Immunoglobulins and Serotonin modulate human macrophage polarization

1 p. Annual Scientific Meeting of the European Society for Clinical Investigation Cluj-Napoca, Romania 27– 30 May 2015Peer reviewe

Growth Hormone Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner

Growth hormone (GH), a pleiotropic hormone secreted by the pituitary gland, regulates immune and inflammatory responses. In this study, we show that GH regulates the phenotypic and functional plasticity of macrophages both in vitro and in vivo. Specifically, GH treatment of GM-CSF–primed monocyte–derived macrophages promotes a significant enrichment of anti-inflammatory genes and dampens the proinflammatory cytokine profile through PI3K-mediated downregulation of activin A and upregulation of MAFB, a critical transcription factor for anti-inflammatory polarization of human macrophages. These in vitro data correlate with improved remission of inflammation and mucosal repair during recovery in the acute dextran sodium sulfate–induced colitis model in GH-overexpressing mice. In this model, in addition to the GH-mediated effects on other immune cells, we observed that macrophages from inflamed gut acquire an anti-inflammatory/reparative profile. Overall, these data indicate that GH reprograms inflammatory macrophages to an anti-inflammatory phenotype and improves resolution during pathologic inflammatory responses.This work was supported in part by grants from the Spanish Ministry of Science, Innovation and Universities (SAF2017-82940-R Agencia Estatal de Investigación/Fondo Europeo de Desarrollo Regional (AEI/FEDER), Unión Europea [UE] [to M.M.], SAF2017-83785-R AEI/FEDER, UE [to Á.L.C.] and FJCI-2016-29990 AEI/FEDER, UE [to B.S.P.]), from the Redes Temáticas de Investigación Cooperativa en Salud Program of Instituto de Salud Carlos III (RD12/0012/0006 and RD12/0012/0007, Red de Investigación en Inflamación y Enfermedades Reumáticas), and the Regional Government of Madrid (B2017/BMD-3804 [to C.M.-A.])

TLR7 activation in M-CSF-dependent monocyte-derived human macrophages potentiates inflammatory responses and prompts neutrophil recruitment

1 p.-4 fig.Toll-like receptor 7 (TLR7) is an endosomal Pathogen-Associated Molecular Pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of SARS-CoV-2-induced severe COVID-19. We previously showed that TLR7 is preferentially expressed by macrophages generated in the presence of M-CSF (M-MØ), whose MAFB-dependent transcriptome resembles pathogenic pulmonary monocyte-derived macrophage subsets in severe COVID-19. We now report that TLR7 activation in M-MØ triggers a weak MAPK, NFkB and STAT1 activation and leads to defective production of type I IFN. Nonetheless, TLR7 engagement re-programs MAFB+ M-MØ towards a distinctive transcriptional profile. Specifically, TLR7-activated M-MØ acquired the expression of genes that characterize inflammatory macrophage subsets in COVID-19 and other inflammatory diseases, including genes encoding neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8) reported as biomarkers for severe COVID-19. Functionally, TLR7-activated M-MØ displayed enhanced proinflammatory responses towards secondary stimulation and a robust production of neutrophil-attracting chemokines (CXCL1, CXCL5, CXCL8), which was dependent on the transcription factors MAFB and AhR. Interestingly, CXCL1 and CXCL5 release from M-MØ was also promoted by SARS-CoV-2 but not by Virus-like particles. As defective TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with severe COVID-19, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.This research work was also funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global)Peer reviewe

Immunophenotyping of peripheral blood monocytes could help identify a baseline pro-inflammatory profile in women with recurrent reproductive failure

