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

Virtualización e impresión 3D de modelos anatómicos aplicados a la docencia y planificación quirúrgica IV

Depto. de Medicina y Cirugía AnimalFac. de VeterinariaFALSEsubmitte

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

Virtualización e impresión 3D de modelos anatómicos aplicados a la docencia en anatomia y cirugía veterinaria II

Este proyecto aplica las técnicas de virtualización de imágenes de piezas anatómicas normales y patológicas con fines de creación de modelos digitales y con impresión 3D para docencia en Anatomía y Cirugía Veterinarias

Mecanismos moleculares de la respuesta antiinflamatoria de macrófagos humanos

Los macrófagos son las células responsables de orquestar la respuesta inmunológica, siendo efectores fundamentales del sistema inmunológico innato y actuando como puente entre este y las células del sistema inmunológico adquirido. Los macrófagos intervienen en un amplio abanico de procesos, que van desde la estimulación y la terminación de la respuesta inflamatoria del huésped a procesos de desarrollo y homeostasis tisular. Esta gran diversidad funcional solo puede desempeñarse gracias al extenso espectro de estados de polarización que los macrófagos pueden adquirir bajo la influencia de señales del microambiente tisular. De hecho, la desregulación de sus funciones efectoras puede dar lugar o favorecer patologías tan relevantes como los tumores, el COVID-19 o diversos trastornos autoinmunes e inflamatorios. Clásicamente, se ha considerado que los macrófagos pueden presentar dos estados de polarización opuestos, si bien esta división es una sobresimplificación. La presencia de factores como TNF-α, IFNγ o GM-CSF dota a los macrófagos de un perfil proinflamatorio, el cual se caracteriza por la expresión de moléculas coestimuladoras y la producción de citoquinas proinflamatorias, como IL-6, IL-12 o TNF-α. Por otra parte, citoquinas como IL-4, IL-13 o M-CSF hacen que los macrófagos adquieran un perfil antiinflamatorio y/o reparador, caracterizado por la producción de citoquinas antiinflamatorias como IL-10 o TGFβ. El estudio de las características transcripcionales de macrófagos antiinflamatorios, y de su respuesta a estímulos patogénicos, constituye el eje central de este trabajo..

MAFB and MAF Transcription Factors as Macrophage Checkpoints for COVID-19 Severity

Defective IFN production and exacerbated inflammatory and pro-fibrotic responses are hallmarks of SARS-CoV-2 infection in severe COVID-19. Based on these hallmarks, and considering the pivotal role of macrophages in COVID-19 pathogenesis, we hypothesize that the transcription factors MAFB and MAF critically contribute to COVID-19 progression by shaping the response of macrophages to SARS-CoV-2. Our proposal stems from the recent identification of pathogenic lung macrophage subsets in severe COVID-19, and takes into consideration the previously reported ability of MAFB to dampen IFN type I production, as well as the critical role of MAFB and MAF in the acquisition and maintenance of the transcriptional signature of M-CSF–conditioned human macrophages. Solid evidences are presented that link overexpression of MAFB and silencing of MAF expression with clinical and biological features of severe COVID-19. As a whole, we propose that a high MAFB/MAF expression ratio in lung macrophages could serve as an accurate diagnostic tool for COVID-19 progression. Indeed, reversing the macrophage MAFB/MAF expression ratio might impair the exacerbated inflammatory and profibrotic responses, and restore the defective IFN type I production, thus becoming a potential strategy to limit severity of COVID-19.This work was supported by grants from Consejo Superior de Investigaciónes Cientı́ficas (202020E228), Ministerio de Economıa y Competitividad (SAF2017-83785-R), and AYUDAS ́ FUNDACIÓ N BBVA A EQUIPOS DE INVESTIGACIÓ N CIENTIFICA SARS-CoV-2 y COVID-19 to MV and AC, Grant ́ 201619.31 from Fundación La Marató/TV3 to AC, and Red de Investigación en Enfermedades Reumáticas (RIER, RD16/0012/ 0007) from Instituto de Salud Carlos III and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Serotonin drives the acquisition of a profibrotic and anti-inflammatory gene profile through the 5-HT7RPKA signaling axis

