RESUMEN
Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.
Asunto(s)
Caveolina 1/metabolismo , Metástasis de la Neoplasia/patología , Neoplasias/patología , Animales , Movimiento Celular , Fibroblastos/patología , Humanos , Melanoma/patología , Ratones , Ratones NoqueadosRESUMEN
Nurr1 is a member of the orphan nuclear receptor family NR4A (nuclear receptor subfamily 4 group A) that modulates inflammation in several cell lineages, both positively and negatively. Macrophages are key regulators of inflammatory responses, yet information about the role of Nurr1 in human macrophages is scarce. Here we examined Nurr1 expression and activity in steady state and activated human macrophages. Pro- and anti-inflammatory macrophages were generated in vitro by culture of blood monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Nurr1 expression was predominant in macrophages with the pro-inflammatory phenotype. Nurr1 activation with the agonists 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) or isoxazolo-pyridinone 7e (IP7e) did not globally modify the polarization status of pro-inflammatory macrophages, but they decreased their production of TNF, IL-1ß, IL-6, IL-8, IL-12 p40, CCL2, IFN-ß, and reactive oxygen species, with variable potencies. Conversely, Nurr1 deficient macrophages increased the expression of transcripts encoding inflammatory mediators, particularly that of IL6, IFNB1, and CCL2. Mechanistically, endogenous Nurr1 interacted with NF-κB p65 in basal conditions and upon lipopolysaccharide (LPS)-mediated activation. C-DIM12 stabilized those complexes in cells exposed to LPS and concurrently decreased NF-κB transcriptional activity and p65 nuclear translocation. Expression of high levels of Nurr1 was associated with a subset of dermal macrophages that display enhanced levels of TNF and lower expression of the anti-inflammatory marker CD163L1 in skin lesions from patients with bullous pemphigoid (BP), a chronic inflammatory autoimmune blistering disorder. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages, both in vivo and in vitro, where it may constitute a brake to attenuate the synthesis of inflammatory mediators.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos , FN-kappa B , Humanos , FN-kappa B/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Antiinflamatorios/metabolismoRESUMEN
During cancer evolution, tumor cells attract and dynamically interact with monocytes/macrophages. To find biomarkers of disease progression in human melanoma, we used unbiased RNA sequencing and secretome analyses of tumor-macrophage co-cultures. Pathway analysis of genes differentially modulated in human macrophages exposed to melanoma cells revealed a general upregulation of inflammatory hallmark gene sets, particularly chemokines. A selective group of chemokines, including CCL8, CCL15, and CCL20, was actively secreted upon melanoma-macrophage co-culture. Because we previously described the role of CCL20 in melanoma, we focused our study on CCL8 and CCL15 and confirmed that in vitro both chemokines contributed to melanoma survival, proliferation, and 3D invasion through CCR1 signaling. In vivo, both chemokines enhanced primary tumor growth, spontaneous lung metastasis, and circulating tumor cell survival and lung colonization in mouse xenograft models. Finally, we explored the clinical significance of CCL8 and CCL15 expression in human skin melanoma, screening a collection of 67 primary melanoma samples, using multicolor fluorescence and quantitative image analysis of chemokine-chemokine receptor content at the single-cell level. Primary skin melanomas displayed high CCR1 expression, but there was no difference in its level of expression between metastatic and nonmetastatic cases. By contrast, comparative analysis of these two clinically divergent groups showed a highly significant difference in the cancer cell content of CCL8 (p = 0.025) and CCL15 (p < 0.0001). Kaplan-Meier curves showed that a high content of CCL8 or CCL15 in cancer cells correlated with shorter disease-free and overall survival (log-rank test, p < 0.001). Our results highlight the role of CCL8 and CCL15, which are highly induced by melanoma-macrophage interactions in biologically aggressive primary melanomas and could be clinically applicable biomarkers for patient profiling. © 2024 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Ratones , Animales , Melanoma/genética , Pronóstico , Neoplasias Cutáneas/genética , Quimiocinas/metabolismo , Macrófagos/metabolismo , Biomarcadores , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Proteínas Inflamatorias de Macrófagos , Quimiocinas CC/genéticaRESUMEN
BACKGROUND: The invariant TCR ζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its 3 immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. OBJECTIVE: We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs. RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247. CONCLUSIONS: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.
