ABSTRACT
A microRNA miR-200c-3p is a regulator of epithelial-mesenchymal transition to control adhesion and migration of epithelial and mesenchymal cells. However, little is known about whether miR-200c-3p affects lymphocyte adhesion and migration mediated by integrins. Using TK-1 (a T lymphoblast cell) as a model of T cell, here we show that repressed expression of miR-200c-3p upregulated α4 integrin-mediated adhesion to and migration across mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Conversely, overexpression of miR-200c-3p downregulated α4 integrin-mediated adhesion and migration. Unlike in epithelial cells, miR-200c-3p did not target talin, a conformation activator of integrin, but, targeted E26-transformation-specific sequence 1 (ETS1), a transcriptional activator of α4 integrin, in T cells. Treatment of the miR-200c-3p-low-expressing TK-1 cells that possessed elevated α4 integrin with ETS1 small interfering RNA (siRNA) resulted in the reversion of the α4 integrin expression, supporting that ETS1 is a target of miR-200c-3p. A potential proinflammatory immune-modulator retinoic acid (RA) treatment of TK-1 cells elicited a significant reduction of miR-200c-3p and simultaneously a marked increase in ETS1 and α4 integrin expression. An anti-inflammatory cytokine TGF-ß1 treatment elevated miR-200c-3p, thereby downregulating ETS1 and α4 integrin expression. These results suggest that miR-200c-3p is an important regulator of α4 integrin expression and functions and may be controlled by RA and TGF-ß1 in an opposite way. Overexpression of miR-200c-3p could be a novel therapeutic option for treatment of gut inflammation through suppressing α4 integrin-mediated T cell migration.
Subject(s)
Cell Adhesion , Cell Movement , Integrin alpha4 , MicroRNAs , T-Lymphocytes , MicroRNAs/genetics , MicroRNAs/metabolism , Humans , Integrin alpha4/metabolism , Integrin alpha4/genetics , Cell Movement/genetics , Cell Adhesion/genetics , T-Lymphocytes/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Proto-Oncogene Protein c-ets-1/genetics , Mucoproteins/genetics , Mucoproteins/metabolism , Transforming Growth Factor beta1/metabolism , Immunoglobulins/genetics , Immunoglobulins/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/genetics , Cell LineABSTRACT
Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.
Subject(s)
Amphiregulin , Integrin alpha4 , Integrin beta Chains , Lupus Nephritis , Lymphocytes , Lupus Nephritis/immunology , Amphiregulin/immunology , Lymphocytes/immunology , Integrin alpha4/genetics , Integrin alpha4/immunology , Humans , Female , Animals , Mice , Disease Models, Animal , Integrin beta Chains/genetics , Integrin beta Chains/immunology , Cell Adhesion/immunology , Cell Movement/immunology , Kidney/drug effects , Kidney/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Protein Binding/immunology , Interleukin-33/pharmacology , Signal TransductionABSTRACT
OBJECTIVE: To assess the association between the polymorphism of integral protein α4 (ITGA4) and intercellular adhesion molecule 1 (ICAM-1) genes and the risk and clinicopathological characteristics of Crohn's disease (CD) among Chinese patients. METHODS: From January 2010 to January 2021, a total of 215 CD patients and 529 gender- and age-matched healthy controls were enrolled from the Second Affiliated Hospital of Wenzhou Medical University as the study subjects. Genotypes of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Harvey-Bradshaw Index (HBI) was applied to assess the disease activity of CD, and the patients were further divided into subgroups based on the Montreal Classification Criteria of CD. Unconditional logistic regression was employed to analyze the distribution of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) polymorphisms between the patients and healthy controls and their association with the clinicopathological characteristics of