RESUMEN
Monocyte-derived macrophages play a key pathogenic role in inflammatory diseases. In the case of rheumatoid arthritis (RA), the presence of specific synovial tissue-infiltrating macrophage subsets is associated with either active disease or inflammation resolution. JAK inhibitors (JAKi) are the first targeted synthetic disease-modifying antirheumatic drugs (tsDMARD) approved for treatment of RA with comparable efficacy to biologics. However, the effects of JAKi on macrophage specification and differentiation are currently unknown. We have analyzed the transcriptional and functional effects of JAKi on human peripheral blood monocyte subsets from RA patients and on the differentiation of monocyte-derived macrophages promoted by granulocyte-macrophage colony-stimulating factor (GM-CSF), a factor that drives the development and pathogenesis of RA. We now report that JAKi Upadacitinib restores the balance of peripheral blood monocyte subsets in RA patients and skewed macrophages towards the acquisition of an anti-inflammatory transcriptional and functional profile in a dose-dependent manner. Upadacitinib-treated macrophages showed a strong positive enrichment of the genes that define synovial macrophages associated to homeostasis/inflammation resolution. Specifically, Upadacitinib-treated macrophages exhibited significantly elevated expression of MAFB and MAFB-regulated genes, elevated inhibitory phosphorylation of GSK3ß, and higher phagocytic activity and showed an anti-inflammatory cytokine profile upon activation by pathogenic stimuli. These outcomes were also shared by macrophages exposed to other JAKi (baricitinib, tofacitinib), but not in the presence of the TYK2 inhibitor deucravacitinib. As a whole, our results indicate that JAKi promote macrophage re-programming towards the acquisition of a more anti-inflammatory/pro-resolution profile, an effect that correlates with the ability of JAKi to enhance MAFB expression.
Asunto(s)
Artritis Reumatoide , Inhibidores de las Cinasas Janus , Humanos , Inhibidores de las Cinasas Janus/farmacología , Inhibidores de las Cinasas Janus/metabolismo , Inhibidores de las Cinasas Janus/uso terapéutico , Macrófagos/metabolismo , Artritis Reumatoide/patología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Antiinflamatorios/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismoRESUMEN
Monocyte-derived macrophages contribute to pathogenesis in inflammatory diseases and their effector functions greatly depend on the prevailing extracellular milieu. Whereas M-CSF primes macrophages for acquisition of an anti-inflammatory profile, GM-CSF drives the generation of T cell-stimulatory and pro-inflammatory macrophages. Liver X Receptors (LXRα and LXRß) are nuclear receptors that control cholesterol metabolism and regulate differentiation of tissue-resident macrophages. Macrophages from rheumatoid arthritis and other inflammatory pathologies exhibit an enriched LXR pathway, and recent reports have shown that LXR activation raises pro-inflammatory effects and impairs the acquisition of the anti-Inflammatory profile of M-CSF-dependent monocyte-derived macrophages (M-MØ). We now report that LXR inhibition prompts the acquisition of an anti-inflammatory gene and functional profile of macrophages generated within a pathological environment (synovial fluid from Rheumatoid Arthritis patients) as well as during the GM-CSF-dependent differentiation of human monocyte-derived macrophages (GM-MØ). Mechanistically, inhibition of LXR results in macrophages with higher expression of the v-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog B (MAFB) transcription factor, which governs the macrophage anti-inflammatory profile, as well as over-expression of MAFB-regulated genes. Indeed, gene silencing experiments on human macrophages evidenced that MAFB is required for the LXR inhibitor to enhance the anti-inflammatory nature of human macrophages. As a whole, our results demonstrate that LXR inhibition prompts the acquisition of an anti-inflammatory transcriptional and functional profile of human macrophages in a MAFB-dependent manner, and propose the use of LXR antagonists as potential therapeutic alternatives in macrophage re-programming strategies during inflammatory responses.
Asunto(s)
Artritis Reumatoide , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/genética , Regulación hacia Arriba , Macrófagos/metabolismo , Artritis Reumatoide/patología , Antiinflamatorios/metabolismo , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismoRESUMEN
Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.
