Vitamin D improves sunburns by increasing autophagy in M2 macrophages

Abstract

Cutaneous inflammation from UV radiation exposure causes epidermal damage, cellular infiltration, and secretion of pro-inflammatory mediators that exacerbate tissue destruction. Recovery is mediated chiefly by anti-inflammatory M2 macrophages that suppress inflammation and augment epidermal regeneration. Vitamin D enables anti-inflammation to promote tissue repair in response to injury. Since vitamin D enhances cellular macroautophagy/autophagy, we investigated the role of autophagy in vitamin D protection of UV-mediated sunburn and inflammation. Using a UV-mediated acute skin injury mouse model, we demonstrate that a single dose of vitamin D resolves injury with sustained inhibition of inflammatory cytokines associated with enhanced autophagy in myeloid anti-inflammatory M2 macs. Increased MAP1LC3B/LC3 expression corroborated with complete autolysosome formation detected by electron microscopy and correlated with degradation of SQSTM1/p62 in the skin following vitamin D treatment. Specifically, pharmacological inhibition of autophagy increased UV-induced apoptosis, suppressed M2 macs recruitment, and prevented vitamin D downregulation of Tnf and Mmp9 in the skin. Furthermore, selective deletion of autophagy in myeloid cells of atg7 cKO mice abrogated vitamin D-mediated protection and recapitulated UV-induced inflammation. Mechanistically, vitamin D signaling activated M2-autophagy regulators Klf4, Pparg, and Arg1. Lastly, analysis of UV-exposed human skin biopsies detected a similar increase in macrophage autophagy following vitamin D intervention, identifying an essential role for autophagy in vitamin D-mediated protection of skin from UV damage. Abbreviations: ARG1: arginase 1; ATG7 cKO: autophagy related 7 conditional knockout; HPF: high powered field; KLF4: Kruppel like factor 4; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; macs: macrophage; 3-MA: 3-methyladenine; MMP9: matrix metallopeptidase 9; NOS2: nitric oxide synthase 2, inducible; PPARG: peroxisome proliferator activated receptor gamma; SQSTM1/p62: sequestosome 1; TNF: tumor necrosis factor; UV: ultraviolet; VD: vitamin D, 25-hydroxy vitamin D₃; 1,25-VD: 1, 25-dihydroxy vitamin D₃.

Keywords: Autophagy; UV; inflammation; macrophage; vitamin D.

Figure 1.

Vitamin D protects from UV-mediated skin inflammation. C57BL/6 mice were exposed to 100 mJ/cm² UV radiation 48 h following shaving and hair depilation from their dorsal side. 1 h following UV, mice were treated with vitamin D (VD), administered i.p. At indicated time points skin was harvested for histology. (a-f) Wound and parallel histopathology images of UV exposed skin (a-c) and following treatment with VD (d-f) at days 2, 3, and 5 post irradiation. Skin was excised post mortem, sectioned and stained with hematoxylin and eosin for histopathological evaluation. Scale bar: 100 µm. (g) Quantification of the area of redness at the site of UV exposure, p = 0.04 at day 4 post UV using ImageJ software, (n = 4 for UV and n = 5 for UV+VD). (h and i) Evaluation of inflammatory markers Tnf, Nos2 and Mmp9 by qPCR using RNA isolated from skin at 48 h and 72 h following UV exposure (n = 6 for all groups at 48 h, n = 3 for all groups at 72 h, p˂0.005 using a paired t-test).

Figure 2.

Vitamin D enhances macrophage-specific autophagy and diminishes inflammation in an autophagy dependent manner. Skin sections were subject to (a-f) confocal microscopy at 72 h and (g-i) real time PCR at 48 h post UV. (a-f) Representative images of skin tissue sections stained for immunofluorescent detection of ADGRE1 (green), LC3 (red), (yellow, indicative of co-localization, is marked with arrows), and DAPI (blue). Tissue gene expression of anti-inflammatory mediator (g) Pparg, and pro-inflammatory mediators (h) Tnf, and (i) Mmp9 were quantified following blockade of autophagy by pharmacological inhibitor 3-MA, (p ≤ 0.03). For Tnf and Mmp9, n = 6 for all groups except UV+3-MA n = 4, for Pparg n = 6 for all groups except control n = 5 and UV+VD+3-MA n = 10. Scale bar: 20 µm.

Figure 3.

Vitamin D augments autophagic flux in macrophages of UV exposed mouse skin. Skin was harvested 48 h post UV and (a) tissue sections homogenized to prepare protein lysates for detection of LC3-I, LC3-II and SQSTM1 by immunoblot analysis, and (b) quantified by densitometry, (c-e) digested to obtain cells for ex vivo staining of control, UV and UV+VD, for detection of ADGRE1 (green), LC3 (red), and DAPI (blue). Scale bar: 10 µm. Number on the images represent percent positivity of LC3⁺ puncta quantified using the scoring application module of MetaMorph. (f) Graphical representation of quantitated puncta positivity (n = 8 for control and UV, n = 10 for UV+VD). (g-j) Transmission electron microscopy images of skin tissue that were fixed and sectioned for detection of macrophage autophagic activity (indicated by arrows, inset) in (g) control, (h) UV, (i) UV+3-MA and (j) UV+VD treated mice. Scale bar: 1 µm.

Figure 4.

