RESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint destruction and severe morbidity. Cigarette smoking (CS) can exacerbate the incidence and severity of RA. Although Th17 cells and the Aryl hydrocarbon receptor (AhR) have been implicated, the mechanism by which CS induces RA development remains unclear. Here, using transcriptomic analysis, we show that microRNA-132 is specifically induced in Th17 cells in the presence of either AhR agonist or CS-enriched medium. miRNA-132 thus induced is packaged into extracellular vesicles produced by Th17 and acts as a proinflammatory mediator increasing osteoclastogenesis through the down-regulation of COX2. In vivo, articular knockdown of miR-132 in murine arthritis models reduces the number of osteoclasts in the joints. Clinically, RA patients express higher levels of miR-132 than do healthy individuals. This increase is further elevated by cigarette smoking. Together, these results reveal a hitherto unrecognized mechanism by which CS could exacerbate RA and further advance understanding of the impact of environmental factors on the pathogenesis of chronic inflammatory diseases.
Asunto(s)
Artritis Reumatoide/genética , MicroARNs/genética , Osteogénesis/fisiología , Adulto , Anciano , Animales , Artritis Experimental/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Fumar Cigarrillos/efectos adversos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Persona de Mediana Edad , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Humo , Células Th17/efectos de los fármacos , Células Th17/metabolismo , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.
Asunto(s)
Trampas Extracelulares/genética , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Insuficiencia Multiorgánica/genética , Proteínas de Unión a Fosfato/genética , Sepsis/genética , Inhibidores del Acetaldehído Deshidrogenasa/uso terapéutico , Traslado Adoptivo , Anciano , Animales , Células Cultivadas , Disulfiram/uso terapéutico , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Insuficiencia Multiorgánica/patología , Insuficiencia Multiorgánica/terapia , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Sepsis/patología , Sepsis/terapiaRESUMEN
BACKGROUND: Neutrophil extracellular traps (NETs) are innate defense mechanisms that are also implicated in the pathogenesis of organ dysfunction. However, the role of NETs in pediatric sepsis is unknown. METHODS: Infant (2 weeks old) and adult (6 weeks old) mice were submitted to sepsis by intraperitoneal (i.p.) injection of bacteria suspension or lipopolysaccharide (LPS). Neutrophil infiltration, bacteremia, organ injury, and concentrations of cytokine, NETs, and DNase in the plasma were measured. Production of reactive oxygen and nitrogen species and release of NETs by neutrophils were also evaluated. To investigate the functional role of NETs, mice undergoing sepsis were treated with antibiotic plus rhDNase and the survival, organ injury, and levels of inflammatory markers and NETs were determined. Blood samples from pediatric and adult sepsis patients were collected and the concentrations of NETs measured. RESULTS: Infant C57BL/6 mice subjected to sepsis or LPS-induced endotoxemia produced significantly higher levels of NETs than the adult mice. Moreover, compared to that of the adult mice, this outcome was accompanied by increased organ injury and production of inflammatory cytokines. The increased NETs were associated with elevated expression of Padi4 and histone H3 citrullination in the neutrophils. Furthermore, treatment of infant septic mice with rhDNase or a PAD-4 inhibitor markedly attenuated sepsis. Importantly, pediatric septic patients had high levels of NETs, and the severity of pediatric sepsis was positively correlated with the level of NETs. CONCLUSION: This study reveals a hitherto unrecognized mechanism of pediatric sepsis susceptibility and suggests that NETs represents a potential target to improve clinical outcomes of sepsis.