Asunto(s)
Metotrexato , Monocitos , Humanos , Metotrexato/farmacología , Metotrexato/metabolismo , Monocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Empalme Alternativo , Precursores del ARN/metabolismo , Ácido Fólico/farmacología , Ácido Fólico/metabolismo , Macrófagos/metabolismo , Expresión Génica , Péptido Sintasas/genéticaRESUMEN
CD28 expression is generally considered to be T lymphocyte specific. We have previously shown CD28 mRNA expression in M-CSF-dependent anti-inflammatory monocyte-derived macrophages (M-MØ), and now demonstrate that CD28 cell surface expression is higher in M-MØ than in GM-CSF-dependent macrophages, and that macrophage CD28 expression is regulated by MAFB and activin A. In vivo, CD28 was found in tumor-associated macrophages and, to a lower extent, in pro-inflammatory synovial fluid macrophages from rheumatoid arthritis patients. Analysis of mouse macrophages confirmed Cd28 expression in bone-marrow derived M-MØ. Indeed, anti-CD28 antibodies triggered ERK1/2 phosphorylation in mouse M-MØ. At the functional level, Cd28KO M-MØ exhibited a significantly higher capacity to activate the OVA-specific proliferation of OT-II CD4+ T cells than WT M-MØ, as well as enhanced LPS-induced IL-6 production. Besides, the Cd28KO M-MØ transcriptome was significantly different from WT M-MØ regarding the expression IFN response, inflammatory response, and TGF-ß signaling related gene sets. Therefore, defective CD28 expression in mouse macrophages associates to changes in gene expression profile, what might contribute to the altered functionality displayed by Cd28KO M-MØ. Thus, CD28 expression appears as a hallmark of anti-inflammatory macrophages and might be a target for immunotherapy.
Asunto(s)
Antígenos CD28/inmunología , Inflamación/inmunología , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Linfocitos T/inmunología , Activinas/genética , Activinas/inmunología , Activinas/metabolismo , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Células Cultivadas , Expresión Génica/inmunología , Perfilación de la Expresión Génica/métodos , Humanos , Inflamación/genética , Inflamación/metabolismo , Activación de Linfocitos/genética , Macrófagos/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/inmunología , Factor de Transcripción MafB/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional (cTr) B cells have been described in patients with long-standing RA, thus our objective was to examine the frequency and evolution of cTr B cells in the peripheral blood of early RA (ERA) patients. METHODS: Freshly isolated peripheral blood mononuclear cells from 48 steroid- and DMARD-naïve ERA patients with a disease duration of <24 weeks and 48 healthy controls (HCs) were examined by flow cytometry. Co-cultures of isolated memory B cells were established with autologous T cells in the absence or presence of Tr B cells. RESULTS: As compared with HCs, ERA patients demonstrated an increased frequency of cTr B cells. cTr B cells of ERA patients and HCs displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFN-γ and IL-21 production, together with ACPA secretion in autologous B/T cell co-cultures. Basal frequencies of cTr B cells above the median value observed in HCs were associated with a good EULAR response to MTX at 12 months [relative risk 2.91 (95% CI 1.37, 6.47)]. A significant reduction of cTr B cells was observed 12 months after initiating MTX, when the cTr B cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone. CONCLUSION: An increased frequency of regulatory cTr B cells is apparent in untreated ERA and the baseline cTr B cell frequency is associated with the clinical response to MTX at 12 months.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B Reguladores , Metotrexato/uso terapéutico , ADP-Ribosil Ciclasa 1/sangre , Adulto , Anticuerpos Antiproteína Citrulinada/metabolismo , Antígenos CD19/sangre , Linfocitos B Reguladores/química , Linfocitos B Reguladores/citología , Biomarcadores/sangre , Antígeno CD24/sangre , Estudios de Casos y Controles , Técnicas de Cocultivo , Regulación hacia Abajo , Femenino , Humanos , Interferón gamma/metabolismo , Interleucinas/metabolismo , Masculino , Glicoproteínas de Membrana/sangre , Persona de Mediana Edad , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: A novel population of B helper cells, phenotypically CD4+CXCR5-PD-1hi, has been described in the synovial tissues and peripheral blood of seropositive RA patients, and termed 'peripheral helper T' (Tph) cells. Contrary to CD4+CXCR5+PD-1hi follicular helper T (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Our objective was to study the frequency of circulating Tph (cTph) and circulating Tfh cell counterparts (cTfh) in