1,25-dihydroxyvitamin D3 enhances the ability of transferred CD4+ CD25+ cells to modulate T helper type 2-driven asthmatic responses

Abstract

The severity of allergic diseases may be modified by vitamin D. However, the immune pathways modulated by the active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], are yet to be fully elucidated. In this study, naturally occurring CD4(+) CD25(+) cells from the skin-draining lymph nodes (SDLN) of mice treated with topical 1,25(OH)(2)D(3) had an increased ability to suppress T helper type 2 (Th2) -skewed immune responses. CD4(+) CD25(+) cells transferred from mice treated with topical 1,25(OH)(2)D(3) into ovalbumin (OVA) -sensitized mice challenged intranasally with OVA 18 hr later, significantly suppressed the capacity of airway-draining lymph node (ADLN) cells to proliferate and secrete cytokines in response to further OVA stimulation ex vivo. The CD4(+) CD25(+) cells from 1,25(OH)(2)D(3)-treated mice also reduced airway hyperresponsiveness and the proportions of neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF). To test the effect of 1,25(OH)(2)D(3) on cells able to respond to a specific antigen, CD4(+) CD25(+) cells were purified from the SDLN of OVA-T-cell receptor (TCR) transgenic mice treated 4 days earlier with topical 1,25(OH)(2)D(3). CD4(+) CD25(+) cells from OVA-TCR mice treated with 1,25(OH)(2)D(3) were able to alter BALF cell content and suppress ADLN responses to a similar degree to those cells from non-transgenic mice, suggesting that the effect of 1,25(OH)(2)D(3) was not related to TCR signalling. In summary, topical 1,25(OH)(2)D(3) increased the regulatory capacity of CD4(+) CD25(+) cells from the SDLN to suppress Th2-mediated allergic airway disease. This work highlights how local 1,25(OH)(2)D(3) production by lung epithelial cells may modulate the suppressive activity of local regulatory T cells.

Figure 2

CD4⁺ CD25⁺ cells from the skin-draining lymph nodes (SDLN) of mice topically treated with vehicle or 1,25(OH)₂D₃ have similar phenotypes. CD4⁺ CD25⁺ cells were purified from the SDLN of BALB/c mice treated topically 96 hr earlier with vehicle or 125 ng 1,25(OH)₂D₃ (VitD). The proportion of live CD4⁺ CD25⁺ cells in the SDLN (left panels) and of isolated CD4⁺ CD25⁺ cells (right panels) are shown. The proportions of Foxp3⁺ cells of the live CD4⁺ CD25⁺cells from the SDLN are shown in the middle panel with an isotype control also shown (shaded histogram). Results are representative of cells pooled from the SDLN of BALB/c mice from four individual experiments.

Figure 1

Experimental timeline for transfer of CD4⁺ CD25⁺ cells into ovalbumin (OVA) -sensitized mice. CD4⁺ CD25⁺ cells were purified from the skin-draining lymph nodes (SDLN) of ‘donor’ BALB/c or DO11.10 mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells (2·5 × 10⁵) or 0·9% saline (200 μl) were adoptively transferred into ‘recipient’ BALB/c mice intraperitoneally sensitized and boosted earlier with 20 μg OVA and 4 mg Alum adjuvant. These recipient mice were challenged with a 1% OVA in saline aerosol for 30 min, 18 hr after the adoptive transfer. Twenty-four hours later, various asthma outcomes were assessed in the recipient mice.

Figure 3

CD4⁺ CD25⁺ cells from the skin-draining lymph nodes (SDLN) of mice topically treated with 1,25(OH)₂D₃ regulate ex vivo proliferation and cytokine production by airway-draining lymph nodes (ADLN) cells. CD4⁺ CD25⁺ cells were purified from the SDLN of BALB/c mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells (2·5 × 10⁵) or 0·9% saline (200 μl) were adoptively transferred into BALB/c mice sensitized and boosted earlier with ovalbumin (OVA). These recipient mice were challenged with an OVA aerosol 18 hr after the adoptive transfer. After a further 24 hr, ADLN cells were pooled from mice within treatment groups and cultured with 10 μg/ml OVA for 96 hr. [³H]thymidine incorporation by ADLN cells is shown for the last 24 hr of a 96-hr culture with interleukin-5 (IL-5), interferon-γ (IFN-γ) or IL-4 levels in supernatants depicted, which were collected after a 96-hr culture. Results are pooled from three independent experiments (n=4 mice/treatment/experiment) and are shown as mean + SEM (*P < 0·05).

Figure 4

CD4⁺ CD25⁺ cells from the skin-draining lymph nodes (SDLN) of mice topically treated with 1,25(OH)₂D₃ regulate airway hyperresponsiveness. CD4⁺ CD25⁺ cells were purified from the SDLN of BALB/c mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells (2·5 × 10⁵) or 0·9% saline (200 μl) were adoptively transferred into BALB/c mice sensitized and boosted earlier with ovalbumin (OVA). These recipient mice were challenged with an OVA aerosol 18 hr after the adoptive transfer. After a further 24 hr, airway resistance to increasing concentrations of the bronchoconstrictor methacholine was determined. Responses were also measured in age-matched naive BALB/c mice. Baseline = values obtained by measurement of Zrs five times at 1-min intervals after stabilization of the mouse on the ventilator; saline = values obtained by measurement of Zrs five times at 1-min intervals after a 90-second saline aerosol; % saline = % increase of saline values for increasing doses of methacholine. Results are shown as mean + SEM (n=8 mice/treatment, *P = 0·03) relative to baseline levels (100%).

Figure 5

CD4⁺ CD25⁺ cells from the skin-draining lymph nodes (SDLN) of mice topically treated with 1,25(OH)₂D₃ regulate cells in the bronchoalveolar lavage fluid (BALF). CD4⁺ CD25⁺ cells were purified from the SDLN of BALB/c mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells (2·5 × 10⁵) were adoptively transferred into BALB/c mice sensitized and boosted earlier with OVA. These recipient mice were challenged with an OVA aerosol 18 hr after the adoptive transfer. After a further 24 hr, the proportions of lymphocytes, neutrophils, eosinophils and macrophages and the total number of cells were determined in BALF. Results are shown as mean + SEM (n=8 mice/treatment, *P < 0·05).

Figure 6

Topical 1,25(OH)₂D₃ does not alter migration of CD4⁺ CD25⁺ cells to the airway-draining lymph nodes (ADLN). CD4⁺ CD25⁺ cells were purified from the skin-draining lymph nodes (SDLN) of BALB/c mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) and then 1 × 10⁵ intravenously transferred into naive BALB/c mice, which were sensitized 18 hr later with ovalbumin (OVA). Three days after sensitization, (a) the proportions of transferred (CFSE⁺ Foxp3⁺ CD4⁺) cells in the ADLN and SDLN were determined by first gating CFSE⁺ SSC^lo cells and then cells that expressed both Foxp3 and CD4. (b) The percentage of CFSE⁺ Foxp3⁺ CD4⁺ cells detected in the ADLN and SDLN and, (c) the proportion of total cells transferred cells. Results for (b) and (c) are depicted as mean + SEM (n=3 mice/treatment, *P < 0·05).

Figure 7

CD4⁺ CD25⁺ cells from the skin-draining lymph nodes (SDLN) of ovalbumin–T-cell receptor (OVA-TCR) transgenic mice topically treated with 1,25(OH)₂D₃ regulate various asthma parameters. CD4⁺ CD25⁺ cells were purified from the SDLN of OVA-TCR transgenic (DO11.10) mice treated topically 4 days earlier with vehicle or 125 ng 1,25(OH)₂D₃. Cells (2·5 × 10⁵) or 0·9% saline (200 μl) were adoptively transferred into BALB/c mice sensitized and boosted earlier with OVA. These recipient mice were challenged with an OVA aerosol 18 hr after the adoptive transfer. After a further 24 hr, (a) airway-draining lymph node (ADLN) cells were pooled within treatments and cultured with and without (−) 10 μg/ml OVA for 96 hr. [³H]thymidine was added to cultures for the last 24 hr of a 96 hr culture. Interleukin-5 (IL-5), interferon-γ (IFN-γ) or IL-4 concentrations in supernatants were determined after 96 hr of culture. At the same time, (b) the proportions of lymphocytes, neutrophils, eosinophils and macrophages and (c) concentrations of IL-4 and IL-5 were measured in bronchoalveolar lavage fluid (BALF). Results are pooled from two independent experiments (n=5 mice/treatment/experiment) and are shown as mean + SEM (*P < 0·05).