Study On The Mechanism Of PD-L1 In The Multi-course Mouse Model Of Tuberculosis And Research Of The Susceptibility To Tuberculosis In Collaborative Cross Mice

Objective To establish of a new acute infection-latent-relapse tuberculosis infection model in mice,which would be foundation of mechanism research and diagnosis marker finding,and also for pharmacodynamic evaluation of anti-tuberculosis medicines.Methods Four-six weeks Female C57BL/6 mice were infected via caudal vein injection with 106 CFU M.tuberculosis H37Rv.Two weeks postinfection,the mice were administered Isoniazid(10 mg/kg)and continued for four weeks.Among the treatment groups,half of the mice were induced by TNF-a antibody(50 ug/each)for four weeks,and half of the mice were untreated for natrural relapse.At each scheduled time point,bacteria loads and pathological changes in the lung,spleen and liver were quantitatively analyzed,to evaluate whether the establishment of multi-stage infection animal model is success or not.Results The bacteria burden reached 3-5 Lg(CFU/mL),and granuloma lesions were extensive in the lung,spleen and liver in the all infected groups at 2nd week postinfection.Histopathologic examination showed:granuloma can be seen in the lung,which mainly consisted of macrophages and lymphocytes,and the lymphocyte infiltration around the blood vessels.For the histopathological changes in liver,a large number(about 20 granulomas per field of microscope)of scattered granuloma with uniform sizes(2-10mm2)were seen,which were mainly consisted of macrophages,lymphocytes and fewer neutrophils.Pathological changes of splenic white pulp and red pulp areas were destroyed by visible granulomas which was mainly consist of macrophages.After Isoniazid treated,the bacteria burden of the lung,spleen and liver respectively were 0,2.68 ±0.59,2.58±0.51Lg(CFU/mL)in the treatment group,and the bacteria burden in the infection control group were 3.64±0.54,4.06±0.38,3.06± 0.28 Lg(CFU/mL).Compared with the infection control group,the bacteria burden was significantly decreased and the pathological lesions alleviated in the group treated.Histopathologic examination showed:the structure of pulmonary alveoli was normal except for less visible perivascular inflammatory cell infiltration,several smaller granulomas was seen in liver,and only a few visible granuloma seen in white pulp and red pulp.Four weeks after treatment,the group without TNF-a and the TNF-a-induced group had a significantly higher bacteria load,which reached among3.8 to 4.4 LgCFU.While bacteria load of spleen and liver were lower than the infected control group.TNF-?-induced group had a significantly severe pathological lesions compared with the group without TNF-a.Conclusions At the second week postinfection,the bacteria burdens of lung,spleen and liver of mice were significantly high,and the pathological lesions including granuloma and inflammatory cells infiltration which prompted that tuberculosis infection was in the acute stage.After four weeks of isoniazid treatment,the bacteria burdens of the lung,spleen and liver were decreased,and the pathological lesions alleviated,when compared with the infection control group,showing significant differences,which prompted that tuberculosis infection was in the latent infection period.After four weeks TNF-a induction,the bacteria burden of the lung,spleen and liver was increased,and the pathological lesions aggravated when compared with the infection control group,showing significant differences which prompted that tuberculosis infection into relapse stage.Therefore,a new acute infection-latent-relapse tuberculosis infection mice model is created.The period of entire model lasted 10 weeks,which greatly shorten the research cycle,had stable bacteria loads and pathological parameters in the each periods,and suited for TB mechanism research and pharmacodynamic evaluation of anti-tuberculosis medicines.Objective To study the immune role and mechanism of blocking PD1/PD-L1 pathway mediated by macrophage with functional PD-L1(Programmed cell death ligand-1,PD-L1)monoclonal antibody in tuberculosis(TB)and relapse in mice.Methods Four to six weeks old female C57BL/6 mice were infected via caudal vein injection with 106 CFU M.tuberculosis H37Rv to