In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis

Abstract

Background and methods: Cytosin-guanosin dinucleotide (CpG) motifs of bacterial DNA are known to be potent activators of innate immunity. We have shown previously that administration of CpG containing oligodeoxynucleotide (CpG-ODN) to mice before the onset of dextran sodium sulphate induced colitis ameliorated colitis and inhibited induction of proinflammatory cytokines. To investigate the possible involvement of CD4(+) T cells in the prophylactic CpG-ODN effects, we used the SCID transfer model of colitis.

Results: CD4(+)CD62L(+) T cells from CpG-ODN treated donors did not induce significant intestinal inflammation in SCID recipients, in contrast with control cells. Additionally, cotransfer of these cells with CD4(+)CD62L(+) cells from normal mice protected recipient animals from colitis, indicating regulatory activity. Also, CD4(+)CD62L(+) cells from toll-like receptor 9 deficient animals induced a significantly more severe colitis in SCID recipients than cells from wild-type littermate controls, suggesting a similar protective role of "endogenous" bacterial DNA leading to a less "aggressive" phenotype of these cells. There was no detectable difference in regulatory T cell surface markers between aggressive and attenuated cell pools but attenuated cell pools showed reduced proliferation in vitro and in vivo and produced less interferon gamma, interleukin (IL)-5, and IL-6 after anti-CD3 stimulation.

Conclusions: Collectively, our data support the concept that both endogenous bacterial DNA and exogenously supplied CpG motifs of bacterial DNA induce regulatory properties in CD4(+) T cells. Therefore, bacterial DNA derived from the normal gut flora may contribute essentially to the homeostasis between effector and regulatory immune mechanisms in healthy individuals to protect them from chronic intestinal inflammation.

Figure 1

Effects of cytosin-guanosin containing oligodeoxynucleotide (CpG-ODN) treatment of donor animals in the SCID transfer model of colitis on colitis development. Donor animals were treated with either CpG-ODN or GpG-ODN (each 10 µg/day over five days) or left untreated, and CD4⁺CD62L⁺ cells were transferred to SCID recipients. As a negative control, SCID mice were injected with phosphate buffered saline (PBS). (A) Weight change after transfer. (B) Histological score in the different groups was examined at the end of the experiments (eight and 12 weeks after transfer). (C) Representative colonic haematoxylin-eosin sections of non-transferred mice and mice transferred with CD4⁺CD62L⁺ cells from CpG-ODN or GpG-ODN treated (control) or untreated donors (control) are shown (magnification 50-fold). (D) Toll-like receptor 9 (TLR9) deficient or wild-type (Wt) littermate controls were treated with CpG-ODN (10 µg/day over five days), CD4⁺CD62L⁺ cells were transferred to SCID recipients, and the histological score was examined at the end of the experiment. Data presented in (A–C) were derived from 5–8 mice per group and are representative of five independent experiments. Values are mean (SEM). *Significantly different from both groups transferred with CD4⁺CD62L⁺ cells from either GpG-ODN or untreated donor mice. Data presented in (B) (12 week data) and (D) were derived from 5–8 mice per group and are representative of two independent experiments. Values are mean (SEM). *Significantly different.

Figure 2

Effects of cytosin-guanosin containing oligodeoxynucleotide (CpG-ODN) treatment of donor animals in the SCID transfer model of colitis on cytokine production. Donor animals were treated with either CpG-ODN or GpG-ODN (each 10 µg/day daily over five days) or left untreated, and CD4⁺CD62L⁺ cells were transferred to SCID recipients. As a negative control, SCID mice were injected with phosphate buffered saline (PBS). (A) Mesenteric lymph node cells were isolated at the end of the experiment and spontaneous cytokine secretion was measured in overnight cultures by ELISA. IL, interleukin; IFN-γ, interferon γ. (B) mRNA from colonic tissue of individual mice was isolated and expression of different cytokines was quantified by real time polymerase chain reaction, as described in materials and methods. Data presented were derived from five independent experiments with at least 20 mice per group. Values are mean (SEM). *Significantly different from both the group transferred with CD4⁺CD62L⁺ cells from untreated and GpG-ODN treated donor mice if not indicated otherwise.

Figure 3

Transfer of CD4⁺CD62L⁺ cells from toll-like receptor 9 (TLR9) deficient mice versus wild-type mice. CD4⁺CD62L⁺ cells from TLR9 deficient donors or wild-type control littermates were isolated and transferred to SCID recipients. (A) Weight change after transfer. (B) Histological score of the different groups was examined at the end of the experiment (seven weeks after transfer). (C) Mesenteric lymph node cells were isolated at the end of the experiment and cytokine secretion was measured after 24 hours in supernatants by ELISA. IL, interleukin; IFN-γ, interferon γ. *Significantly different from the group transferred with CD4⁺CD62L⁺ cells from wild-type littermates. Data presented were derived from five mice per group and are representative of two independent experiments.

Figure 4

Cotransfer of CD4⁺CD62L⁺ cells from cytosin-guanosin containing oligodeoxynucleotide (CpG-ODN) treated and untreated donor animals in SCID mice. Donor animals were treated with CpG-ODN (10 µg/day over five days) or left untreated and either CD4⁺CD62L⁺ cells from untreated or CpG-DNA treated donors or both were transferred to SCID recipients. As a negative control, SCID mice were injected with phosphate buffered saline (PBS). Histological score (A) and interferon γ (IFN-γ) secretion of isolated mesenteric lymph node cells (B) at the end of the experiment (eight weeks after transfer) are shown. *Significantly different from the group transferred with CD4⁺CD62L⁺ cells from untreated donor mice. Data presented were derived from three independent experiments, with 10–15 mice per group. ND, not detected.

Figure 5

Cotransfer of CD4⁺CD62L⁺ cells from cytosin-guanosin containing oligodeoxynucleotide (CpG-ODN) treated and untreated donor animals in SCID mice. Donor animals were treated with CpG-ODN (10 µg/day over five days) or left untreated and either CD4⁺CD62L⁺ cells from untreated or CpG-DNA treated donors or both were transferred to SCID recipients. In vivo proliferation of cells in mesenteric lymph nodes was detected by BrdU incorporation.

Figure 6

Cytokine secretion and proliferation of anti-CD3 stimulated CD4⁺CD62L⁺ cells isolated from cytosin-guanosin containing oligodeoxynucleotide (CpG-ODN) treated donor mice. Mice were treated with either CpG-ODN or GpG-ODN (each 10 µg/day over five days) or left untreated, and CD4⁺CD62L⁺ cells were isolated and incubated in anti-CD3 coated wells, as described in materials and methods, over 24 hours for cytokine detection (A) or 72 hours for ³H-thymidine incorporation (B). PBS, phosphate buffered saline; IL, interleukin; IFN-γ, interferon γ; TGF-β, transforming growth factor β. *Significantly different from both GpG-ODN or untreated mice. Incubations were performed in quadruplicate and results are representative of three independent experiments.

Figure 7

Cytokine secretion and proliferation of anti-CD3 stimulated CD4⁺CD62L⁺ cells isolated from toll-like receptor 9 (TLR9) deficient mice. CD4⁺CD62L⁺ cells were isolated from TLR9 deficient mice or wild-type littermate controls and incubated in anti-CD3 coated wells, as described in materials and methods, over 24 hours for cytokine detection (A) or 72 hours for ³H-thymidine incorporation (both unstimulated and anti-CD3 stimulated) (B). IL, interleukin; IFN-γ, interferon γ. *Significantly different from littermate controls. Incubations were performed in quadruplicate and results are representative of at least two experiments.