RESUMEN

Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using Mapk12/Mapk13-deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here, we generated a Mapk12D171A/D171A/Mapk13-/- (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to lipopolysaccharide (LPS)-induced septic shock and Candida albicans infection than wild-type (WT) mice. Gene expression analyses in LPS-activated wild-type and p38γ/δKIKO macrophages revealed that p38γ/p38δ-regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of Nos2 and Il1b mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.

Asunto(s)

Lipopolisacáridos , Proteína Quinasa 13 Activada por Mitógenos , Animales , Ratones , Proteína Quinasa 13 Activada por Mitógenos/metabolismo , Proteómica , Inmunidad Innata , Proteína Quinasa 12 Activada por Mitógenos/genética , Proteína Quinasa 12 Activada por Mitógenos/metabolismo , Inflamación

RESUMEN

TPL-2 kinase plays an important role in innate immunity, activating ERK1/2 MAPKs in myeloid cells following TLR stimulation. We investigated how TPL-2 controls transcription in TLR4-stimulated mouse macrophages. TPL-2 activation of ERK1/2 regulated expression of genes encoding transcription factors, cytokines, chemokines, and signaling regulators. Bioinformatics analysis of gene clusters most rapidly induced by TPL-2 suggested that their transcription was mediated by the ternary complex factor (TCF) and FOS transcription factor families. Consistently, TPL-2 induced ERK1/2 phosphorylation of the ELK1 TCF and the expression of TCF target genes. Furthermore, transcriptomic analysis of TCF-deficient macrophages demonstrated that TCFs mediate approximately half of the transcriptional output of TPL-2 signaling, partially via induced expression of secondary transcription factors. TPL-2 signaling and TCFs were required for maximal TLR4-induced FOS expression. Comparative analysis of the transcriptome of TLR4-stimulated Fos -/- macrophages indicated that TPL-2 regulated a significant fraction of genes by controlling FOS expression levels. A key function of this ERK1/2-TCF-FOS pathway was to mediate TPL-2 suppression of type I IFN signaling, which is essential for host resistance against intracellular bacterial infection.

Asunto(s)

Interferón beta/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Animales , Regulación de la Expresión Génica , Interferón beta/metabolismo , Lipopolisacáridos/inmunología , Quinasas Quinasa Quinasa PAM/genética , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción TCF/metabolismo

RESUMEN

Mef2 transcription factors comprise a family of four different isoforms that regulate a number of processes including neuronal and muscle development. While roles for Mef2C and Mef2D have been described in B-cell development their role in immunity has not been extensively studied. In innate immune cells such as macrophages, TLRs drive the production of both pro- and anti-inflammatory cytokines. IL-10 is an important anti-inflammatory cytokine produced by macrophages and it establishes an autocrine feedback loop to inhibit pro-inflammatory cytokine production. We show here that macrophages from Mef2D knockout mice have elevated levels of IL-10 mRNA induction compared with wild-type cells following LPS stimulation. The secretion of IL-10 was also higher from Mef2D knockout macrophages and this correlated to a reduction in the secretion of TNF, IL-6 and IL-12p40. The use of an IL-10 neutralising antibody showed that this reduction in pro-inflammatory cytokine production in the Mef2D knockouts was IL-10 dependent. As the IL-10 promoter has previously been reported to contain a potential binding site for Mef2D, it is possible that the binding of other Mef2 isoforms in the absence of Mef2D may result in a higher activation of the IL-10 gene. Further studies with compound Mef2 isoforms would be required to address this. We also show that Mef2D is highly expressed in the thymus, but that loss of Mef2D does not affect thymic T-cell development or the production of IFNγ from CD8 T cells.

Asunto(s)

Interleucina-10/metabolismo , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Células Cultivadas , Mediadores de Inflamación/metabolismo , Interleucina-10/genética , Lipopolisacáridos/farmacología , Factores de Transcripción MEF2/deficiencia , Factores de Transcripción MEF2/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptores Toll-Like/agonistas , Regulación hacia Arriba

