Heme Metabolic Gene Expression In FLVCR-Knockout Mice During T Cell Development

Abstract 2787

Heme is essential for nearly every organism and cell. However, free heme can induce free radical formation and cellular damage, therefore cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Conditional deletion of Flvcr has been shown to cause progressive anemia in neonatal and adult mice (Science 319:825-8, 2008). Recently, we developed a transplant model in which developing lymphocytes lacked FLVCR while erythroid cells expressed FLVCR, preventing anemia, and found that CD4 and CD8 peripheral T cells were severely decreased while B cell numbers were normal. We further demonstrated that FLVCR-knockout thymocytes were blocked at the CD4CD8 double-positive (DP) stage (Blood [ASH Annual Meeting Abstracts] 114: 913, 2009). We hypothesized that developing T cells lacking FLVCR are arrested at the DP stage because of increased intracellular free heme (IFH). While heme is required for erythroid function, little is known about the role of heme in T cell development. Real-time dynamic quantification of IFH in vivo or from ex vivo tissue is a major challenge in heme biology. We reasoned that by measuring the expression of genes transcriptionally-regulated by heme, we could indirectly assess IFH. Three proteins are key regulators of IFH in non-erythroid cells: aminolevulinic acid synthase-1 (ALAS1) is the rate-limiting enzyme in heme synthesis, FLVCR exports heme, and heme oxygenase-1 (HMOX1) degrades heme. Normal thymic T cell development proceeds from the CD4CD8 double-negative (DN) to the CD4CD8 double-positive (DP) stage, which then go on to either the CD4 single-positive (CD4SP) or CD8 single-positive (CD8SP) stage. We flow-sorted cells from each stage and used multiplex quantitative PCR (qPCR) to determine that all three genes were expressed at higher levels early in normal T cell development during the DN and DP stages and then at lower levels in the CD4SP and CD8SP. Heme binding to the negative regulatory protein BACH1 causes dissociation of BACH1 from the Hmox1 promoter and increased Hmox1 transcription, while expression and stability of Alas1 mRNA is under negative feedback control by heme. Therefore, we predicted that increased IFH in FLVCR-knockout thymocytes would lead to an increase in Hmox1 mRNA and a decrease in Alas1 mRNA levels. We compared expression of heme metabolic genes in FLVCR-knockout and control thymocytes. Flvcr expression was nearly absent in FLVCR-knockout DN and DP cells, however, there was a slight increase in Flvcr expression by the few CD4SP and CD8SP present. To understand this result, we analyzed the extent of genomic Flvcr deletion in FLVCR-knockout thymocytes and peripheral B and T cells by genomic qPCR. DN and DP thymocytes had near complete deletion of Flvcr while CD4SP and CD8SP had slightly less-efficient deletion, likely accounting for the increased Flvcr mRNA levels. Strikingly, Flvcr deletion in the few peripheral T cells present was 50–60% in contrast to peripheral B cells (>90%): only those T cells with incomplete Flvcr deletion survived, further underscoring the absolute requirement for FLVCR in developing T cells. We next examined Hmox1 mRNA expression and found that Hmox1 expression was higher in FLVCR-knockout DP, CD4SP, and CD8SP compared to wild-type FLVCR controls. This supports our hypothesis that FLVCR loss leads to increased IFH during T cell development. Alas1 expression was similar in FLVCR-knockout and control thymocytes, a finding that could be explained because heme regulates ALAS1 activity not only at the transcriptional level but also at the post-transcriptional level. Thus Alas1 expression may not be a good indicator of IFH. In summary, we developed a method to quantify relative free heme levels in developing thymocytes through the measurement of heme metabolic gene expression and found that IFH levels were increased in FLVCR-knockout thymocytes compared to controls. Whether and how excess free heme derails the T cell developmental program, remains to be discovered.