| Nontuberculosis mycobacteria(NTM)are ubiquitous in the environment and induce opportunistic infections that can produce a wide range of diseases, it has gradually aroused people’s concern, but there is little research regarding the host immune mechanisms following infection and the reasons NTMs can persist in the host. NTMs are facultative parasitic bacterias, the interaction mechanism of mycobacterium tuberculosis and macrophages that the the first barrier of body’s defense is particularly important. In addition, T cell immune is very important in the process of mycobacterium tuberculosis infection, and the proportion of each subgroup T lymphocyte differentiation affect the body’s ability to fight the NTMs infection directly. 20 strains of NTMs have been isolated from mammals in our preventive veterinary laboratory(Jilin Agricultural University), we study phagocytosis and proliferation of NTMs in mice bone marrow macrophage, obtain two stronger strains-M.vaccae and M.neoaurum. These two strains were reported in clinical infection, however, there has been little research regarding immune mechanisms during M.vaccae and M.neoaurum infection, The goal of this study was to compare the isolated M.vaccae and M.neoaurum to M. bovis BCG in terms of their infection properties and the host immune mechanisms, especially the immune mechanism associated with macrophages and T lymphocytes.RAW264.7 cells were infected with reference strain M. bovis BCG and isolated M. vaccae, M. neoaurum, the cell apoptosis rate were observed by flow cytometry after 0 h, 4 h, 12 h, 24 h, 48 h, we observed the changes of TLR2 and TLR4 mRNA by qRT-PCR, in addition, the expression of TLR2, TLR4, MHCⅠ, MHC II, CD80, CD86 in RAW264.7 cells were observed by flow cytometry, the cytokines(TNF-α、IL-10、IL-12p40) were quantified using an ELISA kit. The mice were infected with reference strain M. bovis BCG and isolated M. vaccae, M.neoaurum using intraperitoneal injections, we sectioned the lung organs and used hematoxylin and eosin(H&E)and acid-fast stains at weeks 2, 4, 6 and 8, we observed the changes of TLR2, TLR4, MHC, MHCⅡ, CD80, CD86 and cytokines IL-12, TNF-α, IL-10 in the infected macrophage by flow cytometry. In order to explore the differentiation of CD4~+T cell after infection with NTMs, we observed the dynamic of the transcription of T-bet, GATA-3,RORγt and Foxp3 mRNA by qRT-PCR, the dynamic of the percentage of CD4~+T cell,CD8+T cell and Th1,Th2,Th17,Treg cell by using flow cytometry, the cytokines(TNF-α, IFN-γ, IL-12p40, MCP-1, IL-4, IL-17 A, IL-10, TGF-β) were quantified using ELISA kit.Results show that the isolated M. gilvum, M. vaccae, M. neoaurum are devoured by mice bone marrow macrophage, phagocytosis rate of M. vaccae is the highest(26.2 ±6.5%)(P<0.05); we observed a large number of bacterias in mice bone marrow macrophage using inverted fluorescence microscope; M. vaccae and M. neoaurum showed strong proliferation ability than M.gilvum on 4d and 7d after infection(P<0.001), the virulence and susceptibility of M. vaccae and M. neoaurum may be more stronger than M. gilvum. M. vaccae and M. neoaurum infection can promote RAW264.7 cell apoptosis rate, the productions of TLR2, TLR4, MHC, MHCⅡ, CD80, CD86 were higher in the M. vaccae and M. neoaurum-infected cells than in the normal group after 4h(P<0.05);the expression of M. vaccae group were higher than M. neoaurum group(P < 0.05), and the level of apoptosis is lower than the M. neoaurum group(P < 0.05), so the virulence of M. vaccae may be stronger than accae M. neoaurum; we observed that expression of TLR2, TLR4, MHC, MHCⅡ, CD80, CD86 decreased after 24 h, but there was a strong influx of IL-10 in M.vaccae and M. neoaurum-infected cells, we concluded that M.vaccae and M. neoaurum decreased the productions of TLR2, TLR4, MHC, MHCⅡ, CD80, CD86, TNF-α, IFN-γ, IL-12p40 in order to resist the antiseptic effect and inhibition of macrophage cell, including cytokines IL-10 high expression is also one of the reasons.By preparation the mice model of mycobacterium tuberculosis infection, we also found high expression of TLR2, TLR4, MHC, MHCⅡ, CD80, CD86, TNF-α, IFN-γ, IL-12p40 of the lung macrophages in M. vaccae and M. neoaurum-infected mice, compared with normal group(P < 0.05), and decreased at week 6 and 8, but the expression of IL-10 did not decreased, the reactions of M. vaccae and M. neoaurum-infected mice is similar to the results of in infected RAW264.7 cells, it may be helpful with the body’s inflammatory response and organ damage. In addition, In survival studies, M. vaccae and M. neoaurum-infected mice had substantial