RESUMO
Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.
Assuntos
Artrite Juvenil/imunologia , Diferenciação Celular/imunologia , Epigênese Genética/imunologia , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/imunologia , Adolescente , Artrite Juvenil/sangue , Artrite Juvenil/genética , Sistemas CRISPR-Cas/genética , Catepsina G/genética , Catepsina G/metabolismo , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Cromatina/metabolismo , Ensaios Enzimáticos , Epigenômica , Feminino , Técnicas de Inativação de Genes , Humanos , Células Jurkat , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Leucócitos Mononucleares/imunologia , Macrófagos/metabolismo , Masculino , Mieloblastina/genética , Mieloblastina/metabolismo , Cultura Primária de Células , Proteólise , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células THP-1 , Adulto JovemRESUMO
Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.
Assuntos
Cálcio/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Células Mieloides/imunologia , Proteínas de Neoplasias/química , Osteogênese/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Molécula 1 de Interação Estromal/químicaRESUMO
Systemic juvenile idiopathic arthritis (sJIA) is a childhood rheumatic disease of unknown origin. Dysregulated innate immunity is implicated in disease pathology. We investigated if IL-1 inhibition affects circulating cytokines and monocyte gene expression. CD14+ monocytes from patients in the RAPPORT trial were analyzed by RT-PCR for expression of IL1B and transcription factors associated with monocyte activation. Serum IL-1ra decreased with treatment, and IL-18BP transiently increased. Serum levels of IL-1ß, IL-6, IL-10 and IL-18 were unchanged. IRF5 and STAT6 were decreased, and PPARG was increased, independent of clinical response, and may represent a skew toward a PPARG-driven M2-like phenotype. IL1B expression was decreased in early clinical responders. A transient increase in STAT1, and a decrease in SOCS1 preceded the reduction in IL1B in early clinical responders. Changes in IL1B/STAT1/SOCS1 could be associated with crosstalk between IL-1 and IFN pathways in sJIA. These transcriptional changes might be useful as drug response biomarkers.
Assuntos
Artrite Juvenil/tratamento farmacológico , Interleucina-1/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/uso terapêutico , Artrite Juvenil/imunologia , Ensaios Clínicos como Assunto , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interleucina-1/imunologia , Interleucina-1beta/imunologia , Monócitos/imunologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina/imunologiaRESUMO
Phospholipase C gamma-2 (PLCγ2)-dependent calcium (Ca(2+)) oscillations are indispensable for nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) activation and downstream gene transcription driving osteoclastogenesis during skeletal remodeling and pathological bone loss. Here we describe, to our knowledge, the first known function of transmembrane protein 178 (Tmem178), a PLCγ2 downstream target gene, as a critical modulator of the NFATc1 axis. In surprising contrast to the osteopetrotic phenotype of PLCγ2(-/-) mice, Tmem178(-/-) mice are osteopenic in basal conditions and are more susceptible to inflammatory bone loss, owing to enhanced osteoclast formation. Mechanistically, Tmem178 localizes to the ER membrane and regulates RANKL-induced Ca(2+) fluxes, thus controlling NFATc1 induction. Importantly, down-regulation of Tmem178 is observed in human CD14(+) monocytes exposed to plasma from systemic juvenile idiopathic arthritis patients. Similar to the mouse model, reduced Tmem178 expression in human cells correlates with excessive osteoclastogenesis. In sum, these findings identify an essential role for Tmem178 to maintain skeletal mass and limit pathological bone loss.
Assuntos
Retroalimentação Fisiológica , Proteínas de Membrana/fisiologia , Fatores de Transcrição NFATC/fisiologia , Osteoclastos/fisiologia , Osteogênese , Animais , Cálcio/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Fosfolipase C gama/fisiologiaRESUMO
Systemic juvenile idiopathic arthritis (sJIA) is characterized by systemic inflammation and arthritis. Monocytes are implicated in sJIA pathogenesis, but their role in disease is unclear. The response of sJIA monocytes to IFN may be dysregulated. We examined intracellular signaling in response to IFN type I (IFNα) and type II (IFNγ) in monocytes during sJIA activity and quiescence, in 2 patient groups. Independent of disease activity, monocytes from Group 1 (collected between 2002 and 2009) showed defective STAT1 phosphorylation downstream of IFNs, and expressed higher transcript levels of SOCS1, an inhibitor of IFN signaling. In the Group 2 (collected between 2011 and 2014), monocytes of patients with recent disease onset were IFNγ hyporesponsive, but in treated, quiescent subjects, monocytes were hyperresponsive to IFNγ. Recent changes in medication in sJIA may alter the IFN hyporesponsiveness. Impaired IFN/pSTAT1 signaling is consistent with skewing of sJIA monocytes away from an M1 phenotype and may contribute to disease pathology.
