PSGL-1, ADAM8, and selectins as potential biomarkers in the diagnostic process of systemic lupus erythematosus and systemic sclerosis: an observational study.

Background: Early diagnosis and treatment of Systemic lupus erythematosus (SLE) and Systemic sclerosis (SSc) present significant challenges for clinicians. Although various studies have observed changes in serum levels of selectins between healthy donors and patients with autoimmune diseases, including SLE and SSc, their potential as biomarkers has not been thoroughly explored. We aimed to investigate serum profiles of PSGL-1 (sPSGL-1), ADAM8 (sADAM8) and P-, E- and L-selectins (sP-, sE- and sL-selectins) in defined SLE and SSc patient cohorts to identify disease-associated molecular patterns. Methods: We collected blood samples from 64 SLE patients, 58 SSc patients, and 81 healthy donors (HD). Levels of sPSGL-1, sADAM8 and selectins were analyzed by ELISA and leukocyte membrane expression of L-selectin and ADAM8 by flow cytometry. Results: Compared to HD, SLE and SSc patients exhibited elevated sE-selectin and reduced sL-selectin levels. Additionally, SLE patients exhibited elevated sPSGL-1 and sADAM8 levels. Compared to SSc, SLE patients had decreased sL-selectin and increased sADAM8 levels. Furthermore, L-selectin membrane expression was lower in SLE and SSc leukocytes than in HD leukocytes, and ADAM8 membrane expression was lower in SLE neutrophils compared to SSc neutrophils. These alterations associated with some clinical characteristics of each disease. Using logistic regression analysis, the sL-selectin/sADAM8 ratio in SLE, and a combination of sL-selectin/sE-selectin and sE-selectin/sPSGL-1 ratios in SSc were identified and cross-validated as potential serum markers to discriminate these patients from HD. Compared to available diagnostic biomarkers for each disease, both sL-selectin/sADAM8 ratio for SLE and combined ratios for SSc provided higher sensitivity (98% SLE and and 67% SSc correctly classified patients). Importantly, the sADAM8/% ADAM8(+) neutrophils ratio discriminated between SSc and SLE patients with the same sensitivity and specificity than current disease-specific biomarkers. Conclusion: SLE and SSc present specific profiles of sPSGL-1, sE-, sL-selectins, sADAM8 and neutrophil membrane expression which are potentially relevant to their pathogenesis and might aid in their early diagnosis.

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.

The role of P-selectin/PSGL-1 in regulating NETs as a novel mechanism in cerebral ischemic injury.

In recent years, substantial advancements have been made in understanding the pathophysiology of ischemic stroke. Despite these developments, therapeutic options for cerebral ischemia remain limited due to stringent time windows and various contraindications. Consequently, there has been a concentrated effort to elucidate the underlying mechanisms of cerebral ischemic injury. Emerging research indicates that neutrophil extracellular traps (NETs) exacerbate inflammation and damage in ischemic brain tissue, contributing to neuronal cell death. The inhibition of NETs has shown potential in preventing thrombosis and the infiltration of immune cells. Central to the formation of NETs are P-selectin and its ligand, P-selectin glycoprotein ligand-1 (PSGL-1), which represent promising therapeutic targets. This review explores the detrimental impact of P-selectin, PSGL-1, and NETs on cerebral ischemia. Additionally, it delineates the processes by which P-selectin and PSGL-1 stimulate NETs production and provides evidence that blocking these molecules reduces NETs formation. This novel insight highlights a potential therapeutic avenue that warrants further investigation by researchers in the field.

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.

Analogues of the pan-selectin antagonist rivipansel (GMI-1070).

The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.

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.

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.

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, a Strategic Biomarker for Pathological Conditions in HIV Infection: A Hypothesis Review.