the patients. RESULTS: The frequencies of T allele and CT+TT genotypes of ITGA4 (rs7562325) were higher in CD patients than the healthy controls (40.70% vs. 31.57%, P = 0.001; 62.79% vs. 54.36%, P = 0.042). The G variant and AG+GG genotypes of ITGA4 (rs6740847) were less common in patients with moderately to severely active CD compared with those with mildly active CD (31.18% vs. 51.72%, P = 0.002; 55.91% vs. 75.86%, P = 0.042). However, the opposite conclusion was drawn for the G allele (G) and AG+GG genotypes of ICAM-1 (rs5498) (31.45% vs. 17.24%, P = 0.027; 54.30% vs. 31.04%, P = 0.020). Compared with patients with terminal ileal or ileocolic CD, G allele and AG+GG genotypes of ITGA4 (rs6740847) were more prevalent in patients with colonic CD (55.26% vs. 29.38%, P < 0.001; 84.21% vs. 53.11%, P<0.001). The same conclusion could also be drawn for the G allele and AG+GG genotypes of ICAM-1 (rs5498) (42.11% vs. 26.84%, P = 0.008; 73.69% vs. 46.33%, P = 0.002). The frequency of homozygous GG genotype of ICAM-1 (rs5498) was lower in patients with stricturing and penetrating CD than those with non-stricturing and non-penetrating CD (0.00% vs. 12.32%, P = 0.001). The G allele and AG+GG genotypes of the ITGA4 (rs6740847) were more common in patients with perianal lesions than those without (40.28% vs. 30.77%, P = 0.049; 72.22% vs. 51.75%, P = 0.004). CONCLUSION: Variants of the ITGA4 (rs7562325) may be a risk factor for CD, whilst those of the ITGA4 (rs6740847) may be associated with the decline of disease activity and risk for colon involvement and perianal lesions. Variants of the ICAM-1 (rs5498) may increase the risk of disease activity and colonic involvement in CD patients, however, it may be a protective factor for stenosis and penetration. In addition, variants of the ITGA4 (rs6740847) and ICAM-1 (rs5498) may be associated with the early onset of CD.
Subject(s)
Crohn Disease , Integrin alpha4 , Intercellular Adhesion Molecule-1 , Humans , Alleles , Case-Control Studies , Crohn Disease/genetics , Genetic Predisposition to Disease , Genotype , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Polymorphism, Single Nucleotide , Integrin alpha4/geneticsABSTRACT
BACKGROUND: Mesenchymal stem cells (MSCs) have shown immense therapeutic potential for various brain diseases. Intrathecal administration of MSCs may enhance their recruitment to lesions in the central nervous system, but any impact on cerebrospinal fluid (CSF) flow remains unclear. METHODS: Rats with or without middle cerebral artery occlusion (MCAO) received intrathecal injections of 2D cultured MSCs, 3D cultured MSCs or an equal volume of artificial cerebrospinal fluid (ACSF). Ventricle volume was assessed by MRI on Days 2 and 14 post-MCAO surgery. A beam walking test was used to assess fine motor coordination and balance. Aggregation of MSCs was evaluated in CSF and frozen brain tissue. Differential expression of cell adhesion molecules was evaluated by RNA-Seq, flow cytometry and immunofluorescence analyses. The influence of VCAM-1 blockade in mediating the aggregation of 2D MSCs was investigated in vitro by counting cells that passed through a strainer and in vivo by evaluating ventricular dilation. RESULTS: MSC expanded in 2D culture formed aggregates in the CSF and caused ventricular enlargement in both MCAO and normal rats. Aggregates were associated with impaired motor function. 2D MSCs expressed higher levels of integrin α4 and VCAM-1 than 3D MSCs. Blockade of VCAM-1 in 2D MSCs reduced their aggregation in vitro and reduced lateral ventricular enlargement after intrathecal infusion. 3D MSCs exhibited lower cell aggregation and reduced cerebral ventricular dilation after intrathecal transplantation CONCLUSIONS: The aggregation of 2D MSCs, mediated by the interaction of integrin α4 and VCAM-1, is a potential risk for obstruction of CSF flow after intrathecal transplantation.