11 p.-4 fig.-4 tab.Recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) are two well-defined clinical entities, but the role of the monocytes in their pathophysiology needs to be clarified. This study aimed to evaluate the role of the three monocyte subsets (classical, intermediate, and non-classical) and relevant cytokines/chemokines in a cohort of RPL and RIF women to better characterize a baseline proinflammatory profile that could define inflammatory pathophysiology in these two different conditions. We evaluated 108 non-pregnant women: 53 RPL, 24 RIF, and 31 fertile healthy controls (HC). Multiparametric flow cytometry was used to quantify the frequency of surface chemokine receptors (CCR2, CCR5, and CX3CR1) on the monocyte subsets. Cytokines were assessed in plasma samples using a multiplex assay. The CX3CR1+ and CCR5+ intermediate monocytes were significantly higher in RPL and RIF compared to HC. A significant positive correlation was observed between CX3CR1+ intermediate monocytes and IL-17A (P = .03, r = 0.43). The Boruta algorithm followed by a multivariate logistic regression model was used to select the most relevant variables that could help define RPL and RIF: in RPL were CX3CR1 non-classical monocytes, TGF-β1, and CCR5 intermediate monocytes; in RIF: CCR5 intermediate monocytes and TGF-β3. The combination of these variables could predict RPL and RIF with 90 % and 82 %, respectively. Our study suggests that a combination of specific blood monocyte subsets and cytokines could aid in identifying RPL and RIF women with a pro-inflammatory profile. These findings could provide a more integrated understanding of these pathologies. Further investigation and validation in independent cohorts are warranted.The project received a research grant from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain), awarded on the 2016 call under the Health Strategy Action 2016–2017, within the National Research Program oriented to Societal Challenges, within the Technical, Scientific and Innovation Research National Plan 2013–2016, with reference PI16/01428, and was co-supported by The European Regional Development Fund (ERDF).Peer reviewe

The GSK3b-MAFB axis controls the pro-fibrotic gene profile of pathogenic monocyte-derived macrophages in severe COVID-19

1 p.-4 fig.MAF and MAFB are members of the “large MAF” transcription factor family that shape the transcriptome of antiinflammatory and pro-tumoral human macrophages. We have now determined the MAF- and MAFB-dependent gene profile of M-CSF-dependent monocyte-derived macrophages (M-MØ), and found that both factors exhibit overlapping transcriptional outcomes during monocyte-to-M-MØ differentiation, but differentially affect macrophage effector functions like production of monocyte-recruiting chemokines, T-cell activation and immunosuppression. Remarkably, MAFB was found to positively regulate the expression of the genesets that define the pathogenic monocyte-derived pulmonary macrophage subsets in COVID-19, as evidenced through siRNA-mediated silencing and analysis of MAFBoverexpressing M-MØ from a Multicentric Carpotarsal Osteolysis (MCTO) patient. MAFB silencing downregulated theexpression of genes coding for biomarkers of COVID-19 severity, and genome-wide mapping of MAFB-binding elements in M-MØ identified biomarkers of COVID-19 severity (CD163, IL10, HGF and CCL2) as direct MAFB targets. Further, and in line with the GSK3b-dependent expression of MAFB, GSK3b inhibition in M-MØ significantly boosted the expression of genes that characterize pathogenic macrophage subsets in severe COVID-19, an effect that was primarily dependent on MAFB. In addition, we have demonstrated that a large number of MAFB-dependent genes, as well as GSK3b-dependent expression of MAFB genes were modulated by SARS-Cov-2 infection on human macrophages. Globally, our results demonstrate that the GSK3b-MAFB axis controls the transcriptome of pathogenic pulmonary macrophages in COVID-19,and positively regulates the expression of biomarkers for COVID-19 severity. Thus, macrophage re-programming through modulation of GSK3 -MAFB axis has potential therapeutic strategy for COVID-19 and other inflammatory diseases.This research work was also funded by the European Commission– NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global).Peer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Regulación de la expresión y función de las lectinas mieloides DC-SIGN y LSECtin

