The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb

Abstract

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.

Keywords: 1,5-anhydroglucitol-6-phosphate; 1,5AG, 1,5-anhydroglucitol; 1,5AG6P, 1,5-anhydroglucitol-6-phosphate; BM, bone marrow; CFU, colony forming units; Dapagliflozin; G-CSF, granulocyte colony stimulating factor; G6PC3, glucose-6-phosphatase C3; G6PT, glucose-6-phospate translocase; GSDIb, Glycogen Storage Disease type 1b; Glycogen storage disease type 1b; M-CSF, macrophage colony stimulating factor; Mouse model; NET, neutrophil extracellular trap; Neutrophils; PMA, phorbol myristate acetate; PRM, parallel reaction monitoring; Renal sodium-glucose co-transporter-2; SGLT2, sodium-glucose co-transporter-2; TM, tamoxifen; fMLP, N-formyl-L-methionyl-L-leucyl-phenylalanine.

Fig. 1

Evaluation of the impact of dapagliflozin on myeloid cell functions in a GSD1b mouse model. (a) Morphological evaluation of neutrophils and their precursors. Left panel: neutrophil maturation state was determined by microscopy examination (200× magnification). Right panel: results are shown as bar graphs. p-Values of TM-G6PT^+/+ or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice: **p ≤ 0.01. (b) Phagocytosis activity of neutrophils and their precursors. Left panel: uptake of E. coli by neutrophils was evaluated by immunofluorescence after incubation at 37 °C for 60 min. Right panel: results are shown as a bar graph and expressed as percentages of cells internalizing E. coli. p-Values of TM-G6PT^+/+ or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice: *p ≤ 0.05; ***p ≤ 0.001. (c) Chemotactic activity of neutrophils and their precursors measured in response to fMLP and CXCL2 by microscopy examination (40× magnification). Results are shown as a bar graph by counting the number of labelled cells on plate-well membrane. p-Values of TM-G6PT^+/+ or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice: *p ≤ 0.05; **p ≤ 0.01 ***p ≤ 0.001 (d-e). Colony-forming unit (CFU) was evaluated following the stimulation of bone marrow cells with G-CSF (d) or M-CSF (e). Results are shown as a bar graph and expressed as number of scored colonies after 7–10 days of culture. p-Values of TM-G6PT^+/+ or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice *p ≤ 0.05; **p ≤ 0.01 ***p ≤ 0.001. (f) Apoptosis of neutrophils and their precursors. Left panel: cells were stained with TUNEL (green) to reveal apoptotic cells and nuclei were stained with DAPI (blue). Apoptotic cells are indicated by the arrowheads. Right panel: results are shown as a bar graph and expressed as percentages of apoptotic cells. p-Values of TM-G6PT^+/+ (CTR) or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice: *p ≤ 0.05. (g) NETs formation of neutrophils and their precursors. Left panel: neutrophils were treated with the membrane-impermeable DNA binding dye SYTOX green to reveal NET formation. Right panel: NETs were evaluated by counting the percentage of NETs positive cells versus the total number of cells following a 2 h stimulation with 100 nM PMA. p-Values of PMA stimulated cells relative to unstimulated cells derived from TM-G6PT^+/+ and treated (+DAPA) or untreated (-DAPA) TM-G6PT^−/− mice: *p ≤ 0.05. (h) Calcium release in neutrophils and their precursors. Left panel: representative traces of calcium release in Fura-2-loaded-cells derived from TM-G6PT^+/+ and dapagliflozin treated (+DAPA) or untreated (-DAPA) TM-G6PT^−/− mice and stimulated with fMLP. Right panel: results are shown as a bar graph and expressed as quantification of calcium release. The mean ± SEM of the AUC of three different experiments is shown. (i) quantification of 1,5AG and 1,5AG6P in neutrophils and their precursors. Results are shown as a before-after graph and expressed as quantification of both metabolites. The mean ± SEM of the AUC of three different experiments is shown. p-Values of TM-G6PT^+/+ or dapagliflozin treated (+DAPA) relative to untreated (-DAPA) TM-G6PT^−/− mice: *p ≤ 0.05;**p ≤ 0.01. Images shown are representative of three experiments. Each value represents the mean of the measurement of three or four mice.