Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.This work was supported by the grants from the Spanish Ministry of Science and Technology (MICINN; BFU2011-29450 to E.V.) and Ministry of Economy and Competitiveness (MINECO; SAF2014-56716-REDT and BFU2014-59585-R to E.V., SAF2011-25834 to F.S.M., SAF2014-58895-JIN to A.C.A and BFU2014-54181-P to J.L.C.), the Madrid regional government (INDISNET-S2011/BMD-2332 to F.S.M.) and the European Research Council (ERC-2011-AdG 294340-GENTRIS to F.S.M.). We are grateful to the “Centro de Transfusión” of the “Comunidad Autónoma de Madrid” for providing the Buffy Coats

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

INTRODUCTION The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. METHODS We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. RESULTS Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. CONCLUSION We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.This work was supported by grants from Ministerio de Ciencia e Innovación (MCIN) from Spain [SAF2016-78711R and PID202-11794 to EM-N and FJC]; Comunidad de Madrid [B2017/BMD-3727 to EMN and FJC]; Comunidad de Madrid (REACT-UE, ANTICIPA-CM Ref. PR38/21-24) to E.M-N and HORIZON-HLTH-2022-STAYHLTH-02 under agreement No 101095679 to FJC the European Union’s Horizon 2020 research and innovation program [ERC-2016- Consolidator Grant 725091 to DS]; MCIN/AEI/10.13039/ 501100011033 [PID2019-108157RB to DS]; la Caixa Foundation (ID 100010434) [LCF/BQ/PR20/11770008 to SW]; Instituto de Salud Carlos III (ISCIII) [PI18/00348 to VE]; ISCIII [PI21/01641 to RT-R]; Spanish National Research and Development Plan, ISCIII and FEDER [PI17/02303 and PI20/01837 to SR-P]; Proyecto Desarrollo Tecnológico [DTS19/00111 to SR-P], AEI/MICIU EXPLORA Project [BIO2017-91272-EXP to SR-P]; Programa Estratégico Instituto de Biologıa y Gene ́ ́ tica Molecular (IBGM), Junta de Castilla y León (CCVC8485) [PID2019-104218RB-I00 to DB]; NIH [DK088199 to RB] and Universidad Complutense de Madrid (UCM 920631) [CT42/ 18-CT43/18 and EB15/21 to BM-A].S

Linfocitos transinfectados para terapia anti-tumoral

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof.[ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de Madrid, Fundación para la Investigación Biomédica del Hospital de la PrincesaE Solicitud de patente europe

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cientìficas (CSIC)

Linfocitos transinfectados para terapia anti-tumoral

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof.[ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.Consejo Superior de Investigaciones Científicas (España), Universidad Autónoma de Madrid, Fundación para la Investigación Biomédica del Hospital de la PrincesaA1 Solicitud de patente con informe sobre el estado de la técnic

Linfocitos transinfectados para terapia anti-tumoral

[EN] The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device that comprises same for this purpose. Furthermore, it also refers to the transinfection method thereof.[ES] La presente invención se refiere a linfocitos, preferiblemente que han sido transinfectados desde células dendríticas con una bacteria, preferiblemente linfocitos T CD4+, preferiblemente Listeria monocytogenes, donde dicha bacteria comprende un péptido tumoral. También se refiere al uso de los linfocitos para terapia y/o tratamiento de tumores sólidos, preferiblemente melanoma, al kit o dispositivo que los comprende para dicho fin. Además, también se refiereal método de transinfección de los mismos.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de Madrid, Fundación para la Investigación Biomédica del Hospital de la PrincesaA1 Solicitud de patente con informe sobre el estado de la técnic

Optimized Protocol for Characterization of Mouse Gut Innate Lymphoid Cells

© 2020 Valle-Noguera, Gómez-Sánchez, Girard-Madoux and Cruz-Adalia.Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.The present research was supