RESUMEN
Glucocorticoids (GCs) are potent anti-inflammatory agents and are broadly used in treating rheumatoid arthritis (RA) patients, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action. We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CCL17 has a non-redundant role in inflammatory arthritis. Here, we provide molecular evidence that GCs can suppress GM-CSF-mediated upregulation of IRF4 and CCL17 expression via downregulating JMJD3 expression and activity. In mouse models of inflammatory arthritis, GC treatment inhibited CCL17 expression and ameliorated arthritic pain-like behavior and disease. Significantly, GC treatment of RA patient peripheral blood mononuclear cells ex vivo resulted in decreased CCL17 production. This delineated pathway potentially provides new therapeutic options for the treatment of many inflammatory conditions, where GCs are used as an anti-inflammatory drug but without the associated adverse side effects.
RESUMEN
BACKGROUND: The cytokine, interleukin-23 (IL-23), can be critical for the progression of inflammatory diseases, including arthritis, and is often associated with T lymphocyte biology. We previously showed that certain lymphocyte-independent, inflammatory arthritis and pain models have a similar requirement for tumour necrosis factor (TNF), granulocyte macrophage-colony stimulating factor (GM-CSF), and C-C motif ligand 17 (CCL17). Given this correlation in cytokine requirements, we explored whether IL-23 might interact with this cytokine cluster in the control of arthritic and inflammatory pain. METHODS: The role of IL-23 in the development of pain-like behaviour was investigated using mouse arthritis models (zymosan-induced arthritis and GM-CSF-, TNF-, and CCL17-driven monoarticular arthritis) and inflammatory pain models (intraplantar zymosan, GM-CSF, TNF, and CCL17). Additionally, IL-23-induced inflammatory pain was measured in GM-CSF-/-, Tnf-/-, and Ccl17E/E mice and in the presence of indomethacin. Pain-like behaviour and arthritis were assessed by relative weight distribution in hindlimbs and histology, respectively. Cytokine mRNA expression in knees and paw skin was analysed by quantitative PCR. Blood and synovial cell populations were analysed by flow cytometry. RESULTS: We report, using Il23p19-/- mice, that innate immune (zymosan)-driven arthritic pain-like behaviour (herein referred to as pain) was completely dependent upon IL-23; optimal arthritic disease development required IL-23 (P < 0.05). Zymosan-induced inflammatory pain was also completely dependent on IL-23. In addition, we found that exogenous TNF-, GM-CSF-, and CCL17-driven arthritic pain, as well as inflammatory pain driven by each of these cytokines, were absent in Il23p19-/- mice; optimal disease in these mBSA-primed models was dependent on IL-23 (P < 0.05). Supporting this cytokine connection, it was found conversely that IL-23 (200 ng) can induce inflammatory pain at 4 h (P < 0.0001) with a requirement for each of the other cytokines as well as cyclooxygenase activity. CONCLUSIONS: These findings indicate a role for IL-23 in innate immune-mediated arthritic and inflammatory pain with potential links to TNF, GM-CSF, CCL17, and eicosanoid function.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-23 , Animales , Citocinas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ratones , Dolor , Factor de Necrosis Tumoral alfaRESUMEN
The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.
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Dolor Crónico/fisiopatología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Nociceptores/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Comunicación Celular , Células Cultivadas , Dolor Crónico/inducido químicamente , Medios de Cultivo Condicionados/farmacología , Femenino , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/fisiopatología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Nervioso/farmacología , Nociceptores/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Factor de Transcripción STAT5/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Macrophage activation in response to LPS is coupled to profound metabolic changes, typified by accumulation of the TCA cycle intermediates citrate, itaconate, and succinate. We have identified that endogenous type I IFN controls the cellular citrate/α-ketoglutarate ratio and inhibits expression and activity of isocitrate dehydrogenase (IDH); and, via 13C-labeling studies, demonstrated that autocrine type I IFN controls carbon flow through IDH in LPS-activated macrophages. We also found that type I IFN-driven IL-10 contributes to inhibition of IDH activity and itaconate synthesis in LPS-stimulated macrophages. Our findings have identified the autocrine type I IFN pathway as being responsible for the inhibition of IDH in LPS-stimulated macrophages.
