The VISTA/VSIG3/PSGL-1 axis: crosstalk between immune effector cells and cancer cells in invasive ductal breast carcinoma.

A checkpoint protein called the V-domain Ig suppressor of T cell activation (VISTA) is important for controlling immune responses. Immune cells that interact with VISTA have molecules, or receptors, known as VISTA receptors. Immune system activity can be modified by the interaction between VISTA and its receptors. Since targeting VISTA or its receptors may be beneficial in certain conditions, VISTA has been studied in relation to immunotherapy for cancer and autoimmune illnesses. The purpose of this study was to examine the expression levels and interactions between VISTA and its receptors, VSIG3 and PSGL-1, in breast cancer tissues. IHC analysis revealed higher levels of proteins within the VISTA/VSIG3/PSGL-1 axis in cancer tissues than in the reference samples (mastopathies). VISTA was found in breast cancer cells and intratumoral immune cells, with membranous and cytoplasmic staining patterns. VISTA was also linked with pathological grade and VSIG3 and PSGL-1 levels. Furthermore, we discovered that the knockdown of one axis member boosted the expression of the other partners. This highlights the significance of VISTA/VSIG3/PSGL-1 in tumor stroma and microenvironment remodeling. Our findings indicate the importance of the VISTA/VSIG3/PSGL-1 axis in the molecular biology of cancer cells and the immune microenvironment.

The role of immune checkpoint inhibitors: Variable number of tandem repeat (VNTR) polymorphism in the second exon of the P-selectin glycoprotein ligand-1 (PSGL-1) gene polymorphism in multiple myeloma.

Somatic mutations and polymorphisms may play a role in multiple myeloma (MM) susceptibility and survival. One of the immune checkpoint inhibitors is P-selectin glycoprotein ligand-1 (PSGL-1); the majority of tumor-infiltrating leukocytes express PSGL-1, causing T cell and immune inhibition via PSGL-1 mediator molecules. We aimed to investigate the effect of variable number of tandem repeat (VNTR) polymorphism in the second exon of the PSGL-1 gene on MM susceptibility, response to treatment and survival in our patient group. A total of 238 patients diagnosed with MM between January 2010 and January 2021 and 162 healthy individuals as a control group were included in this cross-sectional study. The genotypes of the VNTR polymorphism in the second exon of the PSGL-1 gene were statistically compared between patients and healthy controls; the statistically significant effects of the genotypes on response to first-line treatment and survival were examined. The AC genotype was significantly higher in healthy controls compared to patients diagnosed with MM (p < 0.001). The median PFS in patients with AA/AB/AC was 56 months, while it was 100 months in patients with BB/CC. The hazard ratio of 1.34 for PFS was found to be clinically significant and having the BB/CC genotype could provide a longer PFS compared to others, but it was not statistically significant due to the sample size. Our study results will shed light on new study plans in terms of immune checkpoint target therapies among conventional treatment preferences in MM.

VISTA: A Mediator of Quiescence and a Promising Target in Cancer Immunotherapy.

V-domain Ig suppressor of T cell activation (VISTA) is a B7 family member that maintains T cell and myeloid quiescence and is a promising target for combination cancer immunotherapy. During inflammatory challenges, VISTA activity reprograms macrophages towards reduced production of proinflammatory cytokines and increased production of interleukin (IL)-10 and other anti-inflammatory mediators. The interaction of VISTA with its ligands is regulated by pH, and the acidic pH ~6.0 in the tumor microenvironment (TME) facilitates VISTA binding to P-selectin glycoprotein ligand 1 (PSGL-1). Targeting intratumoral pH might be a way to reduce the immunoinhibitory activity of the VISTA pathway and enhance antitumor immune responses. We review differences among VISTA therapeutics under development as candidate immunotherapies, focusing on VISTA binding partners and the unique structural features of this interaction.

Antibody targeting of surface P-selectin glycoprotein ligand 1 leads to lymphoma apoptosis and tumorigenesis inhibition.