15 p.-6 fig.-1 tab.Peripheral serotonin (5-hydroxytryptamine, 5-HT) regulates cell growth and differentiation in numerous cell types through engagement of seven types of cell surface receptors (HTR1–7). Deregulated 5-HT/ HTR levels contribute to pathology in chronic inflammatory diseases, with macrophages being relevant targets for the physio-pathological effects of 5-HT. In fact, 5-HT skews human macrophage polarization through engagement of 5-HT2BR and 5-HT7R receptors. We now report that 5-HT primes macrophages for reduced pro-inflammatory cytokine production and IFN type I-mediated signaling, and promotes an anti-inflammatory and pro-fibrotic gene signature in human macrophages. The acquisition of the 5-HT-dependent gene profile primarily depends on the 5-HT7R receptor and 5-HT7R-initiated PKAdependent signaling. In line with the transcriptional results, 5-HT upregulates TGFβ1 production by human macrophages in an HTR7- and PKA-dependent manner, whereas the absence of Htr7 in vivo results in diminished macrophage infiltration and collagen deposition in a mouse model of skin fibrosis.Our results indicate that the anti-inflammatory and pro-fibrotic activity of 5-HT is primarily mediated through the 5-HT7R-PKA axis, and that 5-HT7R contributes to pathology in fibrotic diseases.This work was supported by grants from Ministerio de Economía y Competitividad (SAF2011-23801 and SAF2014-52423-R to MAV and ALC, and PI I12/439 to JLP), “Programa de Actividades de I + D” from the Comunidad de Madrid/FEDER (RAPHYME S2010/BMD-2350 to JLP and ALC), and RIER (Red de Investigación en Inflamación y Enfermedades Reumáticas, RD12/09 to ALC and JLP) from the Instituto de Salud Carlos III,Ministerio de Economía y Competitividad, Spain (co-financed by FEDER, European Union). MCE was supported by an FPI predoctoral fellowship (BES-2009-021465) from Ministerio de Economía e Innovación.Peer reviewe

Intravenous immunoglobulins promote an expansion of monocytic myeloid-derived suppressor cells (MDSC) in CVID patients

13 p.-4 fig.Common variable immunodeficiency disorders (CVID), the most common primary immune deficiency, includes heterogeneous syndromes characterized by hypogammaglobulinemia and impaired antibody responses. CVID patients frequently suffer from recurrent infections and inflammatory conditions. Currently, immunoglobulin replacement therapy (IgRT) is the first-line treatment to prevent infections and aminorate immune alterations in CVID patients. Intravenous Immunoglobulin (IVIg), a preparation of highly purified poly-specific IgG, is used for treatment of immunodeficiencies as well as for autoimmune and inflammatory disorders, as IVIg exerts immunoregulatory and anti-inflammatory actions on innate and adaptive immune cells. To determine the mechanism of action of IVIg in CVID in vivo, we determined the effect of IVIg infusion on the transcriptome of peripheral blood mononuclear cells from CVID patients, and found that peripheral blood monocytes are primary targets of IVIg in vivo, and that IVIg triggers the acquisition of an anti-inflammatory gene profile in human monocytes. Moreover, IVIg altered the relative proportions of peripheral blood monocyte subsets and enhanced the proportion of CD14+ cells with a transcriptional, phenotypic, and functional profile that resembles that of monocytic myeloid-derived suppressor cells (MDSC). Therefore, our results indicate that CD14 + MDSC-like cells might contribute to the immunoregulatory effects of IVIg in CVID and other inflammatory disorders.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2017-83785-R)to MAV and ALC, Grant 201619.31 from Fundación La Marató/TV3 to ALC, and Red de Investigación en Enfermedades Reumáticas(RIER, RD16/0012/0007), and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF), to ALC, and grant 19/284-E from Instituto de Salud Carlos III (ISCIII) to SSR. MS-F was funded by a Formación de Personal Investigador predoctoral fellowship from MINECO (grant PRE2018-083396).Peer reviewe

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

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 inflammatory responses. We present evidences that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement re-programs MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role