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Interferón Tipo I/metabolismo , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Macrófagos/inmunología , Macrófagos/metabolismo , Animales , Comunicación Autocrina , Ciclo del Ácido Cítrico , Humanos , Interleucina-10/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Succinatos/metabolismoRESUMEN
Pain is the major symptom of osteoarthritis (OA) and is an important factor in strategies to manage this disease. However, the current standard of care does not provide satisfactory pain relief for many patients. The pathophysiology of OA is complex, and its presentation as a clinical syndrome is associated with pathologies of multiple joint tissues. Inflammation is associated with both OA pain and disease outcome and is therefore a major treatment target for OA and OA pain. Unlike TNF inhibitors and IL-1 inhibitors, established drugs such as glucocorticoids and methotrexate can reduce OA pain. Although central nociceptive pathways contribute to OA pain, crosstalk between the immune system and nociceptive neurons is central to inflammatory pain; therefore, new therapies might target this crosstalk. Newly identified drug targets, including neurotrophins and the granulocyte-macrophage colony-stimulating factor (GM-CSF)-CC-chemokine ligand 17 (CCL17) chemokine axis, offer the hope of better results but require clinical validation.
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Analgésicos/uso terapéutico , Dolor Crónico/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Osteoartritis/tratamiento farmacológico , Dolor Crónico/etiología , Humanos , Inflamación/etiología , Osteoartritis/complicacionesRESUMEN
Studies have demonstrated the importance of a GM-CSFâIFN regulatory factor 4 (IRF4)âCCL17 pathway, first identified in monocytes/macrophages, for arthritic pain and disease development. In this study, we further investigated the involvement of this new pathway in shaping the inflammatory response using the zymosan-induced peritonitis (ZIP) model. ZIP (8 mg of zymosan, i.p., day 0) was induced in C57BL/6 wild-type (WT), GM-CSF-/- , Irf4-/- , and Ccl17E/E mice. In comparison with WT mice, GM-CSF-/- and Irf4-/- mice had a reduced ZIP response, as judged by a reduced number of neutrophils and macrophages in the peritoneal cavity. Moreover, the phenotype of the ZIP macrophages was altered by a lack of GM-CSF or IRF4 (increased IL-10 secretion and Arg1 mRNA expression), with IRF4 levels being lower in GM-CSF-/- ZIP macrophages than in the WT cells. In addition, GM-CSF ̶IRF4 signaling upregulated MHC class II expression in ZIP macrophages and bone marrow-derived macrophages. Although Ccl17 mRNA expression was reduced in ZIP macrophages in the absence of either GM-CSF or IRF4, thus supporting the presence of the new pathway in inflammatory macrophages, CCL17 did not modulate the inflammatory response, both in terms of number of myeloid cells or the macrophage phenotype. Thus, during an inflammatory response, both macrophage numbers and their phenotype can depend on GM-CSF- and IRF4-dependent signaling independently of CCL17.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factores Reguladores del Interferón/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Animales , Quimiocina CCL17/genética , Quimiocina CCL17/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Factores Reguladores del Interferón/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Transducción de Señal/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Pain is one of the most debilitating symptoms in many diseases for which there is inadequate management and understanding. CSF-1, also known as M-CSF, acts via its receptor (CSF-1R, c-Fms) to regulate the development of the monocyte/macrophage lineage and to act locally in tissues to control macrophage numbers and function. It has been implicated in the control of neuropathic pain via a central action on microglia. We report in this study that systemic administration of a neutralizing anti-CSF-1R or CSF-1 mAb inhibits the development of inflammatory pain induced by zymosan, GM-CSF, and TNF in mice. This approach also prevented but did not ameliorate the development of arthritic pain and optimal disease driven by the three stimuli in mice, suggesting that CSF-1 may only be relevant when the driving inflammatory insults in tissues are acute and/or periodic. Systemic CSF-1 administration rapidly induced pain and enhanced the arthritis in an inflamed mouse joint, albeit via a different pathway(s) from that used by systemic GM-CSF and TNF. It is concluded that CSF-1 can function peripherally during the generation of inflammatory pain and hence may be a target for such pain and associated disease, including when the clinically important cytokines, TNF and GM-CSF, are involved. Our findings have ramifications for the selection and design of anti-CSF-1R/CSF-1 trials.
