BHBA Regulates The Mechanism Of NETs In Neutrophils Of Dairy Cows Through GPR109A-mTOR Signaling Pathway

One of the ways in which neutrophils defense against microorganisms is through by neutrophil extracellular traps(NETs).NETs is a newly discovered extracellular defense mechanism of innate immune cells that is present in most immune cells,such as macrophages,monocytes,and mast cells.The fibrous network consisting of its own DNA,histones and a variety of granules to kill pathogens,which can capture or even kill bacteria,fungi or viruses.Cow ketosis is a high metabolic disease in peripartum cows and characterized by hyperketonemia.Ketosis increases the risk of peripartum disease in dairy cows,especially for underlying diseases risk,such as fatty liver,atrophic gastritis,mastitis,and endometritis.It is generally believed that the direct result of ketosis is related to the inhibition of innate immunity.Recent studies have shown that in vitro ?-hydroxybutyric acid(BHBA)-treated neutrophils can reduce E.coli-induced neutrophil extracellular trapping network by 10 fold and inhibit antibacterial activity.But what is the effect of hyperketoemia in ketosis cows on the production of NETs? It is unclear what kind of signaling pathway mechanism regulates the production of NETs by BHBA.First,the NETs from ketosis cows under hyperketoemia was studied.Laser confocal microscopy observations showed that neutrophils isolated from ketosis cows can release fibrous structures(NETs)out of the cell.At the same time,immunofluorescence experiments were performed about protein particles on NETs.Expressions of acetylated histone,citrulline histone,and myeloperoxidase(MPO)on NETs were observed by laser confocal microscopy.Quantification of the free DNA in NETs showed that the amount of NETs released from neutrophils was significantly higher in ketosis cows than in normal cows.Western blotting showed that the protein expression levels of the key molecules in the neutrophil GPR109A-mTOR signaling pathway increased.It can be considered that ketosis cows spontaneously induce NETs through GPR109A-mTOR signaling pathway in neutrophils,resulting in cellular NETosis,which may cause autoimmune reactions and innate immunosuppression.Secondly,it was confirmed that the addition of BHBA in vitro can induce the production of NETs in dairy cows by laser confocal microscopy and scanning electron microscopy,and has a certain time and concentration dependence on BHBA;BHBA increases the expression levels of acetylation and citrulline histone in neutrophils,which induces NETosis in neutrophisl from dairy cows.Immunofluorescence experiments confirmed that BHBA can enhance the accumulation of autophagy in neutrophils from dairy cows,while autophagy can regulate the release of NETs.On this basis,this experiment further clarified whether GPR109A-mTOR can regulate BHBA-induced NETs release.After treated neutrophils with GPR109 A inhibitor pertussis toxin and BHBA,compared with the BHBA alone treated group,the release of NETs from the mixed treatment group was significantly decreased,the activity of other protein granzymes was significantly reduced,and the autophagy was significantly reduced with a significant decrease trend in NETosis.Flow cytometry was used to prove that BHBA can increase ROS in neutrophils.After treatment of neutrophils with DPI and BHBA,compared with BHBA alone treatment group,not only the production of ROS and the release of NETs were reducted significantly,but also the intracellular autophagy accumulation was decreased significantly,while inhibiting citrulline histone expression and NETosis.In addition,the inhibition of ROS production can downregulate GPR109 A expression;when autophagy is inhibited,intracellular ROS levels are significantly reduced,indicating that ROS is involved in BHBA-induced neutrophil autophagy regulation and NETs release in dairy cows.Taken together,these results demonstrate that BHBA can regulate the release of NETs from dairy neutrophils via GPR109A-mTOR signaling pathway.The key point is the potential function of autophagy,and this process needs to depend on the production of ROS.Finally,whether the GPR109A-mTOR signaling pathway can regulate the release of NETs in neutrophils from dairy cows when in respond to external pathogenic stimuli.Therefore,treatment of neutrophils with BHBA and LPS to observe the expression of key molecules of GPR109A-mTOR and release of NETs in vitro.The results showed that LPS can inhibit the release of NETs induced by BHBA through the GPR109A-mTOR signaling pathway and inhibit NETosis;while BHBA can also inhibit the release of NETs induced by LPS through the GPR109A-mTOR signaling pathway and suppress NETosis.In conclusion,BHBA can induce NETs production through the GPR109A-mTOR signaling pathway,causing an autoimmune response,resulting in innate immune suppression.BHBA can inhibit LPS-induced neutrophil NETs release through the GPR109A-mTOR signaling pathway,reducing the body's resistance to pathogens.The results provide a theoretical basis for revealing the mechanism of innate immune suppression in ketosis cows and enhancing the immune function of perinatal dairy cows.