Subject(s)
Infarction, Middle Cerebral Artery , Integrin alpha4 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Vascular Cell Adhesion Molecule-1 , Animals , Rats , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/therapy , Integrin alpha4/genetics , Integrin alpha4/metabolism , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Objective: To explore the correlation of CD49d expression patterns with molecular genetics and hotspot gene mutants in patients with chronic lymphocytic leukemia. Methods: The expression of CD49d was detected by flow cytometry and grouped into homogeneous, bimodal, negative and positive expression. Panel fluorescence in situ hybridization (FISH) was used for molecular genetics analysis and next-generation sequencing (NGS) was conducted for gene mutation detection. Results: There were 43 patients (23.89% ) with positive CD49d expression, 137 patients (76.11% ) with negative CD49d expression, 96 patients (53.33% ) with homogeneous CD49d expression and 84 patients (46.67% ) with bimodal CD49d expression. Compared with patients in the CD49d negative group, patients in the CD49d positive group had higher Rai stage (P=0.048) and higher proportion of spleen enlargement (P=0.030) . Compared with patients with homogeneous expression of CD49d, patients with bimodal expression of CD49d had a higher proportion of spleen enlargement (P=0.009) . The expression rate of 11q22- in bimodal CD49d(-) group was significantly higher than that in homogeneous CD49d(-) group (24.29% vs 10.45% , P=0.043) . The incidence of +12 in homogeneous CD49d group was higher than that in bimodal CD49d group (16.67% vs 5.95% , P=0.035) . The incidence of +12 in homogeneous CD49d(+) group was higher than that in bimodal CD49d(-) group (17.24% vs 4.29% , P=0.045) . The incidence of +12 in homogeneous CD49d(-) group was higher than that in bimodal CD49d(-) group (16.42% vs 4.29% , P=0.024) . BIRC3 mutation rate in CD49d positive group was higher than that in CD49d negative group (11.63% vs 2.92% , P=0.037) . Conclusion: There were significant correlations between CD49d and 11q22-, +12 and BIRC3 gene mutation. Patients with bimodal CD49d were more correlated with poor prognosis indexes.
Subject(s)
Integrin alpha4 , Leukemia, Lymphocytic, Chronic, B-Cell , Baculoviral IAP Repeat-Containing 3 Protein/genetics , Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Biomarkers, Tumor/metabolism , Humans , In Situ Hybridization, Fluorescence , Integrin alpha4/genetics , Integrin alpha4/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Molecular Biology , PrognosisABSTRACT
This study was carried out to assess the prognostic power of low CD49d expression (≥10%) in newly diagnosed CLL patients using a previously described cohort. Eighty-five patients were included. Median age at diagnosis; 70 years (43-88); CD49d was expressed in 33/85 (38.8%); 23/33 (69.7%) at ≥30% referred to as 'HiCD49d' and 10/33 (30.3%) between 10 and 30% with a bimodal pattern on scatterplot analysis referred to as 'LoCD49d'. Eleven patients (12.9%) presented as Binet stage B, of whom 8 (72.7%) were CD49d+ (HiCD49d 7/8; LoCD49d 1/8). Seven of 81 patients (8.6%) were NOTCH1 mutated and all were CD49d+ (p ≤ .01). IgVH analysis was performed on 29 (87.8%) of the CD49d+ cases, of whom 21 (72.4%) were unmutated and 8 (27.6%) were mutated. CD38+/CD49d+ accounted for 11/20 (55%) (CD38+/HiCD49D: 9/11; CD38+/LoCD49D: 2/11). At 42 months, treatment had been initiated in 18/85 (21%) patients, of these 10/33 (30.3%) were CD49d+ versus 8/52 (15.4%) of the CD49d- group. The median treatment free interval for the CD49d+ group was 11 months (HiCD49d; 14.5 months, LoCD49d; 11 months) compared to 21.5 months for the CD49d- group. These findings suggest that the predictive value of CD49d expression is retained at expression levels down to 10%.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , ADP-ribosyl Cyclase 1 , Adult , Aged , Aged, 80 and over , Cohort Studies , Humans , Integrin alpha4/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Middle Aged , PrognosisABSTRACT
Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Microphthalmia-Associated Transcription Factor/immunology , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , Cells, Cultured , Dendritic Cells/metabolism , Flow Cytometry , Gene Expression Regulation/immunology , Hyperplasia , Integrin alpha4/genetics , Integrin alpha4/immunology , Integrin alpha4/metabolism , Mice, Inbred C57BL , Mice, Knockout , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Thymus Gland/pathologySubject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Integrin alpha4/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Mutation , Telomere Homeostasis , Tumor Suppressor Protein p53/genetics , Biomarkers, Tumor/analysis , Cyclophosphamide/administration & dosage , Follow-Up Studies , Humans , Integrin alpha4/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Prognosis , Rituximab/administration & dosage , Survival Rate , Vidarabine/administration & dosage , Vidarabine/analogs & derivativesABSTRACT
Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.