RESUMEN
The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.
Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Sinapsis Inmunológicas/inmunología , FN-kappa B/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/inmunología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Humanos , Immunoblotting , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.
Asunto(s)
Movimiento Celular , Polaridad Celular , Cromatina/metabolismo , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/metabolismo , Cromatina/genética , Cromatina/ultraestructura , Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Microscopía de Fuerza Atómica , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genéticaRESUMEN
BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, we detected a new immunofluorescence (IF) pattern in serum autoantibody (autoAb) screening of laboratory-confirmed COVID-19 patients. METHODS: The IF pattern was composed of liver and gastric mucosa staining on rat kidney/liver/stomach sections. RESULTS: We describe 12 patients positive for the cross-reactive antibody, compared with a negative group of 43 hospitalized COVID-19 patients, finding association with either neurologic or thrombotic complications. In sequential pre- and post-COVID-19 serum samples, we confirmed autoAb seroconversion. CONCLUSIONS: Our data indicate that autoAb screening in COVID-19 patients may be easily performed by IF and alert for autoreactive-mediated complications such as thrombotic or neurologic events.
Asunto(s)
Autoanticuerpos/sangre , Betacoronavirus , Infecciones por Coronavirus/inmunología , Enfermedades del Sistema Nervioso/inmunología , Neumonía Viral/inmunología , Trombosis/inmunología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , COVID-19 , Estudios de Casos y Controles , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/complicaciones , Reacciones Cruzadas/inmunología , Femenino , Ferritinas/sangre , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Persona de Mediana Edad , Enfermedades del Sistema Nervioso/virología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/complicaciones , Ratas , SARS-CoV-2 , Seroconversión , Pruebas Serológicas , Trombosis/virología , Adulto JovenRESUMEN
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
Macrophage phenotypic and functional heterogeneity derives from tissue-specific transcriptional signatures shaped by the local microenvironment. Most studies addressing the molecular basis for macrophage heterogeneity have focused on murine cells, whereas the factors controlling the functional specialization of human macrophages are less known. M-CSF drives the generation of human monocyte-derived macrophages with a potent anti-inflammatory activity upon stimulation. We now report that knockdown of MAFB impairs the acquisition of the anti-inflammatory profile of human macrophages, identify the MAFB-dependent gene signature in human macrophages and illustrate the coexpression of MAFB and MAFB-target genes in CD163+ tissue-resident and tumor-associated macrophages. The contribution of MAFB to the homeostatic/anti-inflammatory macrophage profile is further supported by the skewed polarization of monocyte-derived macrophages from multicentric carpotarsal osteolysis (Online Mendelian Inheritance in Man #166300), a pathology caused by mutations in the MAFB gene. Our results demonstrate that MAFB critically determines the acquisition of the anti-inflammatory transcriptional and functional profiles of human macrophages.