Lymphomas are a heterogeneous group of diseases that originate from T, B or natural killer cells. Lymphoma treatment is based on chemotherapy, radiotherapy, and monoclonal antibody (mAb) or other immunotherapies. The P-selectin glycoprotein ligand 1 (PSGL-1) is expressed at the surface of hematological malignant cells and has been shown to have a pro-oncogenic role in multiple myeloma and lymphoma. Here, we investigated the expression and therapeutic potential of PSGL-1 in T and B cell lymphomas. By flow cytometry analysis, we found that PSGL-1 was expressed in both T and B cell-derived lymphoma cell lines but generally at higher levels in T cell lymphoma cell lines. For most T and B cell-derived lymphoma cell lines, in vitro targeting with the PL1 mAb, which recognizes the PSGL-1 N-terminal extracellular region and blocks functional interactions with selectins, resulted in reduced cell viability. The PL1 mAb pro-apoptotic activity was shown to be dose-dependent, to be linked to increased ERK kinase phosphorylation, and to be dependent on the MAP kinase signaling pathway. Importantly, anti-PSGL-1 treatment of mice xenografted with the HUT-78 cutaneous T-cell lymphoma cell line resulted in decreased tumor growth, had no effect on in vivo proliferation, but increased the levels of apoptosis in tumors. Anti-PSGL-1 treatment of mice xenografted with a Burkitt lymphoma cell line that was resistant to anti-PSGL-1 treatment in vitro, had no impact on tumorigenesis. These findings show that PSGL-1 antibody targeting triggers lymphoma cell apoptosis and substantiates PSGL-1 as a potential target for lymphoma therapy.

RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation.

BACKGROUND: Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. METHODS: A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. RESULTS: Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1ß. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. CONCLUSIONS: These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites.

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.

Correlation between plasma PSGL-1 and FIGO stage, tumor metastasis, and survival in epithelial ovarian cancer.

Plasma circulating P-selectin glycoprotein ligand-1 (PSGL-1) levels and its clinical correlation in patients with epithelial ovarian cancer (EOC) are unknown. The study determined plasma PSGL-1 levels in EOC patients and investigated its relationship with clinicopathological factors and prognosis. Plasma PSGL-1 levels were measured using ELISA in 69 patients with EOC, 34 patients with benign ovarian cystadenoma, and 36 healthy controls. Subsequently, the relationship between PSGL-1 levels and clinicopathological characteristics of patients, as well as the prognosis of EOC patients, was examined. Additionally, the specificity and sensitivity of plasma PSGL-1 were assessed through ROC curve analysis. Plasma PSGL-1 was upregulated in EOC patients compared with healthy subjects and/or patients with benign ovarian cystadenoma (p < 0.01). Elevated levels of PSGL-1 in the plasma were positively associated with advanced FIGO stage (p < 0.001), tumor size (p = 0.001), tumor metastasis (p = 0.036), and tumor recurrence (p = 0.013), while was negatively correlated with residual tumor size (p < 0.001). Kaplan-Meier survival analysis showed that high plasma PSGL-1 levels were associated with progression-free survival (p = 0.0345). In univariate and multivariate Cox regression analyses, PSGL-1 (HR = 1.456, p = 0.009) was an independent prognostic marker. Plasma PSGL-1 levels distinguished EOC patients and healthy individuals (AUC = 0.905), patients at late and early FIGO stages (AUC = 0.886), and metastatic and non-metastatic EOC (AUC = 0.722). The expression of plasma PSGL-1 is significantly increased in patients with EOC, serving as a reliable biomarker to differentiate between healthy individuals and those with EOC. Furthermore, PSGL-1 in patients is correlated with prognostic indicators, such as advanced FIGO stage, tumor lymph node metastasis, and progression-free survival.

Surface Geometry of Cargo-less Gold Nanoparticles Is a Driving Force for Selective Targeting of Activated Neutrophils to Reduce Thrombosis in Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by recurrent arterial, venous, and microvascular thrombosis where activated neutrophils play a determinant role. However, neutrophils are challenging to target given their short lifespan in circulation and spontaneous activation. Screening of a small library of gold nanoparticles (AuNPs) led to the discovery of a formulation capable of targeting activated neutrophil attachment and has demonstrated that star-shaped, anti-PSGL-1-antibody-coated AuNPs (aPSGL-1-AuNPs) were more efficacious compared with other shapes of AuNPs. Our findings further revealed an exciting and safe targeting mode toward activated neutrophils in the APS mouse model induced by human-patient-derived antiphospholipid IgGs. Our studies demonstrate that targeting is dependent on the specific topographical features of the highly segregated PSGL-1 on the activated neutrophil surface for which a high affinity shape-driven nanomedicine can be designed and implemented. As such, star-shaped aPSGL-1-AuNPs serve as a promising nanoimmunotherapy for immunothrombosis associated with neutrophil adhesion in APS.

Impact of P-selectin-PSGL-1 Axis on Platelet-Endothelium-Leukocyte Interactions in Fatal COVID-19.

