Screening And Functional Verification Of Bovine Milk-derived Exosomal Inflammation-related MicroRNA

Bovine mastitis is the most serious disease restricts the dairy industry,and Staphylococcus aureus(S.aureus)is one of the most common pathogens responsible for sub-clinical mastitis.In mammary tissue,mammary epithelial cells are very important secreting cells and play crucial role in innate immune system.Many studies showed that S.aureus could activate immune-related signaling pathways of mammary epithelial cells,and influence the activation,differentiation or migration of immune cells.Meanwhile,miRNAs(microRNAs,miRNAs)act as important regulators in these biological processes.Exosomes,nano-scale extacellular vesicles with intercellular communication functions,have been confirmed to be present in milk and mammary epithelial cell culture fluids.However,little researches focused on the level of miRNAs in milk or mammary epithelial cell-derived exosomes infected by S.aureus,and the communication between mammary epithelial cell-derived exosomes and immune cells is also unclear.Therefore,this study intends to identify miRNAs associated with inflammation in milk-derived exosomes at the transcriptional level using miRNA-seq,and to investigate the regulatory mechanisms of miRNAs in inflammatory responses and intercellular communication between mammary epithelial cell-derived exosomes and macrophages using an in vitro cell model.In this study,exosomes were first obtained from healthy(HH)and mastitis(HM)cow milk,and miRNA-seq was used to screen inflammation-related miRNAs.Then,the role of miR-23 a in the inflammatory process was studied in cells infected with LTA.Meanwhile,the expression of miRNAs in mammary epithelial cell derived exosomes was studied,and the role of exosomes in macrophages.Finally,qRT-PCR,flow cytometry,and miRNA transfection were used to analyze the effect of mammary glande cell-derived exosomal miR-221 on macrophage polarization.The main results were as follows:(1)The healthy and mastitis milk exosomes were pelled by differential centrifugation,and the vesicles were identified by transmission electron microscopy,Nanosight and Western blot.Then,6 miRNA-seq libraries were constructed,and 1472 miRNAs were identified,including 492 known miRNAs and 980 newly predicted miRNAs.Analysis of known miRNAs expression pattern revealed that a small portion of highly expressed miRNAs accounted for about 80% of all miRNAs.A total of 14 differentially expressed miRNAs were identified in HH and HM,and were mainly involved in the regulation of disease,immunity and inflammation.(2)In an LTA concentration gradient test,40 μg/mL LTA was used as the optimal infectious concentration of Mac-T cells.TNF-α and IL-6 mRNA significantly increased after LTA treatment at 6 h,and the concentration of TNF-α and IL-6 increased with time.The levels of TLR2 and MyD88 mRNA were significantly increased after LTA infection at 3 h.Furthermore,protein analysis revealed that LTA could activate the PI3K/AKT signaling pathway through binding to the pattern recognition receptor TLR2.Whereas,LTA failed to activate PI3K/AKT after treatment with OxPAPC,indicating that TLR2 plays an indispensable role in inflammatory responses induced by LTA.(3)Then,the levels of 14 miRNAs in Mac-T-derived exosomes after LTA infection were detected,most of these miRNAs were upregulated at 3 h,and the expression of miR-23 a was the highest.And 11 exosomal miRNAs upregulated,3 miRNAs downregulated at 24 h,and the level of miR-221 was highest.To identify the function of miR-23 a,target genes and KEGG pathway enrichment analysis revealed that miR-23 a mainly involved in inflammation-related signaling pathways such as JAK/STAT,PI3K/AKT and MAPK.The dual luciferase reporter system assay revealed that miR-23 a directly targets the PI3 K 3’ UTR.miR-23 a overexpression and inhibition revealed that miR-23 a could inhibit PI3K/AKT signaling pathway through targeting PI3 K,and then inhibited inflammatory response in Mac-T cells.In addition,exosomes were co-cultured with bovine peripheral blood macrophages,the level of cell surface protein CD11 b was increased,the levels of TNF-α and iNOS mRNA were significantly increased,and the level of Arg-1 mRNA was decreased,indicating that the exosomes can promote macrophage polarization towards M1 type.(4)HC11 and RAW264.7 were used to utilize intracellular conmmunication in virto.The expression of miR-221 in HC11-derived exosomes was significantly increased after LTA infection for 12 h,and miR-221 was highly expressed in macrophages after co-culture with RAW264.7.Meanwhile,the expression of TNF-α mRNA was up-regulated in macrophages,the expression of Arg-1 mRNA was down-regulated,and the expression of CD11 b was increased.After transfection of miR-221 mimic and inhibitor,in HC11-derived exosomes,Exo(mimic)and Exo(inhibitor),the expression of miR-221 was increased and decreased,respectively.In addition,co-culture of Exo(mimic)and Exo(inhibitor)with macrophages promotes and inhibits macrophage polarization to M1,respectively.To investigate the mechanism of miR-221 in macrophages,analysis of target gene found that miR-221 could bind to SOCS1 3’ UTR.After overexpression and inhibition of expression of miR-221 in macrophages,the expression levels of SOCS1 protein were found to decrease and increase,respectively.At the same time,the study found that the protein level of p-STAT1 increased and decreased after overexpression or inhibition of miR-221,while the expression of p-STAT3 protein was opposite to that of p-STAT1.However,the expression of p-STAT6 protein had no changes.These results indicated that miR-221 could participate in the regulation of JAK/STAT signaling pathway by targeting SOCS1,thereby affecting macrophage polarization.