Asunto(s)
Diferenciación Celular , Síndrome de Hajdu-Cheney/inmunología , Macrófagos/fisiología , Factor de Transcripción MafB/metabolismo , Monocitos/fisiología , Animales , Antiinflamatorios , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Microambiente Celular , Citocinas/metabolismo , Técnicas de Silenciamiento del Gen , Ontología de Genes , Síndrome de Hajdu-Cheney/genética , Homeostasis , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor de Transcripción MafB/genética , Ratones , Mutación/genética , Receptores de Superficie Celular/metabolismo , Células Th2/inmunología , TranscriptomaRESUMEN
Tumor-associated macrophages (TAM) are important components of the multiple myeloma (MM) microenvironment that support malignant plasma cell survival and resistance to therapy. It has been proposed that macrophages (MØ) retain the capacity to change in response to stimuli that can restore their antitumor functions. Here, we investigated several approaches to reprogram MØ as a novel therapeutic strategy in MM. First, we found tumor-limiting and tumor-supporting capabilities for monocyte-derived M1-like MØ and M2-like MØ, respectively, when mixed with MM cells, both in vitro and in vivo. Multicolor confocal microscopy revealed that MM-associated MØ displayed a predominant M2-like phenotype in the bone marrow of MM patient samples, and a high expression of the pro-M2 cytokine macrophage migration inhibitory factor (MIF). To reprogram the protumoral M2-like MØ present in MM toward antitumoral M1-like MØ, we tested the pro-M1 cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) plus blockade of the M2 cytokines macrophage colony-stimulating factor or MIF. The combination of GM-CSF plus the MIF inhibitor 4-iodo-6-phenyl-pyrimidine achieved the best reprogramming responses toward an M1 profile, at both gene and protein expression levels, as well as remarkable tumoricidal effects. Furthermore, this combined treatment elicited MØ-dependent therapeutic responses in MM xenograft mouse models, which were linked to upregulation of M1 and reciprocal downregulation of M2 MØ markers. Our results reveal the therapeutic potential of reprogramming MØ in the context of MM.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Técnicas de Reprogramación Celular/métodos , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Macrófagos/patología , Mieloma Múltiple/inmunología , Animales , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Microscopía Confocal , Pirimidinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Estradiol-based contraceptives and hormonal replacement therapy predispose women to Candida albicans infections. Moreover, during the ovulatory phase (high estradiol), neutrophil numbers decrease in the vaginal lumen and increase during the luteal phase (high progesterone). Vaginal secretions contain chemokines that drive neutrophil migration into the lumen. However, their expression during the ovarian cycle or in response to hormonal treatments are controversial and their role in vaginal defense remains unknown.To investigate the transepithelial migration of neutrophils, we used adoptive transfer of Cxcr2(-/-) neutrophils and chemokine immunofluorescence quantitative analysis in response to C. albicans vaginal infection in the presence of hormones.Our data show that the Cxcl1/Cxcr2 axis drives neutrophil transepithelial migration into the vagina. Progesterone promotes the Cxcl1 gradient to favor neutrophil migration. Estradiol disrupts the Cxcl1 gradient and favors neutrophil arrest in the vaginal stroma; as a result, the vagina becomes more vulnerable to pathogens.
Asunto(s)
Quimiocinas/metabolismo , Estrógenos/farmacología , Neutrófilos/inmunología , Neutrófilos/fisiología , Progesterona/farmacología , Vagina/citología , Adulto , Animales , Candida albicans/inmunología , Candidiasis/inmunología , Movimiento Celular , Células Cultivadas , Quimiocinas/genética , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Ratones , Ratones Noqueados , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Vagina/inmunologíaRESUMEN
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.
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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
Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.
Asunto(s)
Activinas/metabolismo , Dioxigenasas/metabolismo , Regulación de la Expresión Génica/inmunología , Inflamación/metabolismo , Macrófagos/enzimología , Activinas/genética , Activinas/inmunología , Western Blotting , Dioxigenasas/genética , Dioxigenasas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inmunohistoquímica , Inflamación/genética , Inflamación/inmunología , Macrófagos/inmunología , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunogenicity of SARS-CoV-2 mRNA vaccines is highly heterogeneous in patients with inborn errors of immunity (IEIs). This case report analyzes the immune response to mRNA COVID-19 two-dose primary vaccination followed by three boosters in an IEI patient with marked CD4+ T-cell cytopenia and diminished thymic output, in comparison with that raised against latent, chronic cytomegalovirus (CMV) infection. Serum IgG antibodies anti-spike (S) protein of SARS-CoV-2 and anti-CMV were both determined by chemiluminescent microparticle immunoassays (CMIAs). SARS-CoV-2 and CMV memory CD4+ T-cell responses were simultaneously evaluated in vitro using an activation-induced marker (AIM) assay via multicolor flow cytometry. Throughout the 2-year follow-up that included the administration of five doses of SARS-CoV-2 mRNA vaccines, cellular anti-SARS-CoV-2-specific responses remained consistently negative, with extremely weak humoral responses, while the patient showed in vitro persistent CD4+ T-cell reactivity to CMV peptides and high-IgG CMV-specific titers. The assessment of immune responses to vaccines and prevalent viruses is essential in IEI patients in order to take adequate preventive measures.
