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Ultraviolet (UV) irradiation has profound effects on the skin and the systemic immune system. Several effects of UV radiation on Dendritic cells (DCs) functions have been described. However, gene expression changes induced by UV radiation in DCs have not been addressed before. In this report, we irradiated human monocyte-derived DCs with solar-simulated UVA/UVB and analyzed regulated genes on human whole genome arrays. Results were validated by RT-PCR and further analyzed by Gene Set Enrichment Analysis (GSEA). Solar-simulated UV radiation up-regulated expression of genes involved in cellular stress and inflammation, and down-regulated genes involved in chemotaxis, vesicular transport and RNA processing. Twenty four genes were selected for comparison by RT-PCR with similarly treated human primary keratinocytes and human melanocytes. Several genes involved in the regulation of the immune response were differentially regulated in UVA/UVB irradiated human monocyte-derived DCs, such as protein tyrosine phosphatase, receptor type E (PTPRE), thrombospondin-1 (THBS1), inducible costimulator ligand (ICOSL), galectins, Src-like adapter protein (SLA), IL-10 and CCR7. These results indicate that UV-exposure triggers the regulation of a complex gene repertoire involved in human-DC–mediated immune responses., Ministerio de Educación yCiencia, Programa de Estimulo para laTransferencia de Resultados de la Investigacion(PETRI), I.F. Cantabria, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Funding Este trabajo fue apoyado por las subvenciones RD16/0011/0012 y PI18/0371 a IG-A, y PI12/01578 a SC del Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) y cofinanciado por el Fondo Europeo de Desarrollo Regional. (FEDER). Actualmente, AT-M cuenta con una beca de doctorado de la Comunidad de Madrid (PEJD-2019-PRE/BMD-16851). Actualmente el NM se encuentra otorgado mediante RD16/0011/0012. El trabajo de ER-V cuenta actualmente con una subvención Rio-Hortega CM19/00149 del Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) y cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER)., Galectin 1 (Gal1) is a lectin with a wide cellular expression that functions as a negative regulator of the immune system in several animal models of autoimmune diseases. Identification of patients with rheumatoid arthritis (RA) has improved during the last decade, although there is still a need for biomarkers allowing an early diagnosis. In this regard, it has been recently proposed that Gal1 serum levels are increased in patients with RA compared to the general population. However, this topic is controversial in the literature. In this work, we provide additional information about the potential usefulness of Gal1 serum levels as a biomarker for RA diagnosis. We studied Gal1 serum and synovial fluid levels and clinical parameters in samples from 62 patients with early arthritis belonging to the PEARL study. In addition, 24 healthy donors were studied. We found that both patients fulfilling RA criteria and patients with undifferentiated arthritis showed higher Gal1 levels than healthy donors. Similar findings were observed in synovial fluid, which showed even higher levels than serum. However, we did not find correlation between Gal1 levels and disease activity or disability. Therefore, our results suggest that Gal1 could be a diagnostic but not a severity biomarker., Ministerio de Economía, Industria y Competitividad (MINECO), Instituto de Salud Carlos III (ISCIII) / FEDER, Comunidad de Madrid, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgements Instituto de Salud Carlos III (AES 2017): PI17/01972 to E.D. Spanish Ministry of Economy and Competitiveness (MINECO): Plan Nacional de Salud SAF2017-82886-R, Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV). Fundación BBVA a equipos de Investigación Científica 2018 and from Caixa Banking Foundation under the project code HR17-00016 to F.S.M. This research has been co-financed by Fondo Europeo de Desarrollo Regional (FEDER)., The interplay between T cells, dendritic cells and keratinocytes is crucial for the development and maintenance of inflammation in psoriasis. GADD45 proteins mediate DNA repair in different cells including keratinocytes. In the immune system, GADD45a and GADD45b regulate the function and activation of both T lymphocytes and dendritic cells and GADD45a links DNA repair and epigenetic regulation through its demethylase activity. Here, we analyzed the expression of GADD45a and GADD45b in the skin, dendritic cells and circulating T cells in a cohort of psoriasis patients and their regulation by inflammatory signals. Thirty patients (17 male/13 female) with plaque psoriasis and 15 controls subjects (7 male/8 female), were enrolled. Psoriasis patients exhibited a lower expression of GADD45a at the epidermis but a higher expression in dermal infiltrating T cells in lesional skin. The expression of GADD45a and GADD45b was also higher in peripheral T cells from psoriasis patients, although no differences were observed in p38 activation. The expression and methylation state of the GADD45a target UCHL1 were evaluated, revealing a hypermethylation of its promoter in lesional skin compared to controls. Furthermore, reduced levels of GADD45a correlated with a lower expression UCHL1 in lesional skin. We propose that the demethylase function of GADD45a may account for its pleiotropic effects, and the complex and heterogeneous pattern of expression observed in psoriatic disease., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

