RESUMO
BACKGROUND: IL-17A plays a pivotal pathogenic role in several immune-mediated inflammatory diseases. Despite sharing 50% sequence homology with IL-17A, the role of IL-17F remains less clear. Clinical findings suggest that dual inhibition of IL-17A and IL-17F in psoriatic disease is more efficacious than IL-17A inhibition alone, positing a pathogenic role for IL-17F. OBJECTIVE: We characterized the regulation of IL-17A and IL-17F in psoriatic disease. METHODS: Using both in vitro systems and lesional skin tissue from patients, we interrogated the chromosomal, transcriptional, and protein expression landscape of IL-17A+ and IL-17F+ TH17 cells. Alongside established assays such as single-cell RNA sequencing, we developed a novel cytokine-capture technique that was combined with chromatin immunoprecipitation sequencing and RNA sequencing. RESULTS: We confirm a preferential elevation of IL-17F over IL-17A in psoriatic disease and show that expression of each isoform predominantly occurs in distinct cell populations. The expression of both IL-17A and IL-17F exhibited a high degree of plasticity, with the balance between the 2 isoforms influenced by proinflammatory signaling and by anti-inflammatory drugs such as methylprednisolone. This plasticity was reflected in a broad H3K4me3 region at the IL17A-F locus, while opposing effects of STAT5/IL-2 signaling were observed for each of the 2 genes. Functionally, higher IL17F expression was linked to greater cell proliferation. CONCLUSION: There are key differences in the regulation of IL-17A and IL-17F in psoriatic disease, leading to distinct inflammatory cell populations. As such, we propose that both IL-17A and IL-17F neutralization may be required to maximally inhibit IL-17-driven pathology.
Assuntos
Interleucina-17 , Fator de Transcrição STAT5 , Humanos , Interleucina-17/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.
Assuntos
Doenças do Sistema Nervoso Central , Encefalomielite Autoimune Experimental , Animais , Camundongos , Cuprizona/toxicidade , Sulfatos/efeitos adversos , Oligodendroglia/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Corpo Caloso/patologia , Doenças do Sistema Nervoso Central/patologia , Heparitina Sulfato/uso terapêutico , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Bainha de Mielina/patologiaRESUMO
The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the ß2 family of integrins as regulators of γδ T cells. ß2-integrin-deficient mice displayed a striking increase in numbers of IL-17-producing Vγ6Vδ1+ γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in ß2-integrin-deficient IL-17+ cells compared to their wild-type counterparts. Indeed, ß2-integrin-deficient Vγ6+ cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in ß2-integrin-deficient tissues. Furthermore, our data revealed an unexpected role for ß2 integrins in promoting the thymic development of the IFNγ-producing CD27+ Vγ4+ γδ T cell subset. Together, our data reveal that ß2 integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17-producing Vγ6Vδ1+ cells and promoting the thymic development of the IFNγ-producing Vγ4+ subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.
Assuntos
Antígenos CD18 , Linfócitos Intraepiteliais , Timo , Animais , Antígenos CD18/genética , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Homeostase/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Linfócitos Intraepiteliais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Timo/crescimento & desenvolvimento , Timo/imunologia , Timo/metabolismoRESUMO
Annexin-A1 has a well-defined anti-inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid-induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin-A1 primarily signals, has also been implicated in these diseases. In contrast, treatment with recombinant annexin-A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin-A1 in health and disease and discuss the potential of this protein as a biomarker and therapeutic target.
