Wiskott–Aldrich Syndrome Protein Deficiency in Innate Immune Cells Leads to Mucosal Immune Dysregulation and Colitis in Mice

doi:10.1053/j.gastro.2012.06.008

Gastroenterology

Volume 143, Issue 3, September 2012, Pages 719-729.e2

https://doi.org/10.1053/j.gastro.2012.06.008 Get rights and content

Background & Aims

Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott–Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell–mediated colitis in mice with WASP-deficient cells of the innate immune system.

Methods

Naïve and/or regulatory CD4⁺ T cells were transferred from 129 SvEv mice into RAG-2–deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays.

Results

Transfer of unfractionated CD4⁺ T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103⁺ tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice.

Conclusions

Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.

Section snippets

Mice

WT, RAG KO, WKO,¹⁵ and WRDKO mice,¹⁶ all on a pure 129 SvEv background, were maintained in specific pathogen-free animal facilities at Massachusetts General Hospital (Boston, MA). Foxp3GFP transgenic mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All experiments were conducted on approval and according to regulations of the Subcommittee on Research Animal Care of the Massachusetts General Hospital.

Please see Supplementary Materials and Methods for information on cell transfer,

WASP-Deficient Innate Immune Cells Induce Colitogenicity in WT CD4⁺ T Cells

We previously reported that, in contrast to WKO mice, WASP/RAG-2 double knockout (WRDKO) mice on a 129 SvEv background do not develop spontaneous colitis.¹⁶ We hypothesized that the colitis in WKO mice, although lymphocyte dependent, might be initiated by defects in the innate immune compartment. To investigate whether WASP-deficient innate immune cells could induce WT CD4⁺ T cells to be colitogenic, we transferred unfractionated WT or WKO CD4⁺ T cells into WRDKO mice (Figure 1A). These

Discussion

WASP deficiency in humans and mice is associated with both immunodeficiency and autoimmune sequelae.¹¹ Inflammatory diseases in the setting of a primary genetic immunodeficiency, while perhaps seeming paradoxical, are quite common.³⁷ Because WASP expression is essential for the function of most leukocyte subtypes, the specific contribution of the adaptive and innate immune system to the pathogenesis of the associated autoimmunity has been unclear. Although Treg dysfunction has been noted in

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Citation Excerpt :
Levels of G-CSF were lower in WKO sera (P < .05), further excluding an increase in granulopoiesis and BM output (see Fig E1, C). Because both defective phagocytosis and a proinflammatory environment coexist in the setting of WAS,29-31 we reasoned that neutrophil accumulation might be due to lack of clearance, causing persistence of inflammatory signals normally ceased by phagocytosis.32-34 To evaluate phagocytosis in vivo, we adoptively transferred CFSE-labeled WT neutrophils into WT or WKO recipients.
Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined.
Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS.
We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens.
Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase.
These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.
Uncovering Pathogenic Mechanisms of Inflammatory Bowel Disease Using Mouse Models of Crohn's Disease–Like Ileitis: What is the Right Model?
2017, Cellular and Molecular Gastroenterology and Hepatology
Crohn’s disease and ulcerative colitis, together known as inflammatory bowel disease, are debilitating chronic disorders of unknown cause and cure. Our evolving understanding of these pathologies is enhanced greatly by the use of animal models of intestinal inflammation that allow in vivo mechanistic studies, preclinical evaluation of new therapies, and investigation into the causative factors that underlie disease pathogenesis. Several animal models, most commonly generated in mice, exist for the study of colitis. The appropriateness of their use often can be determined by their mode of generation (ie, chemical induction, T-cell transfer, targeted genetic manipulation, spontaneously occurring, and so forth), the type of investigation (mechanistic studies, pathogenic experiments, preclinical evaluations, and so forth), and the type of inflammation that occurs in the model (acute vs chronic colitis, tissue injury/repair, and so forth). Although most murine models of inflammatory bowel disease develop inflammation in the colon, Crohn’s disease most commonly occurs in the terminal ileum, where a very limited number of mouse models manifest disease. This review discusses appropriate experimental applications for different mouse models of colitis, and highlights the particular utility of 2 highly relevant models of Crohn’s-like ileitis—the spontaneous SAMP1/YitFc inbred mouse strain and the genetically engineered Tnf^{ΔAU-rich element/+} mouse model of tumor necrosis factor overexpression, both of which bear strong resemblance to the human condition. Similar to patients with Crohn’s disease, SAMP1/YitFc ileitis develops spontaneously, without chemical, genetic, or immunologic manipulation, making this model particularly relevant for studies aimed at identifying the primary defect underlying the occurrence of Crohn’s ileitis, as well as preclinical testing of novel treatment modalities.
Absence of WASp enhances hematopoietic and megakaryocytic differentiation in a human embryonic stem cell model
2016, Molecular Therapy
Citation Excerpt :
Absence of WASp in these cell types alters actin polymerization causing problems on signaling, proliferation, migration, and phagocytosis. WAS-deficient mouse models6,7,8 and cell lines9,10 have been fundamental for the understanding of WASp function in all these cell types. However, WAS knockout (WASKO) mouse models do not mimic the Plts defects found in WAS patients making it difficult to study the function of WASp in MKs and Plts physiology.11
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34⁺CD45⁺ progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34⁺CD41⁺ progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.
Maintaining Intestinal Health: The Genetics and Immunology of Very Early Onset Inflammatory Bowel Disease
2015, Cellular and Molecular Gastroenterology and Hepatology
Citation Excerpt :
Nguyen et al91 identified that intestinal inflammation in WASP-deficient mice was critically dependent upon inflammatory T cells, which may result from impaired development of regulatory T cells (Tregs) in the thymus and periphery.92 Surprisingly, these defects are likely occurring in a cell-extrinsic manner, as the absence of WASP in cells of the innate immune system directly contributed to the development of inflammatory T-cell responses in mice.93 The causes of intestinal inflammation in other similar patient populations are less well understood, but defects in regulatory T cells, IgA, and abnormal selection of T-cell and B-cell specificities likely contribute.
Inflammatory bowel disease (IBD) is a multifactoral disease caused by dysregulated immune responses to commensal or pathogenic microbes in the intestine, resulting in chronic intestinal inflammation. An emerging population of patients with IBD younger than 5 years of age represent a unique form of disease, termed very early onset IBD (VEO-IBD), which is phenotypically and genetically distinct from older-onset IBD. VEO-IBD is associated with increased disease severity, aggressive progression, and poor responsiveness to most conventional therapies. Further investigation into the causes and pathogenesis of VEO-IBD will help improve treatment strategies and may lead to a better understanding of the mechanisms that are essential to maintain intestinal health or provoke the development of targeted therapeutic strategies to limit intestinal inflammation and promote tissue repair. Here, we discuss the phenotypic nature of VEO-IBD, the recent identification of novel gene variants associated with disease, and functional immunologic studies interrogating the contribution of specific genetic variants to the development of chronic intestinal inflammation.
Loss of estrogen-mediated immunoprotection underlies female gender bias in experimental Crohn's-like ileitis
2014, Mucosal Immunology
The incidence and severity of Crohn's disease (CD) are increased in female patients. Using SAMP1/YitFc (SAMP) mice, a spontaneous model of chronic intestinal inflammation that displays histologic and pathogenic similarities to human CD, we investigated the potential mechanism(s) contributing to sex differences observed in CD. Similar to gender differences observed in CD patients, SAMP female (SAMP-F) mice displayed an earlier onset and more severe ileitis compared with SAMP male (SAMP-M) mice. Furthermore, T-regulatory cells (Tregs) from gut-associated lymphoid tissue (GALT) of SAMP-F mice were reduced in frequency and impaired in their in vitro and in vivo suppressive functions compared with that of SAMP-M mice. Given the interaction between sex hormones and Treg function, we investigated the possible role of estrogen (E2) in SAMP ileitis. SAMP-M mice responded to exogenous E2 administration by expanding Treg frequency and reducing ileal inflammation, whereas SAMP-F mice were resistant. Conventional T cells and Tregs responded differentially to estrogen signaling, leading to distinct immunoprotective effects mediated by distinct estrogen receptor (ER) isoforms. These mechanisms were impaired in T cells from SAMP-F mice. Thus, hormone signaling influences the expansion and function of GALT Tregs in an ER-dependent manner and contributes to gender-based differences in experimental CD.
Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function
2014, Immunity
Citation Excerpt :
These results define a unique and nonredundant role for IL-10R signaling in innate immune cell control of intestinal mucosal homeostasis. We have recently reported that aberrant interactions between innate immune cells devoid of the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASP) and WT CD4+ T cells lead to colitis development (Nguyen et al., 2012a). In this model, Was−/−Rag2−/− mice develop severe intestinal inflammation following WT CD4+ T cell transfer, characterized by reduced production of IL-10; colitis development can be prevented by exogenous administration of IL-10Ig.
Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4⁺ T cell transfer, Rag2^−/−Il10rb^−/− mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb^−/− anti-inflammatory macrophages ameliorated colitis induction by WT CD4⁺ T cells in Rag2^−/−Il10rb^−/− mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by grants from the National Institutes of Health to S.B.S. (HL59561, DK034854, and AI50950), to D.D.N. (DK080516 and DK083430), and to B.H.H. (AI52267), and grants from the Crohn's and Colitis Foundation of America to D.D.N. (CDA 2193) and M.-A.W. (CDA 2821), and the National Institutes of Health–sponsored Harvard Digestive Disease Center (P30-DK034854).

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Original ResearchBasic and Translational—Alimentary TractWiskott–Aldrich Syndrome Protein Deficiency in Innate Immune Cells Leads to Mucosal Immune Dysregulation and Colitis in Mice

Background & Aims

Methods

Results

Conclusions

Section snippets

Mice

WASP-Deficient Innate Immune Cells Induce Colitogenicity in WT CD4+ T Cells

Discussion

Adv Drug Deliv Rev

Immunity

Immunity

Cancer Cell

Immunity

Immunity

Curr Opin Immunol

Gastroenterology

Clin Immunol

Blood

Blood

Blood

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Wiskott-Aldrich syndrome and inflammatory bowel disease

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Original Research
Basic and Translational—Alimentary Tract
Wiskott–Aldrich Syndrome Protein Deficiency in Innate Immune Cells Leads to Mucosal Immune Dysregulation and Colitis in Mice

WASP-Deficient Innate Immune Cells Induce Colitogenicity in WT CD4⁺ T Cells