Functional Study Of Exosomes Derived From Brain Microvascular Endothelial Cells In Meningitic Escherichia Coli Infection

Bacterial meningitis is an important fatal infectious disease.Meningitis Escherichia coli(E.coli)is one of the main pathogens involving in bacterial meningitis,and the blood-brain barrier(BBB)disruption is a key step during this process.BBB is mainly composed of brain microvascular endothelial cells(BMECs),astrocytes and pericytes,which protects the central nervous system(CNS)from toxins in the blood and the invasive pathogens by controlling their entry and exit.Materials exchange as well as information exchange between the above types of cells are essential for maintaining the integrity of the BBB structure.Exosomes are microvesicles formed by secondary cell endocytosis and are loaded with proteins,nucleic acids and lipid which playing important roles in cell-to-cell information transmission.Meningitic E.coli can cause structural loss and functional changes of the BBB by interacting with BMECs,but it is unclear so far whether the BMECs-derived exosomes play certain roles during this process.In this study,we challenged the BMECs with meningitic E.coli PCN033,isolated and identified the BMECs-derived exosomes,and preliminarily explored the function of these exosomes in the pathogenesis of meningitic E.coli.The exosomal miRNAs were moreover extracted and analyzed via miRNA sequencing.We found that the infected-BMECs-derived exosomes could be intaken by both BMECs and astrocytes U251,indicating the potential roles of exosomes in the regulation of BBB stability.By the treatment of exosomes isolating from infected BMECs,we found that the transcellular resistance of BMECs significantly decreased,and the tight junction proteins ZO-1 and Occludin were significantly downregulated.The BMECs activation marker ICAM-1 was significantly upregulated,while the astrocytes activative marker GFAP was significantly downregulated.Via exosomal miRNA sequencing as well as real-time PCR verification,we identified mir-146a-5p,mir-497-5p,mir-5008-3p,mir-6854-5p,mir-4449 and novel-183 were found being significantly upregulated in exosomes of BMECs infected with PCN033.Based on their target genes prediction and KEGG analysis,we found the candidate target genes of these above miRNAs are involved in various biological processes and signaling pathways,including cell proliferation,growth and survival,and inflammatory and enzymatic activities.By carrying these upregulated miRNAs,the exosomes may affect the barrier function of BMECs and the inflammatory response of the astrocytes.