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, 204 (7), 1519-24

CD11b Facilitates the Development of Peripheral Tolerance by Suppressing Th17 Differentiation

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CD11b Facilitates the Development of Peripheral Tolerance by Suppressing Th17 Differentiation

Driss Ehirchiou et al. J Exp Med.

Abstract

Antigen-induced immune suppression, like T cell activation, requires antigen-presenting cells (APCs); however, the role of APCs in mediating these opposing effects is not well understood, especially in vivo. We report that genetic inactivation of CD11b, which is a CD18 subfamily of integrin receptors that is highly expressed on APCs, abolishes orally induced peripheral immune tolerance (oral tolerance) without compromising APC maturation or antigen-specific immune activation. The defective oral tolerance in CD11b(-/-) mice can be restored by adoptive transfer of wild-type APCs. CD11b deficiency leads to enhanced interleukin (IL) 6 production by APCs, which subsequently promotes preferential differentiation of naive T cells to T helper 17 (Th17) cells, which are a T cell lineage characterized by their production of IL-17. Consequently, antigen feeding and immunization of CD11b(-/-) mice results in significant production of IL-17 within the draining lymph nodes that interferes with the establishment of oral tolerance. Together, we conclude that CD11b facilitates oral tolerance by suppressing Th17 immune differentiation.

Figures

Figure 1.
Figure 1.
CD11b is required for induction of oral tolerance. (a) CD11b deficiency prevents orally induced peripheral tolerance as determined by DTH. WT C57BL/6 or CD11b−/− mice were fed daily with PBS or 1 mg OVA for 7 d, and then immunized with OVA/CFA. After 7 d, these mice were challenged with OVA in the left footpad and saline in the right footpad. Footpad thickness was measured after 24 h, and the increase in thickness (swelling) between the left and right footpads is given. *, P = 0.0015, WT versus CD11b−/−. n = 3–6. (b) CD11b deficiency prevents tolerance in OVA-stimulated lymphocyte proliferation. Total LN cells from the OVA-fed and immunized WT and CD11b−/− mice were stimulated with graded concentrations of OVA, and proliferation was measured after 72 h by [3H]thymidine incorporation. P = 0.00003, WT versus CD11b−/−. n = 4. (c) Restoration of oral tolerance in CD11b−/− mice by passive transfer of WT APCs. CD11b−/− mice were injected i.v. with splenocytes or adhesion-enriched APCs from naive WT mice, and then fed with PBS or 1 mg OVA per day for 7 d. Establishment of oral tolerance in recipient CD11b−/− mice is determined by DTH response to OVA. *, P = 0.04, PBS versus OVA. n = 3. Data shown are means ± the SEM and are representative of three to four independent experiments.
Figure 2.
Figure 2.
CD11b deficiency leads to Th17 immune deviation under the condition of oral tolerance induction. (a) Cytokine production in response to antigen restimulation. Singe-cell suspension, obtained from the iLNs of PBS- or OVA-fed and immunized WT (open bar) or CD11b−/− (shaded bar) mice, were restimulated in vitro with 100 μg/ml OVA for 72 h, and the cytokine concentrations in the supernatants were determined by microbead multiplex assay. *, P < 0.001, WT versus CD11b−/−. n = 4. (b) Identification of Th17 cells by intracellular staining. Total CD4+ T cells obtained from PBS- or OVA-fed and immunized mice were stimulated with PMA and ionomycin in the presence of GolgiPlug for 5 h, and then analyzed for intracellular expression of IL-17 and IFNγ by three-color flow cytometry. Plots were gated on CD4+ cells (top, gate R3) and the percentages of cells staining positive for IL-17 or IFNγ were shown. (c) Existence of a Th17 cell population in CD11b−/− mice under conditions of oral tolerance induction. The percentages of Th17 cells, identified by their production of IL-17, but not IFNγ, within CD4+ subpopulation (gate R3), were determined by flow cytometry. Data shown are the means ± the SEM and are representative of three independent experiments. *, P = 0.0001, WT versus CD11b−/−. n = 12.
Figure 3.
Figure 3.
Existence of a mature Th17 population in antigen-fed and immunized CD11b−/− mice. (a) The IL-17–expressing cells represent a mature and stable Th17 population. Total CD4+ T cells, which were purified by MACS from the iLNs of OVA-fed and immunized WT or CD11b−/− mice, were cocultured with their corresponding irradiated APCs in the presence of anti-CD3 mAbs with or without 2 ng/ml TGFβ or 20 ng/ml IL-23 plus anti–IL-4 and anti-IFNγ for 3 d. They were then restimulated with PMA and ionomycin in the presence of GolgiPlug for 5 h to retain the cytokines intracellularly. The restimulated cells were analyzed by intracellular staining with anti–IL-17 and anti-IFNγ, and the percentages of CD17+IFNγ cells within CD4+ population were determined by three-color flow cytometry and shown on the right. *, P < 0.0001, WT versus CD11b−/−. n = 4. (b) Th17 cells from CD11b−/− mice are capable of maintaining a stable Th17 phenotype under different polarizing conditions. Purified CD4+ T cells from OVA-fed and immunized WT or CD11b−/− mice were cocultured with their irradiated APCs in the presence of anti-CD3 under either Th1 (IFNγ plus anti-IL-4), Th2 (IL-4 plus anti-IFNγ), or Th17 (TGFβ plus IL-6) polarizing conditions for 3 d. The percentage of CD17+IFNγ CD4+ T cells was determined by intracellular cytokine staining. Data shown are representative of three mice per group; these experiments were repeated three times, with similar results.
Figure 4.
Figure 4.
IL-17 administration in WT mice interferes with the establishment of oral tolerance. WT mice were injected i.v. with PBS or IL-17 (60 pg/mouse) during OVA feeding, immunized with OVA/CFA, and challenged. Development of immune tolerance was determined by inhibition of footpad swelling in the DTH response, as in Fig. 1 a. *, P = 0.003, PBS versus IL-17. n = 5–6 mice per group. Data shown are means ± the SEM and are representative of two independent experiments.

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