The Effect Of M. Bovis And BCG Infection On Murine Dendrtic Cell Arginine Metabolism

Tuberculosis is an important zoonotic disease which is caused by Mycobacterium tuberculosis(M tb) complex and leads to a great pandemic to humans and cattle. An important reason for TB eradiction is that persistent infection of M.tuberculosis or M.bovis. Once M.tb intrudes into the host cells, the host immune system will make response to the pathogen including cellular immunity in the early stage and humoral immunity in the later stage. M.tuberculosis is the most successful bacillus in the world due to unique mechanism of immune evasion and latent persistence which is only partially known such as changing style of metabolism, impeding the formation of phagolysosomes, affecting T cell recognition and induction of cell apoptosis, etc.In order to explore the potential molecular mechanism of M. bovis persistent infection, the previous study used murine whole genome microarray to screen the differential gene expression of DCs post-infection by both virulent M. bovis and BCG and discovered 36 differential transcripts with over 2 fold difference. Among them, some genes including Slc7A2 and Slc7A1 are related to the arginine metabolism. The arginine metabolism will produce reactive nitrogen intermediates (RNI) and RNI, the cytotoxic effector molecules, are critical to antibacterial capability of the host cells. Therefore, this study aimed to study arginine metabolism of dendritic cells after virulent M.bovis and its attenuated strain BCG infection, revealing the NO (one of RNI) production and the effect on infected cells and intracellular bacteria, and finally provide evidence to elucidate the mechanism of immune evasion and persistence for M.bovis.The main results are as follows:1. The effect on NO production of dendrtic cell after infectionThe concentration of NO in the supernatants was detected by Griess Reagent System. The results have shown that the concentration of NO was highly increased after infection. And this increasing was amplified along with increased post-infection time. After 24h post-infection, the concentration of NO in BCG group was highly than M.bovis with 12.57μM and 12.04μM respectively.2. The effect on transcription of cationic amino acid transporter (CAT) and iNOS after infectionThe qRT-PCR was used to detect the related genes transcriptions in arginine metabolism. The results shown that the transcriptions of Slc7A2、Slc7Al and iNOS in both infected groups were increased compared uninfected group. The Slc7A2 transcription was 4.263 folds in BCG group higher than 3.14 folds in M.bovis group. The iNOS transcription was 5.328 folds in BCG group higher than 1.247 folds in M.bovis group. The similar result was obtained for Slc7A1.The addition of cytokine IFN-y (10ng/mL) in the culture greatly increased the iNOS and Slc7A2 transcription. The fold of up-regulation for iNOS was 13.362 and 15.345 respectively in BCG+IFN-y and M.bovis+IFN-ygroup, while 5.696 folds and 12.247 folds respectively for Slc7A2.3. The effect on transcription of arginase after infectionTwo isoforms of mammalian arginases exists in DCs. But the level of arginase I mRNA was greatly higher than arginaseⅡ. Compared to uninfected group, arginase I transcription was 29.925 folds and 9.812 folds respectively in BCG group and M.bovis group, while 0.231 folds and 0.432 folds respectively for arginaseⅡ. It can be seen that murine dendritic cells was mainly expressed arginase type I in DCs infected with different virulence M.bovis.4. Flow cytometry analysis for apoptosisThe apoptosis of DCs was detected by flow cytometry analysis following PI staining. (Only for BCG group), besides TLR4 antibody treatment group, the proportion of apoptosis in other groups was lower than BCG infected group. The apoptosis of DCs was descended from 21.73% to 12.7% in BCG infection by PDTC treatment group compared to BCG infected group. The changes in other group as follow,16.82% for TLR2 antibody treatment group,16.625% for TLR2 and TLR4 antibodies co-treatment group. However, the proportion increased to 27.05% in the TLR4 antibody treatment group.5. The result of intracellular bacterium survivalThe bacterial plating assay showed that BCG replicated better than M.bovis in DCs during the period of 24 h post-infection. The amount of associated bacteria for BCG at 24h post-infection was 139500±28723 CFU/well, while that of M. bovis was 82500±27000 CFU/well. There was a significant difference between them (P=0.0276). However, at the presence of IFN-y (100 ng/ml), the difference in the numbers of associated bacteria for both strains remained statistically significant (P=0.0366). The bacterial numbers were significantly decreased with the P values of 0.0351 for BCG (82500±30740 CFU/well) and 0.0234 for M. bovis (31000+1000 CFU/well) respectively. When the inhibitor L-NMMA was added, the number for each bacillus was increased. Since L-NMMA inhibited NO production, this result confirmed the NO level is conversely correlated to the bacterial replication.