ABSTRACT
OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.
Subject(s)
Anemia, Sideroblastic/genetics , Anti-Inflammatory Agents/therapeutic use , Genetic Diseases, X-Linked/genetics , Immunologic Deficiency Syndromes/genetics , Mutation , Nucleotidyltransferases/genetics , RNA, Transfer/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Adult , Anemia, Sideroblastic/blood , Child , Child, Preschool , Cytokines/blood , Cytokines/genetics , Developmental Disabilities/genetics , Female , Genetic Diseases, X-Linked/blood , Humans , Immunophenotyping , Male , Pedigree , Phenotype , Tumor Necrosis Factor-alpha/analysis , Exome SequencingABSTRACT
We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11c(high)CD8 alpha(high)CD11b(low) dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 microg) and Flt3-L (3 microg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-gamma, eosinophilia and substantially increased IL-10 and the number of CD4(+)CD25(+) Forkhead winged helix transcription factor box P3 (Foxp3(+)) IL-10(+) T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-gamma levels, and decreased BALF IL-10 levels and the number of CD4(+)CD25(+)Foxp3(+)IL-10(+) T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4(+)CD25(+) T cells isolated from lungs of Flt3-L-treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4(+)CD25(+) T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4(+)CD25(+)Foxp3(+)IL-10(+)ICOS(+) T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/metabolism , CD4 Antigens/metabolism , Forkhead Transcription Factors/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Lung/immunology , Membrane Proteins/pharmacology , T-Lymphocytes/cytology , Animals , Antibodies/pharmacology , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , Cell Count , Cell Separation , Cockroaches/immunology , Flow Cytometry , Forkhead Transcription Factors/genetics , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Gene Expression Regulation/drug effects , Immunization , Inducible T-Cell Co-Stimulator Protein , L-Selectin/metabolism , Lung/drug effects , Lung/pathology , Methacholine Chloride/pharmacology , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , RNA, Messenger/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Airway inflammation and remodeling are primary characteristics of long-standing asthma. A balance between the T(H)1/T(H)2 cytokines regulates the accumulation and activation of inflammatory cells, including mast cells and eosinophils. Recently, we demonstrated that pUMVC3-hFLex, an active plasmid, mammalian expression vector for the secretion of Flt3-L, reversed established airway hyperresponsiveness (AHR) in a murine model of acute allergic airway inflammation. The present experiments were undertaken to examine the effect of pUMVC3-hFLex in a chronic model of allergic airway inflammation that was established in Balb/c mice by sensitization and challenge with ovalbumin (OVA). pUMVC3-hFLex or the control plasmid, pUMVC3, were administered by injection into the muscle interior tibialis. Treatment with pUMVC3-hFLex completely reversed established AHR (p < 0.05), and this effect continued even after several exposures to the allergen (p < 0.05). pUMVC3-hFLex treatment prevented the development of goblet cell hyperplasia and subepithelial fibrosis, and significantly reduced serum levels of IL-4 and IL-5, and increased serum IL-10 levels (p < 0.05) with no effect on serum IL-13. Serum IgE or serum total and anti-OVA IgG1 and IgG2a levels did not change. Total BALF cellularity and BALF IL-5 levels were reduced (p < 0.05), but there was no significant effect on BALF IL-10 and IL-13. These results suggest that pUMVC3-hFLex treatment can prevent the development of airway remodeling and maintain airway protection in chronic experimental asthma model, and might provide a novel approach for treating chronic asthma.
Subject(s)
Asthma/therapy , Membrane Proteins/genetics , Animals , Asthma/genetics , Asthma/immunology , Asthma/pathology , Collagen/metabolism , Cytokines/blood , Cytokines/immunology , Female , Goblet Cells/drug effects , Goblet Cells/immunology , Goblet Cells/pathology , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Immunoglobulin Isotypes/immunology , Immunotherapy/methods , Membrane Proteins/blood , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Ovalbumin/pharmacology , Plasmids/genetics , Plasmids/immunologyABSTRACT
OBJECTIVE: Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. METHODS: ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. RESULTS: ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. CONCLUSIONS: Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes.
