Resveratrol Ameliorates TNFα-Mediated Suppression of Erythroid Colony Formation in Human CD34+ Cells Via Cellular Pathways Involving Downregulation of NF-κB.

Abstract 2507

Poster Board II-484

Although the etiology of anemia remains unexplained in one third of elderly subjects, increasing evidence implicates chronic inflammation in the pathogenesis of anemia in the elderly population. We hypothesize that in addition to classical anemia of inflammation (AI) mediated by hepcidin, a significant proportion of unexplained anemia involves the overexpression of proinflammatory cytokines like TNFα that promote direct suppression of erythroid progenitors to a degree more prominent than the effects of hepcidin, resulting in the development of unexplained anemia marked by absence of the expected biochemical profile of AI. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop effective targeted, oral therapies for patients. In the current study, we aimed to evaluate whether resveratrol, a flavanol found in high concentration in red wine that appears to possess potent anti-inflammatory properties, ameliorates TNFα-mediated erythroid colony suppression in human CD34+ cells. Human peripheral blood CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium in the presence of IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) and increasing doses of TNFα (0-100ng/ml) and erythroid burst-forming units (BFU-E) measured on day 14. Consistent with prior experiments, the percentage of BFU-E colonies was reduced by 60% at day 14 in the presence of 25 and 100ng/ml TNFα (p < 0.01, t-test), which was reversed to baseline with the addition of the neutralizing anti-TNFα antibody infliximab (p < 0.01, t-test). In order to determine whether the addition of resveratrol moderated TNFα-mediated erythroid colony suppression, human CD34+ cells were initially pre-incubated with increasing doses of resveratrol (0-50μM) for 72 hours and cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), and SCF (50ng/ml) in the presence or absence of 25ng/ml TNFα and varying concentrations of resveratrol (0-50μM) and BFU-E measured on day 14. We noted 50% recovery in BFU-E colony numbers at day 14 in CD34+ cells grown in the presence of 10μM resveratrol and TNFα in comparison with TNFα alone (p < 0.02, t-test). To better characterize TNFα-mediated pathways influenced by resveratrol, we analyzed in parallel CD34+ cells pre-incubated for 72 hours with 10μM resveratrol and grown in serum-free liquid culture containing similar concentrations of IL-3, SCF, and EPO in the presence or absence of 25ng/ml of TNFα and 10μM resveratrol. Total RNA and protein were isolated at multiple timepoints after the addition of TNFα on culture day 4 and TNFα-mediated gene and protein expression as well as hematopoietic microRNAs miR155 and miR451 expression analyzed by real-time RT-PCR and/or Western blot. We found that TNFα-treated CD34+ cells exhibited a 5-fold increased expression of miR155 without change in miR451 expression at 12 hours post TNFα treatment compared with control cells. However, CD34+ cells cultured in the presence of 10μM resveratrol and TNFα revealed downregulation of miR155 expression to baseline levels without change in miR451 expression (p < 0.01, t-test). Resveratrol treatment also resulted in 2-fold increased expression of GATA-1 without an appreciable change in GATA-2 (p < 0.01). Western analysis of whole cell lysates using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38MAPK, phospho-JNK, and phospho-ERK 1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to resveratrol treatment. We next examined TNFα-mediated NF-κB signaling in the presence or absence of resveratrol by Western analysis and found that the addition of resveratrol resulted in a 50% reduction in phospho-NF-κB levels in response to TNFα, with total NF-κB levels unchanged in all conditions. These data indicate that resveratrol significantly ameliorates TNFα-mediated erythroid colony suppression in human CD34+ cells via novel mechanisms that incorporate downregulation of both miR155 and NF-κB-associated signaling pathways, providing important evidence that resveratrol may be a potentially effective agent in the treatment of TNFα-mediated anemia and inflammation.