Silymarin inhibits ultraviolet radiation-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells

Abstract

Silymarin inhibits UVB-induced immunosuppression in mouse skin. To identify the molecular mechanisms underlying this effect, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in XPA-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8(+) or CD4(+) cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8(+) T cells, and reduced secretion of Th2 cytokines by CD4(+) T cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4(+) regulatory T-cell activity, and stimulation of CD8(+) effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1

Effect of silymarin on UVB-induced suppression of the CHS response and DNA damage in C3H/HeN mice. (A), Topical treatment of mice with silymarin improves the ability of DCs to induce the CHS response. Donor mice (C3H/HeN) treated with or without silymarin were UVB-irradiated and sensitized with DNFB 24 h after the last UVB exposure. Mice were sacrificed 24 h after sensitization, single-cell suspensions of the lymph nodes were prepared, and CD11c+ cells were positively selected using MACS system, as detailed in the Materials and methods. Naïve recipient mice were injected subcutaneously with the CD11c+ cells (5× 105) obtained from donor mice. Recipient mice were ear challenged with DNFB 5 days after injection of cells, and the ear thickness was measured before and 24 h after challenge. The change in ear thickness is reported as the mean of millimeters (× 10-2) ± SD, n=5 per group. *Significantly greater CHS response versus recipient of CD11c+ from UVB plus DNFB treated mice, P<0.001; ¶Significantly lower CHS response versus the positive control (DNFB-sensitized) group, P<0.001. (B), Analysis of CPDs by dot-blot assay. Silymarin repairs UVB-induced DNA damage in vitro in BM-DCs obtained from C3H/HeN mice. BM-DCs were exposed to UVB radiation (5 mJ/cm2) with or without pretreatment with silymarin, harvested either immediately or 24 hours later. Genomic DNA from various treatment groups was isolated and subjected to dot-blot analysis using an antibody specific to CPDs. SLM= silymarin. (C), Treatment of mice with silymarin enhanced the repair of UV-induced DNA damage in epidermal DCs (langerin-positive cells). Langerin-positive cells are shown by red fluorescence and CPD-positive cells are shown by green fluorescence. Arrows indicate langerin-positive or double-positive cells (langerin + CPDs). Representative photomicrographs are shown, n=3/group. Magnification, ×400.

Figure 2

Effect of silymarin on UVB-induced suppression of CHS response and DNA damage in XPA-deficient mice. (A), Topical treatment of XPA-deficient mice with silymarin does not improve the ability of DCs to induce CHS in naïve mice. Donor mice (XPA-deficient) were treated with or without silymarin and CD11c+ cells from lymph nodes were positively selected using MACS system as described in Materials and methods. Recipient mice (C3H/HeN) were injected subcutaneously with 5× 105 CD11c+ cells obtained from donor mice (XPA-deficient). Recipient mice were ear challenged with DNFB 5 days after injection of cells, and ear skin thickness was measured before and 24 h after challenge. The change in ear thickness is reported as the mean of millimeters (× 10-2) ± SD, n=5 per group. *No significantly greater CHS response in silymarin treated mice versus recipient of CD11c+ from UVB plus DNFB treated mice. ¶Significantly lower CHS response versus the positive control (DNFB-sensitized) group, P<0.001. (B), Analysis of CPDs by dot-blot assay. Silymarin does not stimulate repair of UVB-induced DNA damage in BM-DCs obtained from XPA-deficient mice. Results are shown from a single experiment that is representative of two independent assays.

Figure 3

Topical treatment of mice with silymarin improves the functionality of DCs from UVB-irradiated wild-type mice and enhances the proliferation of CD4+ T cells, but not in XPAKO mice. CD4+ T cells isolated from the spleens of naïve mice (wild-type) were labeled with CFSE and co-cultured with CD11c+ cells (DCs) isolated from lymph nodes of the different treatment groups of wild-type and XPA-KO mice in the presence of anti-CD3e (5.0 μg/ml). After 4 days of co-culture, cells were harvested and analyzed for their proliferation index using FACS. Representative histograms from one experiment are shown from a total of two independent experiments. Numerical values in different treatment groups indicate percentage of proliferating CD4+ T cells.

Figure 4

Silymarin prevents transferable UVB-induced suppression of CHS through modulation of activity or function of CD8+ T cells. (A), Donor mice (C3H/HeN) that were topically treated with and without silymarin were UVB-irradiated, DNFB sensitized, and sacrificed 5 days later. CD8+ T cells were positively selected from the single-cell suspensions prepared from spleens and lymph nodes cells, as detailed in Materials and methods. The CD8+ T cells (8 × 106) were injected i.v. into naïve mice. The recipient mice were ear challenged immediately and ear swelling response was measured before and 24 h after challenge. (B), Treatment of mice with silymarin enhances the production of IFN! and IL-2 by CD8+ T cells. The treatment groups were as described in Figure. CD8+ T cells were isolated from silymarin treated or untreated UVB-irradiated mice, then CD8+ T cells were co-cultured with DNBS-labeled BM-DC for 48 h. The concentration of cytokines in the cell culture supernatants were estimated by cytokine-specific ELISA kits and are presented as mean ± SD in terms of pg or ng per 2 million cells, n=5/group. *Significant increase versus UVB+DNFB group, P<0.001.

Figure 5

Silymarin prevents transferable UVB-induced suppression of CHS through modulation of activity or function of CD4+ suppressor T cells. (A), The donor mice were treated as described in Figure 4, Panel A. CD4+ T cells were positively selected from the spleens and lymph nodes using the MACS system. CD4+ T cells (8×106) were injected i.v. into naïve recipient mice. The recipient mice were sensitized with DNFB and ear was challenged 5 days after sensitization. The change in ear thickness is reported as the mean of millimeters (mm × 10-2) ±SD, n=5 per group. Those naïve mice that received CD4+ T cells from UVB-exposed donor mice that were treated with silymarin showed a greater CHS response than UVB-exposed mice that were not silymarin treated. *Significantly greater CHS response versus recipient of T cells from UVB plus DNFB treated mice (4th bar), P<0.001; ¶Significantly lower CHS response versus the positive control group (2nd and 3rd bar), P<0.001. (B), Treatment of mice with silymarin decreases the production of IL-4 and IL-10 but increases the secretion of IFNγ and IL-2 by CD4+ T cells. CD4+ T cells were isolated from the different treatment groups, as described in Materials and methods. The CD4+ T cells were then co-cultured with DNBS-labeled BM-DC for 48 h. The concentrations of cytokines in the cell culture supernatants were estimated by ELISA and are presented as mean ± SD in terms of pg/2 million cells, n=5/group. *Significant increase versus positive control, P<0.001. ¶Significant decrease versus UVB+ DNFB group, P<0.001. **Significant increase versus UVB+ DNFB group, P<0.01.