Assuntos
Anexina A1 , Imunidade Adaptativa , Animais , Anexina A1/metabolismo , Anti-Inflamatórios , Humanos , Inflamação , Camundongos , Receptores de Formil Peptídeo/metabolismo , Linfócitos T/metabolismoRESUMO
BACKGROUND: Once bulk RNA-seq data has been processed, i.e. aligned and then expression and differential tables generated, there remains the essential process where the biology is explored, visualized and interpreted. Without the use of a visualisation and interpretation pipeline this step can be time consuming and laborious, and is often completed using R. Though commercial visualisation and interpretation pipelines are comprehensive, freely available pipelines are currently more limited. RESULTS: Here we demonstrate Searchlight, a freely available bulk RNA-seq visualisation and interpretation pipeline. Searchlight provides: a comprehensive statistical and visual analysis, focusing on the global, pathway and single gene levels; compatibility with most differential experimental designs irrespective of organism or experimental complexity, via three workflows; reports; and support for downstream user modification of plots via user-friendly R-scripts and a Shiny app. We show that Searchlight offers greater automation than current best tools (VIPER and BioJupies). We demonstrate in a timed re-analysis study, that alongside a standard bulk RNA-seq processing pipeline, Searchlight can be used to complete bulk RNA-seq projects up to the point of manuscript quality figures, in under 3 h. CONCLUSIONS: Compared to a manual R based analysis or current best freely available pipelines (VIPER and BioJupies), Searchlight can reduce the time and effort needed to complete bulk RNA-seq projects to manuscript level. Searchlight is suitable for bioinformaticians, service providers and bench scientists. https://github.com/Searchlight2/Searchlight2 .
Assuntos
Publicações , Software , RNA-Seq , Sequenciamento do Exoma , Fluxo de TrabalhoRESUMO
OBJECTIVES: Circulating myeloid precursors are responsible for post-natal osteoclast (OC) differentiation and skeletal health, although the exact human precursors have not been defined. Enhanced osteoclastogenesis contributes to joint destruction in rheumatoid arthritis (RA) and tumour necrosis factor (TNF) is a well-known pro-osteoclastogenic factor. Herein, we investigated the interplay between receptor activator of nuclear factor kappa-Β ligand (RANK-L), indispensable for fusion of myeloid precursors and the normal development of OCs, and TNF in directing the differentiation of diverse pre-OC populations derived from human peripheral blood. METHODS: Flow cytometric cell sorting and analysis was used to assess the potential of myeloid populations to differentiate into OCs. Transcriptomic, epigenetic analysis, receptor expression and inhibitor experiments were used to unravel RANK-L and TNF signalling hierarchy. RESULTS: TNF can act as a critical homoeostatic regulator of CD14+ monocyte (MO) differentiation into OCs by inhibiting osteoclastogenesis to favour macrophage development. In contrast, a distinct previously unidentified CD14-CD16-CD11c+ myeloid pre-OC population was exempt from this negative regulation. In healthy CD14+ MOs, TNF drove epigenetic modification of the RANK promoter via a TNFR1-IKKß-dependent pathway and halted osteoclastogenesis. In a subset of patients with RA, CD14+ MOs exhibited an altered epigenetic state that resulted in dysregulated TNF-mediated OC homoeostasis. CONCLUSIONS: These findings fundamentally re-define the relationship between RANK-L and TNF. Moreover, they have identified a novel pool of human circulating non-MO OC precursors that unlike MOs are epigenetically preconditioned to ignore TNF-mediated signalling. In RA, this epigenetic preconditioning occurs in the MO compartment providing a pathological consequence of failure of this pathway.
Assuntos
Artrite Reumatoide , Osteoclastos , Artrite Reumatoide/patologia , Diferenciação Celular/genética , Homeostase , Humanos , Osteoclastos/patologia , Osteogênese , Ligante RANK/metabolismo , Fator de Necrose Tumoral alfaRESUMO
OBJECTIVES: Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. METHODS: Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. RESULTS: APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. CONCLUSION: Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.