Subject(s)
Aminopeptidases/metabolism , HLA-B27 Antigen/metabolism , Minor Histocompatibility Antigens/metabolism , Flow Cytometry , Gene Knockdown Techniques , Genetic Predisposition to Disease , HLA-B27 Antigen/genetics , Humans , Immunoblotting , Immunoprecipitation , Spondylitis, Ankylosing/genetics , Spondylitis, Ankylosing/metabolism , Spondylitis, Ankylosing/pathology , U937 CellsABSTRACT
We have previously reported that fms-like tyrosine kinase 3 ligand (Flt3-L) prevents and reverses established allergic airway inflammation in an ovalbumin (OVA) induced mouse model of asthma. In this study, we investigated the effect of pUMVC3-hFLex, a plasmid, mammalian expression vector for the secretion of Flt3-L on the same mouse model as well as the duration of the effect of the treatment. Allergic airway inflammation to OVA was established in BALB/c mice. OVA-sensitized mice received three intramuscular (i.m.) injections of 200 mug pUMVC3-hFLex over 10 days. The response to pUMVC3-hFLex therapy was assessed based on airway hyperresponsiveness (AHR) to methacholine and inflammation, measured as serum cytokine and immunoglobulins (Ig) levels, and the total and differential cells in bronchoalveolar lavage fluid (BALF). pUMVC3-hFLex treatment completely reversed established AHR (P<0.01) and this effect lasted for at least 24 days after the last treatment injection (P<0.001). pUMVC3-hFLex treatment significantly increased BALF interferon-gamma (IFN-gamma) (P<0.01), serum interleukin (IL)-10 (P<0.01) and anti-OVA IgG2a levels (P<0.01). In contrast, serum IL-4 and IgE levels were significantly reduced (P<0.05). Total BALF cellularity, eosinophiles counts and BALF IL-5 levels were also reduced (P<0.01). pUMVC3-hFLex treatment can reverse established experimental asthma and might provide a novel approach for treating asthma.
Subject(s)
Genetic Therapy , Inflammation/therapy , Membrane Proteins/genetics , Ovalbumin/immunology , Plasmids , Respiratory Hypersensitivity/therapy , Animals , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Dendritic Cells/immunology , Disease Models, Animal , Female , Immunization , Immunoglobulins/immunology , Inflammation/immunology , Interleukin-4/immunology , Interleukin-5/immunology , Mice , Mice, Inbred BALB C , Plasmids/administration & dosage , Respiratory Hypersensitivity/immunologyABSTRACT
OBJECTIVE: Spontaneous inflammatory responses initiated by NLRP3 mutations promote inflammasome-mediated interleukin-1ß (IL-1ß) processing and release and can induce rapid necrotic cell death. The cells that produce IL-1ß in neonatal-onset multisystem inflammatory disease (NOMID) have not been clearly identified, nor have the mechanisms mediating IL-1ß release and cell death been completely elucidated. METHODS: Whole blood cells were stimulated with lipopolysaccharide (LPS) in the presence of cathepsin B and caspase 1 inhibitors, followed by ATP treatment. Supernatants were collected and incubated with IL-1ß-capturing beads. Cells were fixed, permeabilized, and stained for a combination of cell surface and intracellular markers, and a novel flow cytometry bead-based assay was used to measure secreted IL-1ß. LPS-stimulated cells were also evaluated using immunofluorescence microscopy. RESULTS: Monocytes characterized by CD14(high) -CD16(low) expression and intracellular CD83 were increased in NOMID patients and were responsible for the majority of IL-1ß production in response to LPS stimulation. This population of monocytes also underwent a rapid death response with LPS alone that is temporally associated with IL-1ß and ASC release and has characteristic features of pyronecrotic but not pyroptotic cell death. Inhibition of cell death reduced IL-1ß production from NOMID patient cells. In addition, IL-1 triggered cell death in monocytes from NOMID patients. CONCLUSION: Our findings indicate that monocytes are the predominant IL-1ß-producing cell population in the peripheral blood of NOMID patients. Furthermore, they suggest that IL-1 receptor blockade may work in part by preventing pyronecrotic cell death, which may be an important target in NOMID and other forms of cryopyrin-associated periodic syndromes.
