, rolling and extravasation of immune cells. PSGL-1 involvement in core molecular programs, such as SYK, PLCγ2, PI3Kγ or MAPK pathways, suggests additional functions beyond the modulation of cell trafficking. Recently, several studies identified a novel mechanism responsible for PSGL-1-mediated immune suppression in the tumor microenvironment and proved a novel concept of PSGL-1 as a critical checkpoint molecule for tumor immunotherapy. The immunotherapeutic approach has gained an ever-growing interest in the treatment of several hematological malignancies, and in particular, novel targets for immunotherapy are still highly sought-after in T-cell lymphomas. Based on our results obtained through gene expression profiling and immunohistochemical analysis, PSGL-1, already suggested as a potential target in multiple myeloma humoral immunotherapy, could be considered noteworthy among the candidates.Due to the high expression of P-selectin glycoprotein ligand-1 (PSGL-1) in lymphoproliferative disorders and in multiple myeloma, it has been considered as a potential target for humoral immunotherapy, as well as an immune checkpoint inhibitor in T-cells. By investigating the expression of SELPLG in 678 T- and B-cell samples by gene expression profiling (GEP), further supported by tissue microarray and immunohistochemical analysis, we identified anaplastic large T-cell lymphoma (ALCL) as constitutively expressing SELPLG at high levels. Moreover, GEP analysis in CD30+ ALCLs highlighted a positive correlation of SELPLG with TNFRSF8 (CD30-coding gene) and T-cell receptor (TCR)-signaling genes (LCK, LAT, SYK and JUN), suggesting that the common dysregulation of TCR expression in ALCLs may be bypassed by the involvement of PSGL-1 in T-cell activation and survival. Finally, we evaluated the effects elicited by in vitro treatment with two anti-PSGL-1 antibodies (KPL-1 and TB5) on the activation of the complement system and induction of apoptosis in human ALCL cell lines. In conclusion, our data demonstrated that PSGL-1 is specifically enriched in ALCLs, altering cell motility and viability due to its involvement in CD30 and TCR signaling, and it might be considered as a promising candidate for novel immunotherapeutic approaches in ALCLs.

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.

Phenylboronic acid modified nanoparticles simultaneously target pancreatic cancer and its metastasis and alleviate immunosuppression

Pancreatic ductal adenocarcinoma is one of the most lethal malignant tumors, its drug resistance, immunosuppression and metastasis makes the traditional chemotherapy and immunotherapy inefficient. Here we confirmed a 3-aminophenylboronic acid-modified low molecular weight heparin-D-α-tocopheryl succinate micellar nanoparticle (PBA-LMWH-TOS NP, PLT NP) could inhibit orthotopic pancreatic tumor and its spontaneous metastases. The small particle size and high affinity of PBA to sialic acid residue (SA) made PLT/PTX NPs significantly targeted and accumulated in both pancreatic tumor tissues and metastases. The immunosuppressive microenvironment of pancreatic tumor was most caused by the infiltration of immunosuppressive cells, mainly myeloid-derived suppressor cells (MDSCs). We first reported that P-selectin glycoprotein ligand-1 (PSGL-1) was expressed on the surfaces of MDSCs in pancreatic tumor tissues. Meanwhile, we found that LMWH could inhibit the early stage of adhesion cascade between vascular endothelial cells (VECs) and MDSCs by interfering with P-selectin/PSGL-1 binding, thus inhibiting MDSC recruitment to pancreatic tumor tissues. The therapeutic results indicated that PLT/PTX NPs could significantly improve the immune microenvironment of pancreatic tumor and inhibit spontaneous metastases. This nanosystem provides a new immune microenvironment regulation mechanism based on carrier materials in pancreatic tumor, and has high clinical application potential.

A PSGL-1 glycomimetic reduces thrombus burden without affecting hemostasis

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Identification of the SHREK Family of Proteins as Broad-Spectrum Host Antiviral Factors.

Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123) that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirus. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.

