MAP Induces Macrophage Apoptosis Mediated By ERs And Its Effect On M.bovis-infected Mice

Paratuberculosis,also known as Johne's disease,is caused by Mycobacterium avium subspecies paratuberculosis(MAP)and is an important and extremely common ruminant disease.The disease causes serious economic losses.However,due to the difficulty in isolation and purification of the bacteria,the Chinese MAP isolate and its genomic information are still lacking.Macrophages are considered to be the main host cells in the early stage of MAP infection,and the apoptosis of macrophages directly affects the survival of MAP in cells.However,it is not clear whether MAP will cause apoptosis after infection with macrophages.Bovine tuberculosis(bTB)is a chronic infectious bacterial disease found primarily in wildlife,livestock and humans.It is caused by M.bovis,mainly through aerosol infection.About 10%of people worldwide with tuberculosis are caused by M.bovis.M.bovis and MAP can infect the same dairy farm at the same time,but therearerelatively few studies on the mixed infection of these two pathogens.Therefore,studying the immune mechanism of co-infection of MAP and M.bovis can provide a scientific basis for the prevention and treatment of bovine paratuberculosis and tuberculosis.1.The primary task of this study was to isolate Chinese MAP strains.The positive rate of MAP antibody in 19 dairy farms in xx Province was detected by ELISA.The dairy farms with highest positive rate were selected,and the MAP antibody-positive cows were tracked for histopathological observation.The rectal feces were treated with HPC for MAP isolation and the purity was detected by acid-fast staining.The type of MAP strain was identified by PCR-REA,and genome sequencing of MAP isolates were conducted by high-throughput sequencing.The SOAP GapCloser was used to close gaps.Reads were mapped by BWA,and SNPs and indels were screened by SAM tools.A rooted tree was computed using MEGA.This study successfully isolated two strains of MAP from Chinese dairy farms and determined that both strains were MAP-C type,and proved that Chinese MAP isolates 2015WD-1 and 2015WD-2 are both closest to international reference strain MAP K-10.2.Macrophage J774A.1 was infected with MAP 2015WD-1 strain with different MOI(MOI=1,5,10),and the apoptosis of J774A.1 was detected 12h after infection.The results indicate that macrophage apoptosis can be caused when the MOI of MAP is 10.Therefore,we infected J774A.1 with MAP at MOI=10,pretreated macrophages with endoplasmic reticulum stress inhibitor 4-PBA and endoplasmic reticulum agonist Tm to investigate whether endoplasmic reticulum stress is involved in MAP-induced apoptosis of J774A.1 through Western blot,RT-PCR,flow cytometry and transmission electron microscopy techniques.The results demonstrate that MAP can cause endoplasmic reticulum stress in J774A.1,and apoptosis is reduced when pretreatment with 4-PBA,but apoptosis increased when pretreated with Tm,thus demonstrating that endoplasmic reticulum stress is involved in MAP-induced apoptosis from both positive and negative aspects.This study also explored the relationship between macrophage polarization and endoplasmic reticulum stress and apoptosis after infection with MAP(MOI=10).J774A.1 was induced to differentiate into Ml macrophages by treatment with LPS and IFN-?,and M2 macrophages was induced by treatment with IL-4.M1 and M2 were infected(MOI=10)with MAP at different time points,respectively.Apoptosis and endoplasmic reticulum stress were measured.The results indicate that M1 is more likely to undergo apoptosis induced by the endoplasmic reticulum than M2 macrophages.3.This study constructed a mouse model of mixed infection of MAP and M.bovis.The effects of MAP on M.bovis-infected mice were evaluated by observing changes in body weight and death and histopathological observations;The concentrations of cytokinesin serum including TNF-?,IFN-?,IL-1?,IL-2,IL-4,IL-5,IL-6,IL-9,IL-10,IL-22,IL-13,IL-27,IL-23,IL-12P70,GM-CSF,IL-18 and IL-17A were measured by ProcartaPlex(?)Multiplex Immunoassay;Flow cytometry was used to explore CD4 and CD8 T cell subsets,the ratio of Thl/Th2 response,memory T cell phenotype and multifunctional T cells in the spleen;the production of IFN-? in the lungs of mice was messured by immunohistochemistry.The results indicate that infection with MAP in advance can inhibit the progression of M.bovis-infected mice by inducing Thl responses and increased the proportion multifunctional CD4+ T cells in spleen.In addition,less IFN-? was produced in lungs can also contribute to the survival in MAP+M.bovis infected mice than M.bovis infected mice.