Subject(s)
Lymphocytes/metabolism , Nucleic Acid Heteroduplexes/metabolism , Oligonucleotides/metabolism , RNA/metabolism , Administration, Intravenous , Adoptive Transfer , Animals , Demyelinating Diseases/genetics , Demyelinating Diseases/immunology , Demyelinating Diseases/pathology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Endocytosis/drug effects , Female , Gene Expression Regulation , Gene Silencing , Graft vs Host Disease/genetics , Graft vs Host Disease/immunology , Humans , Integrin alpha4/genetics , Integrin alpha4/metabolism , Jurkat Cells , Male , Mice, Inbred C57BL , Nucleic Acid Heteroduplexes/administration & dosage , Nucleic Acid Heteroduplexes/pharmacokinetics , Nucleic Acid Heteroduplexes/pharmacology , Oligonucleotides/administration & dosage , Oligonucleotides/pharmacokinetics , Oligonucleotides/pharmacology , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spinal Cord/pathology , Tissue Distribution/drug effectsABSTRACT
This study set out to check the quantitative and qualitative properties of peripheral CD4+CD25+CD49d- T regulatory (CD49d- Treg) cells in type 2 diabetes mellitus (T2DM) patients. This work comprised 35 newly diagnosed patients and 35 healthy controls (HCs). The frequency of FoxP3 expressing CD49d- Treg cells was determined by flow cytometry. The gene expression of FoxP3 and CD49d was assessed by real-time PCR. Suppression assays with purified CD49d- Treg cells and CD4+CD25- T conventional (Tconv) cells were done by flow cytometry. The supernatants of Tconv/CD49d- Treg co-cultures were tested for IFN-γ, IL-4, IL-17, and IL-10 using ELISA. The frequency of CD49d- Treg cells (by both CD4+CD25+CD49d- and CD4+CD25++CD49d- phenotypes) observed to be reduced in patients versus HCs. In the patients, decreased protein and gene expression of FoxP3 was seen in CD49d- Treg cells. Suppressive potency of CD49d- Treg cells to inhibit Tconv cells proliferation was diminished, and inversely related to fasting plasma glucose and hemoglobin A1c in the patients. Tconv cells from T2DM patients released higher amount of IL-17 and lower concentration of IL-10 versus HCs. In Tconv/CD49d- Tregs co-cultures, decreased IL-17 and increased IL-10 levels were seen in HCs, but not T2DM patients. CD49d- Treg cells from the patients have a fundamental defect and Treg cells fail to inhibit the aggressive inflammatory responses.
Subject(s)
Diabetes Mellitus, Type 2/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Cells, Cultured , Coculture Techniques , Cytokines/immunology , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/genetics , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Gene Expression , Glycated Hemoglobin/analysis , Humans , Integrin alpha4/genetics , Integrin alpha4/immunology , Male , Middle AgedABSTRACT
The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.