144 p.-31 fig.-4 tab.-anexo.El ser humano se encuentra expuesto constantemente a microorganismos y agentes extraños presentes en el ambiente o en su propio cuerpo. El sistema inmunitario es el responsable de realizar la difícil tarea de discriminar lo propio de lo extraño y potencialmente dañino, así como de actuar apropiadamente para eliminar dichos agentes sin dañar los tejidos propios. El sistema inmunitario comprende el sistema innato o natural y el sistema adaptativo o específico, y la coordinación de ambas respuestas permite llevar a cabo su función defensiva de modo eficiente. El sistema innato proporciona la primera línea de defensa frente a organismos extraños, previniendo la infección y en algunos casos llegando a la eliminación del microorganismo. Este sistema, presente en la mayoría de los organismos multicelulares [1], incluye componentes celulares y moleculares que representan mecanismos de resistencia no específicos de antígeno, y entre los que se encuentran la barrera epitelial, células fagocíticas (neutrófilos, macrófagos y células dendríticas), proteínas efectoras circulantes (sistema del complemento, colectinas, proteína C reactiva) y citoquinas. El sistema adaptativo es una ventaja evolutiva desarrollada en vertebrados, principalmente en mamíferos, que supone una herramienta más sofisticada para hacer frente a los microorganismos que escapan a la respuesta innata. Este sistema se caracteriza por ser antígeno-específico y otorgar memoria inmunológica al organismo, lo que le permite responder más eficientemente frente a exposiciones repetidas de un mismo agente. La respuesta inmunitaria adaptativa incluye componentes humorales (anticuerpos) y celulares (linfocitos T), y sus niveles relativos dependen de la naturaleza del microorganismo. En consecuencia, las respuestas innata y adaptativa son componentes de un sistema integrado de defensa del organismo. Existen dos importantes conexiones entre la inmunidad innata y la inmunidad adaptativa. En primer lugar, la respuesta innata tiene efecto sobre la respuesta inmune adaptativa e influye y determina la naturaleza de esta última. Por otra parte, la respuesta adaptativa emplea muchos de los mecanismos efectores de la inmunidad innata para eliminar el microorganismo, modulando sus actividades antimicrobianas. Las células dendríticas (DC) y los macrófagos son los mayores exponentes de dichas conexiones. Las células dendríticas (DC) se identificaron por primera vez en 1868 en la epidermis, donde reciben el nombre de células de Langerhans. En un principio fueron consideradas parte del sistema nervioso, y no fue hasta 1973 cuando Steinman y Cohn las describieron en otros tejidos y denominaron células dendríticas [2]. Las DC son células derivadas de médula ósea que actúan como células presentadoras de antígenos (APC) y, por tanto, poseen la capacidad exclusiva de iniciar la respuesta inmunitaria y relacionar las respuestas innata y adaptativa [3, 4]. Las DC se encuentran distribuidas por todo el organismo, aunque en un número y proporción tan bajo que imposibilitó su estudio hasta que no se desarrollaron técnicas para su generación y cultivo in vitro a partir de progenitores CD34+ de médula ósea o monocitos [5]. El tratamiento de monocitos humanos CD14+ con GM-CSF e IL-4 [6] da lugar a células con fenotipo característico de dendríticas inmaduras. La relevancia fisiológica de la diferenciación de monocito a célula dendrítica in vitro ha sido corroborada in vivo al demostrarse que los monocitos inflamatorios transportan partículas fagocitadas a los nódulos linfáticos, donde adquieren características funcionales de células dendríticas [7, 8].Como centinelas del sistema inmune, las DC expresan una gran cantidad de receptores especializados en reconocimiento de patógenos y captura de antígenos, lo cual les permite muestrear constantemente el medio que les rodea en busca de “señales de peligro”. Además, las DC tienen la capacidad de activar y polarizar a los linfocitos T vírgenes y, dependiendo de su estado de maduración, promover la tolerancia inmunológica o respuesta inmunitaria [9, 10]. Esta ambivalencia funcional ha llevado a la célula dendrítica a ser el foco de atención de protocolos de inmunoterapia en enfermedades autoinmunes, cáncer o transplante [11]. La capacidad de las DC de activar a los linfocitos T vírgenes las diferencia del resto de células presentadoras de antígeno, y es la razón por la que se les denomina APCs profesionales. Las DC tienen la capacidad de controlar el tipo de respuesta inmunitaria que se genera frente a un antígeno, determinando si los linfocitos T vírgenes han de polarizarse hacia Th1, Th2, Th17 o T reguladoras [4]. Los linfocitos Th1 son efectivos frente a patógenos intracelulares, ya que el IFN-γ que producen estimula mecanismos antimicrobianos en macrófagos y células infectadas, mientras que los Th2 (productores de IL-4) son más efectivos en la eliminación de patógenos extracelulares. Este proceso de polarización es de gran relevancia, por cuanto los patógenos pueden manipularla, interferir en el balance Th1/Th2 y, por tanto, perjudicar la generación de una respuesta inmunitaria efectiva. Las células dendríticas son comúnmente clasificadas en dos clases fenotípicas según su grado de parentesco con los linajes mejor definidos [12]: DC mieloides (convencionales), con un fenotipo prototípico CD11c+/CD123-, y DC plasmacitoides (productoras de interferón) definidas por un fenotipo CD11c-/CD123+ [13]. En el caso de las DC mieloides, sus progenitores salen de la médula ósea, entran en la circulación y posteriormente llegan a los tejidos periféricos, donde se convierten en DC inmaduras. Las DC reciben distintos nombres según su localización (células de Langerhans, DC intersticiales, DC dérmicas, DC tímicas,…). En este estadío, las DC expresan gran cantidad de receptores de reconocimineto de patógenos (PRR), lo que las dota de una gran capacidad para detectar “señales de peligro” [14]. La alta capacidad endocítica que presentan estas células les permite capturar y procesar eficientemente los antígenos presentes en el medio extracelular [14]. En este estadío las DC contribuyen al establecimiento de la tolerancia periférica [10]. Sin embargo, las DC inmaduras son extremadamente sensibles a la presencia de patógenos, productos derivados de patógenos o moléculas propias alteradas (“señales de peligro”), que disparan drásticos cambios transcripcionales, morfológicos y funcionales en ellas. Este proceso se ha denominado “maduración” [8, 9] y es clave en la generación de respuestas inmunitarias antígeno-específicas.Aunque se ha demostrado la existencia de un “core” de genes regulados en presencia de cualquier patógeno o estímulo inflamatorio, la maduración de DC es un proceso patógeno-específico, por cuanto el conjunto de cambios génicos que tiene lugar es único y exclusivo para cada patógeno [15]. El nuevo fenotipo de las células dendríticas maduras les permite presentar los antígenos anteriormente capturados y procesados en el contexto del complejo mayor de histocompatibilidad (MHC) [9, 16]. Algunos de los cambios experimentados en el proceso de maduración incluyen la disminución de receptores implicados en captura de antígeno y capacidad endocítica, el aumento de los niveles de moléculas coestimuladoras y MHC, el aumento de la expresión de citoquinas polarizadoras (IL-10, IL-23, IL12p70), la disminución en los niveles de expresión de receptores de quimioquinas inflamatorias y el aumento de la capacidad migratoria en respuesta a quimioquinas como CCL19 y CCL21 [9]. Simultáneamente al proceso de maduración tiene lugar la migración de las células dendríticas hacia los órganos linfoides secundarios, donde promoverán la activación y polarización de linfocitos T vírgenes, orquestándose así la respuesta inmunitaria adaptativa. En resumen, tras el comienzo de un proceso infeccioso, las células dendríticas inmaduras detectan el “peligro” y sufren una reprogramación de su perfil de expresión génica de manera patógeno-específica (maduración). Este proceso de maduración les permitirá promover la polarización de los linfocitos T vírgenes y, por tanto, iniciar la respuesta inmunitaria más efectiva frente a ese microorganismo concreto [15, 19, 20].Peer reviewe

