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1.
J Allergy Clin Immunol ; 140(6): 1604-1615.e5, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28216433

RESUMO

BACKGROUND: A number of food allergies (eg, fish, shellfish, and nuts) are lifelong, without any disease-transforming therapies, and unclear in their underlying immunology. Clinical manifestations of food allergy are largely mediated by IgE. Although persistent IgE titers have been attributed conventionally to long-lived IgE+ plasma cells (PCs), this has not been directly and comprehensively tested. OBJECTIVE: We sought to evaluate mechanisms underlying persistent IgE and allergic responses to food allergens. METHODS: We used a model of peanut allergy and anaphylaxis, various knockout mice, adoptive transfer experiments, and in vitro assays to identify mechanisms underlying persistent IgE humoral immunity over almost the entire lifespan of the mouse (18-20 months). RESULTS: Contrary to conventional paradigms, our data show that clinically relevant lifelong IgE titers are not sustained by long-lived IgE+ PCs. Instead, lifelong reactivity is conferred by allergen-specific long-lived memory B cells that replenish the IgE+ PC compartment. B-cell reactivation requires allergen re-exposure and IL-4 production by CD4 T cells. We define the half-lives of antigen-specific germinal centers (23.3 days), IgE+ and IgG1+ PCs (60 and 234.4 days, respectively), and clinically relevant cell-bound IgE (67.3 days). CONCLUSIONS: These findings can explain lifelong food allergies observed in human subjects as the consequence of allergen exposures that recurrently activate memory B cells and identify these as a therapeutic target with disease-transforming potential.


Assuntos
Anafilaxia/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Hipersensibilidade Alimentar/imunologia , Células Th2/imunologia , Alérgenos/imunologia , Animais , Arachis/imunologia , Células Cultivadas , Humanos , Imunidade Humoral , Imunoglobulina E/metabolismo , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
Biochim Biophys Acta ; 1852(9): 1846-55, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26073433

RESUMO

Constitutive Wnt/ß-catenin signaling is a key contributor to colorectal cancer (CRC). Although inactivation of the tumor suppressor adenomatous polyposis coli (APC) is recognized as an early event in CRC development, it is the accumulation of multiple subsequent oncogenic insults facilitates malignant transformation. One potential contributor to colorectal carcinogenesis is the POZ-ZF transcription factor Kaiso, whose depletion extends lifespan and delays polyp onset in the widely used Apc(Min/+) mouse model of intestinal cancer. These findings suggested that Kaiso potentiates intestinal tumorigenesis, but this was paradoxical as Kaiso was previously implicated as a negative regulator of Wnt/ß-catenin signaling. To resolve Kaiso's role in intestinal tumorigenesis and canonical Wnt signaling, we generated a transgenic mouse model (Kaiso(Tg/+)) expressing an intestinal-specific myc-tagged Kaiso transgene. We then mated Kaiso(Tg/+) and Apc(Min/+) mice to generate Kaiso(Tg/+):Apc(Min/+) mice for further characterization. Kaiso(Tg/+):Apc(Min/+) mice exhibited reduced lifespan and increased polyp multiplicity compared to Apc(Min/+) mice. Consistent with this murine phenotype, we found increased Kaiso expression in human CRC tissue, supporting a role for Kaiso in human CRC. Interestingly, Wnt target gene expression was increased in Kaiso(Tg/+):Apc(Min/+) mice, suggesting that Kaiso's function as a negative regulator of canonical Wnt signaling, as seen in Xenopus, is not maintained in this context. Notably, Kaiso(Tg/+):Apc(Min/+) mice exhibited increased inflammation and activation of NFκB signaling compared to their Apc(Min/+) counterparts. This phenotype was consistent with our previous report that Kaiso(Tg/+) mice exhibit chronic intestinal inflammation. Together our findings highlight a role for Kaiso in promoting Wnt signaling, inflammation and tumorigenesis in the mammalian intestine.

3.
Front Cell Dev Biol ; 10: 998373, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36274850

RESUMO

The function and structure of the mammalian epithelial cell layer is maintained by distinct intercellular adhesion complexes including adherens junctions (AJs), tight junctions, and desmosomes. The AJ is most integral for stabilizing cell-cell adhesion and conserving the structural integrity of epithelial tissues. AJs are comprised of the transmembrane protein E-cadherin and cytoplasmic catenin cofactors (α, ß, γ, and p120-catenin). One organ where malfunction of AJ is a major contributor to disease states is the mammalian intestine. In the intestine, cell-cell adhesion complexes work synergistically to maintain structural integrity and homeostasis of the epithelium and prevent its malfunction. Consequently, when AJ integrity is compromised in the intestinal epithelium, the ensuing homeostatic disruption leads to diseases such as inflammatory bowel disease and colorectal carcinoma. In addition to their function at the plasma membrane, protein components of AJs also have nuclear functions and are thus implicated in regulating gene expression and intracellular signaling. Within the nucleus, AJ proteins have been shown to interact with transcription factors such as TCF/LEF and Kaiso (ZBTB33), which converge on the canonical Wnt signaling pathway. The multifaceted nature of AJ proteins highlights their complexity in modulating homeostasis and emphasizes the importance of their subcellular localization and expression in the mammalian intestine. In this review, we summarize the nuclear roles of AJ proteins in intestinal tissues; their interactions with transcription factors and how this leads to crosstalk with canonical Wnt signaling; and how nuclear AJ proteins are implicated in intestinal homeostasis and disease.

4.
Front Immunol ; 11: 155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32117293

RESUMO

Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the impact of the microbiota on their frequency and function at mucosal sites remains unclear. Here, we investigated the role of the microbiota in the regulation of enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This was associated with changes in the production of enteric signals that regulate eosinophil attraction and survival, and was fully reversed by complex colonization. Additionally, SI eosinophils of GF mice exhibited more cytoplasmic protrusions and less granule content than SPF controls. Lastly, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate, and adaptive immune processes in health and disease.


Assuntos
Eosinófilos/imunologia , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Células Th2/imunologia , Animais , Modelos Animais de Doenças , Eosinofilia , Feminino , Hipersensibilidade Alimentar/sangue , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/microbiologia , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos
5.
PLoS One ; 14(6): e0217220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31199830

RESUMO

Chronic intestinal inflammation contributes to pathologies such as inflammatory bowel disease (IBD) and colon cancer. While the precise etiology remains controversial, IBD is believed to manifest as a result of various factors. We previously reported that intestinal-specific overexpression of the transcription factor Kaiso results in an intestinal inflammatory response; however, the cause of this inflammation is unknown. To elucidate the underlying mechanism(s) of the Kaiso-mediated intestinal inflammatory phenotype, we evaluated two independent transgenic mouse lines that express varying levels of Kaiso (KaisoTg). Histological analyses of KaisoTg mice revealed intestinal damage including thickening of the mucosa, intestinal "lesions" and crypt abscesses, which are reminiscent of IBD pathology. Additionally, higher Kaiso levels induced intestinal neutrophilia as early as 12 weeks, which worsened as the mice aged. Notably, the Kaiso-induced intestinal inflammation correlated with a leaky intestinal barrier and mis-regulation of E-cadherin expression and localization. Interestingly, Kaiso overexpression resulted in reduced proliferation but enhanced migration of intestinal epithelial cells prior to the onset of inflammation. Collectively, these data suggest that Kaiso plays a role in regulating intestinal epithelial cell integrity and function, dysregulation of which contributes to a chronic inflammatory phenotype as mice age.


Assuntos
Caderinas/metabolismo , Regulação da Expressão Gênica , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Fatores de Transcrição/metabolismo , Animais , Humanos , Camundongos , Fatores de Transcrição/genética
6.
PLoS One ; 8(9): e74160, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040197

RESUMO

Since its discovery, several studies have implicated the POZ-ZF protein Kaiso in both developmental and tumorigenic processes. However, most of the information regarding Kaiso's function to date has been gleaned from studies in Xenopus laevis embryos and mammalian cultured cells. To examine Kaiso's role in a relevant, mammalian organ-specific context, we generated and characterized a Kaiso transgenic mouse expressing a murine Kaiso transgene under the control of the intestine-specific villin promoter. Kaiso transgenic mice were viable and fertile but pathological examination of the small intestine revealed distinct morphological changes. Kaiso transgenics (Kaiso(Tg/+)) exhibited a crypt expansion phenotype that was accompanied by increased differentiation of epithelial progenitor cells into secretory cell lineages; this was evidenced by increased cell populations expressing Goblet, Paneth and enteroendocrine markers. Paradoxically however, enhanced differentiation in Kaiso(Tg/+) was accompanied by reduced proliferation, a phenotype reminiscent of Notch inhibition. Indeed, expression of the Notch signalling target HES-1 was decreased in Kaiso(Tg/+) animals. Finally, our Kaiso transgenics exhibited several hallmarks of inflammation, including increased neutrophil infiltration and activation, villi fusion and crypt hyperplasia. Interestingly, the Kaiso binding partner and emerging anti-inflammatory mediator p120(ctn) is recruited to the nucleus in Kaiso(Tg/+) mice intestinal cells suggesting that Kaiso may elicit inflammation by antagonizing p120(ctn) function.


Assuntos
Cateninas/genética , Efeito Fundador , Regulação da Expressão Gênica , Infiltração de Neutrófilos/genética , Fatores de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cateninas/metabolismo , Diferenciação Celular , Proliferação de Células , Enterócitos/metabolismo , Enterócitos/patologia , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Regiões Promotoras Genéticas , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Transcrição HES-1 , Fatores de Transcrição/metabolismo , Transgenes , delta Catenina
7.
J Vis Exp ; (25)2009 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-19287353

RESUMO

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.


Assuntos
Drosophila/embriologia , Microscopia de Fluorescência/métodos , Animais
8.
Autophagy ; 5(3): 329-38, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19066463

RESUMO

The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the downregulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, upregulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.


Assuntos
Autofagia/fisiologia , Caspases/metabolismo , Drosophila melanogaster/fisiologia , Animais , Apoptose , Morte Celular , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Macrófagos/metabolismo , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Mutação , Fagocitose , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo
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