Subject(s)
Colitis/therapy , Gene Silencing , Nanoparticles/chemistry , RNA, Small Interfering/pharmacology , Animals , Colitis/genetics , Gene Expression Regulation/drug effects , Humans , Integrin alpha4/chemistry , Integrin alpha4/genetics , Integrin beta Chains/chemistry , Integrin beta Chains/genetics , Lipids/chemistry , Lipids/pharmacology , Liver/drug effects , Mice , Nanoparticles/therapeutic use , RNA, Small Interfering/geneticsSubject(s)
Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Integrin alpha4/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Aged, 80 and over , Anemia/complications , Gene Expression Regulation, Leukemic , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Lymphocytosis/complications , Male , MutationABSTRACT
BACKGROUND: The spontaneously diabetic "non-obese diabetic" (NOD) mouse is a faithful model of human type-1 diabetes (T1D). METHODS: Given the pivotal role of α4 integrin (CD49d) in other autoimmune diseases, we generated NOD mice with α4-deficient hematopoiesis (NOD.α4-/-) to study the role of α4 integrin in T1D. RESULTS: NOD.α4-/- mice developed islet-specific T-cells and antibodies, albeit quantitatively less than α4+ counterparts. Nevertheless, NOD.α4-/- mice were completely and life-long protected from diabetes and insulitis. Moreover, transplantation with isogeneic α4-/- bone marrow prevented progression to T1D of pre-diabetic NOD.α4+ mice despite significant pre-existing islet cell injury. Transfer of α4+/CD3+, but not α4+/CD4+ splenocytes from diabetic to NOD.α4-/- mice induced diabetes with short latency. Despite an only modest contribution of adoptively transferred α4+/CD3+ cells to peripheral blood, pancreas-infiltrating T-cells were exclusively graft derived, i.e., α4+. Microbiota of diabetes-resistant NOD.α4-/- and pre-diabetic NOD.α4+ mice were identical. Co- housed diabetic NOD.α4+ mice showed the characteristic diabetic dysbiosis, implying causality of diabetes for dysbiosis. Incidentally, NOD.α4-/- mice were protected from autoimmune sialitis. CONCLUSION: α4 is a potential target for primary or secondary prevention of T1D.
Subject(s)
Adaptive Immunity/genetics , Integrin alpha4/genetics , Integrin alpha4/metabolism , Animals , Antibodies, Monoclonal , Antigens/metabolism , Autoimmune Diseases/metabolism , Blood Glucose/metabolism , Diabetes Mellitus, Type 1/metabolism , Immunity, Humoral , Immunotherapy, Adoptive , Islets of Langerhans/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Natalizumab/therapeutic useABSTRACT
Many inflammatory bowel disease (IBD) patients require surgical intervention due to limited pharmacological treatment options. Antibodies targeting α4ß7, a gut-homing integrin, are one of the most promising IBD treatments. As retinoic acid (RA) regulates expression of gut-homing proteins including α4ß7 integrin, we tested if ALDH1A enzymes in the RA synthesis pathway could be targeted for IBD treatment using a potent inhibitor, WIN 18,446. Age- and sex-matched Smad3-/- mice were fed a diet with and without WIN 18,446 for 3 weeks before triggering inflammation with Helicobacter bilis infection. Colitis was evaluated by histopathology one week following the IBD trigger, and T cell subsets were evaluated before and after the IBD trigger. WIN 18,446 treatment significantly reduced IBD severity in Smad3-/- mice and reduced expression of α4ß7 integrin on multiple activated CD4+ T cell subsets. This change was associated with increased ratios of induced regulatory T cells to Th17 cells during the inflammatory response in the draining lymph nodes. These studies indicate that RA reduction via ALDH1A enzyme inhibition is a potential new target for IBD treatment. Further studies are needed to examine its effects on other types of immune cells, to evaluate the efficacy window for this target, and to determine its efficacy in other animal models of IBD.
Subject(s)
Aldehyde Dehydrogenase 1 Family/metabolism , Colitis/drug therapy , Helicobacter/metabolism , Integrin alpha4/genetics , Lymphocyte Activation/drug effects , Retinal Dehydrogenase/metabolism , Aldehyde Dehydrogenase 1 Family/antagonists & inhibitors , Animals , Colitis/etiology , Colitis/microbiology , Diamines/pharmacology , Disease Models, Animal , Female , Helicobacter Infections/complications , Helicobacter Infections/drug therapy , Integrin alpha4/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Dehydrogenase/antagonists & inhibitorsABSTRACT
Arsenic and benzo[α]pyrene (BaP) are widespread carcinogens and important etiology factors for lung cancer. Moreover, arsenic and BaP co-exposure displays a significantly stronger effect in inducing lung cancer than arsenic or BaP exposure alone. This study was performed to investigate the basic mechanism of the synergistic carcinogenic effect of arsenic and BaP co-exposure. It was found that integrin α4 (ITGA4) expression levels are significantly up-regulated and the Hedgehog pathway is highly activated in arsenic plus BaP co-exposure-transformed human bronchial epithelial cells. Either ITGA4 downregulation or Hedgehog pathway inhibition in the co-exposure-transformed cells significantly reduced their cancer stem cell (CSC)-like property and tumorigenicity. It was determined that ITGA4 downregulation leads to the inhibition of the Hedgehog pathway activation, which is achieved by increasing suppressor of fused (SUFU) protein stability through reducing the PI3K/Akt signaling. Moreover, stably overexpressing SUFU in the co-exposure-transformed cells significantly reduces their CSC-like property and tumorigenicity. These findings indicate that ITGA4 up-regulation activates the Hedgehog pathway to enhance the CSC-like property and tumorigenicity of arsenic and BaP co-exposure-transformed cells, offering new mechanistic insight for the synergistic carcinogenic effect of arsenic and BaP co-exposure.
