Intestinal dysbacteriosis induces changes of T lymphocyte subpopulations in Peyer’s patches of mice and orients the immune response towards humoral immunity

We hypothesized that alterations in gut microflora may induce changes of cellular composition and cytokine productions in PPs, and lead to a shift in immune response/tolerance. To test this, we established mouse models with dysbacteriosis through intragastric administration of ceftriaxone sodium. Alterations of lymphocytes in PPs (particularly the T lymphocyte subpopulations) were examined, as was cytokine mRNA expression (Additional file 1: Supplementary material). The population of commensal bacteria was found reduced in mice treated with ceftriaxone sodium and some are even eradicated (Additional file 2: Table S1). The structure of intestinal microflora was seriously disrupted. The population of CD3⁺ T cells, which represents the level of cell-mediated immune response, was detected by flow cytometry. Compared with control mice, the total level of CD3⁺ T cells in PPs of mild dysbacteriosis mice increased to 22.2%, and to 26.9% in PPs of the severe group (Figure 1a). The T lymphocyte subpopulations were also examined. Results in Figure 1b show that CD4⁺ T cells in PPs of mild and severe dysbacteriosis mice increased by 7.37% and 5.15%, respectively. In contrast, the CD8⁺ T cells in PPs of mice with dysbacteriosis decreased from 5.85% to no more than 3.60%, resulting in a higher ratio of CD4⁺/CD8⁺ compared with healthy mice (Figure 1c). Changes in CD4⁺/CD8⁺ ratio are normally considered as an important reference to predict the shifting of immune responses. The increased proportion of CD4⁺/CD8⁺ indicates that intestinal dysbacteriosis has induced humoral immunity in mice. The populations of CD4⁺CD25⁺ T cells in PPs of healthy and dysbacteriosis mice were also detected. CD4⁺CD25⁺ are marker molecules on the surface of regulatory T cells (Treg cells), which are crucial for maintenance of immunological tolerance [8]. In PPs of dysbacteriosis mice groups, the regulatory CD4⁺CD25⁺ T cells were severely decreased (Figure 1c). These striking decreases indicate a significant reduction of immunological tolerance. The transforming growth factor-beta (TGF-β) is a critical regulator for the development of Treg cells, which are crucial for the maintenance of immunological tolerance [9]; we therefore detected the mRNA expression levels of TGF-β gene. As shown in Figure 2, a decreasing pattern was observed in dysbacteriosis mice groups, which suggested a delay of Treg cells development. This is basically correlated with the trends that have seen for the population of CD4⁺CD25⁺ T cells. Th cells represent a functionally heterogeneous population, comprising mainly two distinct subsets (termed Th1 and Th2 cells), defined by their cytokine secretion profiles [20]. Th1 cells produce IL-2, IFN-γ and tumour necrosis factor-beta (TNF-β), and promote both macrophage activation, and the production of complement fixing and opsonising antibodies. Th2 cells, which synthesize cytokines such as IL-4, IL-10 and etc., provide optimal help for antibody production, promote mast cell growth, eosinophil differentiation and activation, resulting in humoral or allergic responses [10]. In the present study, a slight decrease (by 10.7%) of Il2 gene expression was observed in PPs of mice with mild dysbacteriosis, while a dramatic decrease (by 60.9%) was observed in the severe group (Figure 2). The mRNA levels of the Ifng gene also decreased (by 20.4% and 37.4%, respectively). Expression of the Il4 gene in PPs of mice with mild dysbacteriosis increased by 23.6% compared with control, and it was 36.4% higher in the severe group. Expression of the Il10 gene increased dramatically in PPs of both mild (by 98.9%) and severe (by 200.4%) groups. The decrease in mRNA levels of Il2 and Ifng in PPs of dysbacteriosis mice suggest activation and proliferation of Th2 cells, while the elevation of Il4 and Il10 inhibit Th1 cells. Results in Figure 2 thus support the observation that dysbacteriosis in mice has induced a shifting of immune responses in PPs towards humoral immunity.