3375 Chronic inflammation promotes intestinal macrophages to become modulators of the Notch pathway

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.

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Interleukin 7 Transgenic Mice Develop Chronic Colitis with Decreased Interleukin 7 Protein Accumulation in the Colonic Mucosa

Journal of Experimental Medicine ◽

10.1084/jem.187.3.389 ◽

1998 ◽

Vol 187 (3) ◽

pp. 389-402 ◽

Cited By ~ 152

Author(s):

Mamoru Watanabe ◽

Yoshitaka Ueno ◽

Tomoharu Yajima ◽

Susumu Okamoto ◽

Tatsuhiko Hayashi ◽

...

Keyword(s):

T Cells ◽

Epithelial Cells ◽

Transgenic Mice ◽

Chronic Inflammation ◽

Colonic Mucosa ◽

Flow Cytometric Analysis ◽

Receptor Expression ◽

Protein Accumulation ◽

Chronic Colitis ◽

Interleukin 7

We have demonstrated that intestinal epithelial cells produce interleukin 7 (IL-7), and IL-7 serves as a potent regulatory factor for proliferation of intestinal mucosal lymphocytes expressing functional IL-7 receptor. To clarify the mechanism by which locally produced IL-7 regulates the mucosal lymphocytes, we investigated IL-7 transgenic mice. Here we report that transgenic mice expressing murine IL-7 cDNA driver by the SRα promoter developed chronic colitis in concert with the expression of SRα/IL-7 transgene in the colonic mucosa. IL-7 transgenic but not littermate mice developed chronic colitis at 4–12 wk of age, with histopathological similarity to ulcerative colitis in humans. Southern blot hybridization and competitive PCR demonstrated that the expression of IL-7 messenger RNA was increased in the colonic mucosal lymphocytes but not in the colonic epithelial cells. IL-7 protein accumulation was decreased in the goblet cell–depleted colonic epithelium in the transgenic mice. Immunohistochemical and cytokine production analysis showed that lymphoid infiltrates in the lamina propria were dominated by T helper cell type 1 CD4+ T cells. Flow cytometric analysis demonstrated that CD4+ intraepithelial T cells were increased, but T cell receptor γ/δ T cells and CD8α/α cells were not increased in the area of chronic inflammation. Increased IL-7 receptor expression in mucosal lymphocytes was demonstrated in the transgenic mice. These findings suggest that chronic inflammation in the colonic mucosa may be mediated by dysregulation of colonic epithelial cell–derived IL-7, and this murine model of chronic colitis may contribute to the understanding of the pathogenesis of human inflammatory bowel disease.

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Granzyme B skews CD4+ T cell differentiation resulting in increased intestinal pathogenicity

10.1101/2020.12.04.412056 ◽

2020 ◽

Author(s):

Kristen L. Hoek ◽

Michael J. Greer ◽

Kathleen G. McClanahan ◽

Ali Nazmi ◽

M. Blanca Piazuelo ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Immune Responses ◽

Cell Biology ◽

Intestinal Inflammation ◽

Granzyme B ◽

Disease Onset ◽

T Cell Differentiation ◽

Transfer Model

AbstractCD4+ T cell activation and differentiation are important events that set the stage for proper immune responses. Many factors are involved in the activation and differentiation of T cells, and these events are tightly controlled to prevent unwanted and/or exacerbated immune responses that may harm the host. It has been well documented that granzyme B, a potent serine protease involved in cell-mediated cytotoxicity, is readily expressed by certain CD4+ T cells, such as regulatory T cells and CD4+CD8aa+ intestinal intraepithelial lymphocytes, both of which display cytotoxicity associated with granzyme B. However, because not all CD4+ T cells expressing granzyme B are cytotoxic, additional roles for this protease in CD4+ T cell biology remain unknown. Here, using a combination of in vivo and in vitro approaches, we report that granzyme B-deficient CD4+ T cells display increased IL-17 production. In the adoptive transfer model of intestinal inflammation, granzyme B-deficient CD4+ T cells triggered a more rapid disease onset than their WT counterparts, and presented a differential transcription profile. Similar results were also observed in granzyme B-deficient mice infected with Citrobacter rodentium. Our results suggest that granzyme B modulates CD4+ T cell differentiation, providing a new perspective into the biology of this enzyme.

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NK-Like Homeodomain Proteins Regulate NOTCH3-Signaling in T-Cells.

Blood ◽

10.1182/blood.v112.11.1194.1194 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1194-1194

Author(s):

Stefan Nagel ◽

Letizia Venturini ◽

Grzegorz K. Przybylski ◽

Piotr Grabarczyk ◽

Corinna Meyer ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Expression Patterns ◽

Hematopoietic Cells ◽

Notch Pathway ◽

T Cell Differentiation ◽

Homeodomain Proteins

Abstract Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

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Induction of Antigen-Specific Regulatory T Cells following Overexpression of a Notch Ligand by Human B Lymphocytes

Journal of Virology ◽

10.1128/jvi.77.20.10872-10880.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 10872-10880 ◽

Cited By ~ 90

Author(s):

Stéphane Vigouroux ◽

Eric Yvon ◽

Hans-Joachim Wagner ◽

Ettore Biagi ◽

Gianpietro Dotti ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Regulatory T Cells ◽

Cell Activity ◽

Notch Pathway ◽

Antigen Presenting Cells ◽

Cytotoxic T Cell ◽

Inhibition Of Proliferation ◽

Notch Ligand ◽

Antigen Presenting

ABSTRACT In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4+CD25+ and CD8+CD25− T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.

