Inflammatory and Vitamin Bio-Markers Of Iron Trafficking and Distribution In Transfusional Overload: Insights From Comparing Diamond Blackfan Anemia With Sickle Cell Disease and Thalassemia (MCSIO PILOT Study)
Authors: Porter John , Walter Patrick B , Neumayr Lynne D , Evans Patricia , Weyhmiller Marcela G , Harmatz Paul , Wood John C. , Miller Jeffery L , Weiss Guenter , Yuan Quing , Wang Zhiyue J , Grosse Regine , Schoennagel Bjoern P , Meerpohl Joerg , Fischer Roland , Nielsen Peter , Seifert Markus , Vichinsky Elliott 
Published: October 2019

Abstract

Introduction
Complications of transfusional iron overload vary with the underlying hematologic condition and may be affected by inherent differences in non-transferrin bound iron (NTBI) generation between these conditions. We have previously reported high NTBI levels in Diamond Blackfan anemia (DBA) relative to sickle cell (SCD), despite lower serum ferritin and LIC associated with very low sTfR levels in DBA. This suggested that low transferrin iron utilization in DBA relative to SCD and thalassemia major (TM) accounted for high NTBI levels. It is known that pro-inflammatory cytokine IL6 will increase hepcidin synthesis thereby decreasing ferroportin expression on macrophages but it is not known whether high levels of inflammatory cytokines can overcome or limit the effects of iron overload in raising NTBI and transferrin saturation. Here we have examined additional factors that may affect NTBI levels in different forms of inherited anemias. The relationship of inflammatory markers, the hepcidin/ferritin ratio and monocyte ferroportin in these conditions to NTBI levels have been examined. The possible effect of vitamin C or vitamin D deficiency on NTBI has also been examined.

Methods
15 iron-overloaded patients (5 from each group of TM, SCD, and DBA) with ferritin > 1500 g/dl or LIC > 7 mg/g dry wt, age 16, age 0 to 9 at initiation of transfusion and 10 to 20 years of transfusion exposure were enrolled from 3 sites in the US and Europe. 5 non-transfused healthy controls were also enrolled. Fasting, early morning blood samples (including WBC isolation) were obtained one day prior to transfusion. Chelation was held for 72 hours prior to each sample and samples taken pre-transfusion. Measurement of hepcidin, sTfR, GDF-15, transferrin saturation, NTBI and LPI was as described elsewhere. Inflammatory cytokine IL6 and IL10 were measured by multiplex bead fluorimetry. Ferroportin expression in circulating monocytes, a potential marker of its expression in resident macrophages of spleen, liver and bone marrow was measured by western blotting.

Results
Results shown in Table 1 are most notable for the following findings: in DBA, the inflammatory markers IL6 and IL10 are high as in SCD or TM. Furthermore plasma hepcidin levels and hepcidin/ferritin ratios are higher in DBA, than in SCD or TM. Despite these high values of hepcidin, IL6 and IL10, monocyte ferroportin levels and NTBI remain high in DBA. This suggests that chronically high levels of circulating hepcidin do not decrease NTBI in patients with low transferrin iron utilization (as in DBA) and that this latter effect combined with iron overload is the more powerful determinant in raising NTBI levels. If the circulating monocytes are a true reflection of resident tissue macrophages that phagocytize red cells, this would suggest that high hepcidin levels are insufficient to suppress ferroportin expression in the presence of severe iron overload. TGFb was most raised in SCD whereas TNFα, were similar in SCD, Thal and DBA. In SCD ferroportin levels on circulating monocytes were lower than DBA and Thal as well as being lower than control which could contribute to low transferrin saturation and low NTBI levels. Vitamin C levels were also lowest and this could contribute to iron retention within macrophages and hence the low NTBI levels observed in SCD.

Conclusions
Taken with our previous findings (ASH abstract 2012), our results are consistent with NTBI and Tf saturation being determined by an interaction of opposing effects, which vary depending on the underlying cause of anemia. NTBI and Tf saturation are increased by (a) iron overload, (b) low erythropoiesis though low utilization of transferrin iron. The findings in this study extension suggest that vitamin C deficiency in SCD could be a contributing factor to low NTBI and this requires further investigation. However the presence of a raised inflammatory cytokine and raised hepcidin appears to be insufficient to abrogate the combined effects of low utilization of transferrin iron and iron overload on raising NTBI in DBA.

Disclosures:
Porter: Novartis: Consultancy, Research Funding; Celgene: Consultancy; Shire: Consultancy. Walter:Novartis: Research Funding. Harmatz:Novartis: Research Funding; Shire: Consultancy, Honoraria, Research Funding, travel, travel Other. Vichinsky:Novartis: Honoraria, Research Funding.