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Axial spondyloarthritis (axSpA) is characterized by type-17 immune-driven joint inflammation, and intestinal inflammation is present in around 70% of patients. In this study, we asked whether axSpA stool contained Th17-associated cytokines and whether this related to systemic Th17 activation. We measured stool cytokine and calprotectin levels by ELISA and found that patients with axSpA have increased stool IL-17A, IL-23, GM-CSF, and calprotectin. We further identified increased levels of circulating IL-17A+ and IL-17F+ T-helper cell lymphocytes in patients with axSpA compared to healthy donors. We finally assessed stool metabolites by unbiased nuclear magnetic resonance spectroscopy and found that multiple stool amino acids were negatively correlated with stool IL-23 concentrations. These data provide evidence of type-17 immunity in the intestinal lumen, and suggest its association with microbial metabolism in the intestine.
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HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyzed ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8+ T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4ß7, and CCR6. In addition, we found an expansion of YeiH-specific CD8+ T cells in axSpA and B27AAU blood compared with that from individuals acting as healthy controls, whereas influenza-specific CD8+ T cells were equivalent across groups. Finally, we demonstrated the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8+ T cells may occur in enteric organs.
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Espondiloartritis Axial , Antígeno HLA-B27 , Receptores CCR6 , Uveítis Anterior , Humanos , Uveítis Anterior/inmunología , Antígeno HLA-B27/genética , Antígeno HLA-B27/inmunología , Receptores CCR6/genética , Receptores CCR6/metabolismo , Receptores CCR6/inmunología , Espondiloartritis Axial/inmunología , Femenino , Masculino , Adulto , Linfocitos T CD8-positivos/inmunología , Integrinas/metabolismo , Integrinas/inmunología , Persona de Mediana EdadRESUMEN
BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a proinflammatory cytokine overproduced in several inflammatory and autoimmune diseases, including axial spondyloarthritis. Namilumab is a human IgG1 monoclonal anti-GM-CSF antibody that potently neutralises human GM-CSF. We aimed to assess the efficacy of namilumab in participants with moderate-to-severe active axial spondyloarthritis. METHODS: This proof-of-concept, randomised, double-blind, placebo-controlled, phase 2, Bayesian (NAMASTE) trial was done at nine hospitals in the UK. Participants aged 18-75 years with axial spondyloarthritis, meeting the Assessment in SpondyloArthritis international Society (ASAS) criteria and the ASAS-defined MRI criteria, with active disease as defined by a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), were eligible. Those who had inadequately responded or had intolerance to previous treatment with an anti-TNF agent were included. Participants were randomly assigned (6:1) to receive subcutaneous namilumab 150 mg or placebo at weeks 0, 2, 6, and 10. Participants, site staff (except pharmacy staff), and central study staff were masked to treatment assignment. The primary endpoint was the proportion of participants who had an ASAS ≥20% improvement (ASAS20) clinical response at week 12 in the full analysis set (all randomly assigned participants). This trial is registered with ClinicalTrials.gov (NCT03622658). FINDINGS: From Sept 6, 2018, to July 25, 2019, 60 patients with moderate-to-severe active axial spondyloarthritis were assessed for eligibility and 42 were randomly assigned to receive namilumab (n=36) or placebo (n=six). The mean age of participants was 39·5 years (SD 13·3), 17 were women, 25 were men, 39 were White, and seven had previously received anti-TNF therapy. The primary endpoint was not met. At week 12, the proportion of patients who had an ASAS20 clinical response was lower in the namilumab group (14 of 36) than in the placebo group (three of six; estimated between-group difference 6·8%). The Bayesian posterior probability η was 0·72 (>0·927 suggests high clinical significance). The rates of any treatment-emergent adverse events in the namilumab group were similar to those in the placebo group (31 vs five). INTERPRETATION: Namilumab did not show efficacy compared with placebo in patients with active axial spondyloarthritis, but the treatment was generally well tolerated. FUNDING: Izana Bioscience, NIHR Oxford Biomedical Research Centre (BRC), NIHR Birmingham BRC, and Clinical Research Facility.