Assuntos
Osteogênese/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Talidomida/análogos & derivados , Adulto , Idoso , Artrite Psoriásica/tratamento farmacológico , Estudos de Casos e Controles , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Inibidores da Fosfodiesterase 4/uso terapêutico , Cultura Primária de Células , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
Ageing is a major risk factor for the development of cardiovascular disease (CVD) and cancer. Whilst the cumulative effect of exposure to conventional cardiovascular risk factors is important, recent evidence highlights clonal haematopoiesis of indeterminant potential (CHIP) as a further key risk factor. CHIP reflects the accumulation of somatic, potentially pro-leukaemic gene mutations within haematopoietic stem cells over time. The most common mutations associated with CHIP and CVD occur in genes that also play central roles in the regulation of inflammation. While CHIP carriers have a low risk of haematological malignant transformation (<1% per year), their relative risk of mortality is increased by 40% and this reflects an excess of cardiovascular events. Evidence linking CHIP, inflammation and atherosclerotic disease has recently become better defined. However, there is a paucity of information about the role of CHIP in the development and progression of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). While systemic inflammation plays a role in the pathophysiology of both heart failure with reduced and preserved ejection fraction (EF), it may be of greater relevance in the pathophysiology of HFpEF, which is also strongly associated with ageing. This review describes CHIP and its pathogenetic links with ageing, inflammation and CVD, while providing insight into its putative role in HFpEF.
Assuntos
Doenças Cardiovasculares , Hematopoiese Clonal , Insuficiência Cardíaca , Inflamação , Envelhecimento , Humanos , Fatores de Risco , Volume SistólicoRESUMO
T cell protective immunity is associated with multifunctional memory cells that produce several different cytokines. Currently, our understanding of when and how these cells are generated is limited. We have used an influenza virus mouse infection model to investigate whether the cytokine profile of memory T cells is reflective of primary responding cells or skewed toward a distinct profile. We found that, in comparison to primary cells, memory T cells tended to make multiple cytokines simultaneously. Analysis of the timings of release of cytokine by influenza virus-specific T cells, demonstrated that primary responding CD4 T cells from lymphoid organs were unable to produce a sustained cytokine response. In contrast CD8 T cells, memory CD4 T cells, and primary responding CD4 T cells from the lung produced a sustained cytokine response throughout the restimulation period. Moreover, memory CD4 T cells were more resistant than primary responding CD4 T cells to inhibitors that suppress T cell receptor signaling. Together, these data suggest that memory CD4 T cells display superior cytokine responses compared to primary responding cells. These data are key to our ability to identify the cues that drive the generation of protective memory CD4 T cells following infection.
Assuntos
Antígenos Virais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Imunidade Celular/efeitos dos fármacos , Memória Imunológica , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Imunidade Celular/genética , Imunofenotipagem , Vírus da Influenza A/química , Vírus da Influenza A/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell-cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.
Assuntos
Comunicação Celular/fisiologia , Exossomos/fisiologia , Osteoartrite/metabolismo , Cartilagem Articular/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Membrana Sinovial/metabolismoRESUMO
OBJECTIVES: Fatty acid oxidation (FAO) and glycolysis have been implicated in immune regulation and activation of macrophages. However, investigation of human monocyte intracellular metabolism in the context of the hypoxic and inflammatory rheumatoid arthritis (RA) synovium is lacking. We hypothesized that exposure of monocytes to the hypoxic and inflammatory RA environment would have a profound impact on their metabolic state, and potential to contribute to disease pathology. METHODS: Human monocytes were isolated from buffy coats and exposed to hypoxia. Metabolic profiling of monocytes was carried out by LC-MS metabolomics. Inflammatory mediator release after LPS or RA-synovial fluid (RA-SF) stimulation was analysed by ELISA. FAO was inhibited by etomoxir or enhanced with exogenous carnitine supplementation. Transcriptomics of RA blood monocytes and RA-SF macrophages was carried out by microarray. RESULTS: Hypoxia exacerbated monocyte-derived CCL20 and IL-1ß release in response to LPS, and increased glycolytic intermediates at the expense of carnitines. Modulation of carnitine identified a novel role for FAO in the production of CCL20 in response to LPS. Transcriptional analysis of RA blood monocytes and RA-SF macrophages revealed that fatty acid metabolism was altered and CCL20 increased when monocytes enter the synovial environment. In vitro analysis of monocytes showed that RA-SF increases carnitine abundance and CCL20 production in hypoxia, which was exacerbated by exogenous carnitine. CONCLUSION: This work has revealed a novel inflammatory mechanism in RA that links FAO to CCL20 production in human monocytes, which could subsequently contribute to RA disease pathogenesis by promoting the recruitment of Th17 cells and osteoclastogenesis.
