Antibacterial responses by peritoneal macrophages are enhanced following vitamin D supplementation

Abstract

Patients with chronic kidney disease (CKD), who usually display low serum 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D), are at high risk of infection, notably those undergoing peritoneal dialysis (PD). We hypothesized that peritoneal macrophages from PD patients are an important target for vitamin D-induced antibacterial activity. Dialysate effluent fluid was obtained from 27 non-infected PD patients. Flow cytometry indicated that PD cells were mainly monocytic (37.9±17.7% cells CD14+/CD45+). Ex vivo analyses showed that PD cells treated with 25D (100 nM, 6 hrs) or 1,25D (5 nM, 6 hrs) induced mRNA for antibacterial cathelicidin (CAMP) but conversely suppressed mRNA for hepcidin (HAMP). PD cells from patients with peritonitis (n = 3) showed higher baseline expression of CAMP (18-fold±9, p<0.05) and HAMP (64-fold±7) relative to cells from non-infected patients. In 12 non-infected PD patients, oral supplementation with a single dose of vitamin D2 (100,000 IU) increased serum levels of 25D from 18±8 to 41±15 ng/ml (p = 0.002). This had no significant effect on PD cell CD14/CD45 expression, but mRNA for HAMP was suppressed significantly (0.5-fold, p = 0.04). Adjustment for PD cell CD14/CD45 expression using a mixed linear statistical model also revealed increased expression of CAMP (mRNA in PD cells and protein in effluent) in vitamin D-supplemented patients. These data show for the first time that vitamin D supplementation in vitro and in vivo promotes innate immune responses that may enhance macrophage antibacterial responses in patients undergoing PD. This highlights a potentially important function for vitamin D in preventing infection-related complications in CKD.

Figure 1. Vitamin D-related gene expression in CKD patient PD cells at baseline.

Correlation between PD cell mRNA expression of CAMP or HAMP and: A) donor patient serum concentrations of 25D (ng/ml); B) CYP27B1 mRNA; C) VDR mRNA in PD cells isolated from CKD patient dialysate effluents from n = 27 baseline samples (pre-vitamin D supplementation) from 27 different patients. Data for mRNA expression are shown as 1/ΔCt values.

Figure 2. Effect of 25D and 1,25D on vitamin D-related gene expression in baseline PD cells in vitro.

Effect of 1,25D (5 nM, 6 hrs, n = 13) (A) or 25D (100 nM, 6 hrs, n = 8) (B) on expression for mRNA for CYP27B1, VDR, CAMP and HAMP in PD cells cultured in RPMI with 10% human serum and GM-CSF (10 IU/mL). Data are shown as fold-change in mRNA expression relative to vehicle (0.1% ethanol)-treated cells for each gene with vehicle expression = 1 (see dashed line). * = statistically different from vehicle-treated cells, p<0.05. *** = statistically different from vehicle-treated cells, p<0.001.

Figure 3. Baseline expression, and 25D/1,25D-regulation of vitamin D-related genes in PD cells from patients with peritonitis.

(A) Relative expression of mRNA for CYP27B1, VDR, CYP24A1, CAMP and HAMP in PD cells from peritonitis patients (n = 3) compared to PD cells from non-infected patients (n = 8). Data are shown as fold-change in mRNA expression relative to non-infected cells. (B) Effect of 1,25D (5 nM, 6hrs) or 25D (100 nM, 6 hrs) on expression for mRNA for CAMP (grey bars) or HAMP (black bars) in PD cells from peritonitis patients (n = 3) RPMI with 10% human serum and GM-CSF (10 IU/mL). Data are shown as fold-change in mRNA expression relative to non-infected PD cells (3A) or vehicle (0.1% ethanol)-treated cells (3B). * = statistically different from vehicle-treated (0.1% ethanol) cells, p<0.05. ** = statistically different from vehicle-treated cells, p<0.01.