Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib

Abstract

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that works with a liver/kidney/intestine-restricted glucose-6-phosphatase-alpha (G6Pase-alpha) to maintain glucose homeostasis between meals. Clinically, GSD-Ib patients manifest disturbed glucose homeostasis and neutrophil dysfunctions but the cause of the latter is unclear. Neutrophils express the ubiquitously expressed G6PT and G6Pase-beta that together transport G6P into the endoplasmic reticulum (ER) lumen and hydrolyze it to glucose. Because we expected G6PT-deficient neutrophils to be unable to produce endogenous glucose, we hypothesized this would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibited increased production of ER chaperones and oxidative stress, consistent with ER stress, increased annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of proapoptotic effectors, and caspase-9 activation demonstrated the involvement of the intrinsic mitochondrial pathway in these processes. The results demonstrate that G6P translocation and hydrolysis are required for normal neutrophil functions and support the hypothesis that neutrophil dysfunction in GSD-Ib is due, at least in part, to ER stress and increased apoptosis.

Figure 1

Increase in the expression of ER chaperones in neutrophils of GSD-Ib mice. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Hema 3–stained cytospins of peritoneal neutrophils. (B) Western-blot analysis of protein extracts of peritoneal neutrophils using antibodies against gelatinase, Gr-1, β-actin, or GAPDH. Each lane contains 50 μg protein. (C) Western-blot analysis of protein extracts of neutrophils using antibodies against GRP78, GRP170, PDI, or β-actin. Each lane contains 50 μg protein.

Figure 2

GSD-Ib neutrophils display increased rate of apoptosis. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative flow cytometric analysis of neutrophil annexin V (x-axis) binding and PI (y-axis) uptake. Numbers on the plots are percentages of total cells. (B) Quantification of annexin V–positive neutrophils determined by immunofluorescence staining. (C) Quantification of caspase-3–positive neutrophils determined by immunofluorescence staining. (D) The DEVD-cleaving activity of active caspase-3 in protein extracts of peritoneal neutrophils. Data represent the means (± SEM) of 3 independent experiments. **P < .001.

Figure 3

GSD-Ib neutrophils exhibit oxidative stress. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative flow cytometric analysis of neutrophil carboxy-DCF staining. (B) Quantification of carboxy-DCF–positive neutrophils determined by immunofluorescence staining. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (C) Oxyblot analysis of carbonyl groups in oxidative modified proteins. Each lane contains 50 μg protein. The vertical line has been inserted to indicate a repositioned gel lane. (D) Representative flow cytometric analysis of neutrophil R-123 staining. (E) Quantification of R-123–positive neutrophils determined by immunofluorescence staining. Data represent the means (± SEM) of 3 independent experiments. **P < .001. (F) Western blot analysis of protein extracts of neutrophils using antibodies against Mn-SOD or β-actin. Each lane contains 50 μg protein.

Figure 4

Bax activation in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative immunofluorescence of Bax staining (green fluorescence), Hoechst 33342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Bax-positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (B) Western blot analysis of protein extracts of neutrophils using antibodies against Bax, β-actin, or GAPDH. Each lane contains 50 μg protein.

Figure 5

Increased accumulation and release of proapoptotic factors in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative immunofluorescence of Smac/Diablo staining (green fluorescence), Hoechst 33342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Smac/Diablo-positive neutrophils in unaffected and GSD-Ib mice. (B) Western-blot analysis of protein extracts of neutrophils using antibodies against Smac/Diablo, β-actin or GAPDH. Each lane contains 50 μg protein. (C) Representative immunofluorescence of Omi/HtrA2 staining (green fluorescence), Hoechst 33 342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Omi/HtrA2-positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (D) Western blot analysis of protein extracts of neutrophils using antibodies against Omi/HtrA2, β-actin, or GAPDH. Each lane contains 50 μg protein.

Figure 6

Caspase-9 activation in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Immunofluorescence of active caspase-9 staining (green fluorescence) and Hoechst 33342 nuclei staining (blue fluorescence), magnification ×400. (B) Quantification of caspase-9–positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001.