In critically ill patients infected with SARS-CoV-2, early leukocyte recruitment to the respiratory system was found to be orchestrated by leukocyte trafficking molecules accompanied by massive secretion of proinflammatory cytokines and hypercoagulability. Our study aimed to explore the interplay between leukocyte activation and pulmonary endothelium in different disease stages of fatal COVID-19. Our study comprised 10 COVID-19 postmortem lung specimens and 20 control lung samples (5 acute respiratory distress syndrome, 2 viral pneumonia, 3 bacterial pneumonia, and 10 normal), which were stained for antigens representing the different steps of leukocyte migration: E-selectin, P-selectin, PSGL-1, ICAM1, VCAM1, and CD11b. Image analysis software QuPath was used for quantification of positive leukocytes (PSGL-1 and CD11b) and endothelium (E-selectin, P-selectin, ICAM1, VCAM1). Expression of IL-6 and IL-1ß was quantified by RT-qPCR. Expression of P-selectin and PSGL-1 was strongly increased in the COVID-19 cohort compared with all control groups (COVID-19:Controls, 17:23, P < .0001; COVID-19:Controls, 2:75, P < .0001, respectively). Importantly, P-selectin was found in endothelial cells and associated with aggregates of activated platelets adherent to the endothelial surface in COVID-19 cases. In addition, PSGL-1 staining disclosed positive perivascular leukocyte cuffs, reflecting capillaritis. Moreover, CD11b showed a strongly increased positivity in COVID-19 compared with all controls (COVID-19:Controls, 2:89; P = .0002), indicating a proinflammatory immune microenvironment. Of note, CD11b exhibited distinct staining patterns at different stages of COVID-19 disease. Only in cases with very short disease course, high levels of IL-1ß and IL-6 mRNA were observed in lung tissue. The striking upregulation of PSGL-1 and P-selectin reflects the activation of this receptor-ligand pair in COVID-19, increasing the efficiency of initial leukocyte recruitment, thus promoting tissue damage and immunothrombosis. Our results show that endothelial activation and unbalanced leukocyte migration play a central role in COVID-19 involving the P-selectin-PSGL-1 axis.

VISTA and its ligands: the next generation of promising therapeutic targets in immunotherapy.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel negative checkpoint receptor (NCR) primarily involved in maintaining immune tolerance. It has a role in the pathogenesis of autoimmune disorders and cancer and has shown promising results as a therapeutic target. However, there is still some ambiguity regarding the ligands of VISTA and their interactions with each other. While V-Set and Immunoglobulin domain containing 3 (VSIG-3) and P-selectin glycoprotein ligand-1(PSGL-1) have been extensively studied as ligands for VISTA, the others have received less attention. It seems that investigating VISTA ligands, reviewing their functions and roles, as well as outcomes related to their interactions, may allow an understanding of their full functionality and effects within the cell or the microenvironment. It could also help discover alternative approaches to target the VISTA pathway without causing related side effects. In this regard, we summarize current evidence about VISTA, its related ligands, their interactions and effects, as well as their preclinical and clinical targeting agents.

PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

Virion-incorporated PSGL-1 and CD43 inhibit both cell-free infection and transinfection of HIV-1 by preventing virus-cell binding.

HIV-1 particles incorporate various host transmembrane proteins in addition to viral Env glycoprotein during assembly at the plasma membrane. In polarized T cells, HIV-1 structural protein Gag localizes to the plasma membrane of uropod, a rear-end protrusion. Notably, uropod transmembrane proteins PSGL-1 and CD43 cocluster specifically with Gag assembling at the plasma membrane even in cells that do not form uropods. Recent reports have shown that expression of either PSGL-1 or CD43 in virus-producing cells reduces the infectivity of progeny virions and that HIV-1 infection reduces the cell surface expression of these proteins. However, the mechanisms for both processes remain to be determined. In this study, we found that virion incorporation of PSGL-1 and CD43 closely correlates with diminished virion infectivity. PSGL-1 and CD43 inhibited virus attachment to CD4+ cells irrespective of the presence of Env. These proteins also inhibited virion attachment to CD4- lymphoid organ fibroblastic reticular cells that mediate transinfection of CD4+ T cells. Consistent with the possibility that highly extended extracellular domains of these proteins physically block virus-cell attachment, the inhibitory effect of PSGL-1 required its full-length ectodomain. HIV-1 encoding Gag mutants that are defective in either coclustering with these host proteins or ESCRT-dependent particle release failed to reduce PSGL-1 on surface of infected cells. This study reveals an anti-HIV-1 mechanism that suppresses virus-cell attachment and a previously unappreciated process of HIV-1-mediated down-regulation of host antiviral proteins, both of which likely require virion incorporation of these proteins.

Low P-Selectin Glycoprotein Ligand-1 Expression in Neutrophils Associates with Disease Activity and Deregulated NET Formation in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis.

