Epigenetic Regulation Of Delayed IL-12 Production By Mouse Macrophages During Neospora Caninum Infection

Neosporiasis is a protozoiasis caused by Neospora caninum which parasites in the host cells.The disease can cause the pregnant animals abortion,stillbirth,weak fetus and other clinical symptoms.Also,it can cause congenital dyskinesia and congenital neurological disorders of newborn animals.The disease has a wide range of distribution with a large number of host species,which caused huge economic losses to animal industry.At present,because the life cycle of N.caninum is still unknown,there are no effective drugs and vaccines for the prevention and control of Neosporiasis.Macrophage is an important part of innate immunity and adaptive immunity which plays a critical role in pathogens removal.It is important as antigen presenter to T cells that stimulates the proliferation of Th1 which mainly dues to IL-12 secretion.N.caninum is intracellular parasite that can survive and multiple in macrophage.IL-12 is a kind of proinflammatory cytokine that participates in the early immune response.It is mainly secreted by macrophages and other APCs that plays a significant roles in the early immune response,especially caused by intracellular pathogenic microorganisms.IL-12 can activate NK cells caused IFN-? secretion.Some researches show that during N.caninum infection,IL-12 and IFN-? involved in the immune response and activate free oxygen radical,nitric oxid or other metabolites,then interact with these substances to kill N.caninum.Even that,parasites still can envade the host immune response by its multiple activity.Inhibition of the cytokines secretion is one of that.It is found that Toxoplasma gondii can evade the immune attack by delaying the secretion of IL-12 in the early stage of infection.The epigenetic modification,such as mi RNA and the histone modification,can regulate the expression of IL-12.At present the changes of IL-12 of host cells induced by the infection of N.caninum and the mechanism of how epigenetic modification regulates the expression of IL-12 is still unknown.In this study,with the dynamic changes of IL-12 level of macrophage induced by the infection of N.caninum,mi RNAs and histone modification regulated the expression of IL-12,the effect of epigenetic modification on the expression of IL-12 in macrophages infected by N.caninum was elucidated.IL-12 production by macrophages during N.caninum infection.N.caninum tachyzoites were isolated and purified by density gradient centrifugation,then infected mice peritoneal macrophages isolated from mouse abdominal cavity.The cell supernatants were collected after infected at 6h,12 h,18h,24 h,36h,and detected the expression of IL-12 in the supernatant.The results showed that the expression of IL-12 could be detected at 6h,12 h,18h,but the level of expression was low.The expression level of IL-12 began to rise at 24 h,and continued to rise at 36 h.The results showed that the IL-12 expression level of mice macrophage would be inhibited at the first 18 h post infection with N.caninum,The regulation of mi RNA on IL-12p40 production by macrophages during N.caninum infection.Firstly,mice peritoneal macrophages were infected with N.caninum tachyzoites.The total RNA of 6h,12 h,18h,24 h,36h macrophages were extracted and reverse transcribed,the dynamic changes of 6 mi RNAs related to IL-12 secretion(mi R-221 ? mi R-155 ? mi R-146 a ? mi R-10 a ? mi R-21 ? mi R-187)were detected by Real-time PCR.It was found that the change trend of mi R-187 was consistent with the trend of IL-12 secretion.No significant change trend of other mi RNAs was found.Then the macrophages transfected with mi R-187 mimics and the macrophages transfected with mi R-187 inhibitor were infected with N.caninum tachyzoites,respectively.The cell supernatant of 6h,12 h,18h,24 h,36h were collected and detected for the expression of IL-12.The results showed that the secretion of IL-12 had a significant increase in the macrophages transfected with mi R-187 mimics and IL-12 expression of the macrophages transfected with mi R-187 inhibitor decreased slightly.This indicates that mi R-187 can enhance the expression of IL-12 during the process that N.caninum infection.Mi R-187 target gene in NF-k B sequence was predicted.The pmir GLO-nfkbiz-187-mi RNA and pmir GLO-mutnfkbiz-187-mi RNA which included target site and mutational target site were constructed.The recombinant plasmids pmir GLO-nfkbiz-187-mi RNA+mi R-187-mimic and pmir GLO-mut-nfkbiz-187-mi RNA+ mi R-187-mimic were transfected into macrophages cells,respectively.The interaction of nfkbiz with mi R-187 were evaluated using the Dual luciferase reporter assay.The relative luciferase level was significantly reduced in cells transfected with pmir GLO-nfkbiz-187-mi RNA and mi R-187-mimic plasmids when compared to cells transfected with pmir GLO-nfkbiz-187-mi RNA(*P?0.05),whereas luciferase level was not significantly changed in cells transfected with pmir GLO-mut-nfkbiz-87-mi RNA(NF-k B targeted site mutations)and mi R-187-mimic plasmids.Histone H3 modifications associated with IL-12 promoters in macrophages infected by N.caninum.IL-12 promoter associated histone H3 modifications on H3K4me3,H3K27me2 and H3K9/14 ac was evaluated by CHIP at 18 h after N.caninum infection.IL-12p40 promoter associated H3 had a significant upregulation on H3K27me2 after N.caninum infection compared to blank control.No significant changes were found on the levels of H3K4me3,H3K27me2 and H3K9/14 ac in H3 histone associated with IL-12p35 promoter compared to blank control.This indicates that N.caninum could enhance the change of H3K27me2 to inhibit the expression of IL-12p40.In conclusion,the result shows that after the macrophages infected by N.caninum,the delayed IL-12 expression and dynamic changes in the expression of four mi RNAs.and the mi R-187 related with the changes of IL-12 expression.It has been confirmed that N.caninum can induce the expression of mi R-187 and regulate IL-12p40 promoter on H3K27me2 to decrease the secretion of IL-12 in mouse macrophages.From the perspective of epigenetics,this study reveal the mechanism of host immune envasion by delayed the expression of IL-12 during the early stage of N.caninum infection.