Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

Neuregulin 2 (NRG2) is expressed in gliomas and promotes migration of human glioma cells.

INTRODUCTION: Glioma is the most common primary brain tumour in adults. Numerous studies have shown that neuregulins (NRGs) may be involved in the formation of glioma. Although NRG1 has been extensively studied in glioma, the functions of NRG2 in glioma development remain elusive. MATERIAL AND METHODS: In the present study, we investigated the expression of NRG2 in different grades of human glioma samples, and analysed the functional effects of NRG2 in glioma cells mainly using wound healing assay and transmigration assay. RESULTS: We found that NRG2 was differentially expressed in different grades of human glioma/glioblastoma tissues. The data from wound healing assays demonstrated that NRG2 can differentially promote the migration of SHG44 human glioma, and U251 and U-87 MG human glioblastoma cells at different time points. The results of cell transmigration assays showed that, compared with the vehicle control, the number of cells that migrated to the underside of the insert was increased significantly for all the 3 cell lines treated with 5 nM of NRG2 for 12 hours. CONCLUSIONS: In conclusion, our results demonstrated that NRG2 is expressed in gliomas to varying extents, and it may play roles in the migration of glioma cells in vitro. These data suggest that treatment targeting NRG2 signalling may partly reverse the migration-based metastasis of glioma cells.

Lymphatic Endothelial Cell Junctions: Molecular Regulation in Physiology and Diseases.

Lymphatic endothelial cells (LECs) lining lymphatic vessels develop specialized cell-cell junctions that are crucial for the maintenance of vessel integrity and proper lymphatic vascular functions. Successful lymphatic drainage requires a division of labor between lymphatic capillaries that take up lymph via open "button-like" junctions, and collectors that transport lymph to veins, which have tight "zipper-like" junctions that prevent lymph leakage. In recent years, progress has been made in the understanding of these specialized junctions, as a result of the application of state-of-the-art imaging tools and novel transgenic animal models. In this review, we discuss lymphatic development and mechanisms governing junction remodeling between button and zipper-like states in LECs. Understanding lymphatic junction remodeling is important in order to unravel lymphatic drainage regulation in obesity and inflammatory diseases and may pave the way towards future novel therapeutic interventions.

Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma.

Neural cell adhesion molecular L1-like protein (CHL1) is a member of the cell adhesion molecule L1 family and serves an important role in the development and progression of tumors. The cytokine neuregulin 1 (NRG1) has been indicated in the tumorigenesis and promotion of metastasis through the modulation of L1. However, the roles of NRG1 in regulating CHL1 in glioma have not been elucidated. The present study investigated the protein expression levels and roles of CHL1 and the possible correlation between NRG1 and CHL1 protein expression levels in human gliomas, both in vivo and in vitro. Using immunohistochemistry coupled with a human glioma tissue microarray, it was demonstrated that the percentage of CHL1-positive areas was the highest in grade II glioma tissues. Using immunofluorescence staining, a positive correlation was identified between the expression levels of CHL1 and proliferating cell nuclear antigen. In addition, CHL1 downregulation also resulted in increased senescence of U-87 MG human glioblastoma cells. In vitro, administration of NRG1α induced a significant increase in CHL1 protein expression levels in human glioma SHG-44 and U251 cells and in human glioblastoma U-87 MG cells, whereas NRG1ß failed to increase CHL1 expression levels in U251 cells. These findings were further confirmed by the downregulation of NRG1 expression levels using small interfering RNA treatment, which resulted in the reduction of CHL1 protein expression levels in U-87 MG cells. These data indicate that NRG1 can regulate CHL1 protein expression levels in gliomas, that it is correlated with malignancy, and that NRG1 may contribute to malignancy by upregulating CHL1 protein expression levels in glioma/glioblastoma cells.

Ocular Microbiota and Intraocular Inflammation.

The term ocular microbiota refers to all types of commensal and pathogenic microorganisms present on or in the eye. The ocular surface is continuously exposed to the environment and harbors various commensals. Commensal microbes have been demonstrated to regulate host metabolism, development of immune system, and host defense against pathogen invasion. An unbalanced microbiota could lead to pathogenic microbial overgrowth and cause local or systemic inflammation. The specific antigens that irritate the deleterious immune responses in various inflammatory eye diseases remain obscure, while recent evidence implies a microbial etiology of these illnesses. The purpose of this review is to provide an overview of the literature on ocular microbiota and the role of commensal microbes in several eye diseases. In addition, this review will also discuss the interaction between microbial pathogens and host factors involved in intraocular inflammation, and evaluate therapeutic potential of targeting ocular microbiota to treat intraocular inflammation.