Novel Knowledge about Molecular Mechanisms of Heparin-Induced Thrombocytopenia Type II and Treatment Targets.

Heparin-induced thrombocytopenia type II (HIT II), as stated in the literature, occurs in about 3% of all patients and in 0.1-5% of surgical patients. Thrombosis develops in 20-64% of patients with HIT. The mortality rate in HIT II has not decreased using non-heparin treatment with anticoagulants such as argatroban and lepirudin. An improved understanding of the pathophysiology of HIT may help identify targeted therapies to prevent thrombosis without subjecting patients to the risk of intense anticoagulation. The review will summarize the current knowledge about the pathogenesis of HIT II, potential new therapeutic targets related to it, and new treatments being developed. HIT II pathogenesis involves multi-step immune-mediated pathways dependent on the ratio of PF4/heparin and platelet, monocyte, neutrophil, and endothelium activation. For years, only platelets were known to take part in HIT II development. A few years ago, specific receptors and signal-induced pathways in monocytes, neutrophils and endothelium were revealed. It had been shown that the cells that had become active realised different newly formed compounds (platelet-released TF, TNFα, NAP2, CXCL-7, ENA-78, platelet-derived microparticles; monocytes-TF-MPs; neutrophils-NETs), leading to additional cell activation and consequently thrombin generation, resulting in thrombosis. Knowledge about FcγIIa receptors on platelets, monocytes, neutrophils and FcγIIIa on endothelium, chemokine (CXCR-2), and PSGL-1 receptors on neutrophils could allow for the development of a new non-anticoagulant treatment for HIT II. IgG degradation, Syk kinase and NETosis inhibition are in the field of developing new treatment possibilities too. Accordingly, IdeS and DNases-related pathways should be investigated for better understanding of HIT pathogenesis and the possibilities of being the HIT II treatment targets.

GALNT4 primes monocytes adhesion and transmigration by regulating O-Glycosylation of PSGL-1 in atherosclerosis.

Atherosclerosis is a major underlying cause of cardiovascular disease. Genome wide association studies have predicted that GalNAc-T4 (GALNT4), which responsible for initiating step of mucin-type O-glycosylation, plays a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanism remains obscure. Thus, we sought to determine the role and mechanism of GALNT4 in atherosclerosis. Firstly, we found the expression of GALNT4 and protein O-glycosylation were both increased in plaque as atherosclerosis progressed in ApoE-/- mice by immunohistochemistry. And the expression of GALNT4 was also increased in human monocytes treated with ACS (acute coronary syndrome) sera and subjected to LPS and ox-LDL in vitro. Moreover, silencing expression of GALNT4 by shRNA lentivirus alleviated atherosclerotic plaque formation and monocyte/macrophage infiltration in ApoE-/- mice. Functional investigations demonstrate that GALNT4 knockdown inhibited P-selectin-induced activation of ß2 integrin on the surface of monocytes, decreased monocytes adhesion under flow condition with P-selectin stimulation, as well as suppressed monocytes transmigration triggered by monocyte chemotactic protein- 1(MCP-1). In contrast, GALNT4 overexpression enhanced monocytes adhesion and transmigration. Furthermore, Vicia Villosa Lectin (VVL) pull down and PSGL-1 immunoprecipitation assays showed that GALNT4 overexpression increased O-Glycosylation of PSGL-1 and P-selectin induce phosphorylation of Akt/mTOR and IκBα/NFκB on monocytes. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activation of Akt/mTOR and IκBα/NFκB. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Collectively, GALNT4 catalyzed PSGL-1 O-glycosylation that involved in P-selectin induced monocytes adhesion and transmigration via Akt/mTOR and NFκB pathway. Thus, GALNT4 may be a potential therapeutic target for atherosclerosis.

P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells.

BACKGROUND: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown. RESULTS: Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0-250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1. CONCLUSIONS: Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Association of VLA4, 5, 6 and PSGL1 expression levels with engraftment in autologous HPSC transplantation in multiple myeloma patients.

