RESUMEN
The transcription factor IRF5 has been implicated as a therapeutic target for the autoimmune disease systemic lupus erythematosus (SLE). However, IRF5 activation status during the disease course and the effects of IRF5 inhibition after disease onset are unclear. Here, we show that SLE patients in both the active and remission phase have aberrant activation of IRF5 and interferon-stimulated genes. Partial inhibition of IRF5 is superior to full inhibition of type I interferon signaling in suppressing disease in a mouse model of SLE, possibly due to the function of IRF5 in oxidative phosphorylation. We further demonstrate that inhibition of IRF5 via conditional Irf5 deletion and a newly developed small-molecule inhibitor of IRF5 after disease onset suppresses disease progression and is effective for maintenance of remission in mice. These results suggest that IRF5 inhibition might overcome the limitations of current SLE therapies, thus promoting drug discovery research on IRF5 inhibitors.
Asunto(s)
Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Animales , Autoanticuerpos/inmunología , Enfermedades Autoinmunes , Femenino , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inmunoglobulina G , Factores Reguladores del Interferón/efectos de los fármacos , Riñón/patología , Lupus Eritematoso Sistémico/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta , Transducción de Señal , Factores de Transcripción , Familia-src QuinasasRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the roots of Angelica reflexa B.Y.Lee (AR) have been used to treat cough, phlegm, neuralgia, and arthralgia in Northeast Asia. AIM OF THE STUDY: The anti-asthmatic effect of AR root extract (ARE) was determined using a murine airway allergic inflammation model and the primary T cell polarization assay. MATERIALS AND METHODS: To evaluate the anti-asthmatic effect of ARE, inflammatory cell infiltration was determined histologically and inflammatory mediators were measured in bronchoalveolar lavage fluid (BALF). Furthermore, the effects of AREs on Th2 cell differentiation and activation were determined by western blotting and flow cytometry. RESULTS: Asthmatic phenotypes were alleviated by ARE treatment, which reduced mucus production, inflammatory cell infiltration (especially eosinophilia), and type 2 cytokine levels in BALF. ARE administration to mice reduced the number of activated Th2 (CD4+CD25+) cells and level of GATA3 in the lungs. Furthermore, ARE treatment inhibited the differentiation of Th2 cells in primary cell culture systems via interferon regulatory factor 4 (IRF4) signaling. CONCLUSIONS: Our findings indicate that the anti-asthmatic effect of AREs is mediated by the reduction in Th2 cell activation by regulating IRF4.
Asunto(s)
Angelica/química , Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Hipersensibilidad/tratamiento farmacológico , Extractos Vegetales/farmacología , Neumonía/tratamiento farmacológico , Células Th2/efectos de los fármacos , Animales , Antiasmáticos/química , Antiasmáticos/uso terapéutico , Asma/inducido químicamente , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Femenino , Factor de Transcripción GATA3/efectos de los fármacos , Factor de Transcripción GATA3/metabolismo , Hipersensibilidad/inmunología , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/toxicidad , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Raíces de Plantas/química , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología , Eosinofilia Pulmonar/inducido químicamente , Eosinofilia Pulmonar/tratamiento farmacológico , Células RAW 264.7 , Células Th2/inmunologíaRESUMEN
OBJECTIVE: Fibronectin is a matrix protein that is fragmented during cartilage degradation in osteoarthritis (OA). Treatment of chondrocytes with fibronectin fragments (FN-f) has been used to model OA in vitro, but the system has not been fully characterized. This study sought to define the transcriptional response of chondrocytes to FN-f, and directly compare it to responses traditionally observed in OA. DESIGN: Normal human femoral chondrocytes isolated from tissue donors were treated with either FN-f or PBS (control) for 3, 6, or 18 h. RNA-seq libraries were compared between time-matched FN-f and control samples in order to identify changes in gene expression over time. Differentially expressed genes were compared to a published OA gene set and used for pathway, transcription factor motif, and kinome analysis. RESULTS: FN-f treatment resulted in 3,914 differentially expressed genes over the time course. Genes that are up- or downregulated in OA were significantly up- (P < 0.00001) or downregulated (P < 0.0004) in response to FN-f. Early response genes were involved in proinflammatory pathways, whereas many late response genes were involved in ferroptosis. The promoters of upregulated genes were enriched for NF-κB, AP-1, and IRF motifs. Highly upregulated kinases included CAMK1G, IRAK2, and the uncharacterized kinase DYRK3, while growth factor receptors TGFBR2 and FGFR2 were downregulated. CONCLUSIONS: FN-f treatment of normal human articular chondrocytes recapitulated many key aspects of the OA chondrocyte phenotype. This in vitro model is promising for future OA studies, especially considering its compatibility with genomics and genome-editing techniques.