Subject(s)
Arsenic/adverse effects , Benzo(a)pyrene/adverse effects , Cell Transformation, Neoplastic/chemically induced , Integrin alpha4/genetics , Lung Neoplasms/pathology , Up-Regulation , Animals , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Neoplastic , Hedgehog Proteins/metabolism , Humans , Integrin alpha4/metabolism , Lung Neoplasms/chemically induced , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Neoplastic Stem Cells/drug effects , Protein Stability , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Signal Transduction/drug effectsABSTRACT
Endocardium is critically important for proper function of the cardiovascular system. Not only does endocardium connect the heart to blood vasculature, it also plays an important role in heart morphogenesis, valve formation, and ventricular trabeculation. The extracellular protein Fibronectin (Fn1) promotes endocardial differentiation, but the signaling pathways downstream of Fn1 that regulate endocardial development are not understood. Here, we analyzed the role of the Fibronectin receptors Integrin alpha5 (Itga5) and Integrin alpha4 (Itga4) in zebrafish heart development. We show that itga5 mRNA is expressed in both endocardium and myocardium during early stages of heart development. Through analysis of both itga5 single mutants and itga4;itga5 double mutants, we show that loss of both itga5 and itga4 results in enhanced defects in endocardial differentiation and morphogenesis compared to loss of itga5 alone. Loss of both itga5 and itga4 results in cardia bifida and severe myocardial morphology defects. Finally, we find that loss of itga5 and itga4 results in abnormally narrow anterior endodermal sheet morphology. Together, our results support a model in which Itga5 and Itga4 cooperate to promote endocardial differentiation, medial migration of endocardial and myocardial cells, and morphogenesis of anterior endoderm.
Subject(s)
Cell Differentiation , Endocardium/embryology , Integrin alpha4/metabolism , Integrin alpha5/metabolism , Models, Biological , Organogenesis , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Integrin alpha4/genetics , Integrin alpha5/genetics , Mutation , Zebrafish/genetics , Zebrafish Proteins/geneticsSubject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Graft vs Host Disease/drug therapy , Integrin alpha4/antagonists & inhibitors , Integrin beta Chains , Animals , Disease Models, Animal , Graft vs Host Disease/genetics , Graft vs Host Disease/metabolism , Graft vs Host Disease/pathology , Humans , Integrin alpha4/genetics , Integrin alpha4/metabolism , Mice , Mice, KnockoutABSTRACT
Integrins, transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix interactions, are heterodimers that consist of an α- and ß-subunit. The integrin α4 gene (itgα4) is expressed in various type of cells and tissues. Its biochemical functions and physiological roles have been revealed using cultured cell assays. In contrast, the primary effect caused by itgα4 deletion on vertebrate development is poorly understood, because knockout mice exhibit multiple defects that can lead to embryonic lethality in the uterus. Zebrafish are a convenient vertebrate model to investigate morphogenesis during embryogenesis, because of their external fertilization and subsequent development outside the female's body. Here, we generated a zebrafish mutant line named itgα4 ko108 using the CRISPR/Cas9 genome editing system; the mutant genome harbored an approximately 2.0-kb deletion in the itgα4 locus. A truncated transcript was detected in itgα4 (+/-) or (-/-) fish but not in (+/+) fish. The mutant transcript was hypothesized to encode a truncated Itgα4 protein due to a premature stop codon. itgα4 (-/-) embryos obtained from the mating of heterozygous parents exhibited no apparent phenotype during development at 24 hours post-fertilization (hpf). However, approximately half of them exhibited cephalic hemorrhage at 48 hpf. The incidence ratio was significantly higher than that in (+/+) or (+/-) embryos. Embryonic hemorrhage has also been reported previously in Itgα4 knockout mice. In contrast, embryonic lethality with the other defects reported in the knockout mice was not observed in our zebrafish model. Therefore, the mutant line itgα4 ko108 should be a useful model to investigate a physiological function for Itgα4 in the blood circulation system.