RESUMEN

TPL-2 MAP 3-kinase promotes inflammation in numerous mouse disease models and is an attractive anti-inflammatory drug target. However, TPL-2-deficient (Map3k8 -/-) mice develop exacerbated allergic airway inflammation to house dust mite (HDM) compared with wild type controls. Here, we show that Map3k8D270A/D270A mice expressing kinase dead TPL-2 had an unaltered response to HDM, indicating that the severe airway inflammation observed in Map3k8 -/- mice is not due to blockade of TPL-2 signaling and rather reflects a TPL-2 adaptor function. Severe allergic inflammation in TPL-2-deficient mice was likely due to reduced levels of ABIN-2 (TNIP2), whose stability depends on TPL-2 expression. Tnip2E256K knock-in mutation, which reduced ABIN-2 binding to A20, augmented the HDM-induced airway inflammation, but did not affect TPL-2 expression or signaling. These results identify ABIN-2 as a novel negative regulator of allergic airway responses and importantly indicate that TPL-2 inhibitors would not have unwanted allergic comorbidities.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Hipersensibilidad/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Hipersensibilidad/genética , Hipersensibilidad/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Antiinflamatorios/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Antiinflamatorios/síntesis química , Expresión Génica , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-ß-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8(D270A/D270A) mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8(D270A) mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.

Asunto(s)

Macrófagos/enzimología , Proteínas Quinasas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Toll-Like/metabolismo , Animales , Activación Enzimática , Espectrometría de Masas , Ratones

RESUMEN

The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

Asunto(s)

Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular , Humanos , Masculino , Ratones , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

IL-10 is an important anti-inflammatory cytokine that plays important roles in controlling inflammatory responses and keeping the immune system in check following activation. Loss of IL-10 function in mice or humans results in the development of inflammatory bowel disease in response to an elevated immune response to the gut flora. IL-10 also acts to prevent excessive inflammation during the course of infection and has been implicated in a variety of autoimmune conditions. In response to inflammatory signals, IL-10 can be produced by a number of immune cells including T cells, B cells, macrophages, and dendritic cells. Distinct mechanisms control the production of IL-10 in these different cells types. In this review, we describe recent studies that have looked at the signaling pathways that regulate IL-10 production in these cells. Given the number of cell types that produce IL-10, it is perhaps not surprising that the in vivo source of IL-10 can vary in different immune models. We also describe how work using conditional IL-10 knockout mice or adoptive transfer of IL-10-deficient cells has begun to further our understanding regarding which specific immune cells are required for IL-10 production in vivo under different conditions.

Asunto(s)

Regulación de la Expresión Génica , Interleucina-10/genética , Transcripción Genética , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Interleucina-10/metabolismo , Espacio Intracelular/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Especificidad de Órganos/genética , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo

RESUMEN

The protein kinase ERK5 (MAPK7) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role mediating cell proliferation, survival, epithelial-mesenchymal transition, and angiogenesis. To date, no three-dimensional structure has been published that would allow rational design of inhibitors. To address this, we determined the X-ray crystal structure of the human ERK5 kinase domain in complex with a highly specific benzo[e]pyrimido[5,4-b]diazepine-6(11H)-one inhibitor. The structure reveals that specific residue differences in the ATP-binding site, compared to the related ERKs p38s and JNKs, allow for the development of ERK5-specific inhibitors. The selectivity of previously observed ERK5 inhibitors can also be rationalized using this structure, which provides a template for future development of inhibitors with potential for treatment of disease.

Asunto(s)

Azepinas/química , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/química , Pirimidinas/química , Secuencia de Aminoácidos , Azepinas/farmacología , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Conformación Proteica , Pirimidinas/farmacología , Homología de Secuencia de Aminoácido

RESUMEN

Chemokines, including MCP-1, are crucial to mounting an effective immune response due to their ability to recruit other immune cells. We show that sustained LPS or poly(I:C)-stimulated MCP-1 production requires an IFNß-mediated feedback loop. Consistent with this, exogenous IFNß was able to induce MCP-1 transcription in the absence of other stimuli. Blocking IFNß signaling with Ruxolitinib, a JAK inhibitor, inhibited MCP-1 transcription. The MCP-1 promoter contains potential STAT binding sites and we demonstrate that STAT1 is recruited upon IFNß stimulation. Furthermore we find that IL-10 knockout increases MCP-1 production in response to LPS, which may reflect an ability of IL-10 to repress IFNß production. Overall, these results show the importance of the balance between IFNß and IL-10 in the regulation of MCP-1.