Assuntos
Artrite Reumatoide/metabolismo , Microambiente Celular , Quimiocina CCL20/metabolismo , Ácidos Graxos/metabolismo , Hipóxia/metabolismo , Monócitos/metabolismo , Líquido Sinovial , Carnitina/farmacologia , Quimiocina CCL20/efeitos dos fármacos , Cromatografia Líquida , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , Metabolômica , Análise em Microsséries , Monócitos/efeitos dos fármacos , Membrana Sinovial/metabolismoRESUMO
The alarmin S100A8/A9 is implicated in sterile inflammation-induced bone resorption and has been shown to increase the bone-resorptive capacity of mature osteoclasts. Here, we investigated the effects of S100A9 on osteoclast differentiation from human CD14+ circulating precursors. Hereto, human CD14+ monocytes were isolated and differentiated toward osteoclasts with M-CSF and receptor activator of NF-κB (RANK) ligand (RANKL) in the presence or absence of S100A9. Tartrate-resistant acid phosphatase staining showed that exposure to S100A9 during monocyte-to-osteoclast differentiation strongly decreased the numbers of multinucleated osteoclasts. This was underlined by a decreased resorption of a hydroxyapatite-like coating. The thus differentiated cells showed a high mRNA and protein production of proinflammatory factors after 16 h of exposure. In contrast, at d 4, the cells showed a decreased production of the osteoclast-promoting protein TNF-α. Interestingly, S100A9 exposure during the first 16 h of culture only was sufficient to reduce osteoclastogenesis. Using fluorescently labeled RANKL, we showed that, within this time frame, S100A9 inhibited the M-CSF-mediated induction of RANK. Chromatin immunoprecipitation showed that this was associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK was in part recovered by blocking TNF-α but not IL-1. Together, our data show that S100A9 impedes monocyte-to-osteoclast differentiation, probably via a reduction in RANK expression.-Di Ceglie, I., Blom, A. B., Davar, R., Logie, C., Martens, J. H. A., Habibi, E., Böttcher, L.-M., Roth, J., Vogl, T., Goodyear, C. S., van der Kraan, P. M., van Lent, P. L., van den Bosch, M. H. The alarmin S100A9 hampers osteoclast differentiation from human circulating precursors by reducing the expression of RANK.