Myeloid dendritic cell lectins and their role in immune responses

Dendritic cells are bone marrow-derived professional antigen-presenting cells which link innate and adaptive immune responses. Acting as sentinels of the immune system, dendritic cells are loaded with a large array of antigen-capturing and pathogen-recognition receptors which constantly sample the surrounding extracellular environment for 'danger signals'. However, dendritic cells also exhibit a naive T-lymphocyte-activating ability and, depending on their maturation status, promote either immunological tolerance or primary immune responses. This functional ambivalence has brought dendritic cells to the focus of attention for immunotherapy protocols in autoimmune diseases, cancer and transplantation. © The Thomson Corporation.Peer Reviewe

Folate Receptor β (FRβ) Expression in Tissue-Resident and Tumor-Associated Macrophages Associates with and Depends on the Expression of PU.1

© 2020 by the authors.As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor β (FRβ, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the “anti-inflammatory gene set”, which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRβ marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.This research was funded by Instituto de Salud Carlos III/FEDER (PI17/00037, PI17/01324 and RD16/0012/0007), Instituto de Investigación Sanitaria Gregorio Marañón(II-PI-1-2019), Ministerio de Economía y Competitividad (SAF2017-83785-R) and Fundación La Marató/TV3 (201619.31). FEDER: Fondo Europeo de Desarrollo Regional: una manera de hacer Europa.Peer reviewe