| Objective A new mice tuberculosis model of Pdll specific knockout on macrophages(MΦ)was created to clarify the immune role of Pdl1 on MΦ in tuberculosis infection.Methods Pdl1Flox/-mice was constructed,and homozygous and heterozygous mice(Pdl1Flox/Flox with Cre,PdI1Flox/-with Cre)with specific Pdll knockout on MΦs were breeded by the Cre/loxp technique,which were verified by PCR and FACS.Female and male homozygous,heterozygous,and littermate negative mouse were infected with 2 X 106 CFU H37Rv via caudal vein injection,and the mouse were dissected after four weeks.BACTEC MGIT 960 culture system and the Lowenstein-Jeusen plates(L-J plates)culture method were used to detect the bacterial load of mouse lung,spleen and liver tissues,and pathological changes were quantitatively analyzed,to evaluate whether the mice tuberculosis model of Pdll specific knockout on macrophages is success or not,and to study the role of PD-L1 on macrophages in vivo.At the same time,PD-L1 and PD-1 functional antibody were used to interfere with C57BL/6 mice infected with tuberculosis.PD-L1 isotype antibody(250 μpg/piece/week),PD-L1 antibody(250 μg/piece/week),and PD-1 antibody(250 μg/piece/week)were continuously administered on the 3rd day,the 10th day and the 17th day,respectively.Quantitative analysis of bacteria loads and pathological changes in the lung,spleen and liver,to study the role of widely distributed PD-L1 and PD-1 in tuberculosis infection in mice.Results The genotype of progeny mice was successfully detected by PCR,including Pdl1Flox/Flox-Cre,Pdl1Flox/+-Cre and Pdl1Flox/Flox mice.Flow cytometry was used to detect the percentage of PD-L1 expression on macrophages,T cells and B cells of the above-mentioned mice,the percentage of PD-L1 expression on macrophages of Pdl1Flox/Flox-Cre,Pdl1Flox/+-Cre and Pdl1Flox/Flox mice was 4.4 ± 2.8%,40.9 ± 3.5%,and 87.4 ± 3.8%.The percentage of Pdl1Flox/Flox-Cre mice was significantly lower than the latter two(P<0.001),but there is no significant change in PD-L1 expression on T and B cells.PCR and flow cytometry confirmed that specific Pdll knockout on macrophages in mice was successfully prepared.Four weeks after infection,the time to detection(h)in the 960 culture system and colony forming unit(LgCFU/mL)in L-J plates of lung,spleen and liver both show that both PD-1 antibody and PD-L1 antibody can significantly reduce the bacterial load in target organs of C57BL/6 mice(P<0.05,P<0.05).In different subline mice with specific Pdll knockout,the bacterial load(LgCFU/mL)in the lung,spleen and liver tissues of Pdl1Flox/Flox ♀ infected group were 5.22±0.17,5.65±0.11,4.94±0.18,Pdl1Flox/Flox+-Cre and Pdl1Flox/Flox-Cre infected group were both increased.Pdl1Flox/Flox-Cre♀ infected group was increased significantly(P<0.05),which the bacterial load(LgCFU/mL)in the lung,spleen and liver tissues were 5.83±0.12,5.78±0.16,5.35±0.10.In addition,the gender difference of each subline has no obvious effect on the bacterial load.The lesion ratio(%)in lung and spleen,and granulomatous density(number/mm2)in liver of each group showed that PD-1 antibody and PD-L1 antibody can significantly reduce the pathological changes of target organs in C57BL/6 mice(P<0.001,P<0.001).The percentage of lung and spleen lesions(%),and granulomatous density(number/mm2)in liver of PdllFlox/Flox ♀ infected group were 4.61 ±0.17,4.06±0.25,1.98±0.1 1.Pathological changes of target organs in Pdl1Flox/+-Cre and Pdl1Flox/Flox-Cre infected group were both exacerbated.The percentage of lung and spleen lesions(%),and granulomatous density(number/mm2)in liver of Pdl1Flox/Flox-Cre♀ infected group were 10.42±0.53,8.03±0.48,5.09±0.11,which was the most serious(P<0.001).In addition,the gender differences of each sublineage have no significant effect on pathological changes.Conclusions Confirmation of successful preparation of specific knockout mice by PCR and flow cytometry.After tuberculosis infection in knockout mice,tuberculosis-related lesions appeared in the lung,spleen and liver,the bacterial load was maintained at a high level,and the parameters of lesion and bacterial load were stable.Therefore,the new mice tuberculosis model of Pdll specific knockout on macrophages was successfully established.Four weeks after infection,The lung,spleen and liver of Pdl1Flox/Flox-Cre mice were significantly increased in bacterial load,and corresponding pathological changes such as tissue granuloma and inflammatory cell infiltration were observed.However,after extensive intervention with PD-L1 antibody,the bacterial load and lesions in lung,spleen and liver of mice were significantly reduced.Knockouting Pdll and PD-L1 antibody have different effects,which can provide experimental data for the role and mechanism