patients with early RA (eRA). METHODS: Freshly isolated peripheral blood mononuclear cells from 56 DMARD-naïve eRA patients and 56 healthy controls were examined by flow cytometry. Autologous cocultures of naïve or memory B cells were established with isolated peripheral blood Tph or Tfh cells. RESULTS: Seropositive (RF+ and/or ACPA+, n = 38) but not seronegative eRA patients (n = 18) demonstrated increased frequencies and absolute numbers of cTph and cTfh cells. cTph but not cTfh cells expressed CCR2. Those eRA patients who experienced a significant clinical improvement at 12 months demonstrated a marked decrease of their cTph cell numbers whereas their cTfh cell numbers remained unchanged. Both isolated Tph and isolated Tfh cells were able to induce maturation of memory B cells, whereas only Tfh cells could differentiate naïve B cells. CONCLUSION: Two populations of PD-1hiCD4 T cells with distinct phenotype and B cell helping capacity are increased in the peripheral blood of seropositive eRA patients. Whereas cTph cells are present only in patients with an active disease, cTfh cells seem to be constitutively elevated.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos B/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Anciano , Anticuerpos Antiproteína Citrulinada/sangre , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Estudios de Casos y Controles , Técnicas de Cocultivo , Femenino , Humanos , Memoria Inmunológica/inmunología , Inmunofenotipificación , Cooperación Linfocítica/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Factor Reumatoide/sangre , Líquido Sinovial/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacosRESUMEN
OBJECTIVES: Methotrexate (MTX) is the anchor drug for treatment of rheumatoid arthritis (RA), but the mechanism of its anti-inflammatory action is not fully understood. In RA, macrophages display a proinflammatory polarisation profile that resembles granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages and the response to MTX is only observed in thymidylate synthase+ GM-CSF-dependent macrophages. To determine the molecular basis for the MTX anti-inflammatory action, we explored toll-like receptor (TLR), RA synovial fluid (RASF) and tumour necrosis factor receptor (TNFR)-initiated signalling in MTX-exposed GM-CSF-primed macrophages. METHODS: Intracellular responses to TLR ligands, TNFα or RASF stimulation in long-term low-dose MTX-exposed human macrophages were determined through quantitative real-time PCR, western blot, ELISA and siRNA-mediated knockdown approaches. The role of MTX in vivo was assessed in patients with arthritis under MTX monotherapy and in a murine sepsis model. RESULTS: MTX conditioned macrophages towards a tolerant state, diminishing interleukin (IL)-6 and IL-1ß production in LPS, LTA, TNFα or RASF-challenged macrophages. MTX attenuated LPS-induced MAPK and NF-κB activation, and toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF1)-dependent signalling. Conversely, MTX increased the expression of the NF-κB suppressor A20 (TNFAIP3), itself a RA-susceptibility gene. Mechanistically, MTX-induced macrophage tolerance was dependent on A20, as siRNA-mediated knockdown of A20 reversed the MTX-induced reduction of IL-6 expression. In vivo, TNFAIP3 expression was significantly higher in peripheral blood cells of MTX-responsive individuals from a cohort of patients with arthritis under MTX monotherapy, whereas MTX-treated mice exhibited reduced inflammatory responses to LPS. CONCLUSIONS: MTX impairs macrophage proinflammatory responses through upregulation of A20 expression. The A20-mediated MTX-induced innate tolerance might limit inflammation in the RA synovial context, and positions A20 as a potential MTX-response biomarker.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Metotrexato/farmacología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Animales , Artritis Reumatoide/metabolismo , Humanos , Inflamación/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Líquido Sinovial/metabolismo , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND & AIMS: Patients with cirrhosis show recurrent access of bacterial products into the bloodstream inducing a multi-altered immunological status leading to relevant complications. We aimed at evaluating Bifidobacterium pseudocatenulatum CECT7765 effect on the host's macrophage function. PATIENTS & METHODS: Patients with cirrhosis and ascites were included. Granulocyte-macrophage colony-stimulating factor (GM-CSF) monocyte-derived and ascitic fluid (AF) macrophages were cultured with M-CSF, lipopolysaccharide (LPS) and/or the bifidobacterial strain. Pellets and supernatants were evaluated for gene expression of M1 and M2-related