Asunto(s)
Trampas Extracelulares/microbiología , Sepsis/terapia , Animales , Carga Bacteriana/métodos , Brasil , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL/sangre , Ratones Endogámicos C57BL/microbiología , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/patología , Sepsis/mortalidad , Sepsis/patologíaRESUMEN
Rheumatoid arthritis (RA) is an autoimmune arthropathy characterized by chronic articular inflammation. Methotrexate (MTX) remains the first-line therapy for RA and its anti-inflammatory effect is associated with the maintenance of high levels of extracellular adenosine (ADO). Nonetheless, up to 40% of RA patients are resistant to MTX treatment and this is linked to a reduction of CD39 expression, an ectoenzyme involved in the generation of extracellular ADO by ATP metabolism, on circulating regulatory T cells (Tregs). However, the mechanism mediating the reduction of CD39 expression on Tregs is unknown. Here we demonstrated that the impairment in TGF-ß signalling lead to the reduction of CD39 expression on Tregs that accounts for MTX resistance. TGF-ß increases CD39 expression on Tregs via the activation of TGFBRII/TGFBRI, SMAD2 and the transcription factor CREB, which is activated in a p38-dependent manner and induces CD39 expression by promoting ENTPD1 gene transcription. Importantly, unresponsive patients to MTX (UR-MTX) show reduced expression of TGFBR2 and CREB1 and decreased levels of p-SMAD2 and p-CREB in Tregs compared to MTX-responsive patients (R-MTX). Furthermore, RA patients carrying at least one mutant allele for rs1431131 (AT or AA) of the TGFBR2 gene are significantly (pâ¯=â¯0.0006) associated with UR-MTX. Therefore, we have uncovered a molecular mechanism for the reduced CD39 expression on Tregs, and revealed potential targets for therapeutic intervention for MTX resistance.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Artritis Reumatoide/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Resistencia a Medicamentos , Femenino , Regulación de la Expresión Génica , Frecuencia de los Genes , Humanos , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/genética , Proteína Smad2/metabolismoRESUMEN
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39(+)CD4(+)CD25(+)FoxP3(+) Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Resistencia a Medicamentos/inmunología , Metotrexato/uso terapéutico , Linfocitos T Reguladores/inmunología , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/patología , Biomarcadores/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Humanos , Recuento de Linfocitos , Metotrexato/farmacología , Ratones Endogámicos C57BL , Linfocitos T Reguladores/efectos de los fármacos , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
We demonstrate that the interaction between miR-450a-5p and miR-28-5p and signal transducer and activator of transcription 1 (STAT1) mRNA correlates with the osteoblastic differentiation of mesenchymal stem cells from human exfoliated deciduous teeth (shed cells). STAT1 negatively regulates runx-related transcription factor 2 (RUNX2), which is an essential transcription factor in this process. However, the elements that trigger osteoblastic differentiation and therefore pause the inhibitory effect of STAT1 need investigation. Usually, STAT1 can be posttranscriptionally regulated by miRNAs. To test this, we used an in vitro model system in which shed cells were chemically induced toward osteoblastic differentiation and temporally analyzed, comparing undifferentiated cells with their counterparts in the early (2 days) or late (7 or 21 days) periods of induction. The definition of the entire functional genome expression signature demonstrated that the transcriptional activity of a large set of mRNAs and miRNAs changes during this process. Interestingly, STAT1 and RUNX2 mRNAs feature contrasting expression levels during the course of differentiation. While undifferentiated or early differentiating cells express high levels of STAT1 mRNA, which was gradually downregulated, RUNX2 mRNA was upregulated toward differentiation. The reconstruction of miRNA-mRNA interaction networks allowed the identification of six miRNAs (miR-17-3p, miR-28-5p, miR-29b, miR-29c-5p, miR-145-3p, and miR-450a-5p), and we predicted their respective targets, from which we focused on miR-450a-5p and miR-28-5p STAT1 mRNA interactions, whose intracellular occurrence was validated through the luciferase assay. Transfections of undifferentiated shed cells with miR-450a-5p or miR-28-5p mimics or with miR-450a-5p or miR-28-5p antagonists demonstrated that these miRNAs might play a role as posttranscriptional controllers of STAT1 mRNA during osteoblastic differentiation. J. Cell. Biochem. 118: 4045-4062, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Osteoblastos/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción STAT1/metabolismo , Preescolar , Humanos , Masculino , Células Madre Mesenquimatosas/citología , MicroARNs/genética , Osteoblastos/citología , Factor de Transcripción STAT1/genéticaRESUMEN
OBJECTIVE: Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. TREATMENT: CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. METHODS: CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. RESULTS: Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1ß, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes. CONCLUSION: These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Citocinas/metabolismo , Antagonistas de los Receptores de Endotelina , Sulfonamidas/uso terapéutico , Adulto , Animales , Antirreumáticos/uso terapéutico , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Bosentán , Células Cultivadas , Endotelina-1/genética , Endotelina-1/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Leucocitos Mononucleares , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Masculino , Metotrexato/uso terapéutico , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismoRESUMEN
As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3⺠T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4âº/CD8⺠T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/genética , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Timo/patología , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interleucina-4/farmacología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NOD , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Proteínas/metabolismo , Timo/inmunología , Timo/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Transcripción Genética , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed "promiscuous gene expression" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.
