Finding balance between mature and immature neutrophils: The effects of empagliflozin in GSD‐Ib

To the Editor, Glucose-6-phosphate (G6P) translocase (G6PT/SLC37A4) is an ubiquitously expressed enzyme and is required for the conversion of G6P to glucose, thus ensuring glucose production by the liver and kidney [1]. Biallelic loss of function in G6PT/SLC37A4 causes glycogen storage disease type Ib (GSD-Ib), a rare disease with an incidence of ̴1/100,000. GSD-Ib is characterized by severe hypoglycemia, growth retardation, osteoporosis and long-term risk of liver tumours and kidney failure [2]. In addition, individuals with G6PT deficiency also develop neutropenia, together with recurrent bacterial infections, gingivitis, periodontitis, genital and intestinal ulcers as a result of defective phagocytic function [1]. Neutropenia and neutrophil dysfunction in GSD-Ib have been recently ascribed to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5AG6P) that, by inhibiting the activity of hexokinases, limits the phosphorylation of glucose and derails the glycolytic pathway, essential for the immunometabolic activation and the patrolling activity function of these cells [3]. These findings set the stage for testing empagliflozin, an inhibitor of the kidney sodium glucose cotransporter 2 (SGLT2), which also lowers serum 1,5AG6P inGSD-Ibpatients.Data inG6PC3-deficientmice and in a few G6PC3or G6PTdeficient patients have shown that empagliflozin, by lowering serum 1,5-AG and neutrophil 1,5 AG6P, improves neutrophil count and function [4]. This finding is in linewith the deep connection between immune cell function and cellularmetabolism [5]. Neutrophils change their demand of glucose over their maturation from immature to mature cells [6, 7]. Hence, it was speculated that empagliflozin, beyond the mere effect in increasing absolute neutrophil count (ANC), might promote the polarization toward specific neutrophil subtypes in patients affected by GSD-Ib [8]. As mature neutrophils are co-opted in lungs, gastrointestinal tract, and skin [7], investigating the differential neutrophil polarization following the treatment with empagliflozin in patients withG6PTdeficiencymay shed a light into the pharmacological benefit of themechanism of gliflozins in GSD-Ib patients [9]. To this aim, four GSD1b patients (9–16 years old), harboring biallelic SLC37A4 variants and with ongoing granulocyte colonystimulating factor (G-CSF) treatment, were enrolled for this in the study (Table 1). Theywere clinically evaluated at baseline and3months after empagliflozin treatment. Neutrophil subsets distribution in the


Finding balance between mature and immature neutrophils: The effects of empagliflozin in GSD-Ib
To the Editor, Glucose-6-phosphate (G6P) translocase (G6PT/SLC37A4) is an ubiquitously expressed enzyme and is required for the conversion of G6P to glucose, thus ensuring glucose production by the liver and kidney [1]. Biallelic loss of function in G6PT/SLC37A4 causes glycogen storage disease type Ib (GSD-Ib), a rare disease with an incidence of ̴ 1/100,000. GSD-Ib is characterized by severe hypoglycemia, growth retardation, osteoporosis and long-term risk of liver tumours and kidney failure [2].
In addition, individuals with G6PT deficiency also develop neutropenia, together with recurrent bacterial infections, gingivitis, periodontitis, genital and intestinal ulcers as a result of defective phagocytic function [1]. Neutropenia and neutrophil dysfunction in GSD-Ib have been recently ascribed to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5AG6P) that, by inhibiting the activity of hexokinases, limits the phosphorylation of glucose and derails the glycolytic pathway, essential for the immunometabolic activation and the patrolling activity function of these cells [3]. These findings set the stage for testing empagliflozin, an inhibitor of the kidney sodium glucose cotransporter 2 (SGLT2), which also lowers serum 1,5AG6P in GSD-Ib patients. Data in G6PC3-deficient mice and in a few G6PC3-or G6PT-deficient patients have shown that empagliflozin, by lowering serum 1,5-AG and neutrophil 1,5 AG6P, improves neutrophil count and function [4].
This finding is in line with the deep connection between immune cell function and cellular metabolism [5]. Neutrophils change their demand of glucose over their maturation from immature to mature cells [6,7].
Hence, it was speculated that empagliflozin, beyond the mere effect in increasing absolute neutrophil count (ANC), might promote the polarization toward specific neutrophil subtypes in patients affected by GSD-Ib [8]. As mature neutrophils are co-opted in lungs, gastrointestinal tract, and skin [7], investigating the differential neutrophil polarization following the treatment with empagliflozin in patients with G6PT deficiency may shed a light into the pharmacological benefit of the mechanism of gliflozins in GSD-Ib patients [9].
To this aim, four GSD1b patients (9-16 years old), harboring biallelic SLC37A4 variants and with ongoing granulocyte colonystimulating factor (G-CSF) treatment, were enrolled for this in the study (Table 1) Figure 1D). Empagliflozin did not affect circulating pre-neutrophils (p = 0.869; Figure 1E).  and in the reduction of the immature subsets [8]. Our results confirm and extend the findings of Wortmann et al. [8], who proposed that the reduction in the occurrence of infections in G6PT deficient patients treated with empagliflozin could be related to the reduction of 1,5AG6P and to the improvement of glycolysis in mature neutrophils.
Our data confirm Wortmann hypothesis, showing that the improvement in total ANC reflects in the increase of mature neutrophils and results in reduced infections as observed in the only patient in our cohort that had had recurrent infections.
Our data also show the reduction of immature neutrophils following treatment with empagliflozin, which have been found to be increased in G6PT patients [10]. Neutrophil dysfunction has been proposed as a main driver of inflammation in this condition. Recently, it has been reported that empagliflozin may reduce the burden of inflammatory conditions in GSD-Ib patients [4,8] and we observed an improvement in IBD in two patients, it is tempting to speculate that the benefit on inflammation resulting from the treatment with empagliflozin n GSD-Ib patients could be related to the reduction of this specific neutrophil subset, known to increase during either chronic and acute immunoinflammatory stresses [11].
In summary, our report reinforces the knowledge about the beneficial effects of empagliflozin in GSD-Ib patients, highlighting, for the first time, that its clinical efficacy might also be linked to changes in the balance between mature and immature neutrophils. Larger multicenter studies with longer follow-up are required to confirm these findings.

AUTHOR CONTRIBUTIONS
FG, AB and FS contributed to conception and design of the study and wrote the first draft of the manuscript. AB performed neutrophils subsets analysis. All authors contributed to the article and approved the submitted version.