The mechanisms that control the inflammatory–immune response play a key role in tissue remodelling in cardiovascular diseases. T cell activation receptor CD69 binds to oxidized low-density lipoprotein (oxLDL), inducing the expression of anti-inflammatory NR4A nuclear receptors and modulating inflammation in atherosclerosis. To understand the downstream T cell responses triggered by the CD69-oxLDL binding, we incubated CD69-expressing Jurkat T cells with oxLDL. RNA sequencing revealed a differential gene expression profile dependent on the presence of CD69 and the degree of LDL oxidation. CD69-oxLDL binding induced the expression of NR4A receptors (NR4A1 and NR4A3), but also of PD-1. These results were confirmed using oxLDL and a monoclonal antibody against CD69 in CD69-expressing Jurkat and primary CD4 + lymphocytes. CD69-mediated induction of PD-1 and NR4A3 was dependent on NFAT activation. Silencing NR4A3 slightly increased PD-1 levels, suggesting a potential regulation of PD-1 by this receptor. Moreover, expression of PD-1, CD69 and NR4A3 was increased in human arteries with chronic inflammation compared to healthy controls, with a strong correlation between PD-1 and CD69 mRNA expression (r = 0.655 P < 0.0001). Moreover, PD-1 was expressed in areas enriched in CD3 infiltrating T cells. Our results underscore a novel mechanism of PD-1 induction independent of TCR signalling that might contribute to the role of CD69 in the modulation of inflammation and vascular remodelling in cardiovascular diseases.

Objetivo Los mecanismos que controlan la repuesta inmune inflamatoria juegan un papel fundamental en la remodelación del tejido en las enfermedades cardiovasculares. El receptor de activación de células T CD69 une lipoproteínas de baja densidad oxidadas (oxLDL), induciendo la expresión de los receptores nucleares NR4A y modulando la inflamación en aterosclerosis. Sin embargo, los mecanismos subyacentes no se conocen. El objetivo principal de este trabajo es conocer las respuestas desencadenadas con la unión de las oxLDL a CD69 en los linfocitos T. Material y Métodos La línea celular Jurkat expresando CD69 de manera estable se incubó con oxLDL y se realizó un análisis de secuenciación masiva de ARN. Los resultados se comprobaron mediante PCR a tiempo real, citometría de flujo y empleando anticuerpos monoclonales anti-CD69. También se analizaron muestras humanas de arterias abdominales inflamadas mediante inmunohistoquímica, PCR y western-blot. Resultados El perfil de expresión génica mostró diferencias dependiendo de la presencia de CD69 y del grado de oxidación de las LDL. La unión oxLDL-CD69 indujo la expresión de NR4A1 y NR4A3 pero también de la molécula inmunorreguladora PD-1. La inducción de PD-1 y NR4A3 es dependiente del factor de transcripción NFAT. El silenciamiento de NR4A3 indujo un ligero aumento de PD-1, lo que sugiere una potencial regulación de PD-1 por NR4A3. Además, se identificó una elevada expresión de PD-1, CD69 y NR4A3 en muestras de arterias humanas con inflamación crónica en comparación con donantes sanos, encontrando una fuerte correlación entre la expresión de CD69 y PD-1 (r= 0.655 P<0.0001) Conclusiones Nuestros resultados evidencian un nuevo mecanismo de inducción de PD-1 independiente del TCR, señalización que podría contribuir al papel de CD69 en la modulación de la inflamación y el remodelado vascular en las enfermedades cardiovasculares.