Asunto(s)
Envejecimiento/fisiología , Autoantígenos/genética , Enfermedades Autoinmunes/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Regulación de la Expresión Génica/fisiología , Timo/metabolismo , Factores de Transcripción/genética , Animales , Autoantígenos/metabolismo , Enfermedades Autoinmunes/genética , Biomarcadores/metabolismo , Western Blotting , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Diabetes Mellitus Tipo 1/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos NOD , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citología , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
A secondary cervical thymus (CT) is present in the neck region in about 50% of human and mice. CT in mice is an independent and functional organ, which can be colonized by T lymphocyte progenitors and generate thymocytes that are selected by the T cell receptor repertoire following the positive and negative selection. However, CT and the main thoracic thymus (TT) have been shown in mice to have significant functional differences. In this study, we use transcriptional profiling to compare mRNA or miRNAs expression patterns in murine CT and TT. We used these data to perform functional enrichment of the expression signatures and reconstruction of posttranscriptional miRNA-mRNA interaction networks. For this purpose, we compared the transcriptome profiling of paired RNA samples of whole CTs, TTs and parathyroid gland (PT), which was used as an external group, from Foxn1-GFP;Pth-Cre;R26dTomato transgenic mice that differentially label CT and TT. As expected, CT and TT featured comprehensive transcriptome similarity and this suggests that these organs are subjected to correlated transcriptional control. Nevertheless, significant differences were also observed between TT and CT, characterized by 107 differentially expressed (DE) mRNAs, and in 13 DE miRNAs, that in turn established interactions. These results suggest that functional similarity between TT and CT is reflected in their transcriptional activity and that CT functional uniqueness might be under posttranscriptional control.
Asunto(s)
MicroARNs/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , Timocitos/fisiología , Transcriptoma/genética , Animales , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Epidemiologic studies have highlighted the association of environmental factors with the development and progression of autoimmune and chronic inflammatory diseases. Among the environmental factors, smoking has been associated with increased susceptibility and poor prognosis in rheumatoid arthritis (RA). However, the immune and molecular mechanism of smoking-induced arthritis aggravation remains unclear. The transcription factor aryl hydrocarbon receptor (AHR) regulates the generation of Th17 cells, CD4 T cells linked the development of autoimmune diseases. AHR is activated by organic compounds including polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants that are also present in cigarette smoke. In this study, we investigated the role of AHR activation in the aggravation of experiment arthritis induced by exposure to cigarette smoke. METHODS: Mice were exposed to cigarette smoke during the developmental phase of antigen-induced arthritis and collagen-induced arthritis to evaluate the effects of smoking on disease development. Aggravation of articular inflammation was assessed by measuring neutrophil migration to the joints, increase in articular hyperalgesia and changes in the frequencies of Th17 cells. In vitro studies were performed to evaluate the direct effects of cigarette smoke and PAH on Th17 differentiation. We also used mice genetically deficient for AHR (Ahr KO) and IL-17Ra (Il17ra KO) to determine the in vivo mechanism of smoking-induced arthritis aggravation. RESULTS: We found that smoking induces arthritis aggravation and increase in the frequencies of Th17 cells. The absence of IL-17 signaling (Il17ra KO) conferred protection to smoking-induced arthritis aggravation. Moreover, in vitro experiments showed that cigarette smoke can directly increase Th17 differentiation of T cells by inducing AHR activation. Indeed, Ahr KO mice were protected from cigarette smoke-induced arthritis aggravation and did not display increase in TH17 frequencies, suggesting that AHR activation is an important mechanism for cigarette smoke effects on arthritis. Finally, we demonstrate that PAHs are also able to induce arthritis aggravation. CONCLUSIONS: Our data demonstrate that the disease-exacerbating effects of cigarette smoking are AHR dependent and environmental pollutants with AHR agonist activity can induce arthritis aggravation by directly enhancing Th17 cell development.
