Mitochondria Clearance Mechanisms Via Interaction with Macrophages in Erythropoiesis

Adult erythropoiesis involves a series of well-coordinated events resulting in the production of mature red blood cells. One of such events is the mitochondria clearance, which is known to occur cell-autonomously via autophagy-dependent mechanisms. Interestingly, we identified a sequential changes in the transcriptional pattern during terminal erythroid differentiation based on the expression of several macroautophagy (e.g. Atg3, Atg5, Atg7 and Atg10) and non-canonical mitophagy (e.g. Pink1, Park2, Bnip3l/Nix, P62 and Ulk1) genes. Hence we hypothesize that the progressive reduction in mitochondria during terminal erythroid differentiation is directed by distinct transcriptionally-regulated programs. Notably, we revealed a gradual reduction of the expression of lysosome related genes (e.g. Lamp1, CD63, and Atp6v) and lysosomal activities from early to late stages of terminal differentiation. On the other hand, P62-Pink1-Parkin mediated ubiquitin proteasome degradation of mitochondria proteins seems to be more prominent during late stage erythropoiesis. Hence our data suggest that mitochondria clearance is predominantly mediated by canonical autophagy during early stages of terminal differentiation, whereas non-canonical mitophagy pathway seem to play a more predominant role to regulate late stages erythroid maturation.

Next, we discovered mitochondria transfer activities from erythroblasts to their niche. In the context of erythropoiesis, macrophages are known to interact closely with erythroblasts to provide a specialized niche for erythroid precursors to proliferate, differentiate and enucleate. We showed defective erythropoiesis after macrophage depletion in the bone marrow. Subsequently, we identified a tendency for early erythroblasts to associate with macrophages and isolated those erythroblasts from mito-dendra2 mice with trackable mitochondria to establish a murine primary cell co-culture system. Then we report mitochondria transfer activities in the erythroid niche via different modes including direct uptake, micro-vesicle transfer and tunnelling nanotubes (TNT). Interestingly, interchangeable structures between micro-vesicles and TNTs have been observed, suggesting an interplay between cytoskeleton and membrane lipid molecules in the mitochondria transfer mechanisms. Furthermore, mitochondria transfer activities have also been observed in the co-culture of mito-dendra2 erythroid cells with a macrophage cell line, RAW cells, and are significantly enhanced by the activation of the RAW cells via Tfe3 activation.

In summary, our findings may provide insight into the mitochondria clearance machineries that mediates erythroid maturation to fulfil the clinical demand for large scale transfusable blood cell production in vitro.