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Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells

Blood ◽

10.1182/blood-2002-12-3826 ◽

2003 ◽

Vol 102 (10) ◽

pp. 3815-3821 ◽

Cited By ~ 105

Author(s):

Eric S. Yvon ◽

Stephane Vigouroux ◽

Raphael F. Rousseau ◽

Ettore Biagi ◽

Persis Amrolia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Interleukin 2 ◽

Notch Pathway ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Notch Ligand

Abstract Graft-versus-host disease (GVHD) represents one of the major complications of allogeneic hematopoietic stem cell transplantation. Techniques to prevent GVHD have included ex vivo T-cell depletion of the graft or prolonged in vivo immunosuppression. Both reduce the frequency and severity of GVHD but also reduce T-cell-mediated graft-versus-malignancy effect, and increase the risk of infection. A major goal in transplantation is to prevent alloreactivity while preserving activity against tumors and infectious agents. We have used activation of the Notch pathway to try to generate T cells able to specifically regulate alloantigen responses. We used allogeneic Epstein-Barr virus lymphoblastoid B cells (EBV-LCLs) as stimulator cells. Such LCLs are excellent (allo) antigen-presenting cells and can be obtained in large numbers even from donors who have received extensive chemo/radiotherapy. We overexpressed a Notch ligand, Jagged-1, in these cells by adenoviral vector transduction. Stimulation of CD45RA+ naive T cells by Jagged-1 EBV-LCL reduces production of interferon-γ, interleukin-2, and interleukin-5, but up-regulates transforming growth factor-β1 synthesis, consistent with induction of a regulatory T-cell phenotype. Transfer of these T cells to fresh lymphocyte cultures inhibits proliferative and cytotoxic immune responses to the priming alloantigens while sparing responses to third-party stimulator cells. Notch activation in the presence of alloantigen-presenting cells may therefore be a means of inducing specific regulatory T cells while preserving other T-cell functionality. (Blood. 2003;102:3815-3821)

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Ameliorating effect of anti-Fas ligand MAb on wasting disease in murine model of chronic colitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00071.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. G754-G760 ◽

Cited By ~ 10

Author(s):

N. Dan ◽

T. Kanai ◽

T. Totsuka ◽

R. Iiyama ◽

M. Yamazaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lamina Propria ◽

Fas Ligand ◽

Intestinal Inflammation ◽

Therapeutic Effects ◽

Cell Transfer ◽

Chronic Colitis ◽

Wasting Disease ◽

T Cell Transfer

Fas/Fas ligand (FasL) interaction has been implicated in the pathogenesis of various diseases. To clarify the involvement of Fas/FasL in the pathogenesis of intestinal inflammation, we investigated the preventive and therapeutic effects of neutralizing anti-FasL monoclonal antibody (MAb) on the development of chronic colitis induced by adaptive transfer of CD4+CD45RBhigh T cells to SCID mice. Administration of anti-FasL MAb from 1 day after T cell transfer (prevention study) resulted in a significant improvement of clinical manifestations such as wasting and diarrhea. However, histological examination showed that mucosal inflammation in the colon, such as infiltration of T cells and macrophages, was not improved by the anti-FasL MAb treatment. In vitro studies showed that anti-FasL MAb did not inhibit IFN-γ production by anti-CD3/CD28-stimulated lamina propria CD4+ T cells but suppressed TNF-α and IL-1β production by lamina propria mononuclear cells. Therapeutic administration of anti-FasL MAb from 3 wk after T cell transfer also improved ongoing wasting disease but not intestinal inflammation. These results suggest that the Fas/FasL interaction plays a critical role in regulating systemic wasting disease but not local intestinal inflammation.

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Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing

Development ◽

10.1242/dev.124.10.1919 ◽

1997 ◽

Vol 124 (10) ◽

pp. 1919-1928 ◽

Cited By ~ 23

Author(s):

J.F. de Celis ◽

S. Bray ◽

A. Garcia-Bellido

Keyword(s):

Positive Feedback ◽

Notch Pathway ◽

Notch Signalling ◽

Notch Ligand ◽

Asymmetrical Distribution ◽

Drosophila Wing ◽

Split Gene ◽

Enhancer Of Split ◽

Notch Activation ◽

In Cells

The veins in the Drosophila wing have a characteristic width, which is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta is restricted to the developing veins, and coincides with places where Notch transcription is lower. We find that this asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins. In these cells, the expression of the Enhancer of split gene m beta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. We propose that the establishment of vein thickness utilises a combination of mechanisms that include: (1) independent regulation of Notch and Delta expression in intervein and vein territories, (2) Notch activation by Delta in cells where Notch and Delta expression overlaps, (3) positive feedback on Notch transcription in cells where Notch has been activated and (4) repression of veinlet transcription by E(spl)m beta and maintenance of Delta expression by veinlet/torpedo activity.

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RORγt-Expressing Pathogenic CD4+T Cells Cause Brain Inflammation During Chronic Colitis

10.1101/2021.09.01.458634 ◽

2021 ◽

Author(s):

Rajatava Basu ◽

Michel Edwar Mickael ◽

Suniti Bhaumik ◽

Ayanabha Chakraborti ◽

Alan Umfress ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Intestinal Inflammation ◽

Brain Inflammation ◽

Brain Diseases ◽

Transfer Model ◽

Chronic Colitis ◽

Neurobehavioral Disorders ◽

Chronic Intestinal Inflammation ◽

The Brain

Neurobehavioral disorders and brain abnormalities have been extensively reported in both Crohn's Disease (CD) and Ulcerative Colitis (UC) patients. However, the mechanism causing neuropathological disorders in inflammatory bowel disease (IBD) patients remains unknown. Studies have linked the Th17 subset of CD4+T cells to brain diseases associated with neuroinflammation and cognitive impairment, including multiple sclerosis (MS), ischemic brain injury, and Alzheimer's disease. To better understand how CD4+T lymphocytes, contribute to brain pathology in chronic intestinal inflammation, we investigated the development of brain inflammation in the T cell transfer model of chronic colitis. Our findings demonstrate that CD4+T cells infiltrate the brain of colitic Rag1-/- mice in proportional levels to colitis severity. Colitic mice developed hypothalamic astrogliosis that correlated with neurobehavioral disorders. Moreover, the brain-infiltrating CD4+T cells expressed Th17 cell transcription factor RORγt and displayed a pathogenic Th17 cellular phenotype similar to colonic Th17 cells. Adoptive transfer of RORγt-deficient naive CD4+T cells failed to cause brain inflammation and neurobehavioral disorders in Rag1-/- recipients, with significantly less brain infiltration of CD4+T cells. These findings suggest that pathogenic RORγt+CD4+T cells that aggravate colitis migrate preferentially into the brain, contributing to brain inflammation and neurobehavioral disorders, thereby linking colitis severity to neuroinflammation.

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A screen for targets of the Drosophila pseudokinase Tribbles identifies Neuralized and Mindbomb, ubiquitin ligases that mediate Notch signaling

10.1101/406249 ◽

2018 ◽

Author(s):

Anna Shipman ◽

Christopher Nauman ◽

Britney Haymans ◽

Rachel Silverstein ◽

Leonard L. Dobens

Keyword(s):

Cell Differentiation ◽

Family Members ◽

Notch Pathway ◽

Ubiquitin Ligases ◽

Adaptor Proteins ◽

Tissue Growth ◽

E3 Ligases ◽

Tissue Size ◽

Factor C ◽

Notch Activation

ABSTRACTDrosophila Tribbles (Trbl) is the founding member of a family of pseudokinases with conserved roles in antagonizing cell division, tissue growth and cell differentiation. In humans, three Tribbles isoforms serve as adaptor proteins, binding targets such as Cdc25 phosphatase, Akt kinase or the transcription factor C/EBP to block their activity or direct their proteosomal degradation. Mutations in Tribbles family members are associated with susceptibility to diabetes and cancer, notably Notch-induced tumor growth. Trbl misexpression in the fly wing disk leads to a block in mitosis associated with decreased levels of String/Cdc25 and increased levels of Cyclin B leading to reduced overall wing size and reduced trichome density. We show these Trbl growth-restricting phenotypes can be suppressed by manipulating levels of known Trbl targets, and use this sensitized wing system to screen a collection of growth regulating open reading frames (ORFs) to search for enhancers and suppressors affecting cell and tissue size. By precisely measuring morphometric changes in wing phenotypes using a computer-based tool, we detected synthetic interactions with several E3 ubiquitin ligases, and focused our analysis on the Notch pathway components Neuralized (Neur) and Mindbomb1 (Mib1). In the wing, notum and egg chamber epithelia, Trbl misexpression suppressed Neur and Mib1 activities and stabilized the accumulation of both proteins. To understand these interactions, we used yeast two-hybrid assays to show Trbl physically bound to both Neur and Mib1. Our data are consistent with published reports that mammalian Tribbles3 modulates Notch responses by binding and stabilizing Mindbomb and indicate that a wing misexpression approach is useful to identify novel components in a conserved Tribbles signaling pathway.AUTHOR SUMMARYTribbles pseudokinases are adaptor molecules, binding diverse targets regulating cell differentiation, growth and proliferation and directing their proteasomal degradation. To search for novel targets of Drosophila Tribbles, we adopted a wing co-misexpression scheme and measured changes in cell/tissue size to identify enhancers and suppressors of the Tribbles phenotype. We show the Notch pathway components Neuralized and Mindbomb1 E3 ligases act as Tribbles suppressors and demonstrate that Tribbles modulates their levels and activites. Recent demonstration that mammalian Tribbles 3 binds the E3 ligase Mindbomb to promote ligand-mediated Notch activation implies a conserved role for Tribbles family members in Notch activation.

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