Asunto(s)
Artritis Experimental/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Humo/efectos adversos , Células Th17/metabolismo , Animales , Artritis Experimental/etiología , Artritis Experimental/genética , Compuestos Azo/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Pirazoles/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Nicotiana/químicaRESUMEN
Sepsis is a systemic inflammatory response as a result of uncontrolled infections. Neutrophils are the first cells to reach the primary sites of infection, and chemokines play a key role in recruiting neutrophils. However, in sepsis chemokines could also contribute to neutrophil infiltration to vital organs leading to multiple organ failure. ACKR2 is an atypical chemokine receptor, which can remove and degrade inflammatory CC chemokines. The role of ACK2 in sepsis is unknown. Using a model of cecal ligation and puncture (CLP), we demonstrate here that ACKR2 deficient () mice exhibited a significant reduction in the survival rate compared with similarly treated wild-type (WT) mice. However, neutrophil migration to the peritoneal cavity and bacterial load were similar between WT and ACKR2 mice during CLP. In contrast, ACKR2 mice showed increased neutrophil infiltration and elevated CC chemokine levels in the lung, kidney, and heart compared with the WT mice. In addition, ACKR2 mice also showed more severe lesions in the lung and kidney than those in the WT mice. Consistent with these results, WT mice under nonsevere sepsis (90% survival) had higher expression of ACKR2 in these organs than mice under severe sepsis (no survival). Finally, the lungs from septic patients showed increased number of ACKR2 cells compared with those of nonseptic patients. Our data indicate that ACKR2 may have a protective role during sepsis, and the absence of ACKR2 leads to exacerbated chemokine accumulation, neutrophil infiltration, and damage to vital organs.
Asunto(s)
Insuficiencia Multiorgánica/metabolismo , Infiltración Neutrófila , Neutrófilos/metabolismo , Receptores de Quimiocina/metabolismo , Sepsis/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Insuficiencia Multiorgánica/patología , Neutrófilos/patología , Sepsis/patologíaRESUMEN
Patients who survive sepsis can develop long-term immune dysfunction, with expansion of the regulatory T (Treg) cell population. However, how Treg cells proliferate in these patients is not clear. Here we show that IL-33 has a major function in the induction of this immunosuppression. Mice deficient in ST2 (IL-33R) develop attenuated immunosuppression in cases that survive sepsis, whereas treatment of naive wild-type mice with IL-33 induces immunosuppression. IL-33, released during tissue injury in sepsis, activates type 2 innate lymphoid cells, which promote polarization of M2 macrophages, thereby enhancing expansion of the Treg cell population via IL-10. Moreover, sepsis-surviving patients have more Treg cells, IL-33 and IL-10 in their peripheral blood. Our study suggests that targeting IL-33 may be an effective treatment for sepsis-induced immunosuppression.
Asunto(s)
Tolerancia Inmunológica/inmunología , Interleucina-33/inmunología , Sepsis/inmunología , Linfocitos T Reguladores/inmunología , Anciano , Animales , Femenino , Humanos , Tolerancia Inmunológica/genética , Proteína 1 Similar al Receptor de Interleucina-1/deficiencia , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/inmunología , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-33/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Sepsis/genética , Sepsis/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4+ and CD8+ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3+ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs.