obtain active TB.Two weeks postinfection,the mice in different groups were administered isoniazid(10 mg/kg)and isoniazid combined with PD-L1 monoclonal antibody(50 ?g/each),continued for four weeks to obtain latent infection,and the subsequent relapse was monitored.Among the treatment groups,half animals was induced by TNF-a antibody(50 ug/each)for four weeks from the beginning of latent stage,and half animals was monitored without any treatment.At each scheduled time point,bacteria loads and pathological changes in the lung,spleen and liver were quantitatively analyzed,thereby,the in vivo intervention effect of PD-L1 monoclonal antibody on tuberculosis recurrence in mice was revealed.The in vitro experiment was further explored whether knock-down the expression of PD-L1 on the infected macrophages could accerlate the macrophage apoptosis.Results At 2nd week post-infection,the bacteria burden reached 3-4 Lg(CFU/mL),and granuloma lesions were extensive in the lung,spleen and liver in the all infected groups,which appeared as active TB stage.After treated,the bacteria burden of the lung,spleen and liver were decreased,and the pathological lesions alleviated in the group INH and group INH+PD-L1,when compared with the model control group,showing significant differences,but there was no significant difference between the two treatment groups.However,compared with the group INH,the group INH+PD-L1 had a significantly lower bacteria load and milder pathological lesions during the relapse period.Furthermore,knock-down the expression of PD-L1 on macrophages with anti-PD-L1 or PD-L1-siRNA promoted apoptosis of macrophages.Conclusions Blockade of the PD1/PD-L1 pathway by PD-L1 functional antibody can inhibit TB relapse in mice,and knock-down the expression of PD-L1 on macrophages or PD1/PD-L1 pathway with functional antibody can promote apoptosis of macrophages,which together indicate that PD-L1 blockade can effectively promote isoniazid treatment of TB and remarkably inhibit the recurrence of TB in mice.Objective Using the Geneminer database to quantitatively analyse bacterial burden and pathological lesions of collaborative cross mice,to obtain susceptible genes or corresponding SNPs for tuberculosis.Methods C57BL/6 mice and 17 collaborative cross mice were infected via caudal vein injection with 10~6 CFU M.tuberculosis H37 Rv to obtain active TB infection.After four weeks infected,lung,spleen,liver tissue of mice got by removing cervical vertebra to death.By quantitatively analyse for bacteria burden and pathological lesions of mice to certain the susceptible phenotypes for tuberculosis in collaborative cross mice.Using the Geneminer database to analyse for bacteria burden and pathological lesions of collaborative cross mice,to obtain susceptible genes or corresponding SNPs for tuberculosis.Results Bacteria load and the pathological changes of eighteen strains of collaborative cross mice present different numerical values,some strains of mice which charge high bacteria load with lesions value which can be higher or lower.Accordingly,the lesions of infected mice were not consistent with bacteria load.The specific results as follows: bacteria load of lung,spleen and liver from low to high respectively order are:FEW?,LAMA? DET3?,LUS? BOM?,LAT?,PUB?,NUK?,SHE?,TOP?,ROGAN?,PIPING?,DAVIS?,C57?,BOON?,LOT?,SAT?,BEM?;DET3?,SHE?,BOM?,C57?,PUB?,ROGAN?,TOP?,LUSA?,FEW?,NUIC?,LAM?,DAVIS?,LAT?,PIPING?,SAT?,BEM?,LOT?,BOON?;LUS?,SHE?,PUB?,TOP?,BOM?,DET3?,FEW?,DAVIS?,C57?,LAM?,ROGAN?,NUK?,PIPING?,BEM?,SAT?,BOON?,LAT?,LOT?.The lung,spleen and liver respectively located in chromosome 3,4 and 3 and respectively got 143,264 and 0 goals encoding SNPs loci by "MugaQTL(narmalise)" analysis method.Conclusions Mycobacterium tuberculosis have different susceptibility of different mice strains.The same strain doesn't keep same susceptibility with lesions and bacteria load.And the susceptibility of different tissue organs also have differences.According to the different organs or different tissue lesions and bacteria load of mice,to analyze the susceptibility genes.According to the value of bacterial load,geneminer database was used to predict lung and liver susceptibility genes on chromosome 3 and spleen susceptibility genes on chromosome 4.