Impaired G-CSF-induced autophagy as a mechanism contributing to diabetic stem cell mobilopathy

Diabetes is a chronic disease that affects several organs. Hyperglycemia promotes chronic inflammation and impairs angiogenesis due to the increase of myelopoiesis. In diabetic patients a condition called stem cell mobilopathy is developed, causing the incapability to mobilize hematopoietic stem and progenitor cells (HSPCs) after Granulocyte-Colony Stimulating Factor (G-CSF) stimulation, a drug used to mobilize HSPCs for Hematopoietic stem cell transplantation (HSCT). Recently autophagy, a cellular recycling system, has emerged as a novel player in HSPCs mobilization after G-CSF treatment. This project aims to investigate whether an alteration of the autophagic pathway in neutrophils, which are the main target of G-CSF mechanism of action, is involved in diabetic stem cell mobilization in a murine streptozotocin diabetic model of type 1 diabetes (T1D STZ). Furthermore, we have analyzed bulk and single cell RNAseq public dataset of human neutrophils treated with G-CSF to evaluate the transcriptomic profile of autophagy related genes. Autophagy, and in particular autophagosome formation, was investigated using C57Bl6-TG (GFP-LC3) transgenic mice, looking for GFP-LC3 punctae accumulation in bone marrow neutrophils using the Amnis Imagestream MKII imaging flow cytometry platform. For Western Blot (WB) and qPCR analysis C57Bl6/J wild type mice were used to isolate neutrophils. T1D was induced in 3 months old mice with a single intraperitoneal injection of streptozotocin (STZ) 175mg/kg and used after 4 weeks of subsequent hyperglycemia. HSPCs mobilization was induced using rhG-CSF. To block the autophagic flux, cells were treated ex vivo with 80uM chloroquine for one hour. To assess mobilization, we quantified HSPCs in the peripheral blood by flow cytometry and performed a clonogenic colony forming unit (CFU) assay. To induce autophagy or bypass it, diabetic mice were treated with spermidine or β-hydroxybutyrate during G-CSF treatment. We have observed that autophagy-related genes were modulated in unfractioned bone marrow but not in neutrophils, but the results obtained by WB show that ATG5, together with the LC3 lipidated fraction, two important autophagy markers, were upregulated in G-CSF treated mice neutrophils compared to controls. These data were corroborated by the imaging flow cytometry results, in which we could show that in vivo G-CSF increases the number of GFP-LC3 punctae in GR1+ neutrophils of non-diabetic GFP-LC3 mice, confirming the autophagy role in G-CSF mechanism of action. On the other hand, neutrophils from diabetic mice after the treatment with G-CSF did not show any increase in the number of autophagosomes, which supports the hypothesis that autophagy is impaired in diabetes and might be linked to impaired mobilization. Next, we evaluated the effects of spermidine or β-hydroxybutyrate on mobilization in diabetic mice co-treated with G-CSF. By assessing the Lineageˉ c-kit⁺ Sca1⁺ cells in peripheral blood, which are the murine counterpart of human CD34⁺ HSPCs, we have found a partial restoration in G-CSF-derived mobilization. Also, the CFU assay, which can detect functionally competent HSPCs, shows that the mobilization by G-CSF in T1D mice seems partially restored after the co-treatment spermidine or β-hydroxybutyrate treatment. Furthermore, by re-analyzing a publicly available bulk and single cell-RNAseq dataset, sorted human neutrophils we showed that autophagy-related genes were modulated after G-CSF treatment. According to our data, G-CSF is ineffective in regulating autophagy in bone marrow neutrophils in diabetic mice. This may contribute to impaired HSPCs mobilization. However, we found that inducing autophagy with spermidine or bypassing it with β-hydroxybutyrate can partially restore mobilization. Finally, bulk RNAseq and single cell RNAseq analysis on human neutrophils showed how autophagy is also modulated by G-CSF in humans, suggesting the translational potential of our research.