Asunto(s)
Complejo CD3/genética , MicroARNs/genética , Páncreas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Receptores CCR7/genética , Linfocitos T/metabolismo , Regiones no Traducidas 3' , Animales , Sitios de Unión , Análisis por Conglomerados , Biología Computacional/métodos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Procesamiento Postranscripcional del ARN , Reproducibilidad de los Resultados , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Timocitos/inmunología , Timocitos/metabolismo , TranscriptomaRESUMEN
The downregulation of PTA genes in mTECs is associated with the loss of self-tolerance, and the role of miRNAs in this process is not fully understood. Therefore, we studied the expression of mRNAs and miRNAs in mTECs from autoimmune NOD mice during the period when loss of self-tolerance occurs in parallel with non-autoimmune BALB/c mice. Although the expression of the transcriptional regulator Aire was unchanged, we observed downregulation of a set of PTA mRNAs. A set of miRNAs was also differentially expressed in these mice. The reconstruction of miRNA-mRNA interaction networks identified the controller miRNAs and predicted the PTA mRNA targets. Interestingly, the known Aire-dependent PTAs exhibited pronounced refractoriness in the networking interaction with miRNAs. This study reveals the existence of a new mechanism in mTECs, and this mechanism may have importance in the control of self-tolerance.
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Antígenos/genética , Epistasis Genética , Células Epiteliales/metabolismo , MicroARNs/genética , Interferencia de ARN , ARN Mensajero/genética , Timo/citología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Antígenos/inmunología , Análisis por Conglomerados , Células Epiteliales/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Ratones , Autotolerancia , Factores de Transcripción/genética , Transcripción Genética , Proteína AIRERESUMEN
In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire.
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Diabetes Mellitus Tipo 1/inmunología , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Timo/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Animales , Línea Celular , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Diabetes Mellitus Tipo 1/genética , Femenino , Humanos , Insulina/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/genética , Timo/inmunología , Transcriptoma , Proteína AIRERESUMEN
BACKGROUND: Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. METHODOLOGY/PRINCIPAL FINDINGS: To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. CONCLUSIONS/SIGNIFICANCE: This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.
Asunto(s)
Artritis Experimental/genética , Colágeno/administración & dosificación , Susceptibilidad a Enfermedades , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Linfocitos T/metabolismo , Algoritmos , Animales , Artritis Experimental/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos DBA , Análisis de Secuencia por Matrices de Oligonucleótidos , Linfocitos T/inmunologíaRESUMEN
Considering that imbalance of central tolerance in the thymus contributes to aggressive autoimmunity, we compared the expression of peripheral tissue autoantigens (PTA) genes, which are involved in self-representation in the thymic stroma, of two mouse strains; DBA-1/J (MHC-H2(q)) susceptible and DBA-2/J (MHC-H2(d)) resistant to collagen induced arthritis (CIA). We evaluate whether these strains differ in their thymic gene expression, allowing identification of genes that might play a role in susceptibility/resistance to CIA. Microarray profiling showed that 1093 PTA genes were differentially modulated between collagen immunized DBA-1/J and DBA-2/J mice. These genes were assigned to 17 different tissues/organs, including joints/bone, characterizing the promiscuous gene expression (PGE), which is implicated in self-representation. Hierarchical clustering of microarray data and quantitative RT-PCR analysis showed that Aire (autoimmune regulator), an important regulator of the PGE process, Aire-dependent (insulin), Aire-independent (Col2A1 and Gad67), and other 22 joint/bone autoantigen genes were down-regulated in DBA-1/J compared with DBA-2/J in the thymus. Considering the importance of MHC-H2 in peptide-self presentation and autoimmunity susceptibility, we reconstructed transcriptional networks of both strains based on actual microarray data. The networks clearly demonstrated different MHC-H2 transcriptional interactions with PTAs genes. DBA-1/J strain featured MHC-H2 as a node influencing downstream genes. Differently, in DBA-2/J strain network MHC-H2 was exclusively self-regulated and does not control other genes. These findings provide evidence that CIA susceptibility in mice may be a reflex of a cascade-like transcriptional control connecting different genes to MHC-H2 in the thymus.