Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells

Abstract

When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25(+) CD4(+)/CD4(+) cells in the mesenteric lymph node (MLN) and the absolute number of CD25(+) CD4(+) cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25(+) CD4(+) cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25(+) CD4(+) regulatory T (T(reg)) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta than those from the GF mice. When anti-TGF-beta neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25(+) CD4(+) T(reg) cells from the SPF mice was attenuated to the same level as that of the CD25(+) CD4(+) cells from the GF mice. In conclusion, the TGF-beta-producing CD25(+) CD4(+) T(reg) cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25(+) CD4(+) cells from the naive GF mice was much lower than that of the CD25(+) CD4(+) T(reg) cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25(+) CD4(+) T(reg) cells.

Fig. 1

Failure of oral tolerance induction in germ-free mice. For oral tolerance induction, mice were fed 5 mg/day ovalbumin (OVA) (n = 7) or phosphate-buffered saline (n = 5) for consecutive days and were then immunized with OVA in alum at 3 days after the last feeding and every 2 weeks thereafter. Blood samples to measure antibodies in the serum were collected 1 week after the last immunization and were subjected to enzyme-linked immunosorbent assays. (a) OVA-specific immunoglobulin G₁ (IgG1) in serum; (b) OVA-specific IgE in serum. Similar results were obtained in two independent experiments. SPF, specific pathogen-free.

Fig. 2

The expression of forkhead box P3 (FoxP3) in CD25⁺ CD4⁺ cells from specific pathogen-free (SPF) or germ-free (GF) mice. Single-cell suspensions were prepared from the mesenteric lymph node in naïve SPF and GF mice for fluorescence activated cell sorter analysis and were stained with fluorescein isothiocyanate–anti-CD4 antibody, phycoerythrin–anti-FoxP3 antibody and allophycocianin– anti-CD25 antibody. Intracellular FoxP3 expression in cells gated on the CD4⁺ CD25⁺ cells is presented. More than 95% of CD4⁺ CD25⁺ cells in both the SPF mice and GF mice groups expressed intracellular FoxP3. The results are from a representative experiment from three independent experiments.

Fig. 3

Interleukin (IL)-2 production and proliferation of mesenteric lymph node (MLN) and spleen cells in specific pathogen-free (SPF) and germ-free (GF) mice. The MLN or spleen was removed from naive SPF and GF mice and single-cell suspensions were prepared. A total of 250 000 cells were cultured with 1 μg/ml anti-CD3 antibody for 48 h and the supernatants were then collected to measure the IL-2 production. For the proliferation assays, 1 μCi [³H]-thymidine was added to the culture during the last 18 h of culture. (a) IL-2 production; (b) proliferation assay. Five mice were used in each group. The mean and standard deviation are shown in the figures. These results are representative of two independent experiments.

Fig. 4

Suppressive function of CD25⁺ CD4⁺ cells obtained from germ-free (GF) mice is impaired compared with that of CD25⁺ CD4⁺ cells obtained from specific pathogen-free (SPF) mice. Either CD25⁺ CD4⁺ or CD25^– CD4⁺ cells were purified from the mesenteric lymph node in naive SPF or GF mice and antigen presenting cell (APC)-depleted T cells were isolated from the spleens of SPF mice, as described in the Materials and methods. The co-cultured cells were maintained for 48 h and 1 μCi [³H]-thymidine was added to the culture 18 h before harvesting. (a) The titrated CD25⁺ CD4⁺ cells were co-cultured with 2·5 × 10⁵ CD25^– CD4⁺ cells as responder cells, which were obtained from SPF mice in the presence of APC and anti-CD3 antibody. (b) The titrated CD25⁺ CD4⁺ cells were co-cultured with 2·5 × 10⁵ CD25^– CD4⁺ cells derived from GF mice in the presence of APC and anti-CD3 antibody. □: CD25⁺CD4⁺ derived from SPF mice; ▪: CD25⁺ CD4⁺ derived from GF mice. The ratio of CD25^– CD4⁺ : CD25⁺ CD4⁺ is indicated. The mean and standard deviation are shown in the figures and are representative of three experiments. *P < 0·05, **P < 0·01 compared with responder cell proliferation.

Fig. 5

Regulatory cytokine production by CD25⁺ CD4⁺ cells derived from specific pathogen-free (SPF) or germ-free (GF) mice. CD25⁺ CD4⁺ cells were purified from the mesenteric lymph node in naive SPF and GF mice and 5 × 10⁵ CD4⁺ CD25⁺ cells were cultured with plate-bound 1 μg/ml anti-CD3 antibody in 96-well U-bottomed plates. The culture supernatants were collected at 48 h for interleukin-10 and interferon-γ, at 72 h for transforming growth factor-β. The amounts of cytokines in the cultures were measured by enzyme-linked immunosorbent assay. The mean and standard deviation are shown. Similar results were obtained in three independent studies. IL, interleukin; IFN, interferon; TGF, transforming growth factor.

Fig. 6

Neutralization of transforming growth factor (TGF)-β reversed suppressive function of specific pathogen-free (SPF) CD25⁺ CD4⁺ cells. A total of 250 000 CD25⁺ CD4⁺ cells obtained from the mesenteric lymph node (MLN) of naive SPF and germ-free mice were co-cultured with 2 × 10⁵ responder cells purified from the MLN of naive SPF mice and antigen presenting cells and then stimulated with 1 μg/ml anti-CD3 antibody. To neutralize interleukin (IL)-10 and/or TGF-β, 100 μg/ml anti-IL-10 antibody and/or anti-TGF-β antibody was added to the cultures. The co-cultured cells were incubated for 48 h and 1 μCi [³H]-thymidine was added to the culture 18 h before harvest. **P < 0·01.