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Espondiloartritis Axial , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Método Doble Ciego , Femenino , Masculino , Adulto , Persona de Mediana Edad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Espondiloartritis Axial/tratamiento farmacológico , Reino Unido , Resultado del Tratamiento , Adulto Joven , Anciano , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Índice de Severidad de la Enfermedad , Adolescente , Prueba de Estudio ConceptualRESUMEN
Objectives: IL26 levels are elevated in the blood and synovial fluid of patients with inflammatory arthritis. IL26 can be produced by Th17 cells and locally within joints by tissue-resident cells. IL26 induces osteoblast mineralization in vitro. As osteoproliferation and Th17 cells are important factors in the pathogenesis of axial spondyloarthritis (axSpA), we aimed to clarify the cellular sources of IL26 in spondyloarthritis. Methods: Serum, peripheral blood mononuclear cells (n = 15-35) and synovial tissue (n = 3-9) of adult patients with axSpA, psoriatic arthritis (PsA) and rheumatoid arthritis (RA) and healthy controls (HCs, n = 5) were evaluated by ELISA, flow cytometry including PrimeFlow assay, immunohistochemistry and immunofluorescence and quantitative PCR. Results: Synovial tissue of axSpA patients shows significantly more IL26-positive cells than that of HCs (p < 0.01), but numbers are also elevated in PsA and RA patients. Immunofluorescence shows co-localization of IL26 with CD68, but not with CD3, SMA, CD163, cadherin-11, or CD90. IL26 is elevated in the serum of RA and PsA (but not axSpA) patients compared with HCs (p < 0.001 and p < 0.01). However, peripheral blood CD4+ T cells from axSpA and PsA patients show higher positivity for IL26 in the PrimeFlow assay compared with HCs. CD4+ memory T cells from axSpA patients produce more IL26 under Th17-favoring conditions (IL-1ß and IL-23) than cells from PsA and RA patients or HCs. Conclusion: IL26 production is increased in the synovial tissue of SpA and can be localized to CD68+ macrophage-like synoviocytes, whereas circulating IL26+ Th17 cells are only modestly enriched. Considering the osteoproliferative properties of IL26, this offers new therapeutic options independent of Th17 pathways.
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Antígenos CD , Artritis Psoriásica , Interleucinas , Sinoviocitos , Humanos , Artritis Psoriásica/inmunología , Artritis Psoriásica/metabolismo , Sinoviocitos/metabolismo , Sinoviocitos/inmunología , Sinoviocitos/patología , Masculino , Adulto , Femenino , Antígenos CD/metabolismo , Interleucinas/metabolismo , Interleucinas/sangre , Persona de Mediana Edad , Antígenos de Diferenciación Mielomonocítica/metabolismo , Espondiloartritis Axial/inmunología , Células Th17/inmunología , Células Th17/metabolismo , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Articulaciones/patología , Articulaciones/inmunología , Articulaciones/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/sangre , Artritis Reumatoide/patologíaRESUMEN
GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified. Phage-derived loop-binding antibodies recognized the wild-type native receptor to which they had not previously been exposed, generating confidence in the use of chimeric soluble proteins to act as efficient surrogates for membrane protein extracellular loop antigens. This technique provides promise for the rational design of chimeric antigens in facilitating the discovery of specific antibodies to GPCRs.
This technique offers a viable approach for antibody discovery to difficult GPCRs.Structurally relevant, soluble chimeric scaffold proteins of GPR65 were generated.Chimeric antigens were used to identify GPR65-specific antibodies by phage display.
