Erythroid Precursors from Patients with Low-Risk Myelodysplasia Demonstrate Ultrastructural Features of Autophagy.
Authors: Houwerzijl Ewout , Pol Henk-Willem , Blom Nel , Stokroos Ietse , van der Want Johannes , de Wolf Joost , Vellenga Edo 
Published: April 2020

Abstract
Anemia in myelodysplasia (MDS) is partially ascribed to enhanced programmed cell death (PCD) of committed erythroid cells in the bone marrow compartment. Especially, enhanced apoptosis has been described. However, nonapoptotic forms of PCD have been demonstrated in MDS megakaryocytes characterized by the absence of chromatin condensation and caspase-3 and -8 activation (Houwerzijl EJ et al. Blood2005;105:3472–3479). Recent studies have indicated that besides apoptosis, necrosis and autophagic cell death can be recognized as PCD, whereby cells are capable to switch between the different types of PCD dependent on their cellular context. To define in more detail the underlying cell death pathways in MDS erythroblasts, immunohistochemical staining and ultrastructural and immunolabeling analysis were performed on bone marrow samples of low-risk MDS patients and normal controls (n=4). Immunohistochemistry of MDS bone marrow biopsies (n=23) demonstrated no positive staining of the erythroblasts for active caspase -3 and -8. To confirm these results ultrastructural analysis and immunoelectron microscopy was performed on mononuclear cells (MNC) and hematons of a subgroup of these patients (n=9). Hematons are compact hematopoietic complexes in which hematopoietic cells, including erythroblasts, are embedded in their own microenvironment. The ultrastructural analysis revealed that only a small fraction of erythroid cells of the MNC and hematon fraction of both MDS patients and healthy controls demonstrated features of apoptosis (2 ± 2% vs 0%). However, 52 ± 16% of immature and mature MDS erythroblasts contained cytoplasmic vacuoles in contrast to normal erythroblasts, in which vacuoles were only shown in the matured stage (12 ± 3%). These vacuoles were partly double-membraned and stained positive for the lysosomal marker LAMP (lysosome associated membrane protein)-2, catalase and the mitochondrial inner membrane protein (immunogold staining) underscoring the presence of autophagy of mitochondria and other cytoplasmic components. Morphometric analysis confirmed that the vacuolar surface in the cytoplasm of MDS erythroblasts was increased compared to controls (P&lt;0.0001). When bone marrow MNC were cultured in vitro for 24 hours, 27 ± 7% of MDS erythroblasts were apoptotic versus 8% of those from controls which was confirmed with an anti-caspase-3 staining. However, when erythroblasts were cultured in the context of their own microenvironment, i.e. from the hematon fraction, only 6 ± 4% of MDS erythroblasts were apoptotic (P=0.007). In summary, these data indicate that MDS erythroblasts show features of enhanced autophagy at an earlier stage of the erythroid differentiation program than normal controls and that the autophagic process may switch to apoptosis when the appropriate microenvironment is lacking. The enhanced autophagy might be considered as a cell protective mechanism to remove defectively formed and iron-laden mitochondria, but can also have consequences for the erythroid differentiation program due to the premature autophagic degradation of proteins relevant for the maturation process.