Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice

Abstract

Background: Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice.

Results: We have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-gamma.

Conclusion: Our data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.

Figure 1

Comparison of LPS content of mouse pelleted diets. The concentration of LPS was measured to determine the load of microbiota-derived components in mouse diets. The pellets were ground, sonicated in non-pyrogenic water and filtered. LPS concentration in the filtrate was analyzed using the Chromogenic Limulus Amebocyte Lysate (LAL) Test (Cambrex, USA) and is expressed as endotoxin units (EU) per 1 μg of a diet. Results represent the mean (± SE) of four measurements.

Figure 2

The effect of gut microbiota and LPS-rich sterile diet on the weight and cellularity of lymphoid organs. (A) Germ-free mice have smaller spleens compared to conventional mice. (B) Gut microbiota and the LPS-rich diet drive the cellular expansion in MLNs and PPs. Results represent the mean (± SE) of at least 10 mice/group. Statistical analyses were performed using one-way analysis of variance (ANOVA) and a post-hoc comparison test (Tukey-Kramer). * indicates p < 0.05 and ** indicates p < 0.01.

Figure 3

FACS analysis of lymphocyte subpopulations illustrates the stimulating effect of gut microbiota and LPS-rich sterile diet. (A) Both gut microbiota and the LPS-rich diet decrease the proportion of CD19+ B cells in MLNs. (B) LPS-rich diet induces the expansion of CD4+ T cells in MLNs and spleen. (C) Microbial stimulation does not affect the proportion of CD8+ T cells. (D) Both gut microbiota and the LPS-rich sterile diet induce the expansion of Foxp3-expressing CD4+ T cells in MLNs. (E) The ratio of CD4+Foxp3- T cells to CD4+Foxp3+ T cells remains constant in all lymphoid organs. (F) Gut microbiota stimulate the expansion of Foxp3-expressing CD8+ T cells in PPs and MLNs. Results represent the mean (± SE) of at least 10 mice/group. Statistical analyses were performed using one-way analysis of variance (ANOVA) and a post-hoc comparison test (Tukey-Kramer). * indicates p < 0.05 and ** indicates p < 0.01.

Figure 4

In vitro proliferative response of spleen cells is not influenced by gut microbiota or LPS-rich sterile diet. Spleen cells isolated from CV mice and GF mice fed either diet were stained with CFSE fluorescein and stimulated with ConA. After 72 h the cells were analyzed by FACS. Histograms in this figure show the CFSE staining profile of lymphocytes negative for CD19 antigen (B cell marker). Proliferation was measured as the percentage of cells showing decreased staining intensity of CFSE compared to the intensity of the CFSE^bright population. Open histograms represent unstimulated control cells. The presented data are from a representative experiment. Each experiment was repeated at least three times with similar results.

Figure 5

Gut microbiota and the LPS-rich diet influence in vitro cytokine response of spleen cells. Spleen cells from CV and GF mice fed the low LPS diet (AIN-93G) or LPS-rich diet (ST1) were stimulated with ConA or LPS for 48 h. The production of IFNγ, IL-4, IL-10 and IL-12 cytokines was determined by Luminex analyzer. Results represent the mean (± SE) of at least 6 mice/group. Statistical analyses were performed using one-way analysis of variance (ANOVA) and a post-hoc comparison test (Tukey-Kramer). * indicates p < 0.05 and ** indicates p < 0.01.