Appearance of claudin-5+ leukocyte subtypes in the blood and CNS during progression of EAE

Background Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood–brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. Methods To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. Results Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. Conclusion Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.

Maternal nematode infection upregulates expression of Th2/Treg and diapedesis related genes in the neonatal brain

Intestinal nematode infections common during pregnancy have recently been shown to have impacts that extend to their uninfected offspring including altered brain gene expression. If maternal immune signals reach the neonatal brain, they might alter neuroimmune development. We explored expression of genes associated with four distinct types of T cells (Th1, Th2, Th17, Treg) and with leukocyte transendothelial migration and endocytosis transport across the blood–brain barrier (BBB) in the postnatal brain of offspring of nematode-infected mice, through secondary analysis of a whole brain gene expression database. Th1/Th17 expression was lowered by maternal infection as evidenced by down-regulated expression of IL1β, Th1 receptors and related proteins, and of IL22 and several Th17 genes associated with immunopathology. In contrast, Th2/Treg related pathways were upregulated as shown by higher expression of IL4 and TGF-β family genes. Maternal infection also upregulated expression of pathways and integrin genes involved in transport of leukocytes in between endothelial cells but downregulated endosome vesicle formation related genes that are necessary for endocytosis of immunoglobulins across the BBB. Taken together, pup brain gene expression indicates that maternal nematode infection enhanced movement of leukocytes across the neonatal BBB and promoted a Th2/Treg environment that presumably minimizes the proinflammatory Th1 response in the pup brain.

Potential Roles of Extracellular Vesicles as Biomarkers and a Novel Treatment Approach in Multiple Sclerosis

Extracellular vesicles (EVs) are a heterogeneous group of bilayer membrane-wrapped molecules that play an important role in cell-to-cell communication, participating in many physiological processes and in the pathogenesis of several diseases, including multiple sclerosis (MS). In recent years, many studies have focused on EVs, with promising results indicating their potential role as biomarkers in MS and helping us better understand the pathogenesis of the disease. Recent evidence suggests that there are novel subpopulations of EVs according to cell origin, with those derived from cells belonging to the nervous and immune systems providing information regarding inflammation, demyelination, axonal damage, astrocyte and microglia reaction, blood–brain barrier permeability, leukocyte transendothelial migration, and ultimately synaptic loss and neuronal death in MS. These biomarkers can also provide insight into disease activity and progression and can differentiate patients’ disease phenotype. This information can enable new pathways for therapeutic target discovery, and consequently the development of novel treatments. Recent evidence also suggests that current disease modifying treatments (DMTs) for MS modify the levels and content of circulating EVs. EVs might also serve as biomarkers to help monitor the response to DMTs, which could improve medical decisions concerning DMT initiation, choice, escalation, and withdrawal. Furthermore, EVs could act not only as biomarkers but also as treatment for brain repair and immunomodulation in MS. EVs are considered excellent delivery vehicles. Studies in progress show that EVs containing myelin antigens could play a pivotal role in inducing antigen-specific tolerance of autoreactive T cells as a novel strategy for the treatment as “EV-based vaccines” for MS. This review explores the breakthrough role of nervous and immune system cell-derived EVs as markers of pathological disease mechanisms and potential biomarkers of treatment response in MS. In addition, this review explores the novel role of EVs as vehicles for antigen delivery as a therapeutic vaccine to restore immune tolerance in MS autoimmunity.

A Potent Leukocyte Transmigration Blocker: GT-73 Showed a Protective Effect against LPS-Induced ARDS in Mice

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Appearance of Claudin-5+ Leukocyte Subtypes in the Blood and CNS During Progression of EAE

Background: Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood-brain barrier (BBB), is also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. Methods: To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. Results: Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. Conclusion: Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.

Proteomic Analysis Revealed the Characteristics of Key Proteins Involved in the Regulation of Inflammatory Response, Leukocyte Transendothelial Migration, Phagocytosis, and Immune Process during Early Lung Blast Injury

Previous studies found that blast injury caused a significant increased expression of interleukin-1, IL-6, and tumor necrosis factor, a significant decrease in the expression of IL-10, an increase in Evans blue leakage, and a significant increase in inflammatory cell infiltration in the lungs. However, the molecular characteristics of lung injury at different time points after blast exposure have not yet been reported. Therefore, in this study, tandem mass spectrometry (TMT) quantitative proteomics and bioinformatics analysis were used for the first time to gain a deeper understanding of the molecular mechanism of lung blast injury at different time points. Forty-eight male C57BL/6 mice were randomly divided into six groups: control, 12 h, 24 h, 48 h, 72 h, and 1 w after low-intensity blast exposure. TMT quantitative proteomics and bioinformatics analysis were performed to analyze protein expression profiling in the lungs from control and blast-exposed mice, and differential protein expression was verified by Western blotting. The results demonstrated that blast exposure induced severe lung injury, leukocyte infiltration, and the production of inflammatory factors in mice. After analyzing the expression changes in global proteins and inflammation-related proteomes after blast exposure, the results showed that a total of 6861 global proteins and 608 differentially expressed proteins were identified, of which 215, 128, 187, 232, and 65 proteins were identified at 12 h, 24 h, 48 h, 72 h, and 1 week after blast exposure, respectively. Moreover, blast exposure-induced 177 differentially expressed proteins were associated with inflammatory responses, which were enriched in the inflammatory response regulation, leukocyte transendothelial migration, phagocytosis, and immune response. Therefore, blast exposure may induce early inflammatory response of lung tissue by regulating the expression of key proteins in the inflammatory process, suggesting that early inflammatory response may be the initiating factor of lung blast injury. These data can provide potential therapeutic candidates or approaches for the development of future treatment of lung blast injury.