Assuntos
Calgranulina B/fisiologia , Monócitos/efeitos dos fármacos , Osteoclastos/citologia , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Reabsorção Óssea , Calgranulina B/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Código das Histonas , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Interleucina-1/antagonistas & inibidores , Receptores de Lipopolissacarídeos/análise , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/citologia , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND: There is a pressing need in rheumatoid arthritis (RA) to identify patients who will not respond to first-line disease-modifying anti-rheumatic drugs (DMARD). We explored whether differences in genomic architecture represented by a chromosome conformation signature (CCS) in blood taken from early RA patients before methotrexate (MTX) treatment could assist in identifying non-response to DMARD and, whether there is an association between such a signature and RA specific expression quantitative trait loci (eQTL). METHODS: We looked for the presence of a CCS in blood from early RA patients commencing MTX using chromosome conformation capture by EpiSwitch™. Using blood samples from MTX responders, non-responders and healthy controls, a custom designed biomarker discovery array was refined to a 5-marker CCS that could discriminate between responders and non-responders to MTX. We cross-validated the predictive power of the CCS by generating 150 randomized groups of 59 early RA patients (30 responders and 29 non-responders) before MTX treatment. The CCS was validated using a blinded, independent cohort of 19 early RA patients (9 responders and 10 non-responders). Last, the loci of the CCS markers were mapped to RA-specific eQTL. RESULTS: We identified a 5-marker CCS that could identify, at baseline, responders and non-responders to MTX. The CCS consisted of binary chromosome conformations in the genomic regions of IFNAR1, IL-21R, IL-23, CXCL13 and IL-17A. When tested on a cohort of 59 RA patients, the CCS provided a negative predictive value of 90.0% for MTX response. When tested on a blinded independent validation cohort of 19 early RA patients, the signature demonstrated a true negative response rate of 86 and a 90% sensitivity for detection of non-responders to MTX. Only conformations in responders mapped to RA-specific eQTL. CONCLUSIONS: Here we demonstrate that detection of a CCS in blood in early RA is able to predict inadequate response to MTX with a high degree of accuracy. Our results provide a proof of principle that a priori stratification of response to MTX is possible, offering a mechanism to provide alternative treatments for non-responders to MTX earlier in the course of the disease.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Biomarcadores/metabolismo , Cromossomos Humanos/química , Metotrexato/uso terapêutico , Estudos de Coortes , Humanos , Modelos Logísticos , Metotrexato/farmacologia , Locos de Características Quantitativas/genética , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. METHODS: Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. RESULTS: EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect. CONCLUSIONS: Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.
Assuntos
Artrite Psoriásica/patologia , Artrite Reumatoide/patologia , Vesículas Extracelulares/patologia , Osteoclastos/patologia , Adulto , Idoso , Artrite Psoriásica/sangue , Artrite Reumatoide/sangue , Diferenciação Celular , Linhagem Celular , Exossomos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoclastos/citologia , Osteogênese , Ligante RANK/análise , Receptor Ativador de Fator Nuclear kappa-B/análiseRESUMO
Studies of microbial superantigens that target large clonal sets of B cells through conserved antigen-receptor-variable-region sites are providing new insights into the mechanisms of B-cell activation-induced cell death. These investigations have shown differences between the clonal regulation of follicular B cells (B2 cells) and the innate-like marginal-zone B cells and B1 cells, and have also shown how B-cell superantigens can affect specialized host defences against infection. Agents designed to emulate the properties of B-cell superantigens might also provide new approaches for the treatment of B-cell-mediated autoimmune and neoplastic diseases.
Assuntos
Linfócitos B/imunologia , Linfócitos B/microbiologia , Imunidade Celular , Superantígenos/fisiologia , Animais , Linfócitos B/metabolismo , Humanos , Proteína Estafilocócica A/química , Proteína Estafilocócica A/imunologia , Superantígenos/químicaRESUMO
OBJECTIVE: Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis. METHODS: OA was induced in wild-type (WT) and PAR2-deficient (PAR2-/-) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2-/- mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain. RESULTS: Osteophytes formed within 7â days post-DMM in WT mice but osteosclerosis was only evident from 14â days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2-/- mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2-/- mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2-/- mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone. CONCLUSIONS: This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.