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Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Inflamación/inmunología , Articulaciones/inmunología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/fisiología , Monocitos/fisiología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Diferenciación Celular , Linaje de la Célula , Humanos , Factor Estimulante de Colonias de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Dolor , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de SeñalRESUMEN
Interleukin 4 (IL4) is generally viewed as a Th2 cytokine capable of polarizing macrophages into an anti-inflammatory phenotype, whereas granulocyte macrophage-colony-stimulating factor (GM-CSF) is often viewed as a proinflammatory cytokine with part of this function due to its action on monocytes/macrophages. Paradoxically, these two cytokines act additively to enhance the in vitro differentiation of dendritic cells from precursors such as monocytes. One up-regulated marker of an IL4-polarized M2 macrophage is the chemokine (C-C motif) ligand 17 (CCL17), which we have recently reported to be induced by GM-CSF in monocytes/macrophages in an interferon regulatory factor 4 (IRF4)-dependent manner. In this study, we report that IL4 also induces CCL17 production by acting through IRF4 in human monocytes and murine macrophages. Furthermore, evidence is presented that IL4 up-regulates IRF4 expression at the epigenetic level by enhancing the expression and activity of jumonji domain-containing protein 3 (JMJD3) demethylase. Intriguingly, silencing the signal transducer and activator of transcription 6 (STAT6) gene led to a decrease in not only CCL17 formation, but also in that of its upstream regulators, JMJD3 and IRF4. Moreover, IL4 treatment of human monocytes resulted in an increased association of STAT6 to the promoter regions of the CCL17, IRF4, and JMJD3 genes. Thus, despite their vastly different functions, IL4 and GM-CSF appear to share elements of a common signaling pathway in regulating CCL17 production in human monocytes and murine macrophages.
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Quimiocina CCL17/genética , Epigénesis Genética , Interleucina-4/genética , Macrófagos/metabolismo , Monocitos/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Humanos , Factores Reguladores del Interferón/genética , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
Animal models of arthritis enable us to investigate the pathogenesis of the disease and also to evaluate new therapies. Here we describe two different acute inflammatory monoarticular arthritis models (mBSA/IL1ß and mBSA/GM-CSF) providing a more rapid and potentially simplified approach to investigate the pathogenesis.
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Artritis Experimental/genética , Citocinas/toxicidad , Inflamación/genética , Fiebre Reumática/genética , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/patología , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Inflamación/inducido químicamente , Inflamación/patología , Ratones , Fiebre Reumática/inducido químicamente , Fiebre Reumática/patologíaRESUMEN
BACKGROUND: Granulocyte macrophage-colony stimulating factor (GM-CSF) has been implicated in the pathogenesis of a number of inflammatory diseases and in osteoarthritis (OA). We identified previously a new GM-CSFâJmjd3âinterferon regulatory factor 4 (IRF4)âchemokine (c-c motif) ligand 17 (CCL17) pathway, which is important for the development of inflammatory arthritis pain and disease. Tumour necrosis factor (TNF) can also be linked with this pathway. Here we investigated the involvement of the pathway in OA pain and disease development using the GM-CSF-dependent collagenase-induced OA (CiOA) model. METHODS: CiOA was induced in C57BL/6 wild-type (WT), Irf4 -/- , Ccl17 E/E , Ccr4 -/- , Tnf -/- and GM-CSF -/- mice. Additionally, therapeutic targeting of CCL17, Jmjd3 and cyclooxygenase 2 (COX-2) was evaluated. Development of pain (assessment of weight distribution) and OA disease (histologic scoring of synovitis, cartilage destruction and osteophyte size) were assessed. Synovial joint cells, including neutrophils, macrophages, fibroblasts and endothelial cells, were isolated (cell sorting) and gene expression analyzed (quantitative PCR). RESULTS: Studies in the gene-deficient mice indicated that IRF4, CCL17 and the CCL17 receptor, CCR4, but not TNF, were required for CiOA pain and optimal cartilage destruction and osteophyte size. Therapeutic neutralization of CCL17 and Jmjd3 ameliorated both pain and disease, whereas the COX-2 inhibitor only ameliorated pain. In the synovium Ccl17 mRNA was expressed only in the macrophages in a GM-CSF-dependent and IRF4-dependent manner. CONCLUSIONS: The GM-CSFâJmjd3âIRF4âCCL17 pathway is important for the development of CiOA, with CCL17 thus being a potential therapeutic target for the treatment of both OA pain and disease.