genes and cytokine secretion. Cell surface expression molecules were evaluated by flow cytometry. Kupffer cells from bile duct ligated and CCl4 rats were also evaluated. RESULTS: Experiments were run on GM-CSF blood-derived and AF macrophages from 10 patients with cirrhosis and 10 healthy donors. Different macrophage morphology was observed by optical microscopy in cells stimulated with bifidobacteria vs. LPS. M2-like expression of CD206, CD163 and CD16 was significantly increased in macrophages after stimulation with the bifidobacterial strain vs. LPS. B. pseudocatenulatum CECT7765 was able to significantly change the cytokine secretion pattern of blood-derived and AF macrophages and Kupffer cells from bile duct ligated and CCl4 cirrhotic rats compared to that induced by LPS. B. pseudocatenulatum CECT7765 was also effective in inducing a phenotype transition and a functional change from an M1- to an M2-related gene expression and cytokine secretion pattern in AF macrophages even after LPS-pretreatment. B. pseudocatenulatum CECT7765 did not reduce AF macrophage bacterial killing capacity. CONCLUSION: B. pseudocatenulatum CECT7765 induces a morphologic, phenotypic and functional transition towards an anti-inflammatory profile in GM-CSF monocyte-derived and AF macrophages from patients with cirrhosis that may help in controlling sustained inflammation in decompensated cirrhosis.
Asunto(s)
Antiinflamatorios/farmacología , Bifidobacterium , Cirrosis Hepática/inmunología , Macrófagos/inmunología , Anciano , Polaridad Celular , Citocinas/biosíntesis , Femenino , Humanos , Macrófagos del Hígado/inmunología , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
OBJECTIVES: Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MØ) or M-CSF (M-MØ), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively. METHODS: The transcriptomic profile of both human macrophage subtypes exposed to 50â nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints. RESULTS: MTX exclusively modulated gene expression in proinflammatory GM-MØ, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS+ GM-MØ are susceptible to MTX, whereas anti-inflammatory TSlow/- M-MØ and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS+ GM-MØ. Importantly, TS and p53 were found to be expressed by CD163+/TNFα+ GM-CSF-polarised macrophages from RA joints but not from normal synovium. CONCLUSIONS: Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Metotrexato/farmacología , Transducción de Señal/efectos de los fármacos , Humanos , Timidilato Sintasa/metabolismo , Transcriptoma , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis and severity correlates with the presence of macrophage-derived pro-inflammatory cytokines within the inflamed synovium. Macrophage-derived cytokines fuel the pathological processes in RA and are targets of clinically successful therapies. However, although macrophage polarization determines cytokine production, the polarization state of macrophages in RA joints remains poorly defined. To dissect the molecular basis for the tissue-damaging effects of macrophages in RA joints, we undertook the phenotypic and transcriptomic characterization of ex vivo isolated CD14(+) RA synovial fluid (RA-SF) macrophages. Flow cytometry and gene profiling indicated that RA-SF macrophages express pro-inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated with homeostatic and anti-inflammatory polarization (IGF1, HTR2B) and exhibit a transcriptomic profile that resembles the activin A-dependent gene signature of pro-inflammatory in vitro-generated macrophages. In fact, high levels of Smad-activating activin A were found in RA-SF and, accordingly, the Smad signalling pathway was activated in ex vivo-isolated RA-SF macrophages. In vitro experiments on monocytes and macrophages indicated that RA-SF promoted the acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2) but led to a significant reduction in the expression of genes associated with homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming the pro-inflammatory polarization ability of RA-SF. Importantly, the macrophage-polarizing ability of RA-SF was inhibited by an anti-activin A-neutralizing antibody, thus demonstrating that activin A mediates the pro-inflammatory macrophage-polarizing ability of RA-SF. Moreover, and in line with these findings, multicolour immunofluorescence evidenced that macrophages within RA synovial membranes (RA-SM) also express pro-inflammatory polarization markers whose expression is activin A-dependent. Altogether, our results demonstrate that macrophages from RA synovial fluids and membranes exhibit an MMP12(+) EGLN3(+) CCR2(+) pro-inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid.