Assuntos
Artrite Experimental/patologia , Osso e Ossos/patologia , Cartilagem Articular/patologia , Osteoartrite/patologia , Receptor PAR-2/metabolismo , Animais , Artralgia/etiologia , Artralgia/patologia , Artrite Experimental/etiologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Osteoartrite/etiologia , Osteócitos/metabolismoRESUMO
Autoantibodies to glycans present on glycolipids mediate the postinfectious paralytic disease, Guillain-Barré syndrome (GBS). These glycans are also found on lipo-oligosaccharides (LOSs) of GBS-inducing microbes, suggesting molecular mimicry as a mechanism for disease induction. How B lymphocyte tolerance to self-glycans is regulated during the initiation phase of the disease is currently under investigation. The discovery of antiglycolipid antibodies that bind to heteromeric glycolipid complexes has generated new insights in this field. Heteromeric complexes are structurally distinct glycolipids that interact to form new molecular shapes capable of either enhancing or attenuating recognition by autoantibodies. Although the principles emerging from this phenomenon have a substantial impact on diagnostics methods, they also raise intriguing questions about the diversity of innate antibody repertoires, mechanisms of tolerance, and autoantibody targeting of neural membranes.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Glicolipídeos/imunologia , Síndrome de Guillain-Barré/imunologia , Animais , Humanos , Mimetismo MolecularRESUMO
Chemokine-directed leukocyte migration is a critical component of all innate and adaptive immune responses. The atypical chemokine receptor ACKR2 is expressed by lymphatic endothelial cells and scavenges pro-inflammatory CC chemokines to indirectly subdue leukocyte migration. This contributes to the resolution of acute inflammatory responses in vivo. ACKR2 is also universally expressed by innate-like B cells, suppressing their responsiveness to the non-ACKR2 ligand CXCL13, and controlling their distribution in vivo. The role of ACKR2 in autoimmunity remains relatively unexplored, although Ackr2 deficiency reportedly lessens the clinical symptoms of experimental autoimmune encephalomyelitis induced by immunization with encephalogenic peptide (MOG(35-55)). This was attributed to poor T-cell priming stemming from the defective departure of dendritic cells from the site of immunization. However, we report here that Ackr2-deficient mice, on two separate genetic backgrounds, are not less susceptible to autoimmunity induced by immunization, and in some cases develop enhanced clinical symptoms. Moreover, ACKR2 deficiency does not suppress T-cell priming in response to encephalogenic peptide (MOG(35-55)), and responses to protein antigen (collagen or MOG(1-125)) are characterized by elevated interleukin-17 production. Interestingly, after immunization with protein, but not peptide, antigen, Ackr2 deficiency was also associated with an increase in lymph node B cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances T helper type 17 (Th17) cell development and survival. Thus, Ackr2 deficiency does not suppress autoreactive T-cell priming and autoimmune pathology, but can enhance T-cell polarization toward Th17 cells and increase the abundance of GM-CSF(+) B cells in lymph nodes draining the site of immunization.
Assuntos
Autoantígenos/imunologia , Colágeno/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Receptores de Quimiocinas/metabolismo , Células Th17/imunologia , Animais , Anticorpos/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Imunidade , Imunização , Interleucina-17/biossíntese , Articulações/imunologia , Articulações/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Contagem de Linfócitos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Receptores de Quimiocinas/deficiência , Regulação para CimaRESUMO