The most promising therapy for leukemia is hematopoietic stem cell transplantation. Engraftment of HPSCs mainly depends on some factors such as adhesion molecules, including VLAs. This study tried to delineate the relationship between HPSCs engraftment and expression level of PSGL1 and VLA4, 5, and 6 genes in candidate MM patients for autologous bone marrow transplantation. Firstly, the CD 34+ HPSCs were collected from multiple myeloma (MM) patients after five days of G-CSF therapy through apheresis processes. Then, the patients were categorized into two groups of good and bad prognosis depending on engraftment time (Less or more than 18 days). Followingly, the expression of PSGL1 and VLA4, VLA5, and VLA6 genes were assessed by the qRT-PCR technique in each patient. Finally, the correlation between the genes and engraftment time was investigated to determine the prognostic role of each gene on HPSCs transplantation. Our findings demonstrated that there is a significant correlation between VLA4 (P=< 0.0001) and 5 (P = 0.005) levels and HPSCs engraftment time. As the higher levels of VLA4 and 5, the shorter time HPSCs engraftment occurs. In contrast, there was no significant correlation between VLA6 (P = 0.2) and PSGL1 (P = 0.3) genes levels and engraftment time. So that, the patients with a good prognosis had a higher level of VLA4 and VLA5, but no relation was found between VLA6 and PSGL1. It is concluded that VLA4 and VLA5 expression could be considered a significant prognostic factor for HPSC transplantation.

Prediction of renal outcome in Henoch-Schönlein nephritis based on biopsy findings.

BACKGROUND: In Henoch-Schönlein nephritis (HSN), a risk factor for unfavorable outcome is prolonged proteinuria, but the value of renal biopsies in prognosis assessment is debatable. METHODS: We evaluated serial renal biopsies from 26 HSN patients. Follow-up biopsy occurred at median 2.1 years after diagnostic biopsy. Patients formed two groups at the follow-up biopsy: patients without proteinuria (group I; n = 11) and with proteinuria (group II; n = 15). Biopsies underwent evaluation according to three classifications: International Study of Kidney Disease in Children (ISKDC), Oxford (MEST-C), and semiquantitative classification (SQC) including an activity and chronicity score. Analysis also included expression of pro-fibrotic (alpha-smooth muscle actin and vimentin) and inflammatory (P-selectin glycoprotein ligand-1) molecules in the diagnostic biopsy specimens. Definition of unfavorable outcome was active renal disease or reduced renal function at last follow-up. RESULTS: Between the biopsies, SQC chronicity score increased in 22 (85%) patients, whereas activity score and ISKDC grade decreased in 21 (81%) and 17 (65%), respectively. Of the MEST-C parameters, endocapillary proliferation (from 83 to 13%; p < 0.001) and crescents (from 63 to 25%; p = 0.022) showed significant reduction, and segmental glomerulosclerosis (from 38 to 79%; p = 0.006) significant increment. These changes occurred similarly in groups I and II. Expression of the pro-fibrotic and inflammatory molecules showed no clinically significant differences between groups I and II. None in group I and five (33%) patients in group II had unfavorable outcome (p = 0.053). CONCLUSIONS: Our results suggest that follow-up biopsies provide limited additional information to clinical symptoms in HSN outcome prediction.

Biphasic Force-Regulated Phosphorylation Site Exposure and Unligation of ERM Bound with PSGL-1: A Novel Insight into PSGL-1 Signaling via Steered Molecular Dynamics Simulations.

The PSGL-1-actin cytoskeleton linker proteins ezrin/radixin/moesin (ERM), an adaptor between P-selectin glycoprotein ligand-1 (PSGL-1) and spleen tyrosine kinase (Syk), is a key player in PSGL-1 signal, which mediates the adhesion and recruitment of leukocytes to the activated endothelial cells in flow. Binding of PSGL-1 to ERM initials intracellular signaling through inducing phosphorylation of Syk, but effects of tensile force on unligation and phosphorylation site exposure of ERM bound with PSGL-1 remains unclear. To answer this question, we performed a series of so-called "ramp-clamp" steered molecular dynamics (SMD) simulations on the radixin protein FERM domain of ERM bound with intracellular juxtamembrane PSGL-1 peptide. The results showed that, the rupture force of complex pulled with constant velocity was over 250 pN, which prevented the complex from breaking in front of pull-induced exposure of phosphorylation site on immunoreceptor tyrosine activation motif (ITAM)-like motif of ERM; the stretched complex structure under constant tensile forces <100 pN maintained on a stable quasi-equilibrium state, showing a high mechano-stabilization of the clamped complex; and, in consistent with the force-induced allostery at clamped stage, increasing tensile force (<50 pN) would decrease the complex dissociation probability but facilitate the phosphorylation site exposure, suggesting a force-enhanced biophysical connectivity of PSGL-1 signaling. These force-enhanced characters in both phosphorylation and unligation of ERM bound with PSGL-1 should be mediated by a catch-slip bond transition mechanism, in which four residue interactions on binding site were involved. This study might provide a novel insight into the transmembrane PSGL-1 signal, its biophysical connectivity and molecular structural basis for cellular immune responses in mechano-microenvironment, and showed a rational SMD-based computer strategy for predicting structure-function relation of protein under loads.