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Quimiocina CCL17/antagonistas & inhibidores , Quimiocina CCL17/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Dolor/tratamiento farmacológico , Dolor/metabolismo , Animales , Progresión de la Enfermedad , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoartritis/patología , Dolor/patologíaRESUMEN
TNF and granulocyte macrophage-colony stimulating factor (GM-CSF) have proinflammatory activity and both contribute, for example, to rheumatoid arthritis pathogenesis. We previously identified a new GM-CSFâJMJD3 demethylaseâinterferon regulatory factor 4 (IRF4)âCCL17 pathway that is active in monocytes/macrophages in vitro and important for inflammatory pain, as well as for arthritic pain and disease. Here we provide evidence for a nexus between TNF and this pathway, and for TNF and GM-CSF interdependency. We report that the initiation of zymosan-induced inflammatory pain and zymosan-induced arthritic pain and disease are TNF dependent. Once arthritic pain and disease are established, blockade of GM-CSF or CCL17, but not of TNF, is still able to ameliorate them. TNF is required for GM-CSF-driven inflammatory pain and for initiation of GM-CSF-driven arthritic pain and disease, but not once they are established. TNF-driven inflammatory pain and TNF-driven arthritic pain and disease are dependent on GM-CSF and mechanistically require the same downstream pathway involving GM-CSFâCCL17 formation via JMJD3-regulated IRF4 production, indicating that GM-CSF and CCL17 can mediate some of the proinflammatory and algesic actions of TNF. Given we found that TNF appears important only early in arthritic pain and disease progression, targeting a downstream mediator, such as CCL17, which appears to act throughout the course of disease, could be effective at ameliorating chronic inflammatory conditions where TNF is implicated.
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Artritis Reumatoide/metabolismo , Quimiocina CCL17/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Granulocitos , Inflamación/inmunología , Inflamación/patología , Inflamación/fisiopatología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Histona Demetilasas con Dominio de Jumonji , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Dolor/inducido químicamente , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Zimosan/farmacologíaRESUMEN
Mavrilimumab (formerly CAM-3001) is a high-affinity, immunoglobulin G4 monoclonal antibody (mAb) against the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor-α chain. Phase I and II trials in patients with rheumatoid arthritis (RA) treated with mavrilimumab have shown encouraging results with respect to both safety and efficacy. No significant adverse events have so far been noted. The trials have demonstrated significant clinical benefit, meeting primary endpoints. Furthermore, for RA patients treated with mavrilimumab, who were tumour necrosis factor (TNF) inhibitor-inadequate responders, there are encouraging preliminary data indicating benefit and identifying potential biomarkers predictive of patients likely to find benefit. Here, we review the clinical trial data for mavrilimumab and discuss its potential as a treatment for RA in light of the competitive landscape in which it resides.
RESUMEN
Glucocorticoids (GCs) are potent anti-inflammatory drugs whose mode of action is complex and still debatable. One likely cellular target of GCs are monocytes/macrophages. The role of GCs in monocyte survival is also debated. Although both granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) are important regulators of macrophage lineage functions including their survival, the former is often associated with proinflammatory functions while the latter is important in lineage homeostasis. We report here that the GC, dexamethasone, induces apoptosis in GM-CSF-treated human monocytes while having no impact on M-CSF-induced monocyte survival. To understand how GCs, GM-CSF, and M-CSF are regulating monocyte survival and other functions during inflammation, we firstly examined the transcriptomic changes elicited by these three agents in human monocytes, either acting alone or in combination. Transcriptomic and Ingenuity pathway analyses found that dexamethasone differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes, two pathways known to be regulated by ERK1/2 activity. These analyses led us to provide evidence that the GC inhibits ERK1/2 activity selectively in GM-CSF-treated monocytes to induce apoptosis. It is proposed that this inhibition of ERK1/2 activity leads to inactivation of p90 ribosomal-S6 kinase and Bad dephosphorylation leading in turn to enhanced caspase-3 activity and subsequent apoptosis. Furthermore, pharmacological inhibition of GC receptor activity restored the ERK1/2 signaling and prevented the GC-induced apoptosis in GM-CSF-treated monocytes. Increased tissue macrophage numbers, possibly from enhanced survival due to mediators such as GM-CSF, can correlate with inflammatory disease severity; also reduction in these numbers can correlate with the therapeutic benefit of a number of agents, including GCs. We propose that the ERK1/2 signaling pathway promotes survival of GM-CSF-treated proinflammatory monocytes, which can be selectively targeted by GCs as a novel mechanism to reduce local monocyte/macrophage numbers and hence inflammation.