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Técnicas de Visualización de Superficie Celular , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , TecnologíaRESUMEN
INTRODUCTION: Psoriatic arthritis (PsA) affects around 150 000 people in the UK of whom around 50% require treatment with biologics. The most used biologics for PsA target tumour necrosis factor (TNF) or interleukin-17A (IL-17A). About 50% of patients respond to each, but it is not currently possible to predict response for individual patients, necessitating sequential treatment steps. A recent proof of concept study in PsA suggested that using peripheral immunophenotype to choose therapy could improve time to treatment response.This study will test the hypothesis, within an open-label parallel-group biomarker-stratified multicentre randomised controlled trial, which the baseline proportion of CD4+T cells with an activated type 17 immunophenotype (Th17 levels) predicts response to IL-17A or TNF inhibitors in PsA. Additional analyses will identify if the model can be refined by combining additional clinical and immunophenotypic factors. Statistical modelling will be used to predict the likely effectiveness of these approaches compared with standard care. METHODS AND ANALYSIS: Patients with PsA eligible to start their first biologic as part of standard care are recruited and baseline blood tests are taken for immunophenotyping. Participants are stratified equally by Th17 levels and randomised 1:1 to receive either TNF (adalimumab) or IL-17A (secukinumab) inhibitors. The primary analysis will establish the interaction between baseline immunophenotype and treatment on the primary outcome (achievement of minimal disease activity criteria at week 24). In secondary analysis, modelling will identify if this prediction model can be optimised further by incorporating clinical phenotypes and additional immunophenotyping techniques. ETHICS AND DISSEMINATION: Ethical approval for the study was granted by the North West Preston Research Ethics Committee (ref 21/NW/0016). Dissemination will be via conference presentations and peer-reviewed publications, aiming to impact on treatment guidelines. TRIAL REGISTRATION NUMBER: ISRCTN17228602.
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Antirreumáticos , Artritis Psoriásica , Productos Biológicos , Humanos , Artritis Psoriásica/tratamiento farmacológico , Artritis Psoriásica/patología , Antirreumáticos/uso terapéutico , Interleucina-17/uso terapéutico , Medicina de Precisión , Productos Biológicos/uso terapéutico , Resultado del Tratamiento , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios Multicéntricos como AsuntoRESUMEN
Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.
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Artritis Reumatoide , Microbioma Gastrointestinal , Microbiota , Humanos , Microbioma Gastrointestinal/genética , Inflamación , Fenotipo , Redes y Vías MetabólicasRESUMEN
Ankylosing spondylitis (AS) is a common, highly heritable inflammatory arthritis characterized by enthesitis of the spine and sacroiliac joints. Genome-wide association studies (GWASs) have revealed more than 100 genetic associations whose functional effects remain largely unresolved. Here, we present a comprehensive transcriptomic and epigenomic map of disease-relevant blood immune cell subsets from AS patients and healthy controls. We find that, while CD14+ monocytes and CD4+ and CD8+ T cells show disease-specific differences at the RNA level, epigenomic differences are only apparent upon multi-omics integration. The latter reveals enrichment at disease-associated loci in monocytes. We link putative functional SNPs to genes using high-resolution Capture-C at 10 loci, including PTGER4 and ETS1, and show how disease-specific functional genomic data can be integrated with GWASs to enhance therapeutic target discovery. This study combines epigenetic and transcriptional analysis with GWASs to identify disease-relevant cell types and gene regulation of likely pathogenic relevance and prioritize drug targets.
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Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.
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Autoinmunidad , Antígenos HLA-B , Péptidos , Receptores de Antígenos de Linfocitos T , Humanos , Autoantígenos/química , Autoantígenos/inmunología , Autoantígenos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Líquido Sinovial/inmunología , Espondilitis Anquilosante/inmunología , Uveítis Anterior/inmunología , Biblioteca de Péptidos , Reacciones Cruzadas , Secuencias de AminoácidosRESUMEN
Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (ß2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.