Subject(s)
Cryopyrin-Associated Periodic Syndromes/immunology , Interleukin-1beta/immunology , Monocytes/immunology , Necrosis/immunology , Adolescent , Adult , Antigens, CD/immunology , Carrier Proteins/genetics , Caspase Inhibitors/pharmacology , Cathepsin B/antagonists & inhibitors , Cell Death/immunology , Child , Child, Preschool , Cryopyrin-Associated Periodic Syndromes/genetics , Female , GPI-Linked Proteins/immunology , Humans , Immunoglobulins/immunology , Infant , Lipopolysaccharide Receptors/immunology , Lipopolysaccharides/pharmacology , Male , Membrane Glycoproteins/immunology , Monocytes/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Receptors, IgG/immunology , Young Adult , CD83 AntigenABSTRACT
Although previous studies have described CD25 expression and production of interleukin-2 (IL-2) by mature dendritic cells (mDCs), it remains unclear how these molecules participate in the activation of T cells. In search of the mechanisms by which daclizumab, a humanized monoclonal antibody against CD25, inhibits brain inflammation in multiple sclerosis, we observed that although the drug has limited effects on polyclonal T cell activation, it potently inhibits activation of antigen-specific T cells by mDCs. We show that mDCs (and antigen-experienced T cells) secrete IL-2 toward the mDC-T cell interface in an antigen-specific manner, and mDCs 'lend' their CD25 to primed T cells in trans to facilitate early high-affinity IL-2 signaling, which is crucial for subsequent T cell expansion and development of antigen-specific effectors. Our data reveal a previously unknown mechanism for the IL-2 receptor system in DC-mediated activation of T cells.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antigen Presentation , Dendritic Cells/immunology , Immunoglobulin G/pharmacology , Interleukin-2/immunology , T-Lymphocytes/immunology , Antibodies, Monoclonal, Humanized , Antigen Presentation/drug effects , Daclizumab , Dendritic Cells/drug effects , Humans , Immunosuppressive Agents/pharmacology , In Vitro Techniques , Interleukin-2 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-2 Receptor beta Subunit/immunology , Lymphocyte Activation/drug effects , Models, Immunological , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Signal Transduction , T-Lymphocytes/drug effects , Transplantation Tolerance/drug effects , Transplantation Tolerance/immunologyABSTRACT
Flt3 ligand (Flt3-L) is a growth factor for dendritic cells and induces type 1 T cell responses. We recently reported that Flt3-L prevented OVA-induced allergic airway inflammation and suppressed late allergic response and airway hyper-responsiveness (AHR). In the present study we examined whether Flt3-L reversed allergic airway inflammation in an established model of asthma. BALB/c mice were sensitized and challenged with OVA, and AHR to methacholine was established. Then mice with AHR were randomized and treated with PBS or 6 microg of Flt3-L i.p. for 10 days. Pulmonary functions and AHR to methacholine were examined after rechallenge with OVA. Treatment with Flt3-L of presensitized mice significantly suppressed (p < 0.001) the late allergic response, AHR, bronchoalveolar lavage fluid total cellularity, absolute eosinophil counts, and inflammation in the lung tissue. There was a significant decrease in proinflammatory cytokines (TNF-alpha, IL-4, and IL-5) in bronchoalveolar lavage fluid, with a significant increase in serum IL-12 and a decrease in serum IL-5 levels. There was no significant effect of Flt3-L treatment on serum IL-4 and serum total IgE levels. Sensitization with OVA significantly increased CD11b(+)CD11c(+) cells in the lung, and this phenomenon was not significantly affected by Flt3-L treatment. These data suggest that Flt3-L can reverse allergic airway inflammation and associated changes in pulmonary functions in murine asthma model.