Toll-like receptor-4 deficiency enhances repair of UVR-induced cutaneous DNA damage by nucleotide excision repair mechanism

Abstract

UVB-induced DNA damage has a critical role in the development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4(-/-)) and TLR4-competent (TLR4(+/+)) mice were subjected to 90 mJ cm(-2) UVB radiation locally, DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) was repaired more efficiently in the skin and bone marrow-derived dendritic cells (BMDCs) of TLR4(-/-) mice in comparison to TLR4(+/+) mice. Expression of DNA repair gene XPA (xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDCs of TLR4(+/+) mice than TLR4(-/-) mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDCs from UV-irradiated TLR4(-/-) mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (P<0.05) than BMDCs from TLR4(+/+) mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDCs of TLR4(-/-) mice (before UVB exposure) inhibited repair of CPDs, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4(+/+) BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer.

Figure 1. TLR4 deficiency promotes repair of CPD due to increase in XPA expression in mouse skin

(a) Mice were exposed to acute UVB (90 mJ/cm²) radiation and thereafter sacrificed at 0-48 h. Skin samples were obtained and analyzed for CPD. Frozen sections (5 μm thick) were subjected to immunofluorescent staining to detect CPD+ cells that are green as mentioned in the Methods section. CPD were not detected in non-UV-exposed skin. (b) CPD+ cells were counted as a percentage of all epidermal cells in immunofluorescent stained tissue of TLR4^-/- and TLR4^+/+ mice. There were significantly fewer CPD at 24h and 48h post UV-exposure in the skin of TLR4^-/- mice than in the UV-exposed skin of TLR4^+/+ mice. (c) There was an increase in XPA mRNA expression with increasing time post UV exposure, as determined by quantitative real time PCR (qPCR) in the skin of TLR4^-/- mice than in the UV-exposed skin of TLR4^+/+ mice. (d, e) There was increased XPA protein expression with increase in time post UV exposure as determined by western blot analysis, and this increase was more prominent in the skin of TLR4^-/- mice than in the UV-exposed skin of TLR4^+/+ mice. Experiments were conducted and repeated separately in 5 animals in each group with identical results. Scale bar = 50 μm. The experiments were repeated twice with similar results. (*p<0.05, **p<0.01, and ***p<0.001).

Figure 2. TLR4 deficiency enhances repair of CPD in mouse bone marrow-dendritic cells (BMDC) due to increase in XPA expression

(a) BMDC were prepared as described in the Methods section. BMDC were exposed to acute UVB (3 mJ/cm2) radiation and thereafter harvested at 0-48 h and analyzed for CPD. BMDC were subjected to immunofluorescent staining to detect CPD+ cells that are green as mentioned in the Methods section. CPD were not detected in non-UV-exposed BMDC. (b) CPD+ cells were counted as a percentage of BMDCs from TLR4^-/- and TLR4^+/+ mice. There were significantly fewer CPD at 24h and 48h post UV-exposure in the BMDC of TLR4^-/- mice than in the UV-exposed BMDC of TLR4^+/+ mice. (c) There was increased XPA mRNA expression with increase in time post UV exposure as determined by qPCR, and this increase was more prominent in the BMDC of TLR4^-/- mice than in the UV-exposed BMDC of TLR4^+/+ mice. (d, e) There was increased XPA protein expression with increase in time post UV exposure as determined by western blot analysis, and this increase was more prominent in the BMDC of TLR4^-/- mice than in the UV-exposed skin of TLR4^+/+ mice. Experiments were conducted and repeated separately in triplicates in each group with identical results. Scale bar = 50 μm. The experiments were repeated twice with similar results. (*p<0.05, **p<0.01 and ***p<0.001)

Figure 3. TLR4 deficiency augments IL-12 and IL-23 mRNA expression in mouse BMDC

BMDC were prepared as described in the Methods section. BMDC were exposed to acute UVB (3 mJ/cm²) radiation and thereafter harvested at 0-48 h. BMDC were obtained and analyzed for IL-12p35 and IL-23p19 expression as mentioned in the Methods section. There was an increase in (a) IL-12p35 and (b) IL-23p19 mRNA expression with increase in time post UV exposure as determined by quantitative real time PCR (qPCR), and this increase was more prominent in the BMDC of TLR4^-/- mice than in the UV-exposed BMDC of TLR4^+/+ mice. Similar results were obtained for (c) IL-12p35 and (d) IL-23p19 protein expression by ELISA. Experiments were conducted and repeated separately in triplicates in each group with identical results. Scale bar = 50 μm. The experiments were repeated twice with similar results. (**p<0.01 and ***p<0.001)

Figure 4. Neutralization of IL-12 and IL-23 inhibits the repair of BMDC in TLR4 deficient mice

BMDC were prepared as described in the Methods section. BMDC from TLR4 deficient mice were exposed to acute UVB (3 mJ/cm²) radiation and thereafter harvested at 0-24 h. (a) Neutralizing anti-IL-12p35 antibody was added to BMDC cultures before UV exposure. BMDC were exposed to acute UVB (3 mJ/cm²) radiation and thereafter harvested at 0-24 h and analyzed for CPD. BMDC were subjected to immunofluorescent staining to detect CPD+ cells that are green as mentioned in the Methods section. CPD were not detected in non-UV-exposed BMDC. (b) CPD+ cells were counted as a percentage of BMDCs from TLR4^-/- mice. There were significantly more CPD at 24h post UV-exposure in the BMDC that were treated with neutralizing anti-IL-12p35 antibody than those treated with control IgG. (c) There was a decrease in XPA mRNA expression after neutralization with anti-IL-12p35 antibody as determined by quantitative real time PCR (qPCR) in the BMDC of TLR4^-/- mice. (d) Neutralizing anti-IL-23p19 antibody was added separately to BMDC cultures before UV exposure. BMDC were exposed to acute UVB (3 mJ/cm²) radiation and thereafter harvested at 0-24 h and analyzed for CPD. BMDC were subjected to immunofluorescent staining to detect CPD+ cells that are green as mentioned in the Methods section. CPD were not detected in non-UV-exposed BMDC. (e) CPD+ cells were counted as a percentage of BMDCs from TLR4^-/- mice. There were significantly more CPD at 24h post UV-exposure in the BMDC that were treated with neutralizing anti-IL-23p19 antibody than those treated with control IgG. (f) There was a decrease in XPA mRNA expression after neutralization with anti-IL-23p19 antibody as determined by quantitative real time PCR (qPCR) in the BMDC of TLR4^-/- mice. Experiments were conducted and repeated separately in triplicates in each group with identical results. Scale bar = 50 μm. The experiments were repeated twice with similar results. (*p<0.05, **p<0.01 and ***p<0.001).