Asunto(s)
Activinas/metabolismo , Artritis Reumatoide/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Membrana Sinovial/metabolismo , Transcriptoma , Adulto , Anciano , Artritis Reumatoide/patología , Células Cultivadas , Femenino , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Técnicas In Vitro , Inflamación/patología , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/patología , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Persona de Mediana Edad , Fenotipo , Receptores CCR2/metabolismo , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Membrana Sinovial/patologíaRESUMEN
The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF- and M-CSF-polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF-polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF-polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow-derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.
Asunto(s)
Quimiocina CCL2/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Activinas/farmacología , Animales , Quimiocina CCL2/metabolismo , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Análisis por Conglomerados , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , TranscriptomaRESUMEN
Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT(1-7)) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization-associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT(7) mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT(2B) and 5HT(7) receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT(2B) was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT(2B) and 5HT(7), whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.
Asunto(s)
Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptor de Serotonina 5-HT2B/inmunología , Receptores de Serotonina/inmunología , Serotonina/farmacología , Animales , Linaje de la Célula , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Genes Reporteros , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Macrófagos del Hígado/citología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/inmunología , Lipopolisacáridos , Luciferasas , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor de Serotonina 5-HT2B/genética , Receptores de Serotonina/genética , Serotonina/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To examine the frequency and phenotype of Th17 cells in the peripheral blood of patients with early non-radiographic axial SpA (early nrSpA). METHODS: CD4(+) T cells were isolated from the peripheral blood of 30 early nrSpA patients, 11 AS patients and 41 age- and sex-matched healthy controls by Ficoll-Hypaque gradient and magnetic negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells and of cells producing TNF-α or IL-10 was determined by cytometry and concentrations of IL-17, IL-22, IFN-γ, TNF-α, IL-10 and IL-4 were measured by ELISA. RESULTS: Early nrSpA but not AS patients demonstrated a significantly lower percentage of circulating Th17, Th1 and Th17/Th1 cells, together with lower CD4-derived IL-17 and IFN-γ secretion, as compared with controls. In contrast, the percentage of circulating cells producing IL-10 or TNF-α, and the secretion of CD4-derived IL-10, TNF-α, IL-22 and IL-4 in early nrSpA were not different from controls. All Th17 cells were CD45RO(+)CD45RA(-) and CCR6(+). The frequency of circulating Th17, Th1 and Th17/Th1 was negatively correlated with BASDAI, BASFI, ASDAS-CRP, ASDAS-ESR, AS quality of life (ASQOL) and patient's global assessment in HLA-B27(+) but not in HLA-B27(-) early nrSpA patients. A positive correlation between circulating Th17 cells and BASDAI was observed in AS. CONCLUSION: A decreased percentage of Th17, Th1 and Th17/Th1 cells is apparent in peripheral blood CD4(+) T cells from early nrSpA. Th17, Th1 and Th17/Th1 cell numbers are related to disease activity indices in HLA-B27(+), but not in HLA-B27(-), early nrSpA patients.
