Constitutive activation of WASp in X-linked neutropenia renders neutrophils hyperactive
Marton Keszei, … , Scott B. Snapper, Lisa S. Westerberg
Marton Keszei, … , Scott B. Snapper, Lisa S. Westerberg
Published August 20, 2018
Citation Information: J Clin Invest. 2018;128(9):4115-4131. https://doi.org/10.1172/JCI64772.
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Research Article Immunology

Constitutive activation of WASp in X-linked neutropenia renders neutrophils hyperactive

Abstract

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.

Authors

Marton Keszei, Julien Record, Joanna S. Kritikou, Hannah Wurzer, Chiara Geyer, Meike Thiemann, Paul Drescher, Hanna Brauner, Laura Köcher, Jaime James, Minghui He, Marisa A.P. Baptista, Carin I.M. Dahlberg, Amlan Biswas, Sonia Lain, David P. Lane, Wenxia Song, Katrin Pütsep, Peter Vandenberghe, Scott B. Snapper, Lisa S. Westerberg

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Figure 7

Increased phagocytosis rate, normal killing of bacteria, and dysregulated ROS responses in XLN neutrophils.

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Increased phagocytosis rate, normal killing of bacteria, and dysregulate...
E. coli (A) and S.aureus (B) killing capacity of WT and WASp XLN neutrophils were assessed by coincubating serum-opsonized bacteria and neutrophils at a ratio of 1:5 (E. coli/neutrophil) for 60 minutes (A) or 1:1 (S.aureus/neutrophil) for 60 and 90 minutes as shown in upper and lower panels, respectively (B). n = 3 replicates, n = 3 experiments with each bacteria. (C and D) Phagocytosis of Alexa Fluor 488–labeled, serum-opsonized E. coli (C) and S. aureus (D) by WT WASp XLN neutrophils measured by flow cytometry. n = 3 replicates, n = 3 experiments with each bacteria. (E–G) Intracellular ROS (IC ROS) measured with luminol chemiluminescence in murine bone marrow neutrophils upon stimulation with E. coli (n = 8) (E), S.aureus (n = 10) (F), or PMA (n = 18). n = 4. Data are shown as mean ± SD, 2-way ANOVA. (H–J) Extracellular ROS (EC ROS) was measured using a lucigenin chemiluminescence assay in murine bone marrow neutrophils upon stimulation with (H) heat-killed serum-opsonized S. aureus (n = 18), (I) fMLP (n = 4), or (J) PMA (n = 26). n = 4. Data are shown as mean ± SD. Two-way ANOVA. (K) Extracellular ROS was measured upon PMA stimulation of murine (upper panel) and human healthy donor (lower panel) neutrophils with or without pretreatment with jasplakinolide. n = 5. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.