Assuntos
Artrite Juvenil/genética , Interferons/metabolismo , Monócitos/metabolismo , Fator de Transcrição STAT1/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Adolescente , Artrite Juvenil/imunologia , Artrite Juvenil/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Lactente , Interferon gama/farmacologia , Interferons/imunologia , Interferons/farmacologia , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Adulto JovemRESUMO
BACKGROUND: Differentiation of systemic juvenile idiopathic arthritis (SJIA) fever from other childhood fevers is often delayed due to the lack of reliable, specific biomarkers. We hypothesized that PD-L1 expression is dysregulated in SJIA monocytes and compared it to other candidate SJIA biomarkers. METHODS: This pilot study enrolled children with fever without source and compared PD-L1 expression on myeloid cells to C-reactive protein, erythrocyte sedimentation rate, leukocyte counts, S100A12, S100A8, S100A9, calprotectin, and procalcitonin. Logistic regression models were fit to test SJIA diagnosis with each marker used as an independent predictor. Receiver operating characteristic curves and area under curve were calculated. Gene expression profiling on a subset of samples was performed. RESULTS: Twenty subjects (10 active SJIA, 10 febrile non-SJIA) were enrolled. S100 proteins were significantly elevated in SJIA with >80% sensitivity and >90% specificity. PD-L1 expression was significantly lower in SJIA. Other markers were not specific for SJIA. On exploratory gene analysis, 106 genes were significant for SJIA association, and several of these are associated with immune response pathways. CONCLUSION: In this small cohort, S100 proteins were specific diagnostic biomarkers for SJIA in children with fever. Decreased PD-L1 surface expression on circulating myeloid cells in SJIA suggests possible mechanism for loss of peripheral immune regulation.
Assuntos
Artrite Juvenil/sangue , Artrite Juvenil/diagnóstico , Antígeno B7-H1/sangue , Proteínas S100/sangue , Área Sob a Curva , Artrite Juvenil/genética , Biomarcadores/sangue , Análise Química do Sangue , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Projetos Piloto , Valor Preditivo dos Testes , Prognóstico , Curva ROCAssuntos
Antirreumáticos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Síndrome de Ativação Macrofágica/genética , Proteína cdc42 de Ligação ao GTP/genética , Criança , Feminino , Humanos , Lactente , Inflamação/genética , Interleucina-1beta/antagonistas & inibidores , MasculinoRESUMO
Copper(I)-catalyzed azide-alkyne cycloaddition, or CuAAC click chemistry, is an efficient method for bioconjugation aiming at chemical and biological applications. Herein, we demonstrate how the CuAAC method can provide novel phospholipid-protein conjugates with a high potential for the diagnostics and therapy of autoimmune conditions. In doing this, we, for the first time, covalently bind via 1,2,3-triazole linker biologically complementary molecules, namely phosphoethanol amine with human ß2-glycoprotein I and prothrombin. The resulting phospholipid-protein conjugates show high binding affinity and specificity for the autoimmune antibodies against autoimmune complexes. Thus, the development of this work might become a milestone in further diagnostics and therapy of autoimmune diseases that involve the production of autoantibodies against the aforementioned phospholipids and proteins, such as antiphospholipid syndrome and systemic lupus erythematosus.
Assuntos
Antígenos/química , Autoanticorpos/química , Etanolaminas/química , Imunoconjugados/química , Protrombina/química , beta 2-Glicoproteína I/química , Alcinos/química , Complexo Antígeno-Anticorpo/química , Antígenos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Azidas/química , Química Click , Reação de Cicloadição , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Ligação Proteica , Soluções , Triazóis/químicaRESUMO
OBJECTIVE: In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients. METHODS: Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer. RESULTS: Titers of anti-GPI antibodies in reconstituted B6.g7 mice were â¼60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased. CONCLUSION: Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.
Assuntos
Artrite/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/imunologia , 5'-Nucleotidase/metabolismo , Transferência Adotiva , Animais , Artrite/etiologia , Artrite/patologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/patologia , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/imunologia , Células-Tronco Hematopoéticas/patologia , Proteínas de Homeodomínio/genética , Memória Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Superfície Celular/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Timo/citologia , Timo/imunologia , Irradiação Corporal TotalRESUMO
The acute phase protein serum amyloid A (SAA) has been well characterized as an indicator of inflammation. Nevertheless, its functions in pro versus anti-inflammatory processes remain obscure. Here we provide unexpected evidences that SAA induces the proliferation of the tolerogenic subset of regulatory T cells (T(reg)). Intriguingly, SAA reverses T(reg) anergy via its interaction with monocytes to activate distinct mitogenic pathways in T(reg) but not effector T cells. This selective responsiveness of T(reg) correlates with their diminished expression of SOCS3 and is antagonized by T(reg)-specific induction of this regulator of cytokine signaling. Collectively, these evidences suggest a novel anti-inflammatory role of SAA in the induction of a micro-environment that supports T(reg) expansion at sites of infection or tissue injury, likely to curb (auto)-inflammatory responses.