[Background]: Spontaneous Coronary Artery Dissection (SCAD) is an important cause of acute coronary syndromes, particularly in young to middle-aged women. Differentiating acute SCAD from coronary atherothrombosis remains a major clinical challenge. [Methods]: A case-control study was used to explore the usefulness of circulating miRNAs to discriminate both clinical entities. The profile of miRNAs was evaluated using an unbiased human RT-PCR platform and confirmed using individual primers. miRNAs were evaluated in plasma samples from acute SCAD and atherothrombotic acute myocardial infarction (AT-AMI) from two independent cohorts; discovery cohort (SCAD n = 15, AT-AMI n = 15), and validation cohort (SCAD n = 11, AT-AMI n = 41) with 9 healthy control subjects. Plasma levels of IL-8, TGFB1, TGBR1, Endothelin-1 and MMP2 were analysed by ELISA assays. [Findings]: From 15 differentially expressed miRNAs detected in cohort 1, we confirmed in cohort 2 the differential expression of 4 miRNAs: miR-let-7f-5p, miR-146a-5p, miR-151a-3p and miR-223-5p, whose expression was higher in SCAD compared to AT-AMI. The combined expression of these 4 miRNAs showed the best predictive value to distinguish between both entities (AUC: 0.879, 95% CI 0.72–1.0) compared to individual miRNAs. Functional profiling of target genes identified an association with blood vessel biology, TGF-beta pathway and cytoskeletal traction force. ELISA assays showed high plasma levels of IL-8, TGFB1, TGFBR1, Endothelin-1 and MMP2 in SCAD patients compared to AT-AMI. [Interpretation]: We present a novel signature of plasma miRNAs in patients with SCAD. miR-let-7f-5p, miR-146a-5p, miR-151a-3p and miR-223-5p discriminate SCAD from AT-AMI patients and also shed light on the pathological mechanisms underlying this condition. [Funding]: Spanish Ministry of Economy and Competitiveness (MINECO): Plan Nacional de Salud SAF2017-82886-R, Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV). F

Galectin 1 (Gal1) is a lectin with a wide cellular expression that functions as a negative regulator of the immune system in several animal models of autoimmune diseases. Identifcation of patients with rheumatoid arthritis (RA) has improved during the last decade, although there is still a need for biomarkers allowing an early diagnosis. In this regard, it has been recently proposed that Gal1 serum levels are increased in patients with RA compared to the general population. However, this topic is controversial in the literature. In this work, we provide additional information about the potential usefulness of Gal1 serum levels as a biomarker for RA diagnosis. We studied Gal1 serum and synovial fuid levels and clinical parameters in samples from 62 patients with early arthritis belonging to the PEARL study. In addition, 24 healthy donors were studied. We found that both patients fulflling RA criteria and patients with undiferentiated arthritis showed higher Gal1 levels than healthy donors. Similar fndings were observed in synovial fuid, which showed even higher levels than serum. However, we did not fnd correlation between Gal1 levels and disease activity or disability. Therefore, our results suggest that Gal1 could be a diagnostic but not a severity biomarker., Ministerio de Economía y Competitividad (Instituto de Salud Carlos III)/Fondo Europeo de Desarrollo Regional(FEDER), Ministerio de Economía y Competitividad (Instituto de Salud Carlos III)/Fondo Europeo de Desarrollo Regional(FEDER), Comunidad de Madrid, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

SARS‐CoV‐2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID‐19. We investigated whether the specific immune responses in the peripheral blood of 276 patients associated to severity and progression of COVID‐19. At admission, dramatic lymphopenia of T, B and NK cells associated to severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56‐CD16+ NK‐cells increased. Regarding humoral immunity, levels of IgM, IgA and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID‐19 patients, associating severity with an imbalanced humoral response and identifying new targets for therapeutic intervention.