Asunto(s)
Interleucina-10/metabolismo , Espondiloartritis/sangre , Células TH1/inmunología , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Biomarcadores , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Antígeno HLA-B27/inmunología , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Calidad de Vida , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
M-CSF favors the generation of folate receptor ß-positive (FRßâº), IL-10-producing, immunosuppressive, M2-polarized macrophages [M2 (M-CSF)], whereas GM-CSF promotes a proinflammatory, M1-polarized phenotype [M1 (GM-CSF)]. In the present study, we found that activin A was preferentially released by M1 (GM-CSF) macrophages, impaired the acquisition of FRß and other M2 (M-CSF)-specific markers, down-modulated the LPS-induced release of IL-10, and mediated the tumor cell growth-inhibitory activity of M1 (GM-CSF) macrophages, in which Smad2/3 is constitutively phosphorylated. The contribution of activin A to M1 (GM-CSF) macrophage polarization was evidenced by the capacity of a blocking anti-activin A antibody to reduce M1 (GM-CSF) polarization markers expression while enhancing FRß and other M2 (M-CSF) markers mRNA levels. Moreover, an inhibitor of activin receptor-like kinase 4/5/7 (ALK4/5/7 or SB431542) promoted M2 (M-CSF) marker expression but limited the acquisition of M1 (GM-CSF) polarization markers, suggesting a role for Smad2/3 activation in macrophage polarization. In agreement with these results, expression of activin A and M2 (M-CSF)-specific markers was oppositely regulated by tumor ascites. Therefore, activin A contributes to the proinflammatory macrophage polarization triggered by GM-CSF and limits the acquisition of the anti-inflammatory phenotype in a Smad2-dependent manner. Our results demonstrate that activin A-initiated Smad signaling skews macrophage polarization toward the acquisition of a proinflammatory phenotype.
Asunto(s)
Activinas/inmunología , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Activinas/metabolismo , Animales , Biomarcadores/análisis , Western Blotting , Línea Celular , Separación Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica/inmunología , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Smad/metabolismo , TransfecciónRESUMEN
Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF-inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14(+) CD163(+) IL-10-producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewis(x)-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4-dependent) and regulatory (M-CSF-dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.
Asunto(s)
Adenocarcinoma/inmunología , Neoplasias de la Mama/inmunología , Moléculas de Adhesión Celular/biosíntesis , Polaridad Celular/inmunología , Células Dendríticas/inmunología , Interleucina-10/fisiología , Interleucina-6/fisiología , Lectinas Tipo C/biosíntesis , Factor Estimulante de Colonias de Macrófagos/fisiología , Melanoma Experimental/inmunología , Receptores de Superficie Celular/biosíntesis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/metabolismo , Progresión de la Enfermedad , Regulación de la Expresión Génica/inmunología , Humanos , Huésped Inmunocomprometido/genética , Huésped Inmunocomprometido/inmunología , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Lectinas Tipo C/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas de Neoplasias/fisiología , Receptores de Superficie Celular/genética , Células Tumorales CultivadasRESUMEN
Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by macrophage CSF (M-CSF) or granulocyte macrophage CSF (GM-CSF), which promote the generation of antiinflammatory/immunosuppressive MAFB+ (M-MØ) or proinflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and we demonstrate that MAFB regulated the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiated the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulated the expression of profibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Biomarcadores/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismoRESUMEN
Methotrexate (MTX) is an antifolate drug used as a chemotherapeutic agent for acute lymphoblastic leukemia, where MTX improves patients' prognosis. Macrophage reprogramming is being increasingly assessed as an antitumor therapeutic strategy. However, and although MTX limits the pathogenic action of macrophages in chronic inflammatory diseases, its effects on tumor-promoting macrophages have not been previously explored. We now report that MTX shapes the transcriptional and functional profile of M-CSF-dependent human macrophages, whose transcriptome is highly enriched in the gene signature that defines pathogenic tumor-associated macrophages ("large TAM"). Specifically, MTX prompted the acquisition of the gene signature of antitumoral "small TAM" and skewed macrophages toward an IL-6high IFNß1high IL-10low phenotype upon subsequent stimulation. Mechanistically, the MTX-induced macrophage reprogramming effect correlated with a reduction of the M-CSF receptor CSF1R expression and function, as well as a diminished expression of MAF and MAFB transcription factors, primary determinants of pro-tumoral macrophages whose transcriptional activity is dependent on GSK3ß. Indeed, the ability of MTX to transcriptionally reprogram macrophages toward an antitumoral phenotype was abrogated by inhibition of GSK3ß. Globally, our results establish MTX as a macrophage reprogramming drug and indicate that its ability to modulate macrophage polarization may also underlie its therapeutic benefits. Since GSK3ß inhibition abrogates the reprogramming action of MTX, our results suggest that the GSK3ß-MAFB/MAF axis constitutes a target for the macrophage-centered antitumor strategies.
