Vitamin d-directed rheostatic regulation of monocyte antibacterial responses

Abstract

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

Figure 1

Serum hCAP levels do not correlate with serum vitamin D metabolites in normal human donors. A) Effect of donor serum 25OHD concentration (ng/ml) on serum concentration of hCAP (ng/ml). B) Effect of donor serum 1,25(OH)₂D concentration (pg/ml) on serum concentration of hCAP (ng/ml). Correlation coefficient (R) and statistical significance (p value) are shown for each plot.

Figure 2

Leukocyte hCAP mRNA expression does not correlate with serum vitamin D metabolites or serum hCAP levels. A) Effect of donor serum 25OHD concentration (ng/ml) on levels of hCAP mRNA (dCt) in donor leukocytes. B) Effect of donor serum 1,25(OH)₂D concentration (pg/ml) on levels of hCAP mRNA (dCt) in donor leukocytes. C) Relationship between donor leukocyte hCAP mRNA expression (dCt) and serum hCAP levels (ng/ml). Correlation coefficient (R) and statistical significance (p value) are shown for each plot.

Figure 3

Induction of CYP27b1 expression and activity following TLR activation of monocytes cultured in autologous serum conditions. A. Changes in CYP27b1 expression following treatment with ligands to TLR2/1 (19 kDa lipopeptide, 1 ng/ml) or TLR4 (lipopolysaccharide, 100 ng/ml), in monocytes cultured in medium supplemented with autologous serum with or without added 25OHD. Data shown are the mean fold-change in CYP27b1 mRNA expression relative to vehicle-treated cells based on mean ± SD ΔCt values for each different. B) Changes in VDR expression following treatment with ligands to TLR2 in monocytes cultured in medium supplemented with autologous serum with or without added 25OHD. Data shown are the mean fold-change in VDR mRNA expression relative to vehicle-treated cells based on mean ± SD ΔCt values for each different treatment. C) Changes in monocytes synthesis of 1,25(OH)₂D following treatment with ligand to TLR2, in the presence or absence of added 25OHD. * = mean ΔCT values statistically different to ΔCT value for vehicle-treated control monocytes, p<0.05. ** = mean ΔCT values statistically different to ΔCT value for vehicle-treated control monocytes, p<0.01. *** = mean dCT values statistically different to ΔCT value for vehicle-treated control monocytes, p<0.001. # = level of 1,25(OH)₂D statistically different from TLR-treated control p<0.05. # = level of 1,25(OH)₂D statistically different from vehicle-treated control p<0.01.

Figure 4

Regulation of monocyte cathelicidin (hCAP) by TLR ligands and 25OHD. Changes in hCAP expression following treatment with ligands to: A) TLR2/1 (19 kDa lipopeptide, 19 kDa, 1 ng/ml) or B) TLR4 (lipopolysaccharide, 100 ng/ml) in monocytes cultured in medium supplemented with autologous serum with or without added 25OHD (5 or 100 nM). Data shown are the mean fold-change in hCAP mRNA expression relative to vehicle-treated cells based on mean ± SD ΔCt values for each different. *** = mean ΔCT values statistically different to ΔCT value for vehicle-treated control monocytes, p<0.001. # = mean ΔCT values statistically different to ΔCT value for TLR-treated control monocytes, p<0.05. ## = mean ΔCT values statistically different to ΔCT value for TLR-treated control macrophages, p<0.01. ### = mean dCT values statistically different to ΔCT value for TLR-treated control monocytes, p<0.001. Numbers shown in bars indicate mean fold-change values compared to vehicle-treated controls (C).

Figure 5

Monocyte DEFB is induced by TLR ligands but not 25OHD. Changes in DEFB4 expression following treatment with ligands to: TLR2/1 (19 kDa lipopeptide, 19 kDa, 1 ng/ml) or TLR4 (lipopolysaccharide, 100 ng/ml) in monocytes cultured in medium supplemented with autologous serum with or without added 25OHD. Data shown are the mean fold-change in DEFB mRNA expression relative to vehicle-treated cells based on mean ± SD ΔCt values for each different treatment. *** = mean ΔCT values statistically different to ΔCT value for vehicle-treated control macrophages, p<0.001. ^### = mean ΔCt value statistically different from monocytes treated with 25OHD alone, p<0.001. Numbers shown in bars indicate mean fold-change values compared to vehicle-treated controls (C).

Figure 6

TLR2/1-induced cathelicidin (hCAP) in monocytes correlates with levels of serum 25OHD in ex vivo culture supplements. Change in levels of monocyte hCAP mRNA following activation of TLR2/1 by 19 kDa lipopeptide (19 kDa, 1 ng/ml) related to the concentration of: A) 25OHD; B) 1,25(OH)₂D in 10% human serum used for autologous monocyte cultures. Concentrations of 25OHD [25OHD] and 1,25(OH)₂D [1,25(OH)₂D] are shown as ng/ml and pg/ml respectively on the lower x-axis, with a corresponding nM and pM reference point shown on the upper x-axis.

Figure 7

Vitamin D deficiency is associated with decreased expression of monocyte cathelicidin (hCAP) following TLR2/1 challenge. A) Expression of hCAP in ex vivo cultures of human monocytes following 24 hr treatment with the TLR2/1 ligand 19 kDa lipopeptide (1 ng/ml) under autologous serum culture conditions. Data are shown as the mean fold-change in hCAP expression (± SD) of TLR-activated monocytes compared to vehicle treated cells for subjects categorized as vitamin D-sufficient (serum 25OHD greater than 75 nM, n=18) or vitamin D-deficient (serum 25OHD less than 75 nM, n=10). B) Effect of supplementation with vitamin D on serum 25OHD and 1,25(OH)₂D levels in the patients initially categorized as vitamin D-deficient (serum 25OHD less than 75 nM, n=10). C) Effect of vitamin D supplementation of patients (Post-D) initially categorized as vitamin D-deficient (Pre-D) on expression of hCAP in ex vivo cultures of human monocytes treated for 24 hrs with the TLR2/1 ligand 19 kDa lipopeptide under autologous serum culture conditions. Data are shown as the mean fold-change in hCAP expression (± SD) of TLR-activated monocytes compared to vehicle treated cells for vitamin D-insufficient subjects before (Pre-D) and after (Post-D) supplementation with vitamin D. *** = statistically different from values for the <75 nM 25OHD group, p<0.001. * = statistically different from values for the <75 nM 25OHD group, p<0.05. ^## = statistically different from values for the pre-treatment <75 nM 25OHD group, p<0.01.