Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were col...Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination(AI). Based on the day of return of estrus, cows were divided into three groups, pregnant(n = 5), early embryonic mortality(EEM; n = 5) and late embryonic mortality(LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.Results: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d(P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 k Da(ISG15), myxovirus-resistance gene(MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows(P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ(IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT(100 ng/mL) and CCL16(100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA(P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16(P < 0.05).Conclusions: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events.The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as wel as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.展开更多
During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR...During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR8 axis,thereby contributing to liver inflammation.Numerous small molecular receptor antagonists,including R243,have been developed for targeting CCR8;however,these agents face challenges in clinical translation,potentially attributed to their poor pharmacokinetic profiles,lack of target specificity,and potential adverse effects.In this study,we designed four CCR8 antagonizing peptides(AP8i-AP8iv)and performed molecular characterization in silico and therapeutic investigation in vitro and in vivo.Based on in silico docking,molecular dynamic simulation using homology build model and in-vitro(competitive)binding studies,AP8ii(YEWRFYHG)evidenced highly favorable and selective interactions at the CCR8-active site.AP8ii inhibited CCL1-driven chemotaxis and LPS/IFNg-induced pro-inflammatory activation of monocytes-macrophages in vitro.In a CCl4-induced acute liver injury mouse model,AP8ii treatment decreased intrahepatic infiltration of circulating monocytes.Moreover,AP8ii reduced liver inflammation,as indicated by decreased F4/80,IL6 and iNOS expression,diminished ALT levels,and attenuated fibrosis,as indicated by reduced collagen-I expression.In conclusion,we report a novel CCR8-antagonizing peptide that inhibited CCL1-driven intrahepatic monocytes infiltration and differentiation into pro-inflammatory phenotype,consequently ameliorating liver inflammation and fibrogenesis in an acute liver injury mouse model.展开更多
基金supported by Grants-in-Aid for the Research Program on Innovative Technologies for Animal Breeding,Reproduction,and Vaccine Development (REP1001) from the Ministry of Agriculture,Forestry and Fisheries of Japansupported by JSPS KAKENHI Grant Number 17 K08056
文摘Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination(AI). Based on the day of return of estrus, cows were divided into three groups, pregnant(n = 5), early embryonic mortality(EEM; n = 5) and late embryonic mortality(LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.Results: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d(P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 k Da(ISG15), myxovirus-resistance gene(MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows(P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ(IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT(100 ng/mL) and CCL16(100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA(P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16(P < 0.05).Conclusions: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events.The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as wel as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.
文摘During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR8 axis,thereby contributing to liver inflammation.Numerous small molecular receptor antagonists,including R243,have been developed for targeting CCR8;however,these agents face challenges in clinical translation,potentially attributed to their poor pharmacokinetic profiles,lack of target specificity,and potential adverse effects.In this study,we designed four CCR8 antagonizing peptides(AP8i-AP8iv)and performed molecular characterization in silico and therapeutic investigation in vitro and in vivo.Based on in silico docking,molecular dynamic simulation using homology build model and in-vitro(competitive)binding studies,AP8ii(YEWRFYHG)evidenced highly favorable and selective interactions at the CCR8-active site.AP8ii inhibited CCL1-driven chemotaxis and LPS/IFNg-induced pro-inflammatory activation of monocytes-macrophages in vitro.In a CCl4-induced acute liver injury mouse model,AP8ii treatment decreased intrahepatic infiltration of circulating monocytes.Moreover,AP8ii reduced liver inflammation,as indicated by decreased F4/80,IL6 and iNOS expression,diminished ALT levels,and attenuated fibrosis,as indicated by reduced collagen-I expression.In conclusion,we report a novel CCR8-antagonizing peptide that inhibited CCL1-driven intrahepatic monocytes infiltration and differentiation into pro-inflammatory phenotype,consequently ameliorating liver inflammation and fibrogenesis in an acute liver injury mouse model.