Vitamin D repopulates M2 macrophages in the inflamed skin in an autophagy-dependent manner. Cells were harvested from control and irradiated skin, 48 h post exposure, in presence or absence of VD and/or 3-MA for detection of PTPRC (myeloid), ADGRE1 (macrophage), LY6C (M1), and MRC1 (M2). Representative dot plots indicate distribution of PTPRC⁺ ADGRE1⁺ cells into 2 separate populations – LY6C⁺ MRC1⁻ (M1) and LY6C⁻ MRC1⁺ (M2) in (a) control, (b) UV, (c) UV+VD, (d) UV+3-MA, (e) UV+VD+3-MA and graphical representation of (f) ratio of M2 to M1 macrophages. (g–i) Quantification of skin infiltrating myeloid macrophages to include (g) total percent myeloid PTPRC⁺ ADGRE1⁺ macrophages, (h) percent PTPRC⁺ LY6C⁺ or M1 macrophages and (i) percent PTPRC⁺ ADGRE1⁺ MRC1⁺ or M2 macrophages, (n = 26 for control, n = 37 for UV, n = 45 for UV+VD, n = 22 for UV+3-MA, and n = 23 for UV+VD+3-MA), (p ≤ 0.04).

Figure 5.

LC3 expression is selectively upregulated in MRC1⁺ M2 macrophages after treatment with vitamin D. (a–e) Cells were harvested from UV-irradiated skin of all treatment groups. Cells were stained for flow cytometric analysis of PTPRC (myeloid), ADGRE1 (macrophage), LY6C (M1), MRC1 (M2), and LC3 (autophagy). Histograms showing LC3 counts and MFI within M1 and M2 macrophage populations in (a) control, (b) UV, (c) UV+VD, (d) UV+3-MA, and (e) UV+VD+3-MA. Skin tissue sections were subject to immunofluorescent staining to detect LC3 expression in MRC1⁺ M2 macrophages. Representative immunofluorescence images of mouse skin tissue to detect co-localization of MRC1 (green), LC3 (red), and DAPI (blue) in (f) control, (g) UV, (h) UV+VD, (i) UV+3-MA, and (j) UV+VD+3-MA. Arrows point to colocalization. (k) Quantitative representation of MRC1⁺ LC3⁺ cells, p ≤ 0.05, (n = 3 for all groups). Scale bar: 20 µm.

Figure 6.

Vitamin D protects mice from UV induced apoptosis in an autophagy dependent manner. Skin tissue was harvested from mice 72 h following UV exposure and subject to TUNEL staining to evaluate cellular apoptosis. Representative images of TUNEL+ cells in (a) control, (b) UV and (c) UV+VD mice compared with (d) 3-MA, (e) UV+3-MA, and (f) UV+VD+3-MA mice treated with 3-MA. (g) Quantitation of TUNEL+ cells within affected skin tissue, p ≤ 0.04, (n = 4 for control, UV+3-MA, and UV+VD+3-MA, n = 8 for UV and UV+VD, and n = 6 for 3-MA). Scale bar: 100 µm.

Figure 7.

Ablation of autophagy in myeloid cells depletes M2 macrophages and reduces M2:M1 ratio. Skin cells from myeloid autophagy-deficient (atg7 cKO) mice were harvested for flow cytometric analysis of PTPRC (myeloid), ADGRE1 (macrophage), LY6C (M1), and MRC1 (M2). Representative plots illustrate the distribution of PTPRC⁺ ADGRE1+ cells into LY6C⁺ MRC1⁻ (M1) and LY6C⁻ MRC1⁺ (M2) macrophage populations in atg7 cKO and littermates in groups (a) control, (b) UV, and (c) UV+VD. Arrows indicate absence of MRC1⁺ M2 macs in VD-treated atg7 cKO compared to M2 macs restored in VD-treated littermate mice. (d) Quantitation of M2 to M1 ratio in atg7 cKO and littermates. For atg7 cKO n = 14 for control, n = 11 for UV, n = 12 for UV+VD, littermates n = 13 for control and UV, and n = 16 for UV+VD. Number on each plot is its M2:M1 ratio ± sem, p˂0.03. (e) Quantitation of percent M1 and M2 macs comparing atg7 cKO and littermate mice treated with UV+VD, (p = 0.04).

Figure 8.

Vitamin D-enhanced autophagy promotes macrophage polarization via activation of KLF4-PPARG pathway. Bone marrow derived macrophages were treated with 1,25 dihydroxy-vitamin D₃ (VD)+IL4 or VD+LPS+IFNG for 3 to 24 h as indicated to assess for molecular markers that map the autophagy pathway and M2 macrophage polarization. Gene expression was assessed for (a) Klf4 at 3, 6, 9 and 18 h and (b–e) for 24 h for (b) Vdr, (c) Pparg and (d) Arg1 and (e) Cd86 (n = 4), p˂0.05.

Figure 9.

Vitamin D (D3) protection from UV-mediated acute inflammation is associated with enhanced macrophage specific autophagy in human skin. Healthy subjects were treated with a single dose of 200,000 IU D3 following exposure to experimentally induced sunburn. Immunofluorescence microscopy detects for the presence of macrophage marker CD163 (green), autophagy marker LC3 (red), and DAPI (blue) from skin biopsies obtained before (a, b) and after (c, d) D3 treatment. Arrows indicate co-localization (yellow). The dotted box in a and c indicate the areas that have been magnified in b and d. Scale bar: 100 µm for 20X magnification and 20 µm for 63X.