Study On The Immune Function Of Neutrophils Regulated By Orai1 In Dairy Cow Hypocalcemia

Hypocalcemia in dairy cows is a nutritional metabolic disease with high incidence of Ca2+ homeostasis in perinatal dairy cows,which easily leads to inflammatory diseases such as endometritis and mastitis and brings huge economic losses to the dairy industry.As the important second messenger,intracellular Ca2+ regulates cell proliferation,differentiation,hormone secretion,glycogen metabolism and immune function.The protein encoded by the ORAI Ca2+ release-activated calcium modulator 1(ORAI1)gene is a membrane Ca2+ channel subunit that is activated when Ca2+ stores are depleted.Polymorphonuclear neutrophils(PMN)play an important role in resistance to pathogen infection through migration,adhesion,chemotaxis,phagocytosis and production of reactive oxygen species(ROS).Whether the activity of PMNL is affected by hypocalcemia and whether Orai1 is involved is still unknown.In order to explore this,PMNL were isolated at 3 days of calving from dairy cows diagnosed as clinically healthy(n=20,CONTROL,plasma concentration of calcium 2.10-2.80 mmol/L)or with plasma concentration of calcium 1.40-2.0 mmol/L as a criterion for diagnosis of subclinical hypocalcemia(n=20,HYPOCAL).The following assays were performed in both groups of isolated PMNL: plasma and PMNL concentration of nucleosome assembly protein(NAP2)and pro-platelet basic protein(CXCL7)through ELISA;migration,phagocytosis rate,and ROS through fluorescence-activated cell sorting(FACS);adhesion capacity and NETs through confocal;mitochondria through TEM.The results showed PMN migration,adhesion capacity,phagocytosis,ROS and NETs were significantly down-regulated in HYPOCAL.This shows that dairy cows with hypocalcemia could down-regulate PMN immunity function.In order to determine whether ORAI1 regulate PMN immunity function in dairy cows with hypocalcemia,the intracellular Ca2+,Orai1 transcription and ORAI1 protein were measured.Intracellular Ca2+ concentration by Fluo-3,ORAI1 abundance and ROS and phagocytosis by fluorescence activated cell sorting(FACS)were measured in two groups of isolated PMNL.Further,migration and adhering capacity was evaluated with or without the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin,the Ca2+ ionophore Ionomycin,and the blocker of store-operated Ca2+ entry moiety ORAI1 2APB.Some of the in vitro assays also included use of small interfering ORAI1 RNA(si ORAI1).The results showed that the intracellular Ca2+ concentration in HYPOCAL was significantly decreased.In addition,Orai1 was detected in PMNL plasma membrane by FACS,and decreased significantly in HYPOCAL cows.In response to HYPOCAL,the migration,adhesion and phagocytosis of PMNL were lower.Furthermore,plasma and PMNL concentration of nucleosome assembly protein(NAP2)and pro-platelet basic protein(CXCL7)was markedly lower with HYPOCAL.All these changes were associated with lower ROS production by PMNL.Thapsigargin and ionomycin treatment in vitro increased ORAI1 expression,migration of PMNL,adhering capacity,phagocytosis rate and ROS production;conversely,those effects were abrogated by si ORAI1 and ORAI1 inhibitor 2APB treatment.To address the association of plasma Ca2+ and intracellular Ca2+ in dairy cows,the plasma concentration of 1,25(OH)2D3 and PTH were measured by cattle specific ELISA.The PMNL from both groups' of cows were treated with or without 100 n M 1,25(OH)2D3 or 100 n M parathyroid hormone(PTH)to measure ORAI1 surface abundance and intracellular Ca2+ concentration.The results showed plasma concentration of 1,25(OH)2D3 was significantly lower in HYPOCAL,cytosolic Ca2+ concentration and ORAI1 abundance were increased by 1,25(OH)2D3 and PTH supplementation.Overall,the data indicate that failure of PMNL to uptake Ca2+ due to downregulation of ORAI1 during subclinical hypocalcemia is a factor contributing to impaired PMNL function.In addition,plasma PTH or 1,25(OH)2D3 could regulate ORAI1 and also participate in the regulation of PMNL activity.