Interleukin-6 Reduces Erythroid Development and Mitochondrial Membrane Potential In Human Erythroleukemic Cells

Anemia of inflammation or chronic disease is common and is associated with diseases such as cancer, chronic kidney disease, autoimmune disease, and acute and chronic infections. Concentration of the inflammatory cytokine interleukin-6 (IL-6) is known to negatively correlate with hemoglobin concentration in many of these disease states. Whether IL-6 promotes anemia of inflammation outside of the IL-6-hepcidin antimicrobial peptide axis has not been extensively studied. We previously reported that chronic inflammation affected maturation of erythroid progenitors in a mouse model of chronic inflammation but over expression of hepcidin, alone, did not. We hypothesized that IL-6 may directly impair erythroid maturation, independent of iron restriction.

We utilized the human erythroleukemia TF-1 cell line, treated with recombinant human erythropoietin, to model erythroid maturation and exposed TF-1 cells to increasing doses (0, 1, 10, 100ng/ml) of IL-6 over six days. TF-1 erythroid maturation was determined by flow cytometry (based on CD44, CD71, and CD235a expression), and benzidine staining. In addition, expression levels of genes representing early [glycophorin A (GPA)], mid [aminolevulinic acid synthase 2 (ALAS2) and hemoglobin adult major beta chain (HBB)], and late [erythrocyte membrane protein Band 3 (SLC4A1)] stages of erythropoiesis were analyzed by qRT-PCR. Finally, mitochondrial mass, membrane potential, and oxidative stress were measured using fluorescent indicators.

IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation at concentrations at or above 10 ng/ml (p=0.001, Cuzick test). We observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining (p=0.022, Cuzick test), but this did not correspond to detectable decreases in ALAS2 or HBB expression. However, IL-6 down regulated expression of SLCA4A1 which is expressed late in erythropoiesis (p=0.005, Welch t-tes). The mitochondrial membrane potential was decreased, at all IL-6 treatment doses (p=0.05, Student’s t-test), and mitochondrial mass was significantly decreased at the highest dose (p=0.05, Student’s t-test).

These data demonstrate that IL-6 can impair mitochondrial membrane potential, hemoglobin production, and erythroid maturation in an in vitro setting. Our findings suggest that IL-6 affects cells relatively late in erythropoiesis, after they are primed for hemoglobin synthesis. We hypothesize that the effect of IL-6 on the mitochondria of maturing erythroid cells could be a mechanism behind the impaired maturation phenotype that we observed. These results may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and demonstrate potential for the opportunity to develop new therapeutic targets that affect late erythroid development.

Roy:Celgene Corp.: Research Funding.