Autophagy is required for CSF-1–induced macrophagic differentiation and acquisition of phagocytic functions

Abstract

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacologic and genetic evidence indicates that autophagy plays pleiotropic functions in cellular homeostasis, development, survival, and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by colony stimulating factor-1. We show here, using pharmacologic inhibitors, siRNA approaches, and Atg7 ؊/؊ mice, that autophagy initiated by ULK1 is required for proper colony stimulating factor-1-driven differentiation of human and murine monocytes. We also unravel a role for autophagy in macrophage acquisition of phagocytic functions. Collectively, these findings highlight an unexpected and essential role of autophagy during monocyte differentiation and acquisition of macrophage functions. (Blood. 2012;119(19):4527-4531) IntroductionAutophagy (macroautophagy) is a general mechanism for degradation and recycling of macromolecules that is characterized by the formation of double-membrane vesicles called phagosomes that derive from phagophores. 1 Autophagy plays a crucial role in cellular homeostasis, development, differentiation, cell death and survival, and ageing. [2][3][4] Although autophagy functions mainly as a safeguard mechanism during nutrient starvation, excessive autophagy leads to cell death. 5 Monocytes have the unique property to migrate into tissues in response to inflammation where they are subjected to differentiation into morphologically and functionally heterogeneous cells, such as macrophages, myeloid dendritic cells, and osteoclasts, depending on the stimulus. 6 The differentiation of human peripheral blood monocytes into macrophages can be reproduced ex vivo by exposure to colony stimulating factor-1 (CSF-1; also known as M-CSF), a process that requires limited activation of caspase-8 and caspase-3. 7 Binding of CSF-1 to its receptor CSF-1R triggers successive waves of AKT activation, leading to the formation of a caspase-8 activating platform and differentiation of monocytes into macrophages. 7 In the present study, we investigated the implication of autophagy in monocyte differentiation and acquisition of macrophage functions. Methods Human monocyte culture and differentiationHuman peripheral blood monocytes were obtained from healthy donors with informed consent following the Declaration of Helsinki according to recommendations of an independent scientific review board. Experiments on mice were performed with the approval of the ethics committee of the University of Nice. Human and mouse monocytes were enriched with autoMACS Separator (Miltenyi Biotec). Macrophage differentiation was visualized using standard optics (Carl Zeiss), electronic microscopy, flow cytometry, and phagocytic assay. A detailed description is in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Immunoblot assaysWestern blot analysis has been described previo...

The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML

“…More recently we established that the induction of autophagy is also a prerequisite for CSF1-induced monocyte differentiation and the acquisition of phagocytic functions. 7,8 In the same line, it has been previously reported that induction of autophagy is essential for monocyte-macrophage differentiation upon CSF2/GM-CSF stimulation. Indeed, CSF2 induced MAPK8/JNK1 activation to mediate the induction of autophagy and monocyte survival.…”

“…[26][27][28] Notably, the differentiation of hematopoietic cells requires intense energy consumption, membrane remodeling and/or organelles elimination, as exemplified for myeloid, megakaryocytic, or erythroid differentiation. 7,11,29,30 Physiological monocyte differentiation triggered by CSF1R engagement requires the formation of a multimolecular complex consisting of FADD, CFLAR/FLIP, CASP8, RIPK1 and other protein partners; and CASP8 acts as the initiatory caspase to induce the cleavage of key protein substrates (such as RIPK1 and NUCLEOPHOSMIN, among others) that orchestrate the differentiation process. 6,31 Accordingly, inhibition of caspase activation with pancaspase inhibitors or by CASP8 silencing is sufficient to dampen the differentiation of human monocytes into macrophages.…”

“…5,6 More recently, we have established that autophagy also plays a crucial role during CSF1-induced differentiation of monocytes. 7,8 Autophagy (macroautophagy) is an evolutionarily conserved catabolic pathway that delivers cytoplasmic substrates, such as damaged organelles and cytoplasmic proteins, to lysosomes for degradation. 9,10 There is growing evidence that supports a role of autophagy in the rapid cellular changes that are necessary for proper differentiation of different cell types.…”

Autophagy-mediated regulation of macrophages and its applications for cancer

“…When monocytes are stimulated to differentiate into macrophages, autophagy is induced via increased expression and phosphorylation of ULK1. 37,38 Studies involving the inhibition of autophagy by pharmacological agents, siRNA approaches or Atg7 knockout mice show that the CSF1-driven differentiation of monocytes into macrophages is significantly hampered ( Table 1). 37,38 CSF2/GM-CSF (colony-stimulating factor 2 [granulocyte-macrophage]) is another important factor that can drive the differentiation of monocytes into macrophages, and it was demonstrated that autophagy is induced during monocyte differentiation into macrophages triggered by CSF2 in vitro and by thioglycolate in vivo.…”

Doxorubicin resistance mediated by cytoplasmic macrophage colony-stimulating factor is associated with switch from apoptosis to autophagic cell death in MCF-7 breast cancer cells

“…18 Therefore, we propose that the effect of M-CSF on chemoresistance is possibly mediated by autophagy and apoptosis. In this study, we found that cytoplasmic M-CSF-induced doxorubicin (Adriamycin, ADM) resistance is mediated by apoptosis inhibition through activation of the PI3K/Akt/Survivin pathway in MCF-7 cells.…”