Asunto(s)

Comunicación Autocrina/fisiología , Quimiocina CCL2/genética , Retroalimentación Fisiológica/fisiología , Interferón beta/fisiología , Macrófagos/metabolismo , Receptores Toll-Like/fisiología , Animales , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Células Cultivadas , Quimiocina CCL2/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Interferón beta/metabolismo , Interferón beta/farmacología , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-10/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Receptores Toll-Like/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética

RESUMEN

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/farmacología , Interleucina-10/biosíntesis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Línea Celular , AMP Cíclico/metabolismo , Interleucina-10/genética , Ratones , Fenotipo , Fosforilación/efectos de los fármacos , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

Macrophages are an important source of cytokines following infection. Stimulation of macrophages with TLR agonists results in the secretion of TNF-α, IL-6, and IL-12, and the production of these cytokines is controlled by multiple feedback pathways. Macrophages also produce IL-10, which acts to inhibit proinflammatory cytokine production by macrophages via a JAK/STAT3-dependent pathway. We show in this paper that, Ruxolitinib, a recently described selective inhibitor of JAKs, increases TNF, IL-6, and IL-12 secretion in mouse bone marrow-derived macrophages stimulated with LPS. This effect is largely due to its ability to block IL-10-mediated feedback inhibition on cytokine transcription in macrophages. Similar results were also obtained with a second structurally unrelated Jak inhibitor, Tofacitinib. In addition, LPS induced the production of IFN-ß, which was then able to activate JAKs in macrophages, resulting in the stimulation of STAT1 phosphorylation. The initial induction of IL-10 was independent of JAK signaling; however, inhibition of JAKs did reduce IL-10 secretion at later time points. This reflected a requirement for the IFN-ß feedback loop to sustain IL-10 transcription following LPS stimulation. In addition to IL-10, IFN-ß also helped sustain IL-6 and IL-12 transcription. Overall, these results suggest that inhibition of JAKs may increase the inflammatory potential of macrophages stimulated with TLR4 agonists.

Asunto(s)

Células de la Médula Ósea/inmunología , Citocinas/biosíntesis , Retroalimentación , Interleucina-10/antagonistas & inhibidores , Quinasas Janus/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Regulación hacia Arriba/inmunología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/patología , Células Cultivadas , Mediadores de Inflamación/fisiología , Interferón Tipo I/fisiología , Interleucina-10/fisiología , Quinasas Janus/fisiología , Macrófagos/enzimología , Macrófagos/patología , Ratones , Nitrilos , Pirazoles/farmacología , Pirimidinas , Transducción de Señal/inmunología , Receptores Toll-Like/fisiología

RESUMEN

PURPOSE: Several genes that are associated with protection from or susceptibility to trachomatous trichiasis (TT) have been identified through genetic association studies. Yet there have been few studies in which gene expression profiles were assessed in TT cases and disease-free controls. The purpose was to identify genes that are differentially expressed in the upper tarsal conjunctiva of subjects with TT. METHOD: Pathway-focused gene arrays were used to screen conjunctival RNA expression of 226 gene transcripts of interest. The screening was followed by validation of differentially expressed genes by qRT-PCR on an independent set of samples. Three different techniques were then used to test for quantitative differences in the recovered conjunctival protein fraction. RESULTS: Focused arrays identified a set of 13 differentially expressed genes. Validation by qRT-PCR confirmed differential expression in four of these genes (COL1A1, COL7A1, MMP7, and TLR6). Increased expression of MMP7 was the only consistent differentially regulated gene in the conjunctival samples of trichiasis subjects. MMP7 was present in isolated conjunctival proteins and in the tissue culture supernatants of peripheral blood lymphocytes after stimulation. CONCLUSIONS: There is an imbalance in extracellular matrix turnover with minimal contribution of adaptive immune responses at this stage of trichiasis. There was little evidence of broad differential expression in genes characteristic of polar responses of adaptive T cells or macrophages. The control of the MMP7 response and its activity appears significant in the fibrotic changes observed in TT.

Asunto(s)

Enfermedades de la Conjuntiva/genética , Pestañas , Enfermedades del Cabello/genética , Metaloproteinasa 7 de la Matriz/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Tracoma/genética , Activación Transcripcional/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades de la Conjuntiva/epidemiología , ADN Mitocondrial/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Gambia/epidemiología , Enfermedades del Cabello/epidemiología , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tracoma/epidemiología