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Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Inflamación/prevención & control , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Monocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inflamación/enzimología , Inflamación/patología , Factor Estimulante de Colonias de Macrófagos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/enzimología , Monocitos/patología , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Receptor Activador Expresado en Células Mieloides 1/metabolismoRESUMEN
There is burgeoning interest in the interaction between the immune and nervous systems. Pain is mediated by primary sensory neurons (nociceptors) that can respond to a variety of thermal, mechanical and chemical signals. Cytokines are now recognized as important mediators of inflammatory pain. They can induce nociceptor sensitization indirectly via mediators, wherein neurons become primed and thus become more responsive to stimulation; alternatively, there is also evidence that cytokines can directly activate neurons via their specific receptors present on the neuronal cells. We review here the evidence for and against these respective mechanisms, focusing on arthritis and inflammatory skin models. A number of striking inconsistencies amongst the conclusions made in the literature are highlighted and discussed.
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Artritis/inmunología , Citocinas/metabolismo , Inflamación Neurogénica/inmunología , Nociceptores/fisiología , Dolor/inmunología , Receptores de Citocinas/metabolismo , Piel/inmunología , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Porphyromonas gingivalis is one of the bacterial species most closely associated with periodontitis and can shed large numbers of outer membrane vesicles (OMVs), which are increasingly thought to play a significant role in bacterial virulence and pathogenicity. Macrophages are amongst the first immune cells to respond to bacteria and their products, so we sought to directly compare the response of macrophages to P. gingivalis or its purified OMVs. Macrophages stimulated with OMVs produced large amounts of TNFα, IL-12p70, IL-6, IL-10, IFNß, and nitric oxide compared to cells infected with P. gingivalis, which produced very low levels of these mediators. Both P. gingivalis and OMVs induced a shift in macrophage metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, which was supported by enhanced lactate release, decreased mitochondrial oxygen consumption with reduced spare respiratory capacity, as well as increased mitochondrial reactive oxygen species (ROS) production. Corresponding to this metabolic shift, gene expression analysis of macrophages infected with P. gingivalis or stimulated with OMVs revealed a broad transcriptional upregulation of genes critical to glycolysis and a downregulation of genes associated with the TCA cycle. Upon examination of inflammasome signaling and pyroptosis it was found that P. gingivalis did not activate the inflammasome in macrophages as the mature forms of caspase-1, IL-1ß, and IL-18 were not detected and there was no extracellular release of lactate dehydrogenase (LDH) or 7-AAD staining. In comparison, macrophages stimulated with OMVs potently activated caspase-1, produced large amounts of IL-1ß, IL-18, released LDH, and were positive for 7-AAD indicative of pyroptotic cell death. These data directly quantitate the distinct effects of P. gingivalis and its OMVs on macrophage inflammatory phenotype, mitochondrial function, inflammasome activation, and pyroptotic cell death that may have potential implications for their roles in chronic periodontitis.
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Vesículas Extracelulares/metabolismo , Inflamasomas/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Porphyromonas gingivalis/inmunología , Piroptosis , Animales , Caspasa 1/metabolismo , Citocinas/metabolismo , Expresión Génica , Glucólisis , Humanos , Inflamación , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-18 , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , Porphyromonas gingivalis/patogenicidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti-G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti-G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti-G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF-driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Artritis Experimental/terapia , Artritis Reumatoide/terapia , Inmunoterapia/métodos , Neuronas/efectos de los fármacos , Neutrófilos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Animales , Antígenos Ly/inmunología , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Procedimientos de Reducción del Leucocitos , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Manejo del Dolor , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Receptores de Factor Estimulante de Colonias de Granulocito/inmunologíaRESUMEN
