Cholecalciferol supplementation alters calcitriol-responsive monocyte proteins and decreases inflammatory cytokines in ESRD

Abstract

In vitro, monocyte 1alpha-hydroxylase converts 25-hydroxyvitamin D [25(OH)D] to 1,25-dihydroxyvitamin D to regulate local innate immune responses, but whether 25(OH)D repletion affects vitamin D-responsive monocyte pathways in vivo is unknown. Here, we identified seven patients who had 25(OH)D insufficiency and were undergoing long-term hemodialysis and assessed changes after cholecalciferol and paricalcitol therapies in both vitamin D-responsive proteins in circulating monocytes and serum levels of inflammatory cytokines. Cholecalciferol therapy increased serum 25(OH)D levels four-fold, monocyte vitamin D receptor expression three-fold, and 24-hydroxylase expression; therapy decreased monocyte 1alpha-hydroxylase levels. The CD16(+) "inflammatory" monocyte subset responded to 25(OH)D repletion the most, demonstrating the greatest increase in vitamin D receptor expression after cholecalciferol. Cholecalciferol therapy reduced circulating levels of inflammatory cytokines, including IL-8, IL-6, and TNF. These data suggest that nutritional vitamin D therapy has a biologic effect on circulating monocytes and associated inflammatory markers in patients with ESRD.

Figure 1.

Change in the CYP27B1, CYP24, VDR, TLR2, and cathelicidin expression in monocytes in response to cholecalciferol and paricalcitol in patients with ESRD is shown. CD14⁺ cells were isolated from patients (n = 7) at three separate time points as described previously. Monocytes were analyzed by flow cytometry after the addition of fluorescence-labeled antibodies specific for the targets of interest, and the mean fluorescence intensity (MFI) was measured as a quantification of the relative protein expression. (A) The MFI for CYP27B1 decreased with both cholecalciferol (P = 0.11) and paricalcitol (P = 0.06) therapy when compared with the previous time point, with the final postparicalcitol expression being significantly lower than the baseline expression (P < 0.05). CYP24 expression increased with cholecalciferol (P = 0.05) and decreased back to baseline after the discontinuation of this therapy and initiation of paricalcitol (P < 0.05), with no significant difference between postparicalcitol and baseline CYP24 expression. The calculated Cyp27B1-to-CYP24 ratio decreased significantly after 8 wk of cholecalciferol therapy (P < 0.01), with no further change in this ratio after the restart of paricalcitol therapy. (B) VDR expression increased significantly after 25(OH)D repletion with cholecalciferol (P < 0.01) and seemed to increase further after the restart of paricalcitol (P = 0.12). The final postparicalcitol VDR expression was significantly greater than the baseline expression (P = 0.001). Total monocyte TLR2 expression seemed unchanged by both cholecalciferol and paricalcitol therapy, whereas cathelicidin expression seemed to decrease with both of these therapies (NS), with the postparicalcitol cathelicidin expression being significantly lower than the baseline expression (P < 0.05).

Figure 2.

Flow cytometry analysis demonstrates the emergence of a subpopulation of monocytes with extremely high levels of VDR expression in response to cholecalciferol and paricalcitol therapy. (A) Frames 1 through 3 (left to right) illustrate flow cytometry analysis of isolated CD14⁺ monocytes at three collection time points from two separate patients. Time point 1 (precholecalciferol) represents the postwashout collection before the initiation of cholecalciferol therapy. Time point 2 (postcholecalciferol) represents monocytes isolated after 8 wk of cholecalciferol therapy. Time point 3 (postparicalcitol) was taken 2 wk after stopping cholecalciferol and restarting paricalcitol. VDR expression by MFI is plotted on the y axis of each box, with CYP27B1 expression on the x axis. The large, lower population of monocytes (in blue) demonstrates lower levels of VDR expression when compared with the smaller population of cells outlined in the small box (in red), which demonstrates very high levels of VDR expression. After the administration of cholecalciferol, there was a noticeable increase in the number of monocytes expressing high levels of VDR, with a further increase in this population after paricalcitol restart. Similar findings were noted in other study participants. (B) Further subanalysis of these two monocyte populations demonstrating unique degrees of VDR upregulation revealed the population expressing high levels of VDR (in red) to be strongly CD16⁺, whereas the population expressing lower levels of VDR (in blue) contained both CD16⁺ and CD16⁻ cells.

Figure 3.

High- and low-VDR monocyte subpopulations demonstrate unique levels of TLR2, cathelicidin, and CYP24 expression. Our previous finding of the existence of two distinct populations after cholecalciferol therapy prompted us to perform a subanalysis to evaluate the difference in TLR2, cathelicidin, and CYP24 expression within these two monocyte subsets. We found TLR2 expression to be five-fold greater in the high-VDR monocyte subset compared with the low-VDR monocytes (P < 0.001), whereas cathelicidin expression was two-fold greater (P < 0.01) and CYP24 expression was three-fold greater (NS) in the high-VDR monocytes.

Figure 4.

Change in serum IL-8, IL-6, and TNF-α levels after cholecalciferol and paricalcitol therapy. To assess the effects of cholecalciferol and paricalcitol therapies on the inflammatory phenotype of patients with ESRD, we measured serum levels of several inflammatory cytokines that are generated by the monocyte lineage. We observed a 55% decrease in serum IL-8 levels (P = 0.13), a 30% reduction in IL-6 levels (P = 0.05), and a 60% decline in TNF-α levels (P < 0.05) after cholecalciferol therapy. Serum IL-8 and TNF-α levels remained lower after the discontinuation of cholecalciferol and restart of paricalcitol, whereas the IL-6 levels seemed to return to near baseline values (NS, after paricalcitol versus after cholecalciferol).