RESUMEN
Liver X Receptors (LXR) control cholesterol metabolism and exert anti-inflammatory actions but their contribution to human macrophage polarization remains unclear. The LXR pathway is enriched in pro-inflammatory macrophages from rheumatoid arthritis as well as in tumors-associated macrophages from human tumors. We now report that LXR activation inhibits the anti-inflammatory gene and functional profile of M-CSF-dependent human macrophages, and prompts the acquisition of a pro-inflammatory gene signature, with both effects being blocked by an LXR inverse agonist. Mechanistically, the LXR-stimulated macrophage polarization shift correlates with diminished expression of MAFB and MAF, which govern the macrophage anti-inflammatory profile, and with enhanced release of activin A. Indeed, LXR activation impaired macrophage polarization in response to tumor-derived ascitic fluids, as well as the expression of MAF- and MAFB-dependent genes. Our results demonstrate that LXR activation limits the anti-inflammatory human macrophage polarization and prompts the acquisition of an inflammatory transcriptional and functional profile.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos , Macrófagos , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Células Cultivadas , Humanos , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismoRESUMEN
Tumour cell dissemination through corporal fluids (blood, lymph and body cavity fluids) is a distinctive feature of the metastatic process. Tumour cell transition from fluid to adhesive conditions involves an early polarization event and major rearrangements of the submembrane cytoskeleton that remain poorly understood. As regulation of cortical actin-membrane binding might be important in this process, we investigated the role of ezrin and moesin, which are key crosslinking proteins of the ERM (ezrin, radixin, moesin) family. We used short interfering RNA (siRNA) to show that moesin is crucial for invasion by melanoma cells in 3D matrices and in early lung colonization. Using live imaging, we show that following initial adhesion to the endothelium or 3D matrices, moesin is redistributed away from the region of adhesion, thereby generating a polarized cortex: a stable cortical actin dome enriched in moesin and an invasive membrane domain full of blebs. Using Lifeact-GFP, a 17-amino-acid peptide that binds F-actin, we show the initial symmetry breaking of cortical actin cytoskeleton during early attachment of round cells. We also demonstrated that ezrin and moesin are differentially distributed during initial invasion of 3D matrices, and, specifically, that moesin controls adhesion-dependent activation of Rho and subsequent myosin II contractility. Our results reveal that polarized moesin plays a role in orienting Rho activation, myosin II contractility, and cortical actin stability, which is crucial for driving directional vertical migration instead of superficial spreading on the fluid-to-solid tissue interface. We propose that this mechanism of cortical polarization could sustain extravasation of fluid-borne tumour cells during the process of metastasis.
Asunto(s)
Polaridad Celular , Melanoma/fisiopatología , Proteínas de Microfilamentos/metabolismo , Invasividad Neoplásica , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Proteínas de Microfilamentos/genéticaRESUMEN
TAMs constitute a large fraction of infiltrating immune cells in melanoma tissues, but their significance for clinical outcomes remains unclear. We explored diverse TAM parameters in clinically relevant primary cutaneous melanoma samples, including density, location, size, and polarization marker expression; in addition, because cytokine production is a hallmark of macrophages function, we measured CCL20, TNF, and VEGFA intracellular cytokines by single-cell multiparametric confocal microscopy. The Kaplan-Meier method was used to analyze correlation with melanoma-specific disease-free survival and overall survival. No significant correlations with clinical parameters were observed for TAM density, morphology, or location. Significantly, higher contents of the intracellular cytokines CCL20, TNF, and VEGFA were quantified in TAMs infiltrating metastasizing compared to non-metastasizing skin primary melanomas (p < 0.001). To mechanistically explore cytokine up-regulation, we performed in vitro studies with melanoma-conditioned macrophages, using RNA-seq to explore involved pathways and specific inhibitors. We show that p53 and NF-κB coregulate CCL20, TNF, and VEGFA in melanoma-conditioned macrophages. These results delineate a clinically relevant pro-oncogenic cytokine profile of TAMs with prognostic significance in primary melanomas and point to the combined therapeutic targeting of NF-kB/p53 pathways to control the deviation of TAMs in melanoma.