Assuntos
Anergia Clonal/efeitos dos fármacos , Monócitos/fisiologia , Proteína Amiloide A Sérica/farmacologia , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Proteína Amiloide A Sérica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/fisiologiaRESUMO
Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.
Assuntos
Artrite Juvenil , Vesículas Extracelulares , Humanos , Criança , Microscopia Crioeletrônica , Células Endoteliais , MonócitosRESUMO
Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1ß after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.
Assuntos
Artrite Juvenil/imunologia , Monócitos/imunologia , Células Cultivadas , Criança , Citocinas/biossíntese , Citocinas/imunologia , Proteínas Ligadas por GPI/imunologia , Expressão Gênica , Humanos , Receptores de Lipopolissacarídeos/imunologia , Fenótipo , Receptores de IgG/imunologiaRESUMO
BACKGROUND: Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC). METHODS: PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways. RESULTS: Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup. CONCLUSIONS: The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.
Assuntos
Artrite Juvenil/patologia , Patologia Molecular , Sedimentação Sanguínea , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Articulações/patologia , Leucócitos Mononucleares/imunologia , MasculinoRESUMO
OBJECTIVE: Recent observations in systemic juvenile idiopathic arthritis (JIA) suggest an increasing incidence of high-mortality interstitial lung disease often characterized by a variant of pulmonary alveolar proteinosis (PAP). Co-occurrence of macrophage activation syndrome (MAS) and PAP in systemic JIA suggests a shared pathology, but patients with lung disease associated with systemic JIA (designated SJIA-LD) also commonly experience features of drug reaction such as atypical rashes and eosinophilia. This study was undertaken to investigate immunopathology and identify biomarkers in systemic JIA, MAS, and SJIA-LD. METHODS: We used SOMAscan to measure ~1,300 analytes in sera from healthy controls and patients with systemic JIA, MAS, SJIA-LD, or other related diseases. We verified selected findings by enzyme-linked immunosorbent assay and lung immunostaining. Because the proteome of a sample may reflect multiple states (systemic JIA, MAS, or SJIA-LD), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort. RESULTS: Proteome alterations in active systemic JIA and MAS overlapped substantially, including known systemic JIA biomarkers such as serum amyloid A and S100A9, and novel elevations in the levels of heat-shock proteins and glycolytic enzymes. Interleukin-18 levels were elevated in all systemic JIA groups, particularly MAS and SJIA-LD. We also identified an MAS-independent SJIA-LD signature notable for elevated levels of intercellular adhesion molecule 5 (ICAM-5), matrix metalloproteinase 7 (MMP-7), and allergic/eosinophilic chemokines, which have been previously associated with lung damage. Immunohistochemistry localized ICAM-5 and MMP-7 in the lungs of patients with SJIA-LD. The ability of ICAM-5 to distinguish SJIA-LD from systemic JIA/MAS was independently validated. CONCLUSION: Serum proteins support a systemic JIA-to-MAS continuum; help distinguish systemic JIA, systemic JIA/MAS, and SJIA-LD; and suggest etiologic hypotheses. Select biomarkers, such as ICAM-5, could aid in early detection and management of SJIA-LD.
Assuntos
Artrite Juvenil , Pneumopatias , Síndrome de Ativação Macrofágica , Artrite Juvenil/complicações , Biomarcadores , Humanos , Pneumopatias/epidemiologia , Metaloproteinase 7 da Matriz , ProteomaRESUMO
Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.
Assuntos
Artrite Psoriásica/imunologia , Fosforilação/imunologia , Fator de Transcrição STAT3/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Receptores de IgG/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Food allergies are a leading cause of anaphylaxis, and cellular mechanisms involving antigen presentation likely play key roles in their pathogenesis. However, little is known about the response of specific antigen-presenting cell (APC) subsets to food allergens in the setting of food allergies. Here, we show that in peanut-allergic humans, peanut allergen drives the differentiation of CD209+ monocyte-derived dendritic cells (DCs) and CD23+ (FcÑRII) myeloid dendritic cells through the action of allergen-specific CD4+ T cells. CD209+ DCs act reciprocally on the same peanut-specific CD4+ T cell population to reinforce Th2 cytokine expression in a positive feedback loop, which may explain the persistence of established food allergy. In support of this novel model, we show clinically that the initiation of oral immunotherapy (OIT) in peanut-allergic patients is associated with a decrease in CD209+ DCs, suggesting that breaking the cycle of positive feedback is associated with therapeutic effect.