P-selectin glycoprotein ligand-1 (PSGL-1) has been established to be a cell adhesion molecule that is involved in the cellular rolling mechanism and the extravasation cascade, enabling the recruitment of immune cells to sites of inflammation. In recent years, researchers have established that PSGL-1 also functions as an HIV restriction factor. PSGL-1 has been shown to inhibit the HIV reverse transcription process and inhibit the infectivity of HIV virions produced by cells expressing PSGL-1. Cumulative evidence gleaned from contemporary literature suggests that PSGL-1 expression negatively affects the functions of immune cells, particularly T-cells, which are critical participants in the defense against HIV infection. Indeed, some researchers have observed that PSGL-1 expression and signaling provokes T-cell exhaustion. Additionally, it has been established that PSGL-1 may also mediate virus capture and subsequent transfer to permissive cells. We therefore believe that, in addition to its beneficial roles, such as its function as a proinflammatory molecule and an HIV restriction factor, PSGL-1 expression during HIV infection may be disadvantageous and may potentially predict HIV disease progression. In this hypothesis review, we provide substantial discussions with respect to the possibility of using PSGL-1 to predict the potential development of particular pathological conditions commonly seen during HIV infection. Specifically, we speculate that PSGL-1 may possibly be a reliable biomarker for immunological status, inflammation/translocation, cell exhaustion, and the development of HIV-related cancers. Future investigations directed towards our hypotheses may help to evolve innovative strategies for the monitoring and/or treatment of HIV-infected individuals.

A non-human primate derived anti-P-selectin glycoprotein ligand-1 antibody curtails acute pancreatitis by alleviating the inflammatory responses.

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo, we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l-arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

PSGL-1 is an evolutionarily conserved antiviral restriction factor.

IMPORTANCE: Studying the co-evolution between viruses and humans is important for understanding why we are what we are now as well as for developing future antiviral drugs. Here we pinned down an evolutionary arms race between retroviruses and mammalian hosts at the molecular level by identifying the antagonism between a host antiviral restriction factor PSGL-1 and viral accessory proteins. We show that this antagonism is conserved from mouse to human and from mouse retrovirus to HIV. Further studying this antagonism might provide opportunities for developing new antiviral therapies.

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.

PSGL-1: a novel immune checkpoint driving T-cell dysfunction in obstructive sleep apnea.

Introduction: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy. Methods: The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models. Results: Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia. Discussion: In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients.

NMR indicates the N-termini of PSGL1 and CCR7 bind competitively to the chemokine CCL21.

Chemokines are from a family of secreted cytokines that direct the trafficking of immune cells to coordinate immune responses. Chemokines are involved in numerous disease states, including responding to infections, autoimmune disorders, and cancer metastasis. Ther are chemokines, like CCL21, that signal for cellular migration through the activation of G protein-coupled receptors, like CCR7, through interaction with the receptor's extracellular N-terminus, loops, and core of the receptor. CCL21 is involved in routine immune surveillance but can also attract metastasizing cancer cells to lymph nodes. P-selectin glycoprotein ligand 1 (PSGL1) has a role in cellular adhesion during chemotaxis and is a transmembrane signaling molecule. PSGL1 expression enhances chemotactic responses of T cells to CCL21. Here NMR studies indicate the binding sites on CCL21 for the N-termini or PSGL1 and CCR7 overlap, and binding of the N-termini of PSGL1 and CCR7 is competitive.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

Exploring dysregulated immune response genes and endothelial dysfunction biomarkers as predictors of severe COVID-19.

Identifying individuals and factors associated with severe cases of COVID-19 is crucial as the pandemic continues to spread globally. Effective biomarkers for predicting severe cases are essential for optimizing clinical management, therapy, and preventing unfavorable outcomes. This exploratory observational study aimed to investigate the expression of dysregulated immune response genes (ARG1, NOS2, ITGA4, and SELPLG) in total leukocytes, plasmatic levels of P-selectin and PSGL-1, and their clinical associations in patients with mild and severe COVID-19. Data from 117 confirmed COVID-19 patients (severe = 58, mild = 59) were collected upon admission. Gene expression was measured using RT-qPCR, and plasma protein levels assessed with ELISA assay. The severe COVID-19 patient group had a higher median age of 62.0 (p = 0.0001), a higher proportion of black individuals (86.2%, p < 0.0001), and more males (65.5%, p = 0.007). The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were significantly higher in the severe COVID-19 patient group (p < 0.0001), indicating ongoing systemic inflammation. Severe COVID-19 patients also exhibited increased expression of ARG1 (p < 0.05) and SELPLG (p < 0.0001) genes, as well as higher concentrations of soluble P-selectin (p < 0.005) and PSGL-1 (p < 0.05) proteins. Multivariate analysis revealed that NLR, PLR, the expression of SELPLG and sPSGL-1 were independent predictors of COVID-19 severity. In conclusion, this study suggests that biomarkers of endothelial dysfunction and dysregulated leukocyte responses are associated with COVID-19 severity, serving as promising predictive tools for optimizing clinical management and patient monitoring.

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.