Protective Role of ?-arrestin2 in Colitis Through Modulation of T-cell Activation.
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ABSTRACT:
PROVIDER: S-EPMC4654649 | BioStudies |
REPOSITORIES: biostudies
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Project description:MicroRNAs (miRNAs) are epigenetically involved in regulating gene expression. They may be of importance in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to determine the role of miRNAs by their specific blocking in the CD4+CB45RBhi T-cell transfer model of chronic experimental colitis.Colitis caused by transfer of WT CD4+CD45RBhi T cells in severe combined immunodeficiency (SCID) mice shares many features with human IBD. Colonic miRNA expression levels were measured at three time points in colitic mice, where a time-dependent upregulation of multiple miRNAs was seen. To inhibit these miRNAs, specific locked-nucleic-acid-modified (LNA) oligonucleotides were administered in further experiments at the moment the mice demonstrated the first signs of colitis. As controls, PBS and a scrambled sequence of anti-miRNA were used. Genome-wide expression analyses were also performed in order to detect candidate target genes of miR-142-5p, of which inhibition resulted in most effective amelioration of colitis.Anti-miR-142-5p reduced colitis and related wasting disease when administered in the T-cell transfer model, reflected in reduced weight loss and a lower disease activity index (DAI). In further validation experiments we also observed a higher survival rate and less colonic histological inflammation in the antagomir-treated mice. Moreover, by genome-wide expression analyses, we found downstream activation of the anti-inflammatory IL10RA pathway, including three genes also found in the top-20 candidate target genes of miR-142-5p.In conclusion, CD4+CD45RBhi-transfer colitis induces miR-142-5p. Blocking miR-142-5p reduced colitis and prevented wasting disease, possibly by activation of the IL10RA pathway.
| S-EPMC5653202 | BioStudies
Project description:Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ?-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ?-arrestin2 (?arr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ?arr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ?arr2-knockout (?arr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ?arr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ?arr2-/- cells. In order to assess the effect of acute loss of ?arr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ?arr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ?arr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ?arr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.
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Project description:Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide. The disease does not present early clinical symptoms and is commonly diagnosed at an advanced stage. Limited molecular drivers have been identified for RCC, resulting in the lack of effective treatment for patients with progressive disease. Ubiquitous ?Arrestin2 (?Arr2) is well established for its function in the desensitization and trafficking of G protein-coupled receptors. More recently, ?Arr2 has been implicated in the regulation of fundamental cellular functions, including proliferation and invasion. We used bioinformatic and genetic approaches to determine role of ?Arr2 in RCC tumor growth. Analysis of published human datasets shows that ARRB2 (gene encoding ?Arr2) expression is increased in RCC tumor compared to normal tissue and that high levels of ARRB2 correlate with worse patient survival. Experimentally, we show that knockout of ARRB2 decreases rate of RCC cell proliferation and migration in vitro and xenograft tumor growth in animals. Mechanistically, ?Arr2 regulates c-Src activity, Cyclin A expression and cell cycle progression that are involved in tumor growth. These results show that ?Arr2 is a critical regulator of RCC tumor growth and suggest its utility as a potential marker and drug target to treat advanced disease.
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Project description:Inflammatory bowel disease (IBD) is an immunological disease associated with CD4<sup>+</sup> T cell activation in the intestines. CD81 is a regulator of the immune system with multiple biological functions. Therefore, in this study, we assessed the contribution of CD81 to IBD pathophysiology and the therapeutic efficacy of anti-CD81 antibodies. Expression of CD81 was increased on activated T cells in vitro and in colitic mice in vivo. Therapeutic effects of anti-CD81 antibodies on colitic symptoms and inflammation were evaluated in mice with colitis, including long-term effects of the antibodies. Treatment with anti-CD81 antibodies improved colitis scores, reduced colon shortening, decreased loss of body weight, and resulted in fewer pathological changes of the colon in colitic mice. Moreover, the increased inflammatory markers in the blood of colitic mice were decreased by anti-CD81 antibodies. The anti-CD81 antibody treatment had long-lasting therapeutic effects on colitic mice, even after cessation of treatment. Two different clones of the anti-mouse CD81 antibody were also effective in mice with colitis. Furthermore, anti-CD81 antibodies reduced migration of CD4<sup>+</sup> T cells both in colitic mice and in vitro. Thus, CD81 contributes to IBD pathology and treatment with anti-CD81 antibodies may be a potential novel therapy for IBD patients.
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Project description:Homeostasis in the ileum, which is commonly disrupted in patients with Crohn's disease, involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4+T cell responses, we used TCR transgenic tools to breed mice that spontaneously produced CD4+T cells reactive to an antigen expressed in the ileum. At an early age, the ilea of these mice exhibit crypt hyperplasia and accumulate increased numbers of TH17 cells bearing non-transgenic clonotypes. Half of these mice subsequently developed colitis linked to broad mucosal infiltration by TH17 and TH1 cells expressing non-transgenic clonotypes, chronic wasting disease and loss of ileal crypt hyperplasia. By contrast, adult mice with normal growth continued to exhibit TH17-associated ileal crypt hyperplasia and additionally accumulated ileal-reactive Treg cells. Both IL-17A and IFN? were protective, as their deficiency precluded ileal-reactive Treg accumulation and exacerbated colitic disease. IL-23R blockade prevented progression to colitis, whereas nTreg cell transfers prevented colitic disease, ileal crypt hyperplasia and ileal-reactive Treg accumulation. Thus, our studies identify an IL-17A and IFN?-dependent homeostatic process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.
| S-EPMC6571016 | BioStudies
Project description:Endothelial cell nitric oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in endothelial cells, is regulated by complex posttranslational mechanisms. Sinusoidal portal hypertension, a disorder characterized by liver sinusoidal endothelial cell (SEC) injury with resultant reduced eNOS activity and NO production within the liver, has been associated with defects in eNOS protein-protein interactions and posttranslational modifications. We and others have previously identified novel eNOS interactors, including G protein-coupled receptor (GPCR) kinase interactor 1 (GIT1), which we found to play an unexpected stimulatory role in GPCR-mediated eNOS signaling. Here we report that ?-arrestin 2 (?-Arr2), a canonical GPCR signaling partner, localizes in SECs with eNOS in a GIT1/eNOS/NO signaling module. Most importantly, we show that ?-Arr2 stimulates eNOS activity, and that ?-Arr2 expression is reduced and formation of the GIT1/eNOS/NO signaling module is interrupted during liver injury. In ?-Arr2-deficient mice, bile duct ligation injury (BDL) led to significantly reduced eNOS activity and to a dramatic increase in portal hypertension compared to BDL in wild-type mice. Overexpression of ?-Arr2 in injured or ?-Arr2-deficient SECs rescued eNOS function by increasing eNOS complex formation and NO production. We also found that ?-Arr2-mediated GIT1/eNOS complex formation is dependent on Erk1/2 and Src, two kinases known to interact with and be activated by ?-Arr2 in response to GCPR activation. Our data emphasize that ?-Arr2 is an integral component of the GIT1/eNOS/NO signaling pathway and have implications for the pathogenesis of sinusoidal portal hypertension.
| S-EPMC7261012 | BioStudies
Project description:?-Arrestin2 (?arr2) is an adaptor protein that interacts with numerous signaling molecules and regulates insulin sensitivity. We reported previously that ?arr2 was abundantly expressed in mouse pancreatic ?-cells, and loss of ?arr2 leads to impairment of acute- and late-phase insulin secretion. In the present study, we examined the dynamic changes of ?-cell mass in <i>?arr2</i>-deficient (<i>?arr2<sup>-/-</sup></i>) mice <i>in vivo</i> and explored the underlying mechanisms involved. <i>?arr2<sup>-/-</sup></i> mice with exclusively luciferase overexpression in ?-cells were generated and fed a high-fat diet (HFD). ?-Cell mass was determined by <i>in vivo</i> noninvasive bioluminescence imaging from 4 to 20 wks of age. Proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation and fluorescence-activated cell sorter analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were conducted for gene and protein expression. We found that ?-cell mass was reduced dramatically in <i>?arr2<sup>-/-</sup></i> mice at 12 wks old compared with that of their respective HFD-fed controls. The percentage of BrdU- and Ki67-positive cells reduced in islets from <i>?arr2<sup>-/-</sup></i> mice. Exposure of <i>?arr2<sup>-/-</sup></i> islets to high levels of glucose and free fatty acids (FFAs) exacerbated cell death, which was associated with upregulation of the JNK pathway in these islets. Conversely, overexpression of ?arr2 amplified ?-cell proliferation with a concomitant increase in cyclinD2 expression and a decrease in p21 expression and protected ?-cells from glucose- and FFA-induced cell death through JNK-activation inhibition. In conclusion, ?arr2 plays roles in regulation of pancreatic ?-cell mass through the modulation of cell cycle regulatory genes and the inhibition of JNK activation induced by glucolipotoxity, which implicates a role for ?arr2 in the development of type 2 diabetes.
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Project description:Inflammatory bowel disease (IBD) is a chronic inflammatory condition with complex pathogenesis that currently has no cure. ?7 nicotinic acetylcholine receptor (?7nAChR) is known to regulate multiple aspects of immune function. The present study aimed to evaluate the protective effects of PNU282987 and SHP099, which are a selective agonist of ?7nAChR and an SHP2 inhibitor, respectively, in dextran sulfate sodium (DSS)?induced colitis in mice. Acute colitis was induced in mice using 3% DSS, and weight loss, colonic histology and cytokine production from colonic lamina propria were analyzed to evaluate disease severity. Bone marrow?derived macrophages were treated with lipopolysaccharide (LPS) to induce an inflammatory response. Cytokine expression and reactive oxygen species (ROS) levels were quantified. The ?7nAChR agonist, PNU282987, and the SHP2 inhibitor, SHP099, were administered alone or in combination to LPS?induced macrophages or to colitic model mice to evaluate the inflammatory response and protective efficacy in colitis. ?7nAChR protein levels were found to be markedly increased in the colon of DSS?induced colitic mice, and were found to co?localize with macrophages. Consistently, ?7nAChR mRNA and protein levels were upregulated with colitis progression in DSS?induced colitic mice. Colonic inflammation was attenuated by PNU282987 treatment in DSS?induced mice, as evidenced by reduced weight loss and alleviated colonic epithelial cell disruption. These effects of PNU282987 on colitis were enhanced when it was combined with SHP099. Cytokine production and ROS levels induced by LPS in macrophages were decreased by a combination treatment of PNU282987 and SHP099. These findings identified ?7nAChR as an essential element in the role of intestinal macrophages in colonic repair and demonstrated a synergistic effect of PNU282987 and SHP099, suggesting a new potential therapy for IBD.
| S-EPMC7411392 | BioStudies
Project description:Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E. coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E. coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.
| S-EPMC4310462 | BioStudies
Project description:Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4+ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103+ CD11b- DCs and increased CD103- CD11b+ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1?, IL-6, IL-17, TNF?, and IFN? combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103+ CD11b+ DCs in the distal colon. CD103+ CD11b+ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103- CD11b+ DCs from colitic Muc2-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103+ CD11b+ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.
| S-EPMC4487685 | BioStudies