BACKGROUND: Although the role of Th17 and regulatory T cells in the progression of atherosclerosis has been highlighted in recent years, their molecular mediators remain elusive. We aimed to evaluate the association between the CD69 receptor, a regulator of Th17/regulatory T cell immunity, and atherosclerosis development in animal models and in patients with subclinical disease. METHODS: Low-density lipoprotein receptor-deficient chimeric mice expressing or not expressing CD69 on either myeloid or lymphoid cells were subjected to a high fat diet. In vitro functional assays with human T cells were performed to decipher the mechanism of the observed phenotypes. Expression of CD69 and NR4A nuclear receptors was evaluated by reverse transcription-polymerase chain reaction in 305 male participants of the PESA study (Progression of Early Subclinical Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical atherosclerosis and without disease (n=122). RESULTS: After a high fat diet, mice lacking CD69 on lymphoid cells developed large atheroma plaque along with an increased Th17/regulatory T cell ratio in blood. Oxidized low-density lipoprotein was shown to bind specifically and functionally to CD69 on human T lymphocytes, inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA downregulation in peripheral blood leukocytes compared with participants without disease. The expression of CD69 remained associated with the risk of subclinical atherosclerosis in an adjusted multivariable logistic regression model (odds ratio, 0.62; 95% CI, 0.40-0.94; P=0.006) after adjustment for traditional risk factors, the expression of NR4A1, the level of oxidized low-density lipoprotein, and the counts of different leucocyte subsets. CONCLUSIONS: CD69 depletion from the lymphoid compartment promotes a Th17/regulatory T cell imbala

Funding This work is supported by grants SAF2011-25834, ERC-2011-ADG_20110310, Cardiovascular Network RD12-0042-0056 (Instituto de Salud Carlos III), PIE-13-00041 and INDISNET S2011-BMD-2332. L.F.M. is supported by the Juan de la Cierva grant (Spanish Ministry of Economy and Competitivity) and E.R.G. by the Spanish Ministry of Health., The immune system is composed of different cell types localised throughout the organism to sense and respond to pathological situations while maintaining homeostasis under physiological conditions. Intercellular communication between immune cells is essential to coordinate an effective immune response and involves both cell contact dependent and independent processes that ensure the transfer of information between bystander and distant cells. There is a rapidly growing body of evidence on the pivotal role of extracellular vesicles (EVs) in cell communication and these structures are emerging as important mediators for immune modulation upon delivery of their molecular cargo. In the last decade, EVs have been shown to be efficient carriers of genetic information, including microRNAs (miRNAs), that can be transferred between cells and regulate gene expression and function on the recipient cell. Here, we review the current knowledge of intercellular functional transfer of EV‐delivered miRNAs and their putative role in immune regulation., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and function. © 2014, American Society for Microbiology.

This work was supported by grants MEICA from Genoma España Foundation, SAF 2008-02635 from Ministerio de Ciencia e Innovación, INSINET from Comunidad Autónoma de Madrid, FONCICYT-95395, ALA/127249, and RECAVA RD06/0014-0030 from Instituto de Salud Carlos III to FSM and SAF2008-02719 from Ministerio de Ciencia e Innovación to PM. GMH is supported by Instituto de Salud Carlos III. CNIC is supported by the Spanish Ministry of Health and Consumer Affairs and the Pro-CNIC Foundation. We thank Simon Bartlett for editing the manuscript., In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69–/– DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69–/– DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P1) by CD69–/– DCs, whereas S1P3 expression levels were similar in wild-type and CD69–/– DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P1 function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69–/– DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69–/– DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P1) by CD69–/– DCs, whereas S1P3 expression levels were similar in wild-type and CD69–/– DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P1 function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery

