| Objective: By comparing the differences of liver macrophages,natural killer cells(NK)and their interactions between neonatal and adult mice infected with rhesuse rotavirus(RRV).To clarify the role of liver macrophages and NK cells in RRV-induced biliary atresia in mice.Methods: After infecting the extrahepatic biliary duct epithelial cells(BEC)with RRV in vitro,and co-cultured with liver macrophages and/or NK cells of different ages in mice.The expression of functional proteins in mice of different ages was detected by flow cytometry and Elisa.The expressions of iNOS,Arg-1 and Perforin in the liver of mice of different ages after intraperitoneal injection of RRV were detected by Western blot,RT-qPCR and paraffin section immunofluorescence.Results: In vitro RRV-infected BEC system,the expression of CD69 of NK cells in neonatal mice was higher than that in adult mice.The macrophages in adult mice were more likely to stimulate the expression of CD69 and IFN-γ on the surface of NK cells.The expression of CD54,iNOS and Arg-1 in neonatal macrophages were lower than those in adult mice.The Rae-1-Nkg2 d interaction between macrophages and NK cells in neonatal mice was weaker than that in adult mice.Through Transwell co-culture of primary cells,the secretion of cytokines IL-12 and IL-18 and chemokines CCL-5 and CXCL-9 by neonatal macrophages was lower than that of adult macrophages,and the secretion of IL-4 and IL-13 by neonatal macrophages showed no significant difference compared with adult macrophages.After RRV infection,the M1-type macrophage marker iNOS was highly expressed in the liver tissue of neonatal mice,which was opposite to the M2-type macrophage marker Arg-1 in adult mice.At 24 h after injection of RRV,the expression of Perforin in liver tissue of neonatal mice was lower than that of adult mice,but higher at 48 h and 72 h.Conclusions: In vitro RRV-infected BEC system,NK cells of neonatal mice were more easily activated.Adult macrophages are more likely to stimulate NK cells to activate them,and newborn macrophages’ function was weaker.The interaction between macrophages and NK cells in neonatal mice is weaker than that of adult mice,and this interaction may promote the expression of IFN-γ in NK cells.The ability of macrophages in neonatal mice to recruit effector cells to the site of infection and trigger NK cells to produce antiviral and proliferative effects was insufficient.After RRV infection,the M1-type macrophage marker iNOS was highly expressed in the liver tissue of neonatal mice,which was opposite to the M2-type macrophage marker Arg-1 in adult mice.Perforin,an effecting molecule produced by NK cells in response to pathogen stimulation in neonatal mice liver tissue,cannot reach a high level quickly.Part one: Differences in the functions of liver macrophages and natural killer cells in mice of different agesObjective: To study the differences in the expression of liver macrophages,NKand their connectivity related molecules after co-culture of the primary liver macrophages and the system of RRV infected BEC,and co-cultured with NK cells of mice at different age.Methods: The primary liver macrophages,liver NK cells,and BEC of mice were extracted,cultured and identified.In vitro,after the RRV infection with BEC,liver macrophages and/or NK cells of mice at different age after purification,flow cytometry detection the expression of iNOS,Arg-1,CD54 and CD11 b in each group of macrophages,the expression of CD69,IFN-γ in each group of NK cells and the expression of CD40,CD154,Rae-1,Nkg2 d named cell connectivity related molecules.Results: Three primary cells can meet the subsequent experimental needs through their specific markers identification,purity and quantity.In the system of RRV-infected BEC in vitro,the expression of CD69 in NK cells of newborn mice was higher than that of adult mice,and the difference was statistically significant(P<0.05);the expression of iNOS and Arg-1 in macrophages of adult mice was higher than that of the newborn mice,and the difference was statistically significant(P<0.05);NK cells of newborn mice were respectively co-cultured with macrophages of newborn or adult,and the expression of CD69 and IFN-γ in NK cells was higher in the group of co-cultured with liver macrophages of adult,and the difference was statistically significant(P<0.05);the difference of the expression of CD40 in NK cells in each group of mouse liver macrophages and NK cells at different ages cocultured was not statistically significant(P>0.05);NK cells of newborn mice were respectively co-cultured with liver macrophages of newborn or adult,and the expression of Nkg2 d in NK cells was higher in the group of co-cultured with macrophages of adult,and the difference was statistically significant(P<0.05);similarly,NK cells of adult mice were respectively co-cultured with liver macrophages of newborn or adult,and the expression of Nkg2 d in NK cells was higher in the group of co-cultured with liver macrophages of newborn,and the difference was statistically significant(P<0.05);macrophages of newborn mice were respectively co-cultured with NK cells of newborn or adult,and the expression of Rae-1in macrophages was higher in the group of co-cultured with NK cells of adult;while macrophages of adult mice were respectively co-cultured with NK cells of newborn or adult,and the expression of Rae-1in macrophages was higher in the group of co-cultured with NK cells of adult,and the difference was statistically significant(P<0.05).Conclusions: The techniques in the culture of three primary cells are stable.The NK cells of newborn mice were more likely to be activated by the system of RRV-infected BEC in vitro compared with that of adult.The effect of neonatal macrophages on pathogen stimulation was weaker than that of adult mice.And the macrophages of adult mice are more likely to stimulate NK cells and activate them.The Rae-1-Nkg2 d interaction between neonatal macrophages and NK cells was weaker than that of adult mice.Part two: Differences of cytokines secreted by liver macrophages in mice of different ages and their effects on the function of natural killer cellsObjective: To study the difference of cytokines secreted by liver macrophages and the effect on NK cells function after the primary liver macrophages of mice of different ages were cocultured with the system of RRV-infected