RESUMEN
Monocytes are versatile cells that can express different functional programs in response to microenvironmental signals. We show that primary blood monocytes secrete the CXCL12 chemokine, and express the CXCR4 and CXCR7 receptors, leading to an autocrine/paracrine loop that contribute to shape monocyte differentiation to a distinct type of macrophages, with an enhanced expression of CD4, CD14, and CD163, or dendritic cells, with a reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The in vivo relevance of CXCL12 production by mononuclear phagocytes was studied in metastatic melanoma tissues by a thoroughly immunofluorescence phenotyping of CXCL12(high) expressing cells, which were CD45(+), coexpressed the macrophage antigens CD68, CD163, and CD209 and constituted the 60%-90% of tumor-associated macrophages. Microarray analysis of primary monocytes revealed that the vascular endothelial growth factor and the angiogenic chemokine CCL1 mRNA levels were up-regulated in response to CXCL12, leading to enhanced expression of both proteins. In addition, we found that CXCL12 autocrine/paracrine signaling down-regulates the expression of the transcription factor RUNX3 and contributes to maintain the long-term CD4 and CD14 expression in monocytes/macrophages. Together, these results suggest that autocrine CXCL12 production modulates differentiation of monocytes toward a distinct program with proangiogenic and immunosuppressive functions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Macrófagos/citología , Monocitos/citología , Biomarcadores/metabolismo , Western Blotting , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagocitos/inmunología , Fagocitos/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
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
BACKGROUND: Radioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under clinical scrutiny to maximize efficacy and avoid toxicity. Local immunotherapy, by injecting various pathogen-associated molecular patterns, has shown efficacy both preclinically and clinically. BO-112 is a viral mimetic based on nanoplexed double-stranded RNA (poly I:C) which exerts immune-mediated antitumor effects in mice and humans on intratumoral delivery. BO-112 and focal irradiation were used to make the proof-of-concept for local immunotherapy plus radiation therapy combinations. METHODS: Murine transplantable tumor cell lines (TS/A, MC38 and B16-OVA) were used to show increased immunogenic features under irradiation, as well as in bilateral tumor models in which only one of the lesions was irradiated or/and injected with BO-112. Flow cytometry and multiplex tissue immunofluorescence were used to determine the effects on antitumor immunity. Depletions of immune cell populations and knockout mice for the IFNAR and BATF-3 genes were used to delineate the immune system requirements for efficacy. RESULTS: In cultures of TS/A breast cancer cells, the combination of irradiation and BO-112 showed more prominent features of immunogenic tumor cell death in terms of calreticulin exposure. Injection of BO-112 into the tumor lesion receiving radiation achieved excellent control of the treated tumor and modest delays in contralateral tumor progression. Local effects were associated with more prominent infiltrates of antitumor cytotoxic tumor lymphocytes (CTLs). Importantly, local irradiation plus BO-112 in one of the tumor lesions that enhanced the therapeutic effects of radiotherapy on distant irradiated lesions that were not injected with BO-112. Hence, this beneficial effect of local irradiation plus BO-112 on a tumor lesion enhanced the therapeutic response to radiotherapy on distant non-injected lesions. CONCLUSION: This study demonstrates that local BO-112 immunotherapy and focal irradiation may act in synergy to achieve local tumor control. Irradiation plus BO-112 in one of the tumor lesions enhanced the therapeutic effects on distant irradiated lesions that were not injected with BO-112, suggesting strategies to treat oligometastatic patients with lesions susceptible to radiotherapy and with at least one tumor accessible for repeated BO-112 intratumoral injections.