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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 Article has an altmetric score of 2

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

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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 6

Increased in vitro migration, membrane translocation, and Btk-dependent phosphorylation of WASp in XLN neutrophils.

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Increased in vitro migration, membrane translocation, and Btk-dependent ...
(A–C) Migration of WT vs. WASp L272P and WASp I296T bone marrow neutrophils toward the indicated chemoattractants in Transwell plates. Measured with flow cytometry. n = 3 replicates, n = 3 experiments/chemoattractant. (D and E) WT (CD45.1) and WASp XLN (CD45.2) neutrophils were mixed at a 1:1 ratio in the upper well of a Transwell plate and incubated for 10 minutes with inhibitors. Migration rate toward C5a or CXCL1 was plotted. n = 2 experiments. (F) WASp intracellular expression was measured by imaging flow cytometry. Ratios of plasma membrane proximal (determined by Ly6G surface staining) WASp and WASp in the total cell was plotted. Data are shown as mean + SEM of at least 198 neutrophils. n = 3 experiments. (G) WASp and phospho-Y293 WASp in SRC1- and Wortmannin-pretreated neutrophils were measured by Western blot. n = 2. (H) WASp and phospho-Y293 WASp in WASp L272P neutrophils before and after ibrutinib treatment. n = 3. (I) WASp Y293 phosphorylation upon CXCL1 activation with or without ibrutinib treatment (flow cytometry). n = 3. (J) Transwell migration of WT and WASp I296T neutrophils toward C5a (0.125 μg/ml), fMLP (3 μM), and CXCL1 (0.25 μg/ml) after incubation with ibrutinib. n = 3. (K) Ratio of motile cells among adherent cells on rmP-selectin, rmICAM-1, and rmCXCL-1 under flow as in Figure 5G. (L) Numbers of arrested cells as percentages of arrested cells at 0.1 dyn/cm2 as in Figure 5E. (M) Ratio of spread cells at increasing flow rate as in Figure 5F. n = 3 replicates, n = 2 experiments. Data are represented as mean ± SEM. Two-way ANOVA was used for statistical significance. **P < 0.01; ***P < 0.001; ****P < 0.0001.

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