BEC,and the primary NK cells of mice of different ages were co-cultured with Transwell.Methods: The purified macrophages of mouse in different ages and/or NK cells of different ages were co-cultured with the system of RRV-infected BEC in transwell,and the expression of CD69,IFN-γ and TNF-α in NK cells of each group was detected by flow cytometry.The concentrations of CCL-5,CXCL-9,IL-4,IL-10,IL-12,IL-13 and IL-18 in cell culture medium of each group were detected by Elisa.Results: In the system of RRV-infected BEC in vitro,when non-con-cultured with macrophages in transwell,the expression of CD69 and TNF-α in NK cells of newborn mice was higher than that of the adult mice,and the difference was statistically significant(P<0.05);NK cells of newborn mice were respectively co-cultured in transwell with macrophages of newborn or adult,the expression of CD69 in NK cells was higher in the group of co-cultured with macrophages of adult,similarly,NK cells of adult mice were respectively co-cultured in transwell with liver macrophages of newborn or adult,and the expression of CD69 in NK cells was higher in the group of co-cultured with liver macrophages of newborn,and the difference was statistically significant(P<0.05);NK cells of newborn mice were respectively co-cultured in transwell with macrophages of newborn or adult,and there was no significant difference in the expression of IFN-γ between them(P>0.05);when non-co-cultured in transwell with NK cells,the concentration of IL-12,IL-18 and CXCL-9 secreted by the macrophages of newborn mice was lower than that of adult mice,and the difference was statistically significant(P<0.05);NK cells of newborn mice were respectively co-cultured in transwell with macrophages of newborn or adult,the concentration of IL-12 and CCL-5 secreted by the macrophages of newborn mice was lower than that of adult mice,and the difference was statistically significant(P<0.05);macrophages of newborn mice were respectively co-cultured in transwell with NK cells of newborn or adult,and there was no significant difference in the concentration of IL-4 and IL-13 secreted by the macrophages between them(P>0.05);macrophages of adult mice were respectively co-cultured in transwell with NK cells of newborn or adult,and there was no significant difference in the concentration of IL-4 and IL-13 secreted by the macrophages between them(P>0.05).Conclusions: The Rae-1-Nkg2 d connection between macrophages and NK cells may promote the production of IFN-γ in NK cells.The ability of macrophages of newborn mice to secrete IL-12,IL-18,CCL-5,CXCL-9 is lower than that of adult.However,there was no significant difference in the concentrations of IL-4 and IL-13 secreted by macrophages of mice of different ages.Part three: Differences in the functions of macrophages and natural killer cells after intraperitoneal injection of rotavirus in mice of different agesObjective: To explore the functional differences of macrophages and NK cells in liver of mice of different ages after intraperitoneal injection of RRV at different time points.Methods: After the titer of RRV was measured,newborn and adult mice were intraperitoneally injected with RRV,while the control groups were injected with normal saline(NS).Liver tissues of different groups of mice were collected in 24 h,48 h and 72 h after injection.The mRNA levels of Nos2,Arg-1 and Prf1 in liver tissues were detected by RT-qPCR,and the expression of protein of iNOS,Arg-1 and Perforin in liver tissues were detected by Western blot.Immunofluorescence showed the expression of iNOS,Arg-1 and Perforin in liver tissue sections of each group.Results: In terms of the expression of mRNA and protein,24 hours after intraperitoneal injection,the expression of Perforin in liver of newborn mice with RRV was higher than that of control group,and the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Arg-1 and iNOS between them(P>0.05);the expression of Perforin in liver of adult mice with RRV was higher than that of control group,and the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Arg-1 and iNOS between them(P>0.05);the expression of Arg-1 and Perforin in liver of newborn mice with RRV was significantly lower than that of adult mice with RRV,while the expression of iNOS in liver were significantly higher than that of adult mice with RRV,and the difference was statistically significant(P<0.05).48 hours after intraperitoneal injection,the expression of Perforin in liver of newborn mice with RRV was higher than that of control group,and the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Arg-1 and iNOS between them(P>0.05);the expression of Arg-1 in liver of adult mice with RRV was higher than that of control group,and the expression of iNOS in liver of adult mice with RRV was lower than that of control group,the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Perforin between them(P>0.05);the expression of iNOS and Perforin in liver of newborn mice with RRV was significantly higher than that of adult mice with RRV,while the expression of Arg-1 in liver were significantly lower than that of adult mice with RRV,and the difference was statistically significant(P<0.05).72 hours after intraperitoneal injection,the expression of Perforin and iNOS in liver of newborn mice with RRV was higher than that of control group,and the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Arg-1 between them(P>0.05);the expression of Arg-1 in liver of adult mice with RRV was lower than that of control group,and the difference was statistically significant(P<0.05),while there was no significant difference in the expression of Perforin and iNOS between them(P>0.05);the expression of Arg-1 and Perforin in liver of newborn mice with RRV was significantly lower than that of adult mice with RRV,while the expression of iNOS in liver were significantly higher than that of adult mice with RRV,and the difference was statistically significant(P<0.05).Conclusions: After newborn mice was infected with RRV,iNOS,a M1-type macrophage marker,was highly expressed in liver,while after adult mice was infected with RRV,Arg-1,a M2-type macrophage marker,was highly expressed in liver.The expression of Perforin as the effector molecule of NK cells on pathogen stimulation cannot reach a high level quickly in newborn mice liver. |
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