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC–MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl− transport in CF.

Neutrophil transendothelial migration hotspots – mechanisms and implications

ABSTRACTDuring inflammation, leukocytes circulating in the blood stream exit the vasculature in a process called leukocyte transendothelial migration (TEM). The current paradigm of this process comprises several well-established steps, including rolling, adhesion, crawling, diapedesis and sub-endothelial crawling. Nowadays, the role of the endothelium in transmigration is increasingly appreciated. It has been established that leukocyte exit sites on the endothelium and in the pericyte layer are in fact not random but instead may be specifically recognized by migrating leukocytes. Here, we review the concept of transmigration hotspots, specific sites in the endothelial and pericyte layer where most transmigration events take place. Chemokine cues, adhesion molecules and membrane protrusions as well as physical factors, such as endothelial junction stability, substrate stiffness, the presence of pericytes and basement membrane composition, may all contribute to local hotspot formation to facilitate leukocytes exiting the vasculature. In this Review, we discuss the biological relevance of such hotspots and put forward multiple mechanisms and factors that determine a functional TEM hotspot.

Elucidating the Biomechanics of Leukocyte Transendothelial Migration by Quantitative Imaging

Leukocyte transendothelial migration is crucial for innate immunity and inflammation. Upon tissue damage or infection, leukocytes exit blood vessels by adhering to and probing vascular endothelial cells (VECs), breaching endothelial cell-cell junctions, and transmigrating across the endothelium. Transendothelial migration is a critical rate-limiting step in this process. Thus, leukocytes must quickly identify the most efficient route through VEC monolayers to facilitate a prompt innate immune response. Biomechanics play a decisive role in transendothelial migration, which involves intimate physical contact and force transmission between the leukocytes and the VECs. While quantifying these forces is still challenging, recent advances in imaging, microfabrication, and computation now make it possible to study how cellular forces regulate VEC monolayer integrity, enable efficient pathfinding, and drive leukocyte transmigration. Here we review these recent advances, paying particular attention to leukocyte adhesion to the VEC monolayer, leukocyte probing of endothelial barrier gaps, and transmigration itself. To offer a practical perspective, we will discuss the current views on how biomechanics govern these processes and the force microscopy technologies that have enabled their quantitative analysis, thus contributing to an improved understanding of leukocyte migration in inflammatory diseases.

Bioinformatics analysis to screen and identify key biomarkers of papillary renal cell carcinoma

Background: Papillary renal cell carcinoma (PRCC) is the second most common type of renal cell carcinoma after clear cell renal cell carcinoma(ccRCC). Its pathological classification is controversial and its molecular mechanism is poorly understood. Therefore, the identification of key genes and their biological pathways is of great significance to elucidate the molecular mechanisms of PRCC occurrence and progression. Methods:Downloaded PRCC-related datasets GSE7023, GSE48352 and GSE15641 from the Gene Expression Omnibus (GEO) database. Differential expression genes (DEG) were identified and gene ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis were performed. Cytoscape and STRING are used to construct the Protein-Protein Interaction Network (PPI) and module analysis to find hub genes and key pathways. Hierarchical clustering of hub genes was constructed using UCSC. Overall survival and relapse-free survival of Hub genes were analyzed using Kaplan-Meier plotter. UALCAN was applied to analyze genes expression in primary organizations, different stages ,different subtypes and races.Results: A total of 214 DEG genes were identified, including 205 down-regulated genes and 9 up-regulated genes. DEG is mainly concentrated in angiogenesis, kidney development, oxidation-reduction process, metabolic pathways, etc. 17 hub gene enrichment mainly in angiogenesis, cell adhesion, platelet degranulation, Leukocyte transendothelial migration biological processes, etc. 17 hub genes were screened out, which were mainly enriched in the biological processes of angiogenesis, cell adhesion, platelet degranulation, and leukocyte transendothelial migration. Survival analysis showed that EGF, KDR, CXCL12, REN, PECAM1, CDH5, THY1, WT1, PLAU and DCN may be related to the carcinogenesis, metastasis or recurrence of PRCC. Conclusions: DEG and hub genes identified in present study provide clues to the specific molecular mechanisms of PRCC occurrence and development, and may be potential molecular markers and therapeutic targets for accurate classification and efficient diagnosis and treatment of PRCC.