BACKGROUND: Blockade of granulocyte macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFRα) is being successfully tested in trials in rheumatoid arthritis (RA) with clinical results equivalent to those found with neutralization of the current therapeutic targets, TNF and IL-6. To explore further the role of GM-CSF as a pro-inflammatory cytokine, we examined the effect of anti-GM-CSFRα neutralization on myeloid cell populations in antigen-driven arthritis and inflammation models and also compared its effect with that of anti-TNF and anti-IL-6. METHODS: Cell population changes upon neutralization by monoclonal antibodies (mAbs) in the antigen-induced arthritis (AIA) and antigen-induced peritonitis (AIP) models were monitored by flow cytometry and microarray. Adoptive transfer of monocytes into the AIP cavity was used to assess the GM-CSF dependence of the development of macrophages and monocyte-derived dendritic cells (Mo-DCs) at a site of inflammation. RESULTS: Therapeutic administration of a neutralizing anti-GM-CSF mAb, but not of an anti-colony-stimulating factor (anti-CSF)-1 or an anti-CSF-1R mAb, ameliorated AIA disease. Using the anti-GM-CSFRα mAb, the relative surface expression of different inflammatory myeloid populations was found to be similar in the inflamed tissues in both the AIA and AIP models; however, the GM-CSFRα mAb, but not neutralizing anti-TNF and anti-IL-6 mAbs, preferentially depleted Mo-DCs from these sites. In addition, we were able to show that locally acting GM-CSF upregulated macrophage/Mo-DC numbers via GM-CSFR signalling in donor monocytes. CONCLUSIONS: Our findings suggest that GM-CSF blockade modulates inflammatory responses differently to TNF and IL-6 blockade and may provide additional insight into how targeting the GM-CSF/GM-CSFRα system is providing efficacy in RA.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inflamación/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Traslado Adoptivo , Animales , Separación Celular , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF; also known as CSF1), granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3) can each play a part in the host response to injury and infection, and there is burgeoning interest in targeting these CSFs in inflammatory and autoimmune disorders, as well as in cancer. For success in clinical medicine, therapeutic targeting will need to be delineated from current strategies. The individual CSFs have unique biological roles, suggesting that they could be used to target specific conditions. This Review compares the CSFs, with a focus on how they could be targeted, discusses the relevant clinical trial data and summarizes the potential clinical applications of targeting each CSF. Importantly, we discuss the novelty of CSF biology and attempt to clarify some of the surrounding misconceptions and issues that can affect therapeutic decisions.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/metabolismo , Factores Estimulantes de Colonias/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Animales , Ensayos Clínicos como Asunto , Humanos , Interleucina-3/metabolismoRESUMEN
Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4-dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.
Asunto(s)
Quimiocina CCL17/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Inflamación , Factores Reguladores del Interferón/metabolismo , Animales , Artritis/metabolismo , Células de la Médula Ósea/metabolismo , Silenciador del Gen , Heterocigoto , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Macrófagos/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Dolor , Manejo del Dolor , Peritonitis/metabolismoRESUMEN
The K/BxN serum-transfer arthritis (STA) model is a murine model in which the immunological mechanisms occurring in rheumatoid arthritis (RA) and other arthritides can be studied. To induce K/BxN STA, serum from arthritic transgenic K/BxN mice is transferred to naive mice and manifestations of arthritis occur a few days later. The inflammatory response in the model is driven by autoantibodies against the ubiquitously expressed self-antigen, glucose-6-phosphate isomerase (G6PI), leading to the formation of immune complexes that drive the activation of different innate immune cells such as neutrophils, macrophages, and possibly mast cells. The pathogenesis further involves a range of immune mediators including cytokines, chemokines, complement factors, Toll-like receptors, Fc receptors, and integrins, as well as factors involved in pain and bone erosion. Hence, even though the K/BxN STA model mimics only the effector phase of RA, it still involves a wide range of relevant disease mediators. Additionally, as a murine model for arthritis, the K/BxN STA model has some obvious advantages. First, it has a rapid and robust onset of arthritis with 100% incidence in genetically identical animals. Second, it can be induced in a wide range of strain backgrounds and can therefore also be induced in gene-deficient strains to study the specific importance of disease mediators. Even though G6PI might not be an essential autoantigen, for example, in RA, the K/BxN STA model is a useful tool to understand how autoantibodies, in general, drive the progression of arthritis by interacting with downstream components of the innate immune system. Finally, the model has also proven useful as a model wherein arthritic pain can be studied. Taken together, these features make the K/BxN STA model a relevant one for RA, and it is a potentially valuable tool, especially for the preclinical screening of new therapeutic targets for RA and perhaps other forms of inflammatory arthritis. Here, we describe the molecular and cellular pathways in the development of K/BxN STA focusing on the recent advances in the understanding of the important mechanisms. Additionally, this review provides a comparison of the K/BxN STA model to some other arthritis models.