Asunto(s)
Cartílago Articular/citología , Condrocitos/efectos de los fármacos , Fibronectinas/farmacología , Expresión Génica/efectos de los fármacos , Osteoartritis/genética , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/efectos de los fármacos , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Condrocitos/metabolismo , Fémur , Expresión Génica/genética , Humanos , Técnicas In Vitro , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/genética , Quinasas Asociadas a Receptores de Interleucina-1/efectos de los fármacos , Quinasas Asociadas a Receptores de Interleucina-1/genética , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Osteoartritis/metabolismo , Fragmentos de Péptidos/farmacología , Fenotipo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/efectos de los fármacos , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/genéticaRESUMEN
Interferon regulatory factor-8 (IRF-8) is critical for Th1 cell differentiation and negatively regulates myeloid cell development including myeloid-derived suppressor cells (MDSC). MDSC expand during infection with various pathogens including the gastrointestinal (GI) nematode Heligmosomoides polygyrus bakeri (Hpb). We investigated if IRF-8 contributes to Th2 immunity to Hpb infection. Irf8 expression was down-regulated in MDSC from Hpb-infected C57BL/6 (B6) mice. IRF-8 deficient Irf8-/- and BXH-2 mice had significantly higher adult worm burdens than B6 mice after primary or challenge Hpb infection. During primary infection, MDSC expanded to a significantly greater extent in mesenteric lymph nodes (MLN) and spleens of Irf8-/- and BXH-2 than B6 mice. CD4+GATA3+ T cells numbers were comparable in MLN of infected B6 and IRF-8 deficient mice, but MLN cells from infected IRF-8 deficient mice secreted significantly less parasite-specific IL-4 ex vivo. The numbers of alternatively activated macrophages in MLN and serum levels of Hpb-specific IgG1 and IgE were also significantly less in infected Irf8-/- than B6 mice. The frequencies of antigen-experienced CD4+CD11ahiCD49dhi cells that were CD44hiCD62L- were similar in MLN of infected Irf8-/- and B6 mice, but the proportions of CD4+GATA3+ and CD4+IL-4+ T cells were lower in infected Irf8-/- mice. CD11b+Gr1+ cells from naïve or infected Irf8-/- mice suppressed CD4+ T cell proliferation and parasite-specific IL-4 secretion in vitro albeit less efficiently than B6 mice. Surprisingly, there were significantly more CD4+ T cells in infected Irf8-/- mice, with a higher frequency of CD4+CD25+Foxp3+ T (Tregs) cells and significantly higher numbers of Tregs than B6 mice. In vivo depletion of MDSC and/or Tregs in Irf8-/- mice did not affect adult worm burdens, but Treg depletion resulted in higher egg production and enhanced parasite-specific IL-5, IL-13, and IL-6 secretion ex vivo. Our data thus provide a previously unrecognized role for IRF-8 in Th2 immunity to a GI nematode.
Asunto(s)
Enfermedades Gastrointestinales/inmunología , Factores Reguladores del Interferón/inmunología , Células Supresoras de Origen Mieloide/inmunología , Infecciones por Nematodos/inmunología , Nematospiroides dubius/inmunología , Células Th2/inmunología , Animales , Células Cultivadas , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/genética , Interleucina-4/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/inmunologíaRESUMEN
OBJECTIVE: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKÉ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. METHODS: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNß reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. RESULTS: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNß promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. CONCLUSION: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKÉ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies.
