The Effects And Mechanisms Of Neutrophil Extracellular Traps On Immunological Damage Of Bovine Mammary Gland With Mastitis

Bovine mastitis is one of the major diseases causing great economic losses in dairy cattle herds,which mainly divided into clinical type and recessive mastitis.No matter which types of mastitis is characterized by decreasing production of milk yield.The damage of of mammary gland epithelial cells,as professional lactationcells,leads to milk yield decline.The cause of damage can be derived from pathogens or large numbers of innate immune cells in mammary duct system.These immune cells can against pathogens by NETs,phagocytosis and degradation,in this process which may also result in mammary gland epithelial cells damage.As a natural immune response mechanism discovered recent years,neutrophil extracellular traps(NETs)can be rapidly degraded after elimination of pathogen.However,excess NETs release or slow degradation will often cause immunopathological damage to the host.When mastitis occurs,neutrophils in the milk of mammary duct system whether could release NETs to kill pathogens,what effects of NETs on mammary gland epithelial cells,and the mechanism underlying these actions has not known.The answers to these issues have great significance for elucidating the detailed pathogenesis of mastitis.In this paper,we established the qualitative and quantitative analysis methods of ETs,confirmed the existence of NETs in mammary acinar,analyzed the damage situation of breast tissue,then preliminarily revealed the damage mechanism of NETs and its main components on mammary gland epithelial cells.Firstly,the qualitative and quantitative methods of NETs were established.The qualitative method of NETs analysis with scanning electron microscope and laser confocal was successfully established by using different types of stimuli.And the quantitative method of NETs analysis with the combination of dye and fluorescence microplate was also successfully established by using different stimuli.These laid a solid experimental technical foundation for the qualitative and quantitative analysis of NETs in bovine mastitis.Secondly,the excistance of NETs in mammary duct system and its intimate connection with mammary gland damage was confirmed.LPS and S.aureus were respectively used to set up clinical and recessive mastitis in mice models.Paraffin sections of breast tissue were made,the pathological changes of mammary tissue were observed by paraffin section.The results showed that both S.aureus and LPS caused mammary gland of edema,gland bubble structure damage and inflammatory cells infiltration.ELISA analysis also showed a significant increasing cytokines.Results of scanning electron microscope and laser confocal analysis confirmed that NETs excisting in mammary gland with mastitis,and these structures are mainly composed of DNA and histones,elastin,and myeloperoxidase which possess the typical morphological characteristics of NETs.However,DNase I treatment could obviously improve S.aureus or LPS induced-mammary gland damage and cytokine expression.Further study in vivo showed that Cl-amidine,the inhibitor of NETs,can inhibit S.aureus and LPS induced-mammary gland damage,which also confirmed the relation between NETs release and mammary gland damage in mastitis of mice.Thirdly,the damage mechanism of NETs on mouse mammary gland epithelial cells in vitro was investigated.NETs was obtained by stimulating with PMA,and NETs or NETs treated with DNase I was cocultured with mouse mammary gland epithelial cells.The results of LDH analysis that showed NETs and its degradation significantly increased the leleves of LDH in culture supernatant,suggesting the toxic effects of NETs and its degradation on mouse mammary gland epithelial cells.Further analysis also confirmed that the major protein compontent of NETs,histone,also increased LDH levels,suggesting the cytotoxic effects of histone on mouse mammary gland epithelial cells.To further confirm the damage effects of histone on mammary gland,histone was injected into mammary ducts to observe the histopathological changes of mammary gland.The results showed that histone could cause inflammatory cells infiltration in mammary gland tissue of mice,the damage of mammary acinar structural integrity,and significantly increased the levels of cytokines IL-1β and IL-6.Mouse mammary epithelial cells were treated with different concentrations of histone in the supernatant culture.Flow cytometry and western blot analysis revealed that histone could cause the damage of mouse mammary epithelial cells by apoptosis,necrosis and pyroptosis.Finally,the damage mechanism of NETs on mammary epithelial cells was further confirmed in bovine.Mammary gland epithelial cells were isolated and cultured by explant and differential digestion method.The primary mammary gland epithelial cell was confirmed by keratin-18 and immunofluorescence analysis.Bovine neutrophils stimulated with PMA were to collect NETs.NETs and its degradation components respectively incubated with mammary gland epithelial cells,the results showed significantly elevated lactate dehydrogenase levels in culture supernatants,suggesting the toxic effects of NETs and its degradation components on bovine mammary gland epithelial cells.Further analysis showed that histone alone stimulated BMEC leading to the growth of cells slowed down,shrink sharply and become round,and lactate dehydrogenase levels increased in culture supernatant,which suggesting the role of hisone in the toxic effects of NETs on BMEC.Then,BMEC were treated with different concentrations of histone,flow cytometry analysis revealed that histone could cause apoptosis(Annexin V+/PI-),necrosis(Annexin V+/PI+)and pyroptosis(Annexin V-/PI+)of BMEC.Western blot analysis showed that histone induced pyroptosis,apoptosis,and necrosis of BMEC by activating caspase-1,NLRP3 and caspase-3.Above results confirmed the results in mouse that NETs leading to BMEC damage was caused by its components histone,and the mechanism is histone induced pyroptosis,apoptosis and necrosis of BMEC by activating caspase-1,NLRP3 and caspase-3.Taken together,the research demonstrated that NETs excisted in mammary duct system,and the damage mechanism was histone of NETs induced mammary epithelial cell pyroptosis,apoptosis and necrosis by activated caspase-1,NLRP3 and caspase-3.Above study supplemented the mechanism of mammary epithelial damage resulting in milk yield decline in the pathogenesis of bovine mastitis,which providing new perspectives for the immunological prevention and treatment of bovine mastitis,and providing theoretical foundation and experiment evidence for effective prophylaxis and treatment of mastitis in veterinary clinical practice.