of PD-L1 in tuberculosis.Objective Study the role and mechanism of PD-L1 on phagocytosis and killing of macrophages infected by tuberculosis.Methods Macrophage cell line(RAW264.7 cell)was infected by Mycobacterium tuberculosis CM/)with different infection multiples of MOI5(the number of bacteria:the number of cell = 5: 1)andMOHO(the number of bacteria: the number of cell = 10:1).After 3 hours,PD-L1 antibody(10μg /mL)was added,and PD-L1 isotype antibody was used as the antibody control(10μg /mL).Flow cytometry was used to detect the fluorescence intensity of FITC-labeled Mth to reflect the effect of PD-L1 on the phagocytosis of macrophages.To investigate the killing function of macrophages by detecting the number of live bacteria and reactive oxygen species(ROS)levels in macrophages infected with tuberculosis.24 h and 48 h after infection,BACTEC MGIT960 culture system and the Lowenstein-Jeusen plates(L-J plates)culture method were used to detect the number of intracellular viable cells in each group.Flow cytometry was used to detect the proportion of fluorescent cells in each group of ceils to reflect ROS level.And then,high-throughput liquid-phase protein chip was used to detect cytokines in the supernatants of infected cells at 24 h and 48 h to explore the molecular mechanism of effect on phagocytosis and killing function.Results After infected with tuberculosis,the proportion of cells containing FITC-M/tb in MOIIO was significantly higher than that in MOI5(P<0.001).And after adding PD-L1 antibody,the proportion of cells containing FITC-M/6 was also increased significantly in MOIIO(P<0.01).During tuberculosis infection(24h to 48h),PD-L1 antibody can significantly enhance the killing effect of macrophages on intracellular bacteria in MOIIO,and the intracellular ROS content was significantly increased(P<0.01).However in MOI5,the killing effect of macrophages was inhibited,and intracellular ROS production was significantly reduced(P<0.05).High-throughput liquid-phase protein chip assay found that PD-L1 specifically promoted IL-10 secretion in each group of MOI5 and promoted TNF-a secretion in each group of MOIIO at 24h.PD-L1 specifically activates the secretion of IL-I3 and IL-22 in MOI5,and promotes the secretion of GM-CSF,IL-ip,IL-6 and IL-18,inhibits the secretion of IL-4 in MOIIO at 48h.Conclusions PD-L1 antibody promotes the secretion of IL-10,IL-13 and IL-22,and reduces the production of ROS in macrophages to inhibit the phagocytosis and killing of macrophages in MOI5,while in MOIIO,it promotes the secretion of IL-1p,IL-6,IL-18,GM-CSF and TTMF-α inhibits the secretion of IL-4,and increases the intracellular ROS level,thereby enhancing the phagocytosis and killing effect of macrophages on tuberculosis bacteria.Objective To analyze the transcription profile of Mycobacterium tuberculosis(Mtb)infected macrophages that knocked out with gene Pdil,and to screen out PD-L1 related candidate genes in Mtb infected macrophage(MO).Methods The peritoneal MOs were isolated from homozygous and heterozygous mice(Pdl1Flox/Flox- with Cre,PdllHoK/~ with Cre)with specific Pdll knockout on MOs,and that from littermate negative mice(Pdl1Flox/Flox-)were used as controls.The transcriptome sequencing technology was used to detect the 24 h transcript spectrum of macrophages infected by tuberculosis bacteria with MOI5 and MOIIO,and bioinformatics analysis was performed.Results The Pdl1Flox/Flox- with Cre,PdllHox " with Cre and Pdl1Flox)S Mox infected groups were compared with their corresponding non-infected groups.Gene Ontology(GO)functional enrichment analysis showed that the most significant enrichment were immune response(GO: 0006955,P < 0.01)and immune system process(GO: 0002376,P < 0.01)in both MOI5 and MOI10 infected groups.The 22 candidate genes screened out were regarded as specific candidate genes related to PD-L1 in Mtb infection.Through Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,the most significantly enriched pathways include the Toll-like receptor signaling pathway,nuclear factor kappa-B(NF-kB)signaling pathway,hypoxia inducible factor_l(H1F-1)signaling pathway and tumor necrosis factor(TNF)signaling pathway(P< 0.01;P< 0.01;P < 0.01;P < 0.01)in both MOI5 and MOI10 infected groups,16 candidate genes related to PD-L1 were screened.Conclusions Through transcription analysis,22 genes related to immune response,including Cxcll 、Ccl2、Qa5>Ill2a,etc,and 16 genes in immune and inflammation-related metabolic pathways,including Ptgs2、 Cd40、 Cardin Bcl3,etc,which were identified as specific candidate genes related to PD-L1 in macrophages infected with Mtb.Except for116,all candidate genes were selected for this research. |
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