Assuntos
Alérgenos/imunologia , Arachis/imunologia , Imunidade/imunologia , Hipersensibilidade a Amendoim/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Moléculas de Adesão Celular/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Retroalimentação , Humanos , Imunoterapia/métodos , Lectinas Tipo C/imunologia , Camundongos , Monócitos/imunologia , Receptores de Superfície Celular/imunologia , Receptores de IgE/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologiaRESUMO
Monocytes are crucial regulators of inflammation, and are characterized by three distinct subsets in humans, of which classical and non-classical are the most abundant. Different subsets carry out different functions and have been previously associated with multiple inflammatory conditions. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and poor reproducibility. Publicly available transcriptome profiles provide an alternative source of data characterized by high statistical power and real-world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in cell levels. Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures maintain their accuracy regardless of disease state in an independent cohort profiled by RNA-sequencing and are specific to their respective subset when compared to other immune cells from both myeloid and lymphoid lineages profiled across 6160 transcriptome profiles. Consequently, we show that these signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.
Assuntos
Biomarcadores , Monócitos/metabolismo , Transcriptoma , Plasticidade Celular , Biologia Computacional , Suscetibilidade a Doenças , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Homeostase , Humanos , Imunofenotipagem , Monócitos/imunologia , Transdução de SinaisRESUMO
Systemic juvenile idiopathic arthritis (SJIA) is a chronic arthritis of children characterized by a combination of arthritis and systemic inflammation. There is usually non-specific laboratory evidence of inflammation at diagnosis but no diagnostic test. Normalized volumes from 89/889 2-D protein spots representing 26 proteins revealed a plasma pattern that distinguishes SJIA flare from quiescence. Highly discriminating spots derived from 15 proteins constitute a robust SJIA flare signature and show specificity for SJIA flare in comparison to active polyarticular juvenile idiopathic arthritis or acute febrile illness. We used 7 available ELISA assays, including one to the complex of S100A8/S100A9, to measure levels of 8 of the15 proteins. Validating our DIGE results, this ELISA panel correctly classified independent SJIA flare samples, and distinguished them from acute febrile illness. Notably, data using the panel suggest its ability to improve on erythrocyte sedimentation rate or C-reactive protein or S100A8/S100A9, either alone or in combination in SJIA F/Q discriminations. Our results also support the panel's potential clinical utility as a predictor of incipient flare (within 9 wk) in SJIA subjects with clinically inactive disease. Pathway analyses of the 15 proteins in the SJIA flare versus quiescence signature corroborate growing evidence for a key role for IL-1 at disease flare.
Assuntos
Artrite Juvenil/sangue , Proteínas Sanguíneas/análise , Adolescente , Artrite Juvenil/diagnóstico , Biomarcadores/sangue , Proteínas Sanguíneas/classificação , Criança , Pré-Escolar , Diagnóstico Diferencial , Humanos , Lactente , Adulto JovemRESUMO
DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.
Assuntos
Alelos , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/metabolismo , Animais , Anticorpos/metabolismo , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos B/metabolismo , Linhagem Celular , Meia-Vida , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Camundongos , Peptídeos/genética , Ligação Proteica , Conformação Proteica , TransfecçãoRESUMO
Juvenile idiopathic arthritis (JIA) encompasses a group of chronic childhood arthritides of unknown etiology. One subtype, systemic JIA (SJIA), is characterized by a combination of arthritis and systemic inflammation. Its systemic nature suggests that clues to SJIA pathogenesis may be found in examination of peripheral blood cells. To determine the immunophenotypic profiles of circulating mononuclear cells in SJIA patients with different degrees of disease activity, we studied PBMC from 31 SJIA patients, 20 polyarticular JIA patients (similar to adult rheumatoid arthritis), and 31 age-matched controls. During SJIA disease flare, blood monocyte numbers were increased, whereas levels of myeloid dendritic cells (DC) and gammadelta T cells were reduced. At both flare and quiescence, increased levels of CD14 and CD16 were found on SJIA monocytes. Levels of CD16-DC were elevated at SJIA quiescence compared both to healthy controls and to SJIA subjects with active disease. Overall, our findings suggest dysregulation of innate immunity in SJIA and raise the possibility that quiescence represents a state of compensated inflammation.