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Antígeno HLA-B27 , Antígenos de Histocompatibilidad Clase I , Presentación de Antígeno , Genes MHC Clase I , Antígeno HLA-B27/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Chaperonas Moleculares/genética , Péptidos/genéticaRESUMEN
Regulatory T cells (Tregs) play an important role in controlling inflammation and limiting autoimmunity, but their phenotypes at inflammatory sites in human disease are poorly understood. We here analyze the single-cell transcriptome of >16,000 Tregs obtained from peripheral blood and synovial fluid of two patients with HLA-B27+ ankylosing spondylitis and three patients with psoriatic arthritis, closely related forms of inflammatory spondyloarthritis. We identify multiple Treg clusters with distinct transcriptomic profiles, including, among others, a regulatory CD8+ subset expressing cytotoxic markers/genes, and a Th17-like RORC+ Treg subset characterized by IL-10 and LAG-3 expression. Synovial Tregs show upregulation of interferon signature and TNF receptor superfamily genes, and marked clonal expansion, consistent with tissue adaptation and antigen contact respectively. Individual synovial Treg clones map to different clusters indicating cell fate divergence. Finally, we demonstrate that LAG-3 directly inhibits IL-12/23 and TNF secretion by patient-derived monocytes, a mechanism with translational potential in SpA. Our detailed characterization of Tregs at an important inflammatory site illustrates the marked specialization of Treg subpopulations.
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Expresión Génica , Espondiloartritis/fisiopatología , Líquido Sinovial/química , Linfocitos T Reguladores/metabolismo , Adulto , Anciano , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de la Célula IndividualRESUMEN
OBJECTIVES: A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. METHODS: Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed. RESULTS: We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF. CONCLUSIONS: Using multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.
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Artritis Psoriásica/inmunología , Células Dendríticas/citología , Macrófagos/citología , Monocitos/citología , Líquido Sinovial/citología , Linfocitos T/citología , Adulto , Artritis Psoriásica/genética , Artritis Psoriásica/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Osteopontina/genética , Osteopontina/inmunología , Osteopontina/metabolismo , RNA-Seq , Análisis de la Célula Individual , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10-15) lies â¼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.
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Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis.
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Artritis Psoriásica/inmunología , Linfocitos T CD8-positivos/inmunología , Selección Clonal Mediada por Antígenos , Receptores Mensajeros de Linfocitos/metabolismo , Líquido Sinovial/inmunología , Artritis Psoriásica/sangre , Linfocitos T CD8-positivos/metabolismo , Perfilación de la Expresión Génica , Humanos , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Mensajeros de Linfocitos/genética , Análisis de la Célula Individual , Membrana Sinovial/inmunologíaRESUMEN
Objectives: GM-CSF is a pro-inflammatory cytokine with multiple actions predominantly on myeloid cells. Enhanced GM-CSF expression by lymphocytes from patients with Ankylosing Spondylitis (AS) has recently been described, however, its potential pathogenic role(s) in AS are unknown. Methods: The effects of GM-CSF on TNF, IL-23, and CCL17 production by blood, PBMCs and isolated CD14+ monocytes from AS patients and healthy controls (HCs) were studied using ELISA. Serum CCL17 and GM-CSF and T cell GM-CSF production were studied in AS patients including pre-and on TNFi therapy. Results: GM-CSF markedly increased TNF production by LPS-stimulated whole blood, peripheral blood mononuclear cells (PBMC) and purified monocytes from AS patients, with 2 h GM-CSF exposure sufficient for monocyte "priming." Blocking of GM-CSF significantly reduced the production of TNF by whole blood from AS patients but not HCs. GM-CSF priming increased IL-23 production from LPS-stimulated AS and HC whole blood 5-fold, with baseline and stimulated IL-23 levels being significantly higher in AS whole blood. GM-CSF also stimulated CCL17 production from AS and HC blood and CCL17 levels were elevated in AS plasma. GM-CSF could be detected in plasma from 14/46 (30%) AS patients compared to 3/18 (17%) HC. Conclusion: We provide evidence that GM-CSF primes TNF and IL-23 responses in myeloid cells from AS patients and HC. We also show CCL17 levels, downstream of GM-CSF, were elevated in plasma samples of AS patients. Taken together these observations are supportive of GM-CSF neutralization as a potential novel therapeutic approach for the treatment of AS.
