Oral Vitamin D Rapidly Attenuates Inflammation from Sunburn: An Interventional Study

Abstract

The diverse immunomodulatory effects of vitamin D are increasingly being recognized. However, the ability of oral vitamin D to modulate acute inflammation in vivo has not been established in humans. In a double-blinded, placebo-controlled interventional trial, 20 healthy adults were randomized to receive either placebo or a high dose of vitamin D₃ (cholecalciferol) one hour after experimental sunburn induced by an erythemogenic dose of UVR. Compared with placebo, participants receiving vitamin D₃ (200,000 international units) demonstrated reduced expression of proinflammatory mediators tumor necrosis factor-α (P = 0.04) and inducible nitric oxide synthase (P = 0.02) in skin biopsy specimens 48 hours after experimental sunburn. A blinded, unsupervised hierarchical clustering of participants based on global gene expression profiles revealed that participants with significantly higher serum vitamin D₃ levels after treatment (P = 0.007) demonstrated increased skin expression of the anti-inflammatory mediator arginase-1 (P = 0.005), and a sustained reduction in skin redness (P = 0.02), correlating with significant expression of genes related to skin barrier repair. In contrast, participants with lower serum vitamin D₃ levels had significant expression of proinflammatory genes. Together the data may have broad implications for the immunotherapeutic properties of vitamin D in skin homeostasis, and implicate arginase-1 upregulation as a previously unreported mechanism by which vitamin D exerts anti-inflammatory effects in humans.

Fig. 1. Study Design and Baseline Characteristics

Panel A depicts the enrollment, allocation, follow-up, and analysis of participants. Panel B depicts a schematic of the parallel phase study design. Skin erythema and thickness were measured 24hr, 48hr, 72hr, and 1 week after experimental sunburn induced by an erythemogenic dose of simulated solar radiation. Participants returned two weeks after conclusion of the control phase of the study for the investigative phase, and were subsequently randomized to receive the study drug as a post-exposure treatment 1hr after experimental sunburn on the contralateral arm. Punch biopsies for tissue TNF-α, iNOS, and microarray analyses were obtained 48hr after experimental sunburn in both phases of the study.

Fig. 2. Primary Outcomes of Randomized Treatment Groups

Panel A shows representative clinical images of irradiation sites of participants in each treatment group in the control and investigative phases of the study. Panel B shows representative hematoxylin and eosin stained histological images obtained from punch biopsies from participants in each treatment group 48hr after irradiation with 3MED. Panel C presents the difference in TNF-α and iNOS mRNA expression obtained from punch biopsies between the investigative and control phases of the study [(RNA_48hr)_invest/(RNA_48hr)_control]. Bars represent the mean, and error bars represent the standard error of the mean for the placebo (n=4), 50,000 IU D₃ (n=5), 100,000 IU D₃ (n=4), and 200,000 IU D₃ (n=5) groups. Two participants were excluded from the placebo analysis given poor RNA sample quality. Panel D presents a heat map depicting global gene expression averages for each treatment group, with dendrogram depicting the unbiased hierarchical clustering of treatment groups based on similarities in gene expression profiles. Red indicates increased gene expression and green indicates decreased gene expression, correlating to a row-wise z-score. Statistical comparisons are made between vitamin D3 treatment groups and the placebo group. Abbreviations: n.s., non-significant. Scale bar 100 μm.

Fig. 3. Unsupervised Clustering of Participants Based on Gene Expression Profiles

Panel A presents a heat map depicting global gene expression profiles for individual participants, with the resulting dendrogram depicting the unsupervised hierarchical clustering of participants based only on similarities in gene expression. Red indicates increased gene expression and green indicates decreased gene expression, correlating to a row-wise z-score. Two unique clusters emerged from this unbiased and blinded analysis. Cluster 1 was characterized by down regulation of arginase-1, and up regulation of genes involved in skin inflammation. Cluster 2 was characterized by up regulation of arginase-1 and genes involved in skin barrier repair. Panel B presents the fold change difference in skin arginase-1 mRNA expression from punch biopsies obtained in the investigative and control phases of the study [(RNA_48hr)_invest/(RNA_48hr)_control]. Data points represent arginase-1 fold change differences for individual participants at each time point. Horizontal lines represent the mean for cluster 1 (n=11) and cluster 2 (n=7). Panel C presents immunofluorescently stained sections from a representative participant receiving 200,000 IU D₃ before and after vitamin D₃ intervention. Nuclear DNA is depicted in blue (DAPI), arginase-1 protein expression is depicted in red, and CD163, a marker of macrophages, is depicted in green. Arginase-1 protein levels are increased primarily within CD163+ macrophages after vitamin D₃ intervention. White scale bar represents 20 micrometers. Abbreviations: FC, fold change.

Fig. 4. Serum Vitamin D₃ and Skin Erythema Following Experimental Sunburn in Cluster 1 and Cluster 2

Panel A presents the serum 25(OH)D₃ levels over time after study drug administration for cluster 1 (n=11) and cluster 2 (n=7). Panel B presents the serum 25(OH)D₃ levels over time after study drug administration for cluster 1 1 (n=7) and cluster 2 (n=7), excluding the participants from cluster 1 who received placebo (n=4). Error bars represent the standard error of the mean for each group at each time point. Panel c presents the change in erythema over time after experimental sunburn with 2MED for each cluster. Data points represent erythema values for individual participants at each time point. Horizontal lines represent the mean for cluster 1 (n=11) and cluster 2 (n=7) at each time point. Statistical comparisons are between cluster 1 and cluster 2 at each time point. * p<0.05; **p<0.01.

Fig. 5. The Effect of Oral Vitamin D₃ Intervention on Skin Inflammation

As depicted in panel A, levels of vitamin D₃ are at baseline levels in the absence of high dose oral vitamin D₃ intervention. In this context, exposure to erythemogenic doses of UVR results in sunburn and the release of pro-inflammatory cytokines and chemokines in the skin, including TNF-α and iNOS, which further propagate tissue inflammation. Increased skin redness and thickness are mediated by vasodilation, an influx of inflammatory cells, and vascular congestion within the skin. The gene expression profile of skin at this time is characterized by increased expression of various pro-inflammatory genes. As depicted in panel B, levels of vitamin D₃ rapidly rise within the serum after high dose oral vitamin D₃ intervention. Arginase-1 is up regulated within the skin, and production of the pro-inflammatory mediators TNF-α and iNOS are attenuated after sunburn. Reduced skin erythema and thickness are observed clinically. The gene expression profile of skin at this time is characterized by increased expression of skin barrier genes, which help to repair the epidermal barrier and attenuate the inflammatory insult.