Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in human hematopoietic stem/progenitor cells

Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF alpha) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNF alpha is also a potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNF alpha/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34(+) hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNF alpha, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNF alpha inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNF alpha- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (IN) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNF alpha and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNF alpha/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNF alpha- and ceramide-induced phosphorylation of mTOR(S2448) and ULK1(S758), inhibited Atg13(S)(355) phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNF alpha and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNF alpha/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.