July-August 2015, Volume 12, Issue 4
Less Autophagy in Myeloid Precursors Boosts Neutrophil Counts
Published on: June 01, 2015
Rožman S, Yousefi S, Oberson K, et al. The generation of neutrophils in the bone marrow is controlled by autophagy. Cell Death Differ. 2015;22:445-456.
Autophagy is the intracellular process by which cytoplasmic contents are targeted for digestion with recycling of macromolecular constituents such as amino acids, nucleosides, and lipids. Cytoplasm and organelles such as mitochondria, endoplasmic reticulum, and peroxisomes that are destined for autophagic removal are enclosed in a double membrane structure termed the autophagosome that subsequently fuses with a lysosome, creating the autolysosome, in which the sequestered cytoplasm and organelles are degraded by lysosomal enzymes. Autophagy has important roles in differentiation of almost all lineages of hematopoietic cells, including required clearance of mitochondria during reticulocyte maturation and lymphocyte differentiation.1 However, Dr. Sasa Rožman and colleagues in the laboratory of Dr. Hans-Uwe Simon report the surprising findings that autophagy inhibits terminal differentiation of murine neutrophilic granulocyte precursors, and deficient autophagy in neutrophilic precursors increases neutrophil production rates and steady-state numbers.
In mice with a conditional knockout in neutrophilic precursor cells of ATG5, which encodes a protein required for the formation of the autophagosome, these investigators found an approximately 50 percent increase in circulating neutrophils and two- to three-fold increases in splenic and lymph node neutrophils, respectively, compared to wild-type mice. DNA labeling of promyelocytes and myelocytes, the last stages of granulocyte precursors that undergo mitosis, showed a peak appearance of neutrophils in the blood in ATG5-knockout mice on the third day compared with the fourth day in wild-type mice. These increases in neutrophil steady state numbers and production rates appeared to be due to increased proliferation of granulocytic precursors that was not associated with increased granulocyte colony-stimulating factor (G-CSF) or granulocyte macrophage–colony-stimulating factor (GM-CSF) or decreased granulocytic cell apoptosis. In vitro assays demonstrated that ATG5-knockout neutrophils had similar functional capacities, including phagocytosis, reactive oxygen species generation, granule release, and bacterial killing, as did wild-type neutrophils. A murine system of granulocytic differentiation in vitro demonstrated autophagy in the promyelocytes and myelocytes that decreased markedly in metamyelocytes, bands, and mature neutrophils. Inhibition of ATG5 expression by shRNAs decreased the early-stage autophagy and increased in vitro differentiation, whereas lentivirus-mediated overexpression of ATG5 enhanced autophagy and retarded differentiation. With this in vitro granulocytic differentiation system, Dr. Rožman and colleagues also showed by inhibitor studies that the p38 MAPK-mTORC1 pathway, which suppresses autophagy, mediates the decreased autophagy during granulocytic differentiation.
Although the reciprocal relationship between autophagy and neutrophil differentiation is surprising when compared with the key role of autophagy in differentiation of other hematopoietic lineages, this reciprocal relationship is consistent with previous studies of neutrophilic differentiation. Primary or azurophilic granules of neutrophils, which are delivered to phagosomes or to the exterior of mature neutrophils, are formed in myeloblasts and promyelocytes.2 In addition to myeloperoxidase and elastase, azurophilic granules contain lysosomal enzymes. A study demonstrating qualitative differences in the membranes of the azurophilic granules and lysosomes also noted the absence of mature lysosomes in fully differentiated neutrophils3-- a finding consistent with the decline in autophagy during terminal neutrophil differentiation. Thus, promyelocytes have active autophagy that requires lysosomes, while they are also forming the azurophilic granules, which contain lysosomal as well as other degradative enzymes. Autophagy normally decreases markedly in the later stages of neutrophilic precursor differentiation, but inhibition of autophagy beginning in the promyelocytic stage appears to hasten their differentiation and proliferation into neutrophils. Conversely, enhancement of autophagy in immature precursors retards their differentiation.
Reliable increases in neutrophil production in the clinical setting are currently limited to administration of G-CSF or GM-CSF. Dr. Rožman and colleagues demonstrate that inhibiting autophagy by activating the p38 MAPK-mTORC1 pathway can increase production of functional neutrophils by a mechanism other than increasing G-CSF or GM-CSF. Inhibiting autophagy may adversely affect other hematopoietic and non-hematopoietic cells, but increasing neutrophil numbers by a means independent of these granulopoietic factors would be of considerable interest to hematologists. Indeed, autophagy inhibition combined with G-CSF or GM-CSF administration has the potential to hasten recovery of neutrophils in patients with chemotherapy-induced neutropenia. Similarly, patients with congenital or acquired neutropenias, especially those who do not respond to G-CSF treatment, might benefit from suppression of autophagy, if it can be demonstrated to increase their neutrophil production.
Mizushima N, Levine B. Autophagy in mammalian development and differentiation. Nat Cell Biol. 2010;12:823-830.
Häger M, Cowland JB, Borregaard N. Neutrophil granules in health and disease.. J Intern Med. 2010;268:25-34.
Cieutat AM, Lobel P, August JT, et al. Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition marker. Blood. 1998;91:1044-1058.
Conflict of Interests
Dr. Koury indicated no relevant conflicts of interest.
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