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Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Mediadores de Inflamación/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Espondilitis Anquilosante/etiología , Espondilitis Anquilosante/metabolismo , Biomarcadores , Estudios de Casos y Controles , Quimiocina CCL17/biosíntesis , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/inmunología , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/patología , Receptores Toll-Like/agonistas , Inhibidores del Factor de Necrosis Tumoral/farmacología , Inhibidores del Factor de Necrosis Tumoral/uso terapéuticoRESUMEN
Ankylosing spondylitis (AS) is a common immune-mediated inflammatory arthritis with a strong genetic predisposition. We review recent data from genetic and animal studies highlighting the importance of Type 17 immune responses. Furthermore, the efficacy (or lack thereof) of different anti-cytokine monoclonal antibodies has highlighted the diversity of Type 17 immune cells and cytokines critical to AS and related spondyloarthritis pathogenesis. Recent studies have strongly implicated the gut microbiome in AS. Finally, we propose that the local metabolic environment of the joint may have a key role in driving AS, and present a novel model of AS pathogenesis.
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Inflamación/inmunología , Espondilitis Anquilosante/inmunología , Células Th17/inmunología , Animales , Microbioma Gastrointestinal/inmunología , HumanosRESUMEN
Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position Ω). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to â¼40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.
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Aminopeptidasas/metabolismo , Síndrome de Behçet/metabolismo , Antígenos HLA-B/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Péptidos/metabolismo , Proteoma/metabolismo , Aminoácidos/metabolismo , Membrana Celular/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Unión ProteicaRESUMEN
T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.
Asunto(s)
Benzazepinas/farmacología , Histona Demetilasas/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Pirimidinas/farmacología , Células Th17/metabolismo , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Benzazepinas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Código de Histonas/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Humanos , Interleucina-17/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Cultivo Primario de Células , Pirimidinas/uso terapéutico , RNA-Seq , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factores de Transcripción/metabolismoRESUMEN
Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here, we examined the ability of the eight most prevalent HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers compared with all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position 59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine 59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine 67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features, such as peptide binding and antigen presentation, as pivotal mechanisms for disease pathogenesis.
Asunto(s)
Antígeno HLA-B27/metabolismo , Alelos , Línea Celular , Cristalografía por Rayos X , Dimerización , Genotipo , Antígeno HLA-B27/química , Antígeno HLA-B27/genética , Humanos , Mutagénesis Sitio-Dirigida , Polimorfismo Genético , Estabilidad Proteica , Estructura Terciaria de Proteína , Espondilitis Anquilosante/metabolismo , Espondilitis Anquilosante/patologíaRESUMEN
IL-7 is a key factor in T cell immunity and common variants at IL7R, encoding its receptor, are associated with autoimmune disease susceptibility. IL7R mRNA is induced in stimulated monocytes, yet a function for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level in healthy individuals, and find that monocyte surface and soluble IL7R (sIL7R) are markedly induced by lipopolysaccharide. In monocytes, both surface IL7R and sIL7R expression strongly associate with allelic carriage of rs6897932, a disease-associated IL7R polymorphism. Monocytes produce more sIL7R than CD4 + T cells, and the amount is additionally correlated with the expression of DDX39A, encoding a splicing factor. Synovial fluid-derived monocytes from patients with spondyloarthritis are enriched for IL7R+ cells with a unique transcriptional profile that overlaps with IL-7-induced gene sets. Our data thus suggest a previously unappreciated function for monocytes in IL-7 biology and IL7R-associated diseases.