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The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Key Points Wiskott–Aldrich Syndrome protein (WASP) is an important regulator of the actin cytoskeleton in haematopoietic cells. WASP-deficiency gives rise to the human disease Wiskott–Aldrich Syndrome (WAS), an X-linked primary immunodeficiency. Constitutively active mutations of WASP have recently been described to give rise to a distinct human disease: X-linked neutropenia. WASP activity is attenuated by multiple signalling pathways downstream of surface receptors. Conformational change, phosphorylation and degradation are important mechanisms. Although WASP does not seem to have a key role in haematopoiesis, WASP confers selective advantage for many mature haematopoietic cell types and is emerging as a key regulator of lymphocyte homeostasis. WASP is required for a diverse range of cell functions in innate and adaptive immune cells. These relate both to the role of WASP in cytoskelatal rearrangement and as an intrinsic signalling molecule. Autoimmunity is an important feature of WAS which is poorly understood. Recent studies suggest that defective regulatory T cell function is an important component. Insights into the basic mechanisms of WASP-associated disease are advancing our understanding of immune regulation with wider application, and this allows the potential for future therapeutic benefit. As a key regulator of the actin cytoskeleton, Wiskott–Aldrich syndrome protein (WASP) is involved in diverse immune responses, including leukocyte migration and activation. This Review describes how various mutations in mice and humans have led us to a greater appreciation of the many immunological functions of WASP. The Wiskott–Aldrich syndrome protein (WASP) is an important regulator of the actin cytoskeleton that is required for many haematopoietic and immune cell functions, including effective migration, phagocytosis and immune synapse formation. Loss of WASP activity leads to Wiskott–Aldrich syndrome, an X-linked disease that is associated with defects in a broad range of cellular processes, resulting in complex immunodeficiency, autoimmunity and microthrombocytopenia. Intriguingly, gain of function mutations cause a separate disease that is mainly characterized by neutropenia. Here, we describe recent insights into the cellular mechanisms of these two related, but distinct, human diseases and discuss their wider implications for haematopoiesis, immune function and autoimmunity.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.

Wiskott-Aldrich syndrome protein (WASp) is essential for optimal T cell activation. Patients with WAS exhibit both immunodeficiency and a marked susceptibility to systemic autoimmunity. We investigated whether alterations in Treg function might explain these paradoxical observations. While WASp-deficient (WASp(-/-)) mice exhibited normal thymic Treg generation, the competitive fitness of peripheral Tregs was severely compromised. The total percentage of forkhead box P3-positive (Foxp3(+)) Tregs among CD4(+) T cells was reduced, and WASp(-/-) Tregs were rapidly outcompeted by WASp(+) Tregs in vivo. These findings correlated with reduced expression of markers associated with self-antigen-driven peripheral Treg activation and homing to inflamed tissue. Consistent with these findings, WASp(-/-) Tregs showed a reduced ability to control aberrant T cell activation and autoimmune pathology in Foxp3(-/-)Scurfy (sf) mice. Finally, WASp(+) Tregs exhibited a marked selective advantage in vivo in a WAS patient with a spontaneous revertant mutation, indicating that altered Treg fitness likely explains the autoimmune features in human WAS.

Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott–Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative “tumor-suppressor” protein in normal T and B cells, and “oncoprotein” in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.

In T lymphocytes, the Wiskott–Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase–positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation. Although IL-23 is expressed by psoriatic keratinocytes as well as immune cells, only the immune cell derived IL-23 is thought to be important for the development of psoriasis. Here the authors provide evidence that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation.

Wiskott–Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8 + T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFNγ-producing CD8 + T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8 + T cells at the expense of CD4 + T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8 + T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells. Wiskott–Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency syndrome. Here, the authors show that loss of WAS gene in dendritic cells results in increased activity of Rac2, increase of phagosomal pH, and more efficient cross-presentation.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

Comprehensive knowledge of the host factors required for picornavirus infection would facilitate antiviral development. Here we demonstrate roles for three human genes, TNK2, WASL, and NCK1, in infection by multiple picornaviruses. CRISPR deletion of TNK2, WASL, or NCK1 reduced encephalomyocarditis virus (EMCV), coxsackievirus B3 (CVB3), poliovirus and enterovirus D68 infection, and chemical inhibitors of TNK2 and WASL decreased EMCV infection. Reduced EMCV lethality was observed in mice lacking TNK2. TNK2, WASL, and NCK1 were important in early stages of the viral lifecycle, and genetic epistasis analysis demonstrated that the three genes function in a common pathway. Mechanistically, reduced internalization of EMCV was observed in TNK2 deficient cells demonstrating that TNK2 functions in EMCV entry. Domain analysis of WASL demonstrated that its actin nucleation activity was necessary to facilitate viral infection. Together, these data support a model wherein TNK2, WASL, and NCK1 comprise a pathway important for multiple picornaviruses.