Vitamin D3 inhibits p38 MAPK and senescence-associated inflammatory mediator secretion by senescent fibroblasts that impacts immune responses during ageing

Abstract

Vitamin D₃ replacement in older insufficient adults significantly improves their antigen-specific varicella zoster virus (VZV) cutaneous immunity. However, the mechanisms involved in this enhancement of cutaneous immunity are not known. Here, we show for the first time that vitamin D₃ blocks the senescence-associated secretory phenotype (SASP) production by senescent fibroblasts by partially inhibiting the p38 MAPK pathway. Furthermore, transcriptomic analysis of skin biopsies from older subjects after vitamin D₃ supplementation shows that vitamin D₃ inhibits the same inflammatory pathways in response to saline as the specific p38 inhibitor, losmapimod, which also enhances immunity in the skin of older subjects. Vitamin D₃ supplementation therefore may enhance immunity during ageing in part by blocking p38 MAPK signalling and in turn inhibit SASP production from senescent cells in vivo.

Keywords: SASP; ageing; p38 MAPK; senescence; skin; vitamin D.

FIGURE 1

Induction of senescent fibroblasts in vitro. Human dermal fibroblasts were isolated from skin biopsies, and senescence was induced using 10 Gy x‐ray ionising radiation. (a) Senescence was evaluated using an SA‐β‐galactosidase assay. (b) Expression of p21 and γ‐H2Ax was evaluated using immunofluorescence (Mean fluorescence intensity shown, representative of three independent experiments) and (c) p16 using western blot (n = 3). (d) CCL2, IL‐6 and IL‐8 were quantified in culture supernatants using cytometric bead array (n = 6). Data are represented as mean ± SEM and compared using either a paired t test or Wilcoxon matched‐pairs test. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001.

FIGURE 2

1,25(OH)₂D₃ inhibits senescence‐associated mediators in fibroblasts and blocks p38 MAPK. Non‐senescent and senescent fibroblasts were treated with 10 nM 1,25(OH)₂D₃ for 7 days. (a) Quantification of CCL2, IL‐6 and IL‐8 was performed using cytometric bead array (n = 7). Cell numbers of both non‐senescent and senescent cells were counted after 7 days and the cell number and concentrations in the supernatants of senescent cells were adjusted proportionally to the cell number of their non‐senescent controls. (b) Representative blot of phospho‐p38 MAPK (p‐p38), total p38 MAPK and GAPDH with (c) the cumulative data (n = 9) using western blot. Data are represented as mean ± SEM and compared using a one‐way ANOVA with Dunn's multiple comparisons test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

FIGURE 3

Vitamin D mimics the mechanism of action of losmapimod, a specific p38 inhibitor in vivo. (a) Schematic representation of biopsies collection for RNA‐Seq analysis. Biopsies collected 6 hrs post‐saline injection, before and after anti‐inflammatory drug supplementation. All data were represented as delta change of saline‐injected skin as compared to normal skin. (b) GSEA was performed on saline‐injected versus normal skin biopsies to identify pathways induced by saline injection. Circles represent activated hallmark gene sets (NES > 0) in comparison to normal skin, with the radius proportional to NES. Circle colors represent adjusted p‐values for each term. (c) Analysis of previously curated pathways from WikiPathways using ‘Inflammatory response’ (WP453), ‘p38 MAPK signaling’ (WP400) and ‘Vitamin D in inflammatory diseases’ (WP4482) pathways, performed on saline‐injected versus normal skin biopsies before and after vitamin D₃ supplementation. (d) Saline‐specific module and monocyte‐specific module in saline‐injected skin pre and post either vitamin D₃ supplementation (grey) or losmapimod treatment (green). Data are compared using a paired t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.