Cell-Type Specific Requirement of Glucose Catabolism in Erythropoiesis and Thymopoiesis

Glucose catabolism through glycolysis is central to cellular life. In glycolysis, pyruvate is converted to lactate by lactate dehydrogenase (LDH) or oxidized into the tricarboxylic acid (TCA) cycle via acetyl-CoA by pyruvate dehydrogenase (PDH). However, the two enzymes LDH and PDH required for completing glucose catabolism have never been ablated simultaneously in any tissue in vivo to test if glycolysis is required for cell survival and function. Ldha and Ldhb, the genes coding for LDH have not been ablated simultaneously in in the erythroid lineage to test if LDH plays a significant role in erythropoiesis. It is also unknown if erythoid progenitor cells, which unlike mature erythrocytes contain mitochondria, rely on LDH or are able to oxidize glucose via PDH to support erythroid development. To address these questions, we have generated conditional genetic knockouts (KO) of Ldha, Ldhb and PDH (Pdha1) in the hematopoietic system to study the requirement of glucose catabolism in erythropoiesis.

We observed that Ldha^Δ/Δ, and Ldha^Δ/Δ;Ldhb^Δ/Δ but not Ldhb^Δ/Δ mice had anemia, consistent with an essential role for LDHA in erythrocytes or erythropoiesis. Anemia was more severe in Ldha^Δ/Δ;Pdha1^Δ mice as compared to Ldha^Δ/Δ mice suggesting LDH and PDH are required in concert to maintain erythroid development in vivo. Ldha^Δ/Δ mice accumulated CD71⁺Ter119⁺ erythroid progenitors but Ldha^Δ/Δ;Pdha1^Δ knockout mice had a significant decrease in this population. Moreover, there was a more pronounced expansion of upstream CD71^-Ter119^int and CD71⁺Ter119^int cells in Ldha^Δ/Δ;Pdha1^Δ mice. This suggest that the accumulation of CD71⁺Ter119⁺ cells in Ldha^Δ/Δ mice is dependent on PDH and that the presence of either LDHA or PDH is required for the progression from CD71⁺Ter119^int to CD71⁺Ter119⁺ erythroid progenitors. The bone marrow of Ldha^Δ/Δ;Pdha1^Δ showed an accumulation of pre-CFU-E and a depletion of CFU-E cells as compared to single Ldha^Δ/Δ mice. Therefore, the presence of either LDHA or PDH is required to transit from pre-CFU-E to CFU-E stage during erythroid differentiation. Our previous results show that PDH is required for the double positive (DP) stage of T cell development. Analysis of thymus of Ldha^Δ/Δ, and Ldha^Δ/Δ;Ldhb^Δ/Δ mice did not show any changes in thymocytes. However, Ldha^Δ/Δ;Pdha1^Δ mice showed a significant decrease at the double negative (DN1) stage which persisted through all T cell development stages. Given that, PDH is required at the double positive (DP) stage and LDH deficiency alone does not affect T cell development, this suggest that LDH and PDH act redundantly at different stages to support T cell development. Altogether, these results suggest cell-type specific usage of glucose catabolic pathways in different differentiation stages. Our results also suggest that progenitor cells can switch between LDH and PDH for glucose catabolism while mature immune cells rely on either LDH or PDH for survival. To investigate this, we will use metabolomics and isotope tracing methods to analyze the route of glucose catabolism during erythroid and T cell development.