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/metabolismoRESUMEN
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/genéticaRESUMEN
In response to infection by fungal pathogens, the innate immune system recognises specific fungal pathogen associated molecular patterns (PAMPs) via pattern recognition receptors including the C-type lectin dectin-1 and members of the Toll Like Receptor (TLR) family. Stimulation of these receptors leads to the induction of both pro- and anti-inflammatory cytokines. The protein kinases MSK1 and 2 are known to be important in limiting inflammatory cytokine production by macrophages in response to the TLR4 agonist LPS. In this study we show that MSKs are also activated in macrophages by the fungal derived ligand zymosan, as well as the dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38α MAPK pathways. Furthermore, we show that MSKs regulate dectin-1 induced IL-10 production, and that this regulation is dependent on the ability of MSKs to phosphorylate the transcription factor CREB. IL-10 secreted in response to zymosan was able to promote STAT3 phosphorylation via an autocrine feedback loop. Consistent with the decreased IL-10 secretion in MSK1/2 knockout macrophages, these cells also had decreased STAT3 tyrosine phosphorylation relative to wild type controls after stimulation with zymosan. We further show that the reduction in IL-10 production in the MSK1/2 macrophages results in increased secretion of IL-12p40 in response to zymosan relative to wild type controls. The production of high levels of IL-10 but low levels of IL-12 has previously been associated with an M2b or 'regulatory' macrophage phenotype, which was initially described in macrophages stimulated with a combination of immune complexes and LPS. We found that zymosan, via dectin-1 activation, also leads to the expression of SphK1 and LIGHT, markers of a regulatory like phenotype in mouse macrophages. The expression of these makers was further reinforced by the high level of IL-10 secreted in response to zymosan stimulation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Interleucina-10/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Immunoblotting , Lectinas Tipo C/genética , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal/efectos de los fármacos , Zimosan/farmacologíaRESUMEN
Prostaglandin production is catalyzed by cyclooxygenase 2 (cox-2). We demonstrate here that MSK1 and MSK2 (MSK1/2) can exert control on the induction of cox-2 mRNA by Toll-like receptor (TLR) agonists. In the initial phase of cox-2 induction, MSK1/2 knockout macrophages confirmed a role for MSK in the positive regulation of transcription. However, at later time points both lipopolysaccharide (LPS)-induced prostaglandin and cox-2 protein levels were increased in MSK1/2 knockout. Further analysis found that while MSKs promoted cox-2 mRNA transcription, following longer LPS stimulation MSKs also promoted degradation of cox-2 mRNA. This was found to be the result of an interleukin 10 (IL-10) feedback mechanism, with endogenously produced IL-10 promoting cox-2 degradation. The ability of IL-10 to do this was dependent on the mRNA binding protein TTP through a p38/MK2-mediated mechanism. As MSKs regulate IL-10 production in response to LPS, MSK1/2 knockout results in reduced IL-10 secretion and therefore reduced feedback from IL-10 on cox-2 mRNA stability. Following LPS stimulation, this increased mRNA stability correlated to an elevated induction of both of cox-2 protein and prostaglandin secretion in MSK1/2 knockout macrophages relative to that in wild-type cells. This was not restricted to isolated macrophages, as a similar effect of MSK1/2 knockout was seen on plasma prostaglandin E2 (PGE2) levels following intraperitoneal injection of LPS.
Asunto(s)
Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Prostaglandinas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Interleucina-10/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos , Ratones , Ratones Endogámicos C57BL , Prostaglandinas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Estabilidad del ARN , ARN Mensajero/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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 fármacosRESUMEN
Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.
Asunto(s)
Ciclobutanos/farmacología , Indanos/farmacología , Inflamación/inmunología , Macrófagos/inmunología , Morfolinas/farmacología , Compuestos de Fenilurea/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/farmacología , Factores de Transcripción/metabolismo , Análisis de Varianza , Animales , Línea Celular , Ciclobutanos/síntesis química , Citocinas/metabolismo , Cartilla de ADN/genética , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Immunoblotting , Interleucina-10/genética , Interleucina-10/metabolismo , Macrófagos/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Ratones Noqueados , Estructura Molecular , Morfolinas/síntesis química , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/química , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/genética , Proteómica , Pirimidinas/síntesis química , Pirimidinas/química , Interferencia de ARNRESUMEN
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/fisiologíaRESUMEN
On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1ß, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNß production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1ß levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica , Proteína Quinasa 13 Activada por Mitógenos/química , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/química , Animales , Bovinos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Eliminación de Gen , Humanos , Inmunidad Innata , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Isoformas de Proteínas , Choque Séptico/metabolismoRESUMEN
cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein ß-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Especificidad por SustratoRESUMEN
We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related 'long' PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC(50) of 11+/-1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC(50) of 101+/-7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.
Asunto(s)
Encéfalo/enzimología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Evolución Molecular , Regulación Enzimológica de la Expresión Génica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/aislamiento & purificación , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Genoma Humano/genética , Humanos , Datos de Secuencia Molecular , Nucleótidos/genética , Especificidad de Órganos , Fosforilación , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Homología de Secuencia , Factores de TiempoRESUMEN
Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75(NTR)), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75(NTR) to enhance cAMP degradation. The p75(NTR)-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75(NTR)-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75(NTR) regulates degradation of cAMP and perpetuates scar formation after injury.