Protein Kinase C And Autophagy Signal Mediate The Impairment Of Neutrophil Chemotaxis In Ketotic Cows

Ketosis is a metabolic disease that occurs frequently in cows during the perinatal period.Negative energy balance(NEB)occurs in perinatal cows when energy intake cannot meet energy expenditure,lipolysis in cows caused by NEB causes hepatic lipid accumulation and hyperketonemia and leads to ketosis in cows.Ketosis increases the risk of secondary diseases in cows,especially infectious diseases represented by mastitis,which is closely related to natural immunosuppression in cows caused by ketosis.Neutrophils,as the main constituent cells of innate immunity,are active in the first line of innate immunity and play an indispensable role in resisting pathogen invasion.Studies have shown that the phagocytosis of neutrophil and neutrophil extracellular traps(NET)is inhibited in ketotic cows.However,it is not known whether the chemotactic function of neutrophils is affected by ketosis.The purpose of this study was to investigate the effect of ketosis on neutrophil chemotaxis in dairy cows and its regulatory mechanism.(1)In this study,the transcriptome changes of peripheral neutrophils in ketosis and healthy cows were compared.A total of 864 differentially expressed genes were identified by transcriptome sequencing,including 594 up-regulated genes and 270down-regulated genes.GO enrichment analysis showed that the term of biological processes was mainly concentrated in neutrophil immune response,inflammatory response,chemotactic response,cellular calcium signal,protein transport,cell fate and other aspects.The term of molecular function is mainly concentrated in cell chemotaxis,protein activity and Rho GTPase protein activity.The term of cell component is mainly concentrated in the nucleus,cytoplasm,cytoplasm and cytosol.Then GSEA analysis was performed on the GO dataset to obtain 45 statistically significant terms.By comparison,it was found that the Go-GSEA term and GO enrichment analysis term had great overlap in immune response,inflammatory response and chemotaxis,and NES values of other terms were all negative except for Fibronectin binding,which showed an overall upward trend,indicating that these biological processes were in a state of overall inhibition.This is consistent with previous studies on the effects of ketone bodies on immune response,inflammatory response and chemotaxis.On the other hand,KEGG enrichment analysis showed that differentially expressed signaling pathways were mainly involved in cell chemotaxis,adhesion,apoptosis,inflammation,protein degradation,immunity,metabolism,etc.GSEA analysis of KEGG data also obtained15 gene sets,it includes the NF-κB signaling pathway,IL-17 signaling pathway and other inflammatory pathways,as well as intestinal immune network for Ig A production,Toll like receptor signaling pathway and other immune-related signaling pathways and other signaling pathways.In addition to the ether lipid metabolism signal pathway,NES values obtained by GSEA analysis of other signal pathways were all negative,indicating that the inflammatory response and immune function related regulatory signals of neutrophils in ketotic cows were inhibited,which was consistent with the immunosuppressed state of ketotic cows.(2)This study investigated the effect of ketosis(high concentration of BHB)on neutrophil chemotaxis in dairy cows.Scanning electron microscopy(SEM)showed that neutrophils showed "giant tail" during chemotaxis in ketotic cows.Western blotting and quantitative real-time PCR results showed that transcription and protein phosphorylation levels of the tail contractile-effecting molecule MLC2 in neutrophils of cows with ketosis were increased,suggesting that the above "giant tail" phenotype was associated with excessive activation of MLC2.In vitro,3.0 mmol/L BHB was used to simulate clinical ketosis and induce chemotaxis of neutrophils isolated from peripheral blood for 3 h,6 h and 9 h,respectively.It was found that BHB inhibited neutrophils chemotactic distance in a time-dependent manner.Western blotting and quantitative real-time PCR results showed that BHB treatment increased the m RNA abundance and protein phosphorylation levels of PKC and MLC2 in the upstream and increased the expression of Rho A and ROCK1 in the middle.Importantly,Sotra,a PKC inhibitor,mitigated the effects of BHB on neutrophil chemotaxis and PKC signaling pathways.In addition,immunofluorescence showed that Sotra also reversed BHBinduced excessive accumulation of MLC2 in the tail of neutrophils and the " giant tail" phenotype.Finally,in vivo results showed that the PKC phosphorylation level of neutrophils in ketotic cows was significantly higher than that in healthy cows.The above evidence suggests that high concentration of BHB may induce hyperphosphorylation of downstream MLC2 and the accumulation in tail through activation of the PKC signaling pathway,and ultimately shorten the neutrophil chemotactic distance in dairy cows.It’s worth noting that BHB also impairs neutrophil chemotactic directionality in cows,but is not regulated by PKC signaling.(3)This study further explored the molecular mechanism of BHB regulating neutrophil chemotactic directionality impairment in dairy cows.To accurately analyze the state of chemotaxis of the neutrophil,the entire migration tracks of neutrophils were recorded by microscopy with time-lapse photography.Five parameters(total distance traveled,track displacement,track means speed,mean straight line speed,and mean directional change rate.)were used to quantify neutrophil motility and directionality.It was found that neutrophil chemotactic motility and directionality were inhibited in ketotic cows,which was consistent with part(2)of the phenotype.In addition,Western blotting and quantitative real-time PCR results showed that the m RNA abundance of Cdc42 and Rac1,the key effector molecules in the chemotaxis directionality,decreased after BHB treatment,but the total protein level and activation level of Cdc42 and Rac1 were increased.Transmission electron microscopy showed that BHB treatment decreased the number of neutrophil autophagosomes in dairy cows.Western blotting showed that BHB treatment increased the content of Sequestosome-1(p62),while decreased the content of microtubule-associated protein 1 light chain 3-II(LC3-II).It indicated that BHB inhibited the autophagy of neutrophils.These results show that BHB may lead to the accumulation of Cdc42 and Rac1 by inhibiting autophagy.To test this hypothesis,we used in vitro co-stimulation of BHB with the autophagic activator rapamycin(RAPA)and found that the impairment of neutrophil chemotaxis directionality and the effect of BHB on Cdc42 and Rac1 protein accumulation were significantly mitigated.Similarly,in vivo experiments showed significant increases in the content of Cdc42 and Rac1 in ketotic cows.These results confirm that high concentration of BHB leads to the accumulation of Cdc42 and Rac1 through the inhibition of autophagy,and ultimately leads to impaired neutrophil chemotaxis directionality in ketotic cows.In conclusion,compared with healthy cows,the transcription levels of neutrophils in ketotic cows showed a trend of inhibited immune activity and function.High concentration of BHB in ketotic cows impaired neutrophil chemotaxis motility and directionality through PKC-MLC2 and autophagy Cdc42/Rac1 signaling pathways.