1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines

Abstract

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D.

Figure 1. Toll-like receptor regulation of vitamin D metabolism and 1α-hydroxylase expression in colonic cell lines

BBe and Caco-2 colonic cells were treated with vehicle (control, C), ligand to TLR2 (19 kDa lipoprotein, 19 kDa) or ligand to TLR4 (lipopolysaccharide, LPS), or combinations of TLR ligand with 100 nM 25-hydroxylvitamin D (25D) for 24 hrs and then assessed for: A) 24-hydroxylase activity (synthesis of 24,25(OH)₂D) and 1α-hydroxylase activity (synthesis of 1,25(OH)₂D). Data are shown as fmoles metabolite produced/hr/mg protein ± SD for n=4 separate assays. B) Expression of mRNA for 1α-hydroxylase (1α-OHase). RT-PCR data are shown as fold-change in 1α-OHase mRNA relative to vehicle-treated cells. ** = statistically different from vehicle-treated cells, p<0.01.

Figure 2. Toll-like receptor regulation of intracrine induction of antimicrobial agents in colonic cell lines

BBe and Caco-2 colonic cells were treated with vehicle (control, C), ligand to TLR2 (19 kDa lipoprotein, 19) or ligand to TLR4 (lipopolysaccharide, L), or combinations of TLR ligand with 100 nM 25-hydroxylvitamin D (25) for 24 hrs and then assessed for: A) expression of mRNA for cathelicidin (LL37); B) β-defensin-4 (DEFB4). RT-PCR data are shown as fold-change in mRNA relative to vehicle-treated cells. ** = statistically different from vehicle-treated cells, p<0.01. *** = statistically different from vehicle-treated cells, p<0.001.

Figure 3. Mechanisms for involvement of 1α-hydroxylase in antimicrobial responses within different barrier tissues

Schematic representation of mechanisms for intracrine induction of antimicrobial agents such as cathelicidin (LL37) in: A) monocytes; B) skin; C) placenta; D) gastrointestinal tract. Abbreviations: TLR (toll-like receptor); DBP (vitamin D binding protein); 25D₃ (25-hydroxyvitamin D₃); 1,25D3 (1,25-dihydroxyvitamin D₃);1α-OHase (25-hydroxyvitamin D-1α-hydroxylase); VDR (vitamin D receptor); LL37 (cathelicidin); TGFβ (transforming growth factor β); IEC (intestinal epithelial cell); PRR (pathogen recognition receptor); DCs (dendritic cells); APCs (antigen presenting cells).