Asunto(s)
Factor 3 Regulador del Interferón/efectos de los fármacos , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/efectos de los fármacos , Interferón-alfa/efectos de los fármacos , Interferón beta/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Pirimidinas/farmacología , Tiofenos/farmacología , Western Blotting , Niño , Células HEK293 , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Técnicas In Vitro , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Interferón-alfa/inmunología , Interferón beta/inmunología , Leucocitos Mononucleares/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Mutación , Nucleótidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/efectos de los fármacos , Factor de Transcripción STAT1/metabolismoRESUMEN
Acute rejection is a serious and life-threatening complication of liver transplantation (LTx). Tacrolimus (TAC) is a potent immunosuppressant used in experimental and clinical transplantation. Interferon regulatory factor 4 (IRF4) plays key roles as a transcription factor in the immune response. This study explored the role of IRF4 in acute rejection after LTx using TAC treatment. Here, LTx was performed in DA (RT1(n)) and Lewis (LEW) (RT1(l)) rats. The recipients were immunosuppressed with TAC (1.5mg/kg/day subcutaneously) or saline. Liver grafts were harvested 1, 3, 5, 7, and 10 days after LTx for histology, immunohistochemistry, western blotting and real-time PCR. Splenic mononuclear cells were activated with different doses of TAC. The nuclear factor of activated T cells (NFAT) signal pathway and CD4+ T subset-related transcription factors were assessed. The results showed that TAC treatment prolonged the survival of liver allografts in recipients, significantly attenuated hepatic tissue injury and improved liver function. IRF4 expression in grafts was down-regulated after TAC treatment. TAC inhibited the expression of IRF4, NFAT, Foxp3 and RORγt in splenic mononuclear cells in vitro. In conclusions, our studies showed that TAC attenuated acute rejection responses after LTx. This attenuation might depend on the TAC-NFAT-IRF4 signal pathway, which is crucial for the function of T helper subsets (Treg and Th17 cells) in acute rejection after LTx. These findings contribute to our understanding of the immune pharmacological mechanism of TAC to prevent rejection in LTx rats.
Asunto(s)
Rechazo de Injerto/genética , Inmunosupresores/farmacología , Factores Reguladores del Interferón/biosíntesis , Trasplante de Hígado , Tacrolimus/farmacología , Animales , Linfocitos T CD4-Positivos/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Rechazo de Injerto/patología , Factores Reguladores del Interferón/efectos de los fármacos , Hígado/patología , Activación de Macrófagos/efectos de los fármacos , Masculino , Monocitos/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
VirF is an AraC-type transcriptional regulator responsible for activating the transcription of virulence genes required for the intracellular invasion and cell-to-cell spread of Shigella flexneri. Gene disruption studies have validated VirF as a potential target for an anti-virulence therapy to treat shigellosis by determining that VirF is necessary for virulence, but not required for bacterial viability. Using a bacteria-based, ß-galactosidase reporter assay we completed a high-throughput screening (HTS) campaign monitoring VirF activity in the presence of over 140,000 small molecules. From our screening campaign, we identified five lead compounds to pursue in tissue culture-based invasion and cell-to-cell spread assays, and toxicity screens. Our observations of activity in these models for infection have validated our approach of targeting virulence regulation and have allowed us to identify a promising chemical scaffold from our HTS for hit-to-lead development. Interestingly, differential effects on invasion versus cell-to-cell spread suggest that the compounds' efficacies may depend, in part, on the specific promoter that VirF is recognizing.
Asunto(s)
Antibacterianos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Factores Reguladores del Interferón/efectos de los fármacos , Shigella flexneri/efectos de los fármacos , Proteínas Virales/efectos de los fármacos , Factores de Virulencia , Algoritmos , Antibacterianos/aislamiento & purificación , Interpretación Estadística de Datos , Genes Reporteros/genética , Células HeLa , Humanos , Indicadores y Reactivos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Reproducibilidad de los Resultados , Shigella flexneri/genética , Shigella flexneri/crecimiento & desarrollo , Virulencia/genética , beta-Galactosidasa/genéticaRESUMEN
OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings.
Asunto(s)
Extractos Celulares/farmacología , Células Dendríticas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , FN-kappa B/fisiología , Quimiocinas/genética , Quimiocinas/metabolismo , Células Dendríticas/fisiología , Sinergismo Farmacológico , Humanos , Factores Reguladores del Interferón/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Regulación hacia ArribaRESUMEN
BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells. METHODS: PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis. RESULTS: Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study. CONCLUSIONS: This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Herpesviridae/tratamiento farmacológico , Herpesvirus Humano 8/genética , Linfoma de Efusión Primaria/tratamiento farmacológico , Morfolinos/farmacología , Animales , Antivirales/inmunología , Antivirales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Femenino , Infecciones por Herpesviridae/genética , Herpesvirus Humano 8/efectos de los fármacos , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interleucina-6/análisis , Interleucina-6/genética , Interleucina-6/metabolismo , Linfoma de Efusión Primaria/genética , Linfoma de Efusión Primaria/patología , Linfoma de Efusión Primaria/virología , Ratones , Ratones SCID , Terapia Molecular Dirigida , Morfolinos/inmunología , Morfolinos/uso terapéutico , Factor de Transcripción STAT1/efectos de los fármacos , Factor de Transcripción STAT1/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Inmunodeficiencia Combinada Grave/genética , Transfección , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 microg/ml and 290 microg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2-0.9 microg/ml) and enhance CRISPLD2 secretion (range, 1.5-4.2 microg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-alpha and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body's exposure to LPS but also reflect an individual's LPS sensitivity.