Effects Of Bovine Macrophage On Mammary Gland Inflammation Response During Transition Period

The dairy cows are vulnerable to mammary gland inflammation during the perinatal periods,which can lead to higher incidence of mastitis,impairment of animal welfare and reduced production efficiency.The inflammation responses are associated with mammary involution during dry-off and untimely engulfment of apoptotic cells by macrophages.This experiment was conducted to identify the specific receptors used by macrophages to recognize apoptotic cells and signal pathway involved in upregulation of IL-10 through animal and cell experiment.It provides theoretical basis for relieving inflammation responses during perinatal periods.Animal experiment:Lactating cows?n=6?with no mastitis in the last lactation period,dairy milk yield over 20 kg/d,SCC<30×10⁴/m L,no common mastitis-causing pathogens detected from individual quarter and dry-off cows?n=7?with parity?2-4??305-day milk yield?11477±2290??no mastitis in the last lactation period?SCC<30×10⁴/m L?were selected.Cows had ad libitum access to water and respective feeds.Mammary gland tissue and secretion samples were collected at-7,3,7,15d relative to dry off for dry-off cows;mammary gland tissue and milk samples were collected at-8d,3,7,21d relative to calving for early-lactation cows.The results were as follows:?1?Mammary secretion fat percentage didn't change?P>0.05?,while percentage of protein,somatic cell counts?SCC?,total solids increased?P<0.05?,percentage of lactose and urea nitrogen decreased in early dry period?P<0.05?;For early lactating cows,milk percentage of fat didn't change?P>0.05?,while milk percentage of protein?SCC?urea nitrogen and total solids reduced?P<0.05?,percentage of lactose increased in early lactation?P<0.05?.?2?The mammary involution of dairy cows was characterized by the decreasing size of alveoli in early dry period;shrank alveoli gradually expanded,stromal was compressed,preparing for lactating during early lactation.?3?During early dry period,alveolar lumen cells apoptosis rate was highest 3.68%at+3d relative to dry-off?P<0.05?,alveolar and stromal cells proliferation rate was highest at+7d relative to dry-off,7.23%and 7.56%respectively?P<0.05?;During early lactation,alveolar cells apoptosis rate reached highest value at+3d,+8d relative to calving,0.35%and 0.27%respectively?P<0.05?,alveolar an stromal cells proliferation rate reached highest value 8.19%and 5.12%at-8d relative to calving?P<0.05?.?4?Mammary gland expression of gene prolactin receptor?PRLR?decreased at+3d relative and+3d relative to calving?P<0.05?.Expression of gene insulin-like growth factor-1?IGF-1?in mammary gland had no difference in mammary gland during early dry period?P>0.05?,but decreased at+3d relative to calving?P<0.05?;Expression of gene insulin-like growth factor-2?IGF-2?decreased at+3d?+7d relative to dry off?P<0.05?,while decreased at+3d relative+3d relative to calving?P<0.05?;Expression of gene hepatocyte growth factor?HGF?increased at+7d relative to dry off and+21d relative to calving?P<0.05?;Expression of gene vascular endothelial growth factor?VEGFA?was lowest at+3d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?.?5?Mammary gland expression of gene jumonji domain containing 6?JMJD6?increased at+7d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?;Expression of gene integrin?_V?ITGAV?and integrin?₃?ITGB3?increased+3?7d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?;Expression of gene MER proto-oncogene tyrosine-protein kinase?MERTK?and TYRO3 protein tyrosine kinase?TYRO3?increased at+15d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?.?6?Expression of gene tumor necrosis factor?TNF?in mammary gland didn't change during early dry period?P>0.05?,while increased at+15d relative to calving?P<0.05?;Expression of gene interlukin-1??IL-1??increased at+7d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?;Expression of gene interlukin-10?IL-10?upregulated at+15d relative to dry off?P<0.05?,while no difference during early lactation?P>0.05?;Expression of gene mannose receptor C-type1?MRC1?increased at+15d relative to dry-off?P<0.05?,while no difference during early lactation?P>0.05?.Cell experiment:Bovine mammary epithelial cells?BMEC?were cultured and UV radiation was used to induce BMEC apoptosis.50 m L blood samples were collected form 3mid-lactation cows with no mastitis,similar days in milk and parity.Peripheral blood mononuclear cells?PBMC?were isolated from blood.PBMC were grown for eight days until they completely converted into monocyte-derived macrophages,with a media change every three days.The macrophages were divided into three groups:AP group?macrophage+apoptotic BMEC??SB203580 group?macrophage+apoptotic BMEC+5?M SB203580??LPS group?macrophage+1ng/m L LPS?.Supernatants were collected from each group at 4h,8h,12h,24h to measure concentration of TNF-?,IL-1?,IL-10.The results were as follows:?1?Cell mortality rate reached highest value 45.13%when UV radiation for 10 min and sample collected at 18h,which was method for induction of BMEC apoptosis.?2?At different time point,supernatants concentration of IL-10 in AP group was significantly higher than that of SB203580 group?P<0.05?,and supernatants concentration of IL-10 in SB203580 group significantly increased than that of LPS group?P<0.05?.SB203580and AP group had highest supernatants IL-10 concentration at 12h.?3?At different time point,supernatants concentration of TNF-?in LPS group was significantly higher than that of SB203580 group?P<0.05?,and supernatants concentration of TNF-?in SB203580 group significantly increased compared with AP group?P<0.05?.SB203580 and LPS group had highest supernatants TNF-?concentration at 24h.?4?At different time point,supernatants concentration of IL-1?in LPS group was significantly higher than that of SB203580 group?P<0.05?,and supernatants concentration of IL-1?in SB203580 group significantly increased compared with AP group?P<0.05?.SB203580 and LPS group had highest supernatants IL-1?concentration at 24h.In conclusion,cell apoptosis and proliferation are co-regulated by multiply hormone PRL?IGF-1?IGF-2?HGF?VEGFA;For dairy cows,Macrophages use receptor PSR??_V?₃?Mer TK?TYRO3 to recognize and engulf apoptotic BMEC and release IL-10 during mammary involution,which inhibits inflammation responses.