BACKGROUND: The humoral and T-cell responses to booster COVID-19 vaccine types in multidisease immunocompromised individuals who do not generate adequate antibody responses to two COVID-19 vaccine doses, is not fully understood. The OCTAVE DUO trial aimed to determine the value of third vaccinations in a wide range of patients with primary and secondary immunodeficiencies. METHODS: OCTAVE-DUO was a prospective, open-label, multicentre, randomised, controlled, phase 3 trial investigating humoral and T-cell responses in patients who are immunocompromised following a third vaccine dose with BNT162b2 or mRNA-1273, and of NVX-CoV2373 for those with lymphoid malignancies. We recruited patients who were immunocompromised from 11 UK hospitals, aged at least 18 years, with previous sub-optimal responses to two doses of SARS-CoV-2 vaccine. Participants were randomly assigned 1:1 (1:1:1 for those with lymphoid malignancies), stratified by disease, previous vaccination type, and anti-spike antibody response following two doses. Individuals with lived experience of immune susceptibility were involved in the study design and implementation. The primary outcome was vaccine-specific immunity defined by anti-SARS-CoV-2 spike antibodies (Roche Diagnostics UK and Ireland, Burgess Hill, UK) and T-cell responses (Oxford Immunotec, Abingdon, UK) before and 21 days after the third vaccine dose analysed by a modified intention-to-treat analysis. The trial is registered with the ISRCTN registry, ISRCTN 15354495, and the EU Clinical Trials Register, EudraCT 2021-003632-87, and is complete. FINDINGS: Between Aug 4, 2021 and Mar 31, 2022, 804 participants across nine disease cohorts were randomly assigned to receive BNT162b2 (n=377), mRNA-1273 (n=374), or NVX-CoV2373 (n=53). 356 (45%) of 789 participants were women, 433 (55%) were men, and 659 (85%) of 775 were White. Anti-SARS-CoV-2 spike antibodies measured 21 days after the third vaccine dose were significantly higher than baseline pre-third dose titres in the modified intention-to-treat analysis (median 1384 arbitrary units [AU]/mL [IQR 4·3-7990·0] compared with median 11·5 AU/mL [0·4-63·1]; p<0·001). Of participants who were baseline low responders, 380 (90%) of 423 increased their antibody concentrations to more than 400 AU/mL. Conversely, 166 (54%) of 308 baseline non-responders had no response after the third dose. Detectable T-cell responses following the third vaccine dose were seen in 494 (80%) of 616 participants. There were 24 serious adverse events (BNT612b2 eight [33%] of 24, mRNA-1273 12 [50%], NVX-CoV2373 four [17%]), two (8%) of which were categorised as vaccine-related. There were seven deaths (1%) during the trial, none of which were vaccine-related. INTERPRETATION: A third vaccine dose improved the serological and T-cell response in the majority of patients who are immunocompromised. Individuals with chronic renal disease, lymphoid malignancy, on B-cell targeted therapies, or with no serological response after two vaccine doses are at higher risk of poor response to a third vaccine dose. FUNDING: Medical Research Council, Blood Cancer UK.
Assuntos
Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Hospedeiro Imunocomprometido , Imunogenicidade da Vacina , SARS-CoV-2 , Humanos , Feminino , Masculino , COVID-19/prevenção & controle , COVID-19/imunologia , Pessoa de Meia-Idade , Hospedeiro Imunocomprometido/imunologia , SARS-CoV-2/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Idoso , Vacina BNT162/imunologia , Vacina BNT162/administração & dosagem , Anticorpos Antivirais/sangue , Estudos Prospectivos , Imunização Secundária , Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Adulto , Linfócitos T/imunologia , Reino Unido , ChAdOx1 nCoV-19/imunologiaRESUMO
Interleukin (IL)-33, a member of the IL-1 cytokine family, is an important modulator of the immune system associated with several immune-mediated disorders. High levels of IL-33 are expressed by the central nervous system (CNS) suggesting a potential role of IL-33 in autoimmune CNS diseases. We have investigated the expression and function of IL-33 in the development of experimental autoimmune encephalomyelitis (EAE) in mice. We report here that IL-33 and its receptor ST2 (IL-33Rα) are highly expressed in spinal cord tissue, and ST2 expression is markedly increased in the spinal cords of mice with EAE. Furthermore, ST2-deficient (ST2(-/-) ) mice developed exacerbated EAE compared with wild-type (WT) mice while WT, but not ST2(-/-) EAE mice treated with IL-33 developed significantly attenuated disease. IL-33-treated mice had reduced levels of IL-17 and IFN-γ but produced increased amounts of IL-5 and IL-13. Lymph node and splenic macrophages of IL-33-treated mice showed polarization toward an alternatively activated macrophage (M2) phenotype with significantly increased frequency of MR(+) PD-L2(+) cells. Importantly, adoptive transfer of these IL-33-treated macrophages attenuated EAE development. Our data therefore demonstrate that IL-33 plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti-inflammatory M2 macrophages.