weight loss compared with the control group,In particular, the Mycobacterium vaccae-infected group is lower(p<0.05), but M. neoaurum and BCG group is similar(P>0.05). The lung histopathology challenged with M. vaccae and M. neoaurum were more serious than M. bovis BCG group, at week 2, there is a large number of neutrophils in the lungs of M. vaccae–infected mice, at 4w and 6w, with inflammatory cell infiltration and hemorrhage, at 8w, alveolar interval is still thick and alveolar cavity size is differ; But at the 4w, we found the alveolar walls of M. neoaurum group is thicker than M. vaccae group, at week 8, inflammatory cells decreased, alveolar walls are restored; There was no typical tuberculous in the lungs of the two non-tuberculosis mycobacteria infection. These results also showed that the virulence of NTM still exists to the lung injury, which should be pay more attention. It had many acid-fast staining bacilli in lungs of M. vaccae, M. neoaurum, BCG-infected mice, but there are differences between the numbers of bacteria, the bacteria cultured more of M. vaccae group than other groups(P < 0.05).We studied the differentiation situation of T lymphocyte after infection with the mycobacterium tuberculosis bacteria, there was a strong influx of CD3+CD4~+ T cells in M.vaccae and M. neoaurum-infected mice, with the numbers reaching the highest levels at week 4 compared with the other groups(p<0.01) and then declining. However, the M. bovis BCG-infected group did not experience such a rapid decline(P>0.05). There was a rapid influx of CD8 cells, reaching the highest levels on weeks 6 and 8 of M.vaccae and M. neoaurum infection compared with the control group(p<0.01), the M. bovis BCG-infected group did not raise so much(P>0.05). We found that a strong CD4~+ T response was induced by M. vaccae and M. neoaurum on early infection, CD8+ T response was induced on late infection. In contrast, M. bovis BCG induced a CD4~+ T response that did not decline as quickly.We explored the differentiation of CD4~+T cell after infection with the mycobacterium tuberculosis bacteria, we found high expression of T-bet mRNA, RORγt mRNA, IFN-γ, TNF-α, IL-12,IL-17 A of the lungs in M. vaccae and M. neoaurum-infected mice,compared with normal group(P < 0.05),at the same time the proportion of Th1 and Th17 cells also increased(P < 0.05). Th1 response reached the highest at 4w, and then began to decline; and at the time of the 2w Th17 response began to decline. M. vaccae and M. neoaurum inhibited the express of transcription factor GATA- 3 and cytokines IL-4 at the early stage of the infection(P < 0.05), and reduced the proportion of Th2 cells(P < 0.05); at week 6w-8w, Th2 response was enhanced, Th1 and Th17 responses were relatively lower. we found high expression of Foxp3, TGF-β, IL-10 and Treg of the lungs in M. vaccae and M. neoaurum-infected mice, compared with normal group(P < 0.05), Treg response was enhanced, especially at week 6 and 8(P < 0.05), the strong Treg response may be one reason to decrease Th1 and Th17 responses, in addition, at week 6 and 8, the high expression of Th2 reaction also inhibits Th1 and Th17 response. And the Th1, Th2, Th17, Treg responses of M. vaccae group were stronger than M. neoaurum more intense. The Th1 response of BCG group was stable in the whole process of infection, and its Th1 response were inhibited, that made the body injury recovered; at week 4, the expression of Th17 cells, RORγt, Th17 A of BCG group were higher than the normal group(P < 0.05), it had not significant difference with normal group on the rest of the time point(P > 0.05), the Treg response of BCG group was not stronger than NTMs groups, thus the decline of Th1 and Th17 response was weaker than M.vaccae and M. neoaurum groups.Through the above results, we analyzed the process of M.vaccae and M. neoaurum-infected mice, at week 2, antigen was identified by lung macrophages in mice through TLR2 and TLR4, then the antigen was sended to T cells by increasing expression of MHCⅠ, MHCⅡ, CD80/86, and high expression of CD4~+T, Th1 and Th17 was induced; However, at week 6, the Th1 and Th17 responses declined over time, associated with the decline of the TLR2, TLR4, MHCⅠ, MHCⅡ, CD80/86, In addition, strong Th2 and Treg response were found.These results for the study of mycobacterium tuberculosis infection characteristics and host immune mechanism, especially the immune mechanism associated with macrophages and T lymphocytes, at the same time, to provide theoretical basis of NTM and the treatments for NTM disease. |
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