Background: Allergic diseases have a major health care impact in industrialized countries. The development of these diseases is influenced by exposure to allergen and to immunological and genetic factors. However, the molecular mechanisms underlying the inflammatory response that triggers allergy are not well defined. Objective: We have investigated the role of the leukocyte activation antigen CD69 in the regulation of two allergic diseases, asthma and contact dermatitis. Methods: Analysis of two models of allergic diseases in CD69 knockout and wild-type mice: ovalbumin-induced allergic airway inflammation (BALB/c genetic background) and contact hypersensitivity to oxazolone (C57BL/6J genetic background). Results: CD69 deficiency dramatically enhanced the inflammatory response in the ovalbumin-induced asthma model of antigen-induced airway allergy. CD69 knockout mice showed exacerbated pulmonary eosinophil recruitment, high vascular cell adhesion molecule 1 expression levels in lung vasculature, and enhanced TH2 and TH17 cytokines in the bronchoalveolar space and lung tissue. In the hapten-induced cutaneous contact hypersensitivity model, both CD69 deficiency and treatment with anti-CD69 mAb increased inflammation. Treatment with contact allergens induced enhanced TH1 and TH17 responses in CD69 deficient mice, and neutralizing anti–IL-17 antibodies reduced skin inflammation. In both experimental systems, adoptive transfer of lymph node cells from CD69 knockout mice increased the inflammatory response in recipient mice., Ministerio de Ciencia e Innovación (MCIN), Comunidad Autónoma de Madrid, InstitutodeSaludCarlos III/Ministerio de Ciencia e Innovación, Programa Ramón y Cajal, Fondo para la Investigación Científica y Tecnológica (FONCYT), Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Ultraviolet (UV) irradiation has profound effects on the skin and the systemic immune system. Several effects of UV radiation on Dendritic cells (DCs) functions have been described. However, gene expression changes induced by UV radiation in DCs have not been addressed before. In this report, we irradiated human monocyte-derived DCs with solar-simulated UVA/UVB and analyzed regulated genes on human whole genome arrays. Results were validated by RT-PCR and further analyzed by Gene Set Enrichment Analysis (GSEA). Solar-simulated UV radiation up-regulated expression of genes involved in cellular stress and inflammation, and down-regulated genes involved in chemotaxis, vesicular transport and RNA processing. Twenty four genes were selected for comparison by RT-PCR with similarly treated human primary keratinocytes and human melanocytes. Several genes involved in the regulation of the immune response were differentially regulated in UVA/UVB irradiated human monocyte-derived DCs, such as protein tyrosine phosphatase, receptor type E (PTPRE), thrombospondin-1 (THBS1), inducible costimulator ligand (ICOSL), galectins, Src-like adapter protein (SLA), IL-10 and CCR7. These results indicate that UV-exposure triggers the regulation of a complex gene repertoire involved in human-DC–mediated immune responses

Initial adhesive contacts betweenT lymphocytes anddendritic cells (DCs) facilitate recognitionof peptide-MHC complexesbytheTCR. Inthisreport,westudiedthedynamicbehaviorofadhesionandAgreceptorsonDCsduring initialcontactswithT-cells.AdhesionmoleculesLFA-1-andICAM-1,3-GFPaswellasMHCclassII-GFPmoleculeswere veryrapidlyconcentratedattheDCcontactarea.BindingofICAM-3,andICAM-1toalesserextent, toLFA-1expressed bymaturebutnot immatureDC, inducedMHC-IIclusteringintotheimmunesynapse.Also, ICAM-3bindingtoDC inducedtheactivationoftheVav1-Rac1axis,aregulatorypathwayinvolvedinactincytoskeletonreorganization,which wasessentialforMHC-IIclusteringonDCs.OurresultssupportamodelinwhichICAM-mediatedMHC-IIclustering onDCconstitutesaprimingmechanismtoenhanceantigenpresentationtoT-cells., Ministerio de Educación, Cultura y Deporte, Fundación Juan March. Ayuda a la Investigación 2002, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub