| Bovine mastitis is an internal mammary infection caused by a variety of bacteria,which seriously affects the development of dairy industry.Exosomes(Exos)are important participants in intercellular information transmission and substance exchange,and are generally believed to be derived from Mammary epithelial cells(MEC)of dairy cows.MEC is the most important structural cell of mammary tissue and constitutes the "microenvironment" of mammary tissue.Macrophage play an important role in mammary gland innate immunity,it can respond to microen-vironmental factors in tissues and differentiate into M1(typically activated)macrophages,which play a proinflammatory and anti-tumor role.Or M2(alternatingly activated)macrophages,associated with antiinflammatory responses and tissue remodeling.This study intended to conduct a preliminary study on whether mammary epithelial cells mediate macrophage polarization through exosome release in the process of mammary inflammation of dairy cows,thus influencing the process of mammary inflammation.In this study,exosomes derived from milk and MAC-T cells were enriched and purified by differential supercentrifugation.Through nanoparticle tracking analysis(NTA),transmission electron microscope(TEM),western blot,(WB)to identify exosomes;PKH67 labeled the double lipid membrane of exosomes,and fluorescent tracer was used to detect whether exosomes could be absorbed by recipient cells.Reverse transcription-polymerase chain reaction(RT-PCR),and Enzyme Linked immunosorbent assay(ELISA)were used to detect the m RNA and protein levels of pro-inflammatory cytokines and anti-inflammatory cytokines in macrophages treated by exos-omes from different sources,the results are as follows:Identification of inflammatory milk and establishment of mastitis cell model:10milk samples were collected aseptically,after the appraisal,the SCC of 4 samples was low than 200,000 /m L,which were used as control group.The SCC of 3 samples ranged from200,000-2,000,000 /m L,which were in the low somatic cell count mastitis group.The SCC of the other 3 samples was > 2,000,000 /m L,which was used as the high somatic cell count mastitis group.Through bacterial purification culture and 16 S r DNA sequencing analysis,the results showed that four samples with SCC > 200,000 /m L were infected with different bacteria,and all were Gram-negative bacteria.MAC-T cells were treated with 20 μL/m L and 100 μL/m L LPS,respectively,and RTPCR results showed that: Compared to the control group,the expressions of Tumor Necrosis Factor-α(TNF-α)and Interleukin-6(IL-6)in MAC-T cells were significantly increased in both LPS treatment groups,indicating that the model of cellular mastitis was established successfully.Isolation and Identification of Exosomes from Milk and Bovine mammary epithelial cells: The exosomes were isolated from the supernatants of bovine milk and MAC-T cells by ultra fast differential centrifugation,and the particle size of the centrifuged fractions was detected in the range of 30-150 nm using NTA.It was observed by TEM that the centrifugal components had double membrane and typical "cup and plate" structure.WB showed that the centrifuged fractions specifically expressed the sign-ature exosomal proteins CD63 and TSG101,which indicated the successful enrichment and purification of bovine milk and MAC-T cell-derived exosomes.Detection of internalized exosomes in recipient cells: PKH67 fluorescent-labeled exosomes were co-cultured with MAC-T cells and Raw264.7 cells.The results of fluorescence tracer showed that exosomes from different sources could be internalized by the abovementioned cells.In vitro experiments verify the influence of exosomes on inflammatory response:After co-culture of inflammatory exosomes with Raw264.7 cells,RT-PCR results showed that,compared with the control group,Inflammatory exosomes significantly increased the m RNA levels of pro-inflammatory cytokines TNF-α,IL-6 and Interleukin-1β(IL-1β)in Raw264.7cells,while the expression of these pro-inflammatory cytokines m RNA in normal exosomes group was significantly inhibited.These results suggest that exosomes derived from mammary epithelial cells can promote the macrophage-mediated inflammatory response during mammary gland inflammation,while normal exosomes have anti-inflammatory effects.Effect of exosomes on macrophage polarization: Compared with the control group,the m RNA and protein expression levels of pro-inflammatory cytokine TNF-α,IL-1β and IL-6 were significantly increased in Raw264.7 cells after co-culture of inflammatory bovine milk and inflammatory MAC-T cell-derived exosomes with Raw264.7 cells,the protein expression of inducible NOS(i NOS)and mannose-receptor CD206 in macrophages increased significantly.The m RNA and protein levels of anti-inflammatory cytokines Transforming Growth Factor-β(TGF-β)and Interleukin-10(IL-10)decreased.The expression of Arginase-1(ARG-1)m RNA was decreased,while the results of normal milk and normal MAC-T cellderived exosomes were opposite to those of inflammatory exosomes.The above results suggest that mammary epithelial cells,the structural cells of mammary tissue,promote the inflammatory response by exosome-mediated polarization of macrophages toward M1 type during the mammary inflammatory response,which is favorable for the clearance of pathogenic microorganisms at the initial stage of infection,but a sustained inflammatory response will lead to tissue damage and cause inflammation.However,a sustained inflammatory response will lead to tissue damage and cause more serious consequences such as inflammatory factor storm,while exosomes in the healthy state mediate the polarization of macrophages to M2 type and have some anti-inflammatory effect.The results of this experiment provide an example to validate the effect of tissue structural cells on immune cell function and taking macrophages as the target provides a preliminary theoretical basis for the prevention and treatment of dairy mastitis. |
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