The Mineralocorticoid Receptor in Myeloid Cells Promotes Vascular Inflammation and Atherosclerosis via Transcriptional Regulation of Selplg

Elevated plasma aldosterone (Aldo) is associated with increased risk of cardiovascular (CV) ischemia, and inhibition of the Aldo-activated mineralocorticoid receptor (MR) reduces CV-related mortality. Ischemic events including heart attack and stroke are caused by rupture of atherosclerotic plaques with abundant leukocytes. In mouse models of atherosclerosis, MR antagonists reduce plaque size and inflammation. MR in myeloid cells (MyMR) regulates macrophage (Mp) polarization in vitro, but its role in vascular inflammation in vivo is unknown. Here we test the hypothesis that MyMR contributes to vascular inflammation by facilitating leukocyte recruitment. A MyMR-knockout (KO) mouse was created (LysM-Cre/MRfl/fl) and crossed with atheroprone apolipoprotein E-KO (ApoE) mice. Eight-week-old male and female MyMR-intact/ApoE-KO and MyMR-KO/ApoE-KO littermates were fed high fat diet for 8 weeks to accelerate atherogenesis. Aortic root plaque area was modestly decreased with MyMR-KO. FACS analysis of aortic arch plaques revealed fewer Mp (37%, p < 0.01) and T cells (26%, p < 0.05) with MyMR-KO among both sexes. Female plaques were larger but had fewer myeloid (34%, p < 0.05) and T cells (30%, p < 0.01) compared to male plaques. Seven-week-old LysM-Cre/MRfl/fl mice were injected with TNFa intraperitoneally, and leukocyte trafficking was quantified in the mesenteric veins by intravital microscopy four hours later. Male and female MyMR-KO mice had 40% fewer slow-rolling cells (p < 0.0001) and 56% fewer adherent cells (p < 0.01) compared to MyMR-intact controls. There were 39% fewer slow-rolling cells in females versus males (p < 0.01). To explore mechanism, expression of leukocyte adhesion ligands was quantified by FACS in circulating leukocytes and RT-PCR in peritoneal Mp. Surface levels of P-selectin glycoprotein ligand-1 (Selplg) was attenuated on monocytes, but not on granulocytes or T cells, from MyMR-KO mice, particularly among males. Selplg mRNA was decreased by 35% in Mp from MyMR-KO mice (p < 0.05). To determine whether Selplg is transcriptionally regulated by the MR, U937 cells were pre-treated with the MR antagonist spironolactone (1 μM, Spiro) and then treated with Aldo (10 nM). Spiro alone decreased Selplg transcription (p < 0.05) compared to vehicle. Aldo increased Selplg transcription (p < 0.05) compared to vehicle, and this effect was blocked by Spiro (p < 0.0001). MyMR-KO decreases plaque size and inflammation, which may be partially due to a role of MyMR in leukocyte recruitment in early atherosclerosis likely by regulation of Selplg. The sex differences in leukocyte trafficking and plaque inflammation are worth further exploration as a mechanism for female protection from CV disease. This study suggests that MyMR regulation of vascular inflammation is a novel mechanism for the association of Aldo with increased ischemic events and supports testing of MR antagonists to prevent CV ischemia.

Abstract PO014: PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells

Abstract We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO014.

What's New in Shock, February 2021?

What's New in Shock, February 2021?

P-Selectin Glycoprotein Ligand-1 Deficiency Protects Against Aortic Aneurysm Formation Induced by DOCA Plus Salt.

P-selectin glycoprotein ligand-1 (PSGL-1) acts as a crucial regulator for the inflammatory cells infiltration by mediating the adhesion of leukocytes. However, the role of PSGL-1 in aortic aneurysm remains elusive. Here, we investigated the role of PSGL-1 in aortic aneurysm (AA) development. We first detected PSGL-1 expression in samples from aortic aneurysm patients and mouse AA models via western blotting, immunofluorescence, and flow cytometry, and then we used global PSGL-1 knockout mice and their wild type controls to establish an aortic aneurysm model induced by deoxycorticosterone acetate (DOCA) plus high salt (HS). The incidence, fatality rates, and the pathological changes of aortic aneurysm were analyzed in each group. The inflammation, adhesion molecules expression, and PSGL-1 mediated leukocyte-endothelial adhesion and their underlying mechanisms were explored further. Increased PSGL-1 levels were observed in human and mouse aortic aneurysm, and on leukocytes of mice treated with DOCA+HS. PSGL-1 deficiency reduced the incidence and severity of aortic aneurysm significantly, as well as decreased elastin fragmentation, collagen accumulation, and smooth muscle cells degeneration. Mechanistically, the protective effect of PSGL-1 inhibition was mediated by the reduced adhesion molecules, and the subsequently reduced leukocyte-endothelial adhesion through the NF-κB pathway, which finally led to reduced inflammatory cells infiltration and decreased inflammatory factors expression. PSGL-1 deficiency is protective against inflammatory cells migration and recruitment in the condition of AA through attenuation of leukocyte-endothelial adhesion. Inhibition of PSGL-1 may be a potential therapeutic target for the prevention and treatment of human AA.

Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors

Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.