1, 25-Dihydroxyvitamin D Suppresses TNFα-Mediated NF-κB Activation in K562 and Human CD34+ Cells and Promotes Recovery of Erythroid Colony Formation in Human CD34+ Cells

Abstract 2097 Anemia of inflammation (AI) has been widely associated with chronic rheumatologic, infectious, and cardiovascular disorders and comprises one-third of cases of anemia in the elderly. Although the antimicrobial peptide hepcidin is felt to be the prime regulator of AI, emerging evidence supports the premise that subsets of AI may be mediated via hepcidin-independent inflammatory pathways, such as those modulated by tumor necrosis factor alpha (TNFα), that directly suppress erythropoiesis. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop more effective targeted, oral therapies for patients with inflammatory anemia. Our recent analysis of elderly participants in the Third National Health and Nutrition Examination Survey (NHANES III) revealed that vitamin D deficiency was strongly associated with AI in this population, with this subgroup exhibiting a rate of vitamin D deficiency nearly twice that of similarly aged non-anemic individuals. In the current study we aimed to evaluate whether vitamin D treatment may ameliorate TNFα-mediated erythroid colony suppression in humans. Human bone marrow-derived CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) in the presence or absence of TNFα (25 ng/ml) and increasing doses of 1, 25-dihydroxyvitamin D 3 [calcitriol, (0.01-1nM)], and erythroid burst-forming units (BFU-E) measured on day 14. The percentage of BFU-E colonies was reduced by 50% at day 14 in the presence of 25ng/ml TNFα (p 3 in a dose-dependent manner, with 1nM vitamin D 3 resulting in a 50% recovery of BFU-E colony numbers at day 14 (p 3 alone exhibited no significant effect on BFU-E formation at all concentrations tested. Based on studies showing that vitamin D receptor may serve as a negative regulator of NF-κB transcriptional activity, we next investigated whether vitamin D 3 may alter TNFα-induced NF-κB activation in both K562 and human CD34+ cells. K562 cells pre-incubated with 1nM vitamin D 3 for 3, 24, 48 and 72 hours were treated with 5 ng/ml TNFα for 30 minutes, and NF-κB activity in nuclear fraction was determined by an ELISA-format oligonucleotide binding assay. TNFα treatment alone markedly increased the oligonucleotide binding activity of the NF-κB p65 subunit, which was completely blocked by co-incubation with competitor oligonucleotide, confirming the assay specificity. Pre-incubation with vitamin D 3 for 3 – 48 hours showed little effect on TNFα-mediated NF-κB activation, but pre-incubation for 72 hours resulted in suppression of TNFα-induced NF-κB activity by 46% (p 3 for 48 hours and 72 hours exhibited 38% and 84% suppression of TNFα-induced NF-κB activity, respectively (p 3 regulates the NF-κB nuclear translocation independent of IκB. Western analysis of whole cell lysates from both K562 and human CD34+ cells using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38, phospho-JNK, and phospho-ERK1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to vitamin D 3 treatment at all doses tested, suggesting that vitamin D 3 does not inhibit these TNFα-induced signaling cascades. Our study indicates that 1, 25-dihydroxyvitamin D 3 significantly ameliorates TNFα-mediated suppression of erythroid colony development in human CD34+ cells via mechanisms that involve modulation of NF-κB signaling pathways and supports the design of future clinical trials examining whether vitamin D 3 supplementation may prove to be an effective therapy for subgroups of patients with TNFα-mediated inflammatory anemia. Disclosures: No relevant conflicts of interest to declare.