Subject(s)
Embryonic Development/genetics , Integrin alpha4/genetics , Morphogenesis/genetics , Zebrafish/genetics , Animals , CRISPR-Cas Systems/genetics , Cell Communication/genetics , Gene Editing , Gene Expression Regulation, Developmental/genetics , Genome/genetics , Head/growth & development , Head/pathology , Hemorrhage/physiopathology , Heterozygote , Phenotype , Zebrafish/growth & developmentABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline associated with the deposition of amyloid-ß (Aß) plaques, hyperphosphorylation of tau protein, and neuronal loss. Vascular inflammation and leukocyte trafficking may contribute to AD pathogenesis, and a better understanding of these inflammation mechanisms could therefore facilitate the development of new AD therapies. Here we show that T cells extravasate in the proximity of cerebral VCAM-1+ vessels in 3xTg-AD transgenic mice, which develop both Aß and tau pathologies. The counter-ligand of VCAM-1 - α4ß1 integrin, also known as very late antigen-4 (VLA-4) - was more abundant on circulating CD4+ T cells and was also expressed by a significant proportion of blood CD8+ T cells and neutrophils in AD mice. Intravital microscopy of the brain microcirculation revealed that α4 integrins control leukocyte-endothelial interactions in AD mice. Therapeutic targeting of VLA-4 using antibodies that specifically block α4 integrins improved the memory of 3xTg-AD mice compared to an isotype control. These antibodies also reduced neuropathological hallmarks of AD, including microgliosis, Aß load and tau hyperphosphorylation. Our results demonstrate that α4 integrin-dependent leukocyte trafficking promotes cognitive impairment and AD neuropathology, suggesting that the blockade of α4 integrins may offer a new therapeutic strategy in AD.
Subject(s)
Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Cell Communication , Endothelium/metabolism , Integrin alpha4/antagonists & inhibitors , Leukocytes/metabolism , Memory , Alzheimer Disease/diagnosis , Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Biomarkers , Disease Models, Animal , Gene Expression Regulation , Immunohistochemistry , Integrin alpha4/genetics , Integrin alpha4/metabolism , Maze Learning , Mice , Mice, Transgenic , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Treatment Outcome , tau Proteins/metabolismABSTRACT
Plasmacytoid dendritic cells (pDCs) are found in the CNS during neuroinflammation and have been reported to exert regulatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of entry of pDCs into the CNS as well as their phenotype and innate functional properties, once recruited into the CNS, have not been thoroughly examined. Herein, we show that pDCs rapidly accumulate into the brain and spinal cord during the acute phase of EAE, and maintain the expression of numerous phenotypic markers typical of peripheral pDCs. Functionally, CNS-pDCs constitutively expressed IRF7 and were able to rapidly produce type I IFNs and IL-12p40 upon ex vivo TLR-9 stimulation. Using adoptive transfer experiments, we provide evidence that CNS-pDC are recruited from the blood and accumulate into the CNS during the acute phase of EAE. Accumulation of pDCs into the CNS was strongly inhibited in the absence of CD29, but not CD18, suggesting a major role for ß1 but not ß2 integrins. Indeed, blocking the CD49d α4-integrins during acute EAE drastically diminished CNS-pDC numbers. Together, our results demonstrate that circulating pDCs are actively recruited into the CNS during acute EAE through a mechanism largely dependent on CD49d/CD29-integrins.