Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair

Abstract

The immunostimulatory cytokine IL-12 is able to antagonize immunosuppression induced by solar/ultraviolet (UV) radiation via yet unknown mechanisms. IL-12 was recently found to induce deoxyribonucleic acid (DNA) repair. UV-induced DNA damage is an important molecular trigger for UV-mediated immunosuppression. Thus, we initiated studies into immune restoration by IL-12 to discern whether its effects are linked to DNA repair. IL-12 prevented both UV-induced suppression of the induction of contact hypersensitivity and the depletion of Langerhans cells, the primary APC of the skin, in wild-type but not in DNA repair-deficient mice. IL-12 did not prevent the development of UV-induced regulatory T cells in DNA repair-deficient mice. In contrast, IL-12 was able to break established UV-induced tolerance and inhibited the activity of regulatory T cells independent of DNA repair. These data identify a new mechanism by which IL-12 can restore immune responses and also demonstrate a link between DNA repair and the prevention of UV-induced immunosuppression by IL-12.

Figure 1.

IL-12 prevents UV-induced suppression of the induction of CHS in WT but not in Xpa^−/− mice. C57BL/6 (A) or Xpa ^−/− mice (B) were treated daily with UV (1,000 and 400 J/m², respectively) on 4 d on the back and sensitized 24 h after the last exposure through UV-exposed skin (groups 3 and 4). 5 d later, mice were challenged on the left ear and ear swelling was measured 24 h later. Group 4 received 1,000 ng of IL-12 3 h before sensitization. Positive control mice were sensitized and challenged (group 1), negative control animals were only challenged (group 2). Ear swelling is expressed as the difference (cm × 10⁻³, mean ± SD) between the thickness of the challenged and that of the vehicle-treated ear. *P < 0.00001 UV vs. positive control; **P < 0.005 UV vs. UV+IL-12; ***P < 0.00001 UV vs. positive control. n.s., UV vs. UV-IL-12.

Figure 2.

IL-12 breaks established UV-induced tolerance in WT and Xpa^−/− mice. C57BL/6 (A) or Xpa ^−/− mice (B) were treated daily with UV (1,000 and 400 J/m², respectively) on 4 d on the back and sensitized 24 h after the last exposure through UV-exposed skin. 14 d after the first sensitization, mice were resensitized with DNFB applied onto the abdomen (groups 3 and 4). 5 d later, mice were challenged on the right ear and ear swelling was measured 24 h later. Group 4 received 1,000 ng of IL-12 3 h before resensitization. Positive control mice were sensitized and challenged (group 1), negative control animals were only challenged (group 2). *P < 0.05 UV vs. positive control; **P < 0.05 UV vs. UV+IL-12; ***P < 0.0005 UV vs. positive control; ****P < 0.0005 UV vs. UV+IL-12.

Figure 3.

IL-12 inhibits transfer of suppression by UV-induced T reg cells. Naive C57BL/6 (A) or Xpa ^−/− mice (B) were injected i.v. with LN cells obtained from syngeneic donors which were tolerized against DNFB by application of DNFB onto UV-exposed skin. 24 h after injection mice were sensitized against DNFB and 5 d later DNFB challenge was performed on the left ear (group 3). Mice in group 5 received 3 h before and 24 h after cell transfer 1,000 ng IL-12 i.p. Mice in group 4 received cells obtained from UV-exposed donors, which were treated with IL-12 before sensitization through UV-exposed skin, and were sensitized 24 h after cell transfer. *P < 0.001 UV vs. positive control; **P < 0.00001 UV vs. (UV+IL-12); ***P < 0.0001 UV vs. (UV)+IL-12; ****P < 0.0005 UV vs. positive control; *****P < 0.00005 UV vs. (UV)+IL-12; n.s., UV vs. (UV+IL-12).

Figure 4.

IL-12 prevents UV-induced emigration of LC in WT but not in Xpa^−/− mice. Ears of C57BL/6 and Xpa ^−/− mice were exposed to 1,000 J/m² and to 400 J/m², respectively. 24 h later 0.5% DNFB was applied (UV+Sensi.). One group of animals received 1,000 ng IL-12 i.p. 3 h before application of DNFB (UV+Sensi.+IL-12). An additional group was only sensitized (Sensi.). Unirradiated animals served as negative controls (Control). 48 h after hapten application, ears were cut and sheet preparations performed. Sheets were stained with an anti–I-A/I-E Ab, followed by an anti–rat IgG coupled with Texas red and subjected to fluorescence microscopy.

Figure 5.

IL-12 reduces the number of CPD-positive LC in LN draining UV-exposed skin in WT but not in Xpa^−/− mice. C57BL/6 or Xpa ^−/− mice were treated daily with UV on 4 d on the shaved back and sensitized 24 h after the last exposure through UV-exposed skin (UV). One group received 1,000 ng of IL-12 3 h before sensitization (UV+IL-12). As controls LN of untreated and DNFB-sensitized (Sensi.) mice were used. 48 h after sensitization CD11c-positive cells were obtained from draining LN by magnetobead separation. Cells were double stained for Langerin and CPDs and subjected to FACS analysis.