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1.
Elife ; 102021 09 02.
Article in English | MEDLINE | ID: mdl-34473623

ABSTRACT

Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural T-IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to compare the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8+ T cells from lymph nodes. This data exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; upregulated cholesterol and lipid metabolic pathways, leading to high cholesterol levels in T-IEL; suppression of T cell antigen receptor signalling and expression of the transcription factor TOX, reminiscent of chronically activated T cells. These novel findings illustrate how T-IEL integrate multiple tissue-specific signals to maintain their homeostasis and potentially function.


Subject(s)
Cell Lineage , Cellular Microenvironment , Intestinal Mucosa/metabolism , Intraepithelial Lymphocytes/metabolism , Lymphocyte Activation , Proteome , Proteomics , Animals , Biomarkers/metabolism , Chromatography, High Pressure Liquid , Homeostasis , Intestinal Mucosa/immunology , Intraepithelial Lymphocytes/immunology , Male , Mice, Inbred C57BL , Phenotype , Signal Transduction , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
2.
Nat Commun ; 12(1): 4290, 2021 07 13.
Article in English | MEDLINE | ID: mdl-34257288

ABSTRACT

Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.


Subject(s)
Interleukin-15/metabolism , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Granzymes/genetics , Granzymes/metabolism , Humans , Interleukin-15/genetics , Intraepithelial Lymphocytes/metabolism , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism
3.
Mucosal Immunol ; 13(5): 721-731, 2020 09.
Article in English | MEDLINE | ID: mdl-32415229

ABSTRACT

Intraepithelial T lymphocytes (T-IEL) contain subsets of innate-like T cells that evoke innate and adaptive immune responses to provide rapid protection at epithelial barrier sites. In the intestine, T-IEL express variable T cell antigen receptors (TCR), with unknown antigen specificities. Intriguingly, they also express multiple inhibitory receptors, many of which are normally found on exhausted or antigen-experienced T cells. This pattern suggests that T-IEL are antigen-experienced, yet it is not clear where, and in what context, T-IEL encounter TCR ligands. We review recent evidence indicating TCR antigens for intestinal innate-like T-IEL are found on thymic or intestinal epithelium, driving agonist selection of T-IEL. We explore the contributions of the TCR and various co-stimulatory and co-inhibitory receptors in activating T-IEL effector functions. The balance between inhibitory and activating signals may be key to keeping these highly cytotoxic, rapidly activated cells in check, and key to harnessing their immune surveillance potential.


Subject(s)
Immunity, Innate , Intraepithelial Lymphocytes/immunology , Intraepithelial Lymphocytes/metabolism , Lymphocyte Activation/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Animals , Antigens/immunology , Biomarkers , Costimulatory and Inhibitory T-Cell Receptors/metabolism , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Humans , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Receptors, Antigen, T-Cell/metabolism
4.
Methods Mol Biol ; 2121: 141-152, 2020.
Article in English | MEDLINE | ID: mdl-32147793

ABSTRACT

Intestinal intraepithelial lymphocytes (IEL) comprise distinct groups of innate-like and memory T cells that collectively form one of the largest T cell compartments in the body. IEL are located within the intestinal epithelium and are the first immune cells in the gut to interact with the food, microbiota, and pathogens that the gut is continually exposed to. IEL can respond rapidly to external insults to protect the small intestinal epithelium but are also considered regulatory cells that are important to maintain the homeostasis of the gut. However, the mechanisms of IEL activation and their interactions within the epithelium remain largely elusive. Indeed, IEL are not commonly evaluated even in studies of gut immunology, potentially because they are perceived as being difficult to isolate and study. In this protocol, we present a simplified method to isolate IEL from the murine small intestine and provide representative data for flow cytometric analyses of the different IEL subsets. We also outline two procedures for culturing IEL, which can permit functional studies and coculture with epithelial cells. These strategies should make studies of this large but enigmatic T cell compartment more accessible and open up understanding of homeostatic mechanisms in the intestine, and tissue-associated immunity.


Subject(s)
Flow Cytometry/methods , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intraepithelial Lymphocytes/cytology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets/cytology , Animals , Cells, Cultured , Coculture Techniques , Epithelial Cells/cytology , Epithelial Cells/metabolism , Immunity, Innate , Intestinal Mucosa/immunology , Intestine, Small/immunology , Intraepithelial Lymphocytes/immunology , Mice , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
5.
PLoS Biol ; 17(11): e3000540, 2019 11.
Article in English | MEDLINE | ID: mdl-31770366

ABSTRACT

Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22-treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells.


Subject(s)
Adenomatous Polyposis Coli/metabolism , Epithelial Cells/metabolism , Interleukins/metabolism , Adenomatous Polyposis Coli/genetics , Animals , Carcinogenesis/genetics , Colorectal Neoplasms/metabolism , Cytokines/metabolism , Female , Interleukins/genetics , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Intestines/physiology , Male , Mice , Mice, Inbred C57BL , STAT3 Transcription Factor/metabolism , Sequence Analysis, RNA/methods , Signal Transduction , Interleukin-22
6.
PLoS One ; 12(10): e0185786, 2017.
Article in English | MEDLINE | ID: mdl-29020102

ABSTRACT

Vaccinia-H1 Related (VHR) dual-specificity phosphatase, or DUSP3, plays an important role in cell cycle regulation and its expression is altered in several human cancers. In mouse model, DUSP3 deletion prevents neo-angiogenesis and b-FGF-induced microvessel outgrowth. Considering the importance of angiogenesis in metastasis formation, our study aimed to investigate the role of DUSP3 in tumour cell dissemination. Using a Lewis Lung carcinoma (LLC) experimental metastasis model, we observed that DUSP3-/- mice developed larger lung metastases than littermate controls. DUSP3-/- bone marrow transfer to lethally irradiated DUSP3+/+ mice was sufficient to transfer the phenotype to DUSP3+/+ mice, indicating that hematopoietic cells compartment was involved in the increased tumour cell dissemination to lung tissues. Interestingly, we found a higher percentage of tumour-promoting Ly6Cint macrophages in DUSP3-/- LLC-bearing lung homogenates that was at least partially due to a better recruitment of these cells. This was confirmed by 1) the presence of higher number of the Ly6Bhi macrophages in DUSP3-/- lung homogenates and by 2) the better migration of DUSP3-/- bone marrow sorted monocytes, peritoneal macrophages and bone marrow derived macrophages (BMDMs), compared to DUSP3+/+ monocytes, macrophages and BMDMs, in response to LLC-conditioned medium. Our study demonstrates that DUSP3 phosphatase plays a key role in metastatic growth through a mechanism involving the recruitment of macrophages towards LLC-bearing lungs.


Subject(s)
Dual Specificity Phosphatase 3/metabolism , Gene Deletion , Lung Neoplasms/secondary , Macrophages/pathology , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Culture Media, Conditioned/pharmacology , Female , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Lung Neoplasms/pathology , Macrophages/drug effects , Male , Melanoma, Experimental/pathology , Mice, Inbred C57BL , Monocytes/drug effects , Monocytes/pathology
7.
J Immunol ; 199(7): 2515-2527, 2017 10 01.
Article in English | MEDLINE | ID: mdl-28848068

ABSTRACT

Dual-specificity phosphatase 3 (DUSP3) is a small phosphatase with poorly known physiological functions and for which only a few substrates are known. Using knockout mice, we recently reported that DUSP3 deficiency confers resistance to endotoxin- and polymicrobial-induced septic shock. We showed that this protection was macrophage dependent. In this study, we further investigated the role of DUSP3 in sepsis tolerance and showed that the resistance is sex dependent. Using adoptive-transfer experiments and ovariectomized mice, we highlighted the role of female sex hormones in the phenotype. Indeed, in ovariectomized females and in male mice, the dominance of M2-like macrophages observed in DUSP3-/- female mice was reduced, suggesting a role for this cell subset in sepsis tolerance. At the molecular level, DUSP3 deletion was associated with estrogen-dependent decreased phosphorylation of ERK1/2 and Akt in peritoneal macrophages stimulated ex vivo by LPS. Our results demonstrate that estrogens may modulate M2-like responses during endotoxemia in a DUSP3-dependent manner.


Subject(s)
Dual-Specificity Phosphatases/genetics , Dual-Specificity Phosphatases/metabolism , Endotoxemia/enzymology , Endotoxemia/prevention & control , Estrogens/metabolism , Macrophages/physiology , Shock, Septic/prevention & control , Animals , Coinfection/complications , Dual-Specificity Phosphatases/deficiency , Endotoxemia/genetics , Endotoxemia/microbiology , Female , Immune Tolerance , Lipopolysaccharides/immunology , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Knockout , Ovariectomy , Phosphorylation , Sex Characteristics , Signal Transduction
8.
Cancer Res ; 76(9): 2587-99, 2016 05 01.
Article in English | MEDLINE | ID: mdl-26980769

ABSTRACT

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkε in Wnt-driven tumor development. We found that Ikkε was activated in intestinal tumors forming upon loss of the tumor suppressor Apc Genetic ablation of Ikkε in ß-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkε to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkε was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkε-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkε phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkε to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587-99. ©2016 AACR.


Subject(s)
I-kappa B Kinase/metabolism , Interleukin-17/metabolism , Intestinal Neoplasms/pathology , Lipopolysaccharides/metabolism , Signal Transduction/physiology , Wnt Proteins/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Flow Cytometry , Humans , Immunoprecipitation , In Situ Hybridization , Intestinal Neoplasms/metabolism , Mice , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Tumor Microenvironment/physiology
9.
J Immunol ; 194(10): 4951-62, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25876765

ABSTRACT

DUSP3 is a small dual-specificity protein phosphatase with an unknown physiological function. We report that DUSP3 is strongly expressed in human and mouse monocytes and macrophages, and that its deficiency in mice promotes tolerance to LPS-induced endotoxin shock and to polymicrobial septic shock after cecal ligation and puncture. By using adoptive transfer experiments, we demonstrate that resistance to endotoxin is macrophage dependent and transferable, and that this protection is associated with a striking increase of M2-like macrophages in DUSP3(-/-) mice in both the LPS and cecal ligation and puncture models. We show that the altered response of DUSP3(-/-) mice to sepsis is reflected in decreased TNF production and impaired ERK1/2 activation. Our results demonstrate that DUSP3 plays a key and nonredundant role as a regulator of innate immune responses by mechanisms involving the control of ERK1/2 activation, TNF secretion, and macrophage polarization.


Subject(s)
Dual Specificity Phosphatase 3/immunology , Immunity, Innate/immunology , Macrophages/immunology , Shock, Septic/immunology , Signal Transduction/immunology , Adoptive Transfer , Animals , Blotting, Western , Dual Specificity Phosphatase 3/deficiency , Flow Cytometry , Gene Deletion , Humans , Immune Tolerance , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
10.
Circulation ; 131(7): 656-68, 2015 Feb 17.
Article in English | MEDLINE | ID: mdl-25520375

ABSTRACT

BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.


Subject(s)
Dual Specificity Phosphatase 3/antagonists & inhibitors , Dual Specificity Phosphatase 3/deficiency , Platelet Activation/physiology , Pulmonary Embolism/enzymology , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Activation/drug effects , Pulmonary Embolism/blood , Thrombosis/blood , Thrombosis/enzymology
11.
Mol Cancer ; 13: 108, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24886454

ABSTRACT

BACKGROUND: DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies showed that DUSP3 is a negative regulator of ERK and JNK pathways in several cell lines. On the other hand, DUSP3 is implicated in human cancer. It has been alternatively described as having tumor suppressive and oncogenic properties. Thus, the available data suggest that DUSP3 plays complex and contradictory roles in tumorigenesis that could be cell type-dependent. Since most of these studies were performed using recombinant proteins or in cell-transfection based assays, the physiological function of DUSP3 has remained elusive. RESULTS: Using immunohistochemistry on human cervical sections, we observed a strong expression of DUSP3 in endothelial cells (EC) suggesting a contribution for this phosphatase to EC functions. DUSP3 downregulation, using RNA interference, in human EC reduced significantly in vitro tube formation on Matrigel and spheroid angiogenic sprouting. However, this defect was not associated with an altered phosphorylation of the documented in vitro DUSP3 substrates, ERK1/2, JNK1/2 and EGFR but was associated with an increased PKC phosphorylation. To investigate the physiological function of DUSP3, we generated Dusp3-deficient mice by homologous recombination. The obtained DUSP3-/- mice were healthy, fertile, with no spontaneous phenotype and no vascular defect. However, DUSP3 deficiency prevented neo-vascularization of transplanted b-FGF containing Matrigel and LLC xenograft tumors as evidenced by hemoglobin (Hb) and FITC-dextran quantifications. Furthermore, we found that DUSP3 is required for b-FGF-induced microvessel outgrowth in the aortic ring assay. CONCLUSIONS: All together, our data identify DUSP3 as a new important player in angiogenesis.


Subject(s)
Carcinoma, Lewis Lung/genetics , Dual Specificity Phosphatase 3/genetics , Neovascularization, Physiologic/genetics , Animals , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Cell Movement , Cervix Uteri/blood supply , Cervix Uteri/metabolism , Cervix Uteri/pathology , Collagen , Drug Combinations , Dual Specificity Phosphatase 3/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Fibroblast Growth Factors , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Laminin , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Mice , Mice, Knockout , Neovascularization, Pathologic/prevention & control , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C/metabolism , Proteoglycans , Signal Transduction
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