| OBJECTIVE:Based on the principle of "the lung and the large intestine being interior-exteriorly related" in Traditional Chinese Medicine, to investigate the effects of dysbiosis of the mice infected with influenza virus on the balance of immune responses mediated by helper T cell subsets, and to explore the specific intestinal commensal bacteria species that exert great influence on the differentiation of pro-and anti-inflammatory T lymphocytes, guiding the principle of clinical antibiotic treatment for diverse inflammatory diseases.METHODS:1. Establishment of dysbiosis in mice modelMetronidazole, cephradine, and neomycin were used for administration into the mice gastrointestinal tract, producing the over-growth and colonizing predominance of certain bacteria species, such as aerobic bacteria, gram-positive and negative bacteria. Intervened with antibiotics for4weeks, the mice stools were collected for Colony-Forming Units(CFU) counting.2. Mice infection with influenza virusFollowing4weeks administration of antibiotics, mice were inoculated intranasally with FM1influenza virus for4days. Lung biopsy were applied for determination of mice infection.3. Pathological section and Flow cytometryLung and spleen tissues were made for pathological section after the mice demise. T lymphocytes were obtained from mice spleen by using Lymphocyte separation medium(ficoll), and then helper T cell subsets were detected by using flow cytometer(FCM).4. Statistical analysisStatistical analysis was performed by2-sample, two-tailed Student’s t test(two groups) or one-way ANOVA(more than two groups) with Graphpad Prism software or SPSS13.0. P values less than0.05was considered significant.RESULTS:1. Dysbiosis in mice model was successfully established by the administration of Metronidazole, cephradine, and neomycin, confirmed by the colony counts on the bacteria culture plate. Compared with the normal group, bifidobacteria, enterococci and anaerobic bacteria in the neomycin treatment group increased significantly, while enterobacter reduced apparently; by the treatment of metronidazole, bifidobacteria and anaerobic bacteria decreased obviously; and bifidobacterium in the cephradine the treatment group reduced significantly, the differences were all statistically significant (P<0.05). When compared with the control group, enterobacter and anaerobic bacteria in the virus model group reduced significantly, but bifidobacteria and lactobacilli evidently increased(P<0.05).Compared with the virus model group, enterobacter in the metronidazole group increased significantly, whereas bifidobacteria and lactobacilli reduced with a significant difference(P<0.05); enterobacter and anaerobic bacteria in the cephradine treatment group increased significantly, bifidobacteria and lactobacilli was obviously reduced(P<0.05); in the neomycin treatment group, lactobacilli were significantly decreased, while anaerobic bacteria were significantly increased(P<0.05).2. Experimental mice were intranasally infected with influenza virus. Pathological observation was applied to verify. From the microscopic observation in the lung tissue, the normal tissue structure of alveoli, alveolar ducts, and alveolar septa mostly disappeared with widened alveolar septum and varying degrees of vascular congestion and expansion, and the infiltration and exudation of a large number of lymphocytes and other inflammatory cells can be seen from the entire field of view. Mice pathological and the normal group were compared to determine the establishment of mice-infected model.3. Following the detection by flow cytometer, Thl/Th2/Treg cells obtained from mice spleen were found intimately connected with the variation in the makeup of mice commensal microbiota. The results of the number of each type of T lymphocytes in the proportion of CD4-positive T lymphocytes in each antibiotic-treated group showed that, after metronidazole or cephradine treatment, Thl proliferation does not change significantly, compared with control groups(P>0.05); While after oral treatment of neomycin, the proportion of Thl increased significantly, compared with the virus model group, the metronidazole group as well as the cephradine group(P<0.05or P<0.01); in the metronidazole or cephradine treatment group, the proportion of Th2was significantly reduced compared to the normal group and virus model groups(P<0.001); Moreover, we found that in mice fed by neomycin treatment, the proliferation of Th2, when compared to the normal group and the virus model group showed no significant statistical difference(P>0.05), while compared to the metronidazole group and cephradine group, a significant difference was found obviously(P<0.001); After metronidazole or cephradine treatment, the number of regulatory T lymphocytes(Treg) showed no significant proliferation, significantly less than the normal group and the virus model group; in the neomycin group, Treg proliferation in mice, compared to the virus model group, showed no significant different (P>0.05), while having a significant difference compared with the normal group(P<0.001), when compared with the metronidazole group and cephradine group, significant differences were evidently found in the proliferation of Treg(P<0.001). However, during the experiment, the proliferation of Th17lymphocytes was almost not detected in any group. Moreover, when we compared the two control groups(the blank normal group and the virus model group), there is no significant difference between these two groups(P>0.05) in the proportion of T lymphocyte proliferation.CONCLUSION:1. Observing the dysbiosis in mice stool induced by antibiotic treatment, we have found that the composition of enterobacter, enterococcus, bifidobacteria, Lactobacillus, and anaerobic bacteria in the mice intestine differed vastly, providing the experimental basis for the evidence of antibiotics inducing the body dysbiosis in the common clinical practice.2. However, with the comparison between the two control groups(including the blank control group and the control group with the flu infection), there was no significant difference, indicating that influenza virus was seemingly not involved in the mediation of immune responses by helper T cell subsets.3. By analyzing the makeup of helper T cell subsets in each experimental group, we come to the conclusion that certain antibiotic-sensitive bacteria in the mice gut affect differentially the Thl/Th2/Treg cells, with obvious statistical difference, and commensal microbiota in mice gut were tightly connected with regulation of immune system in mice, elucidating the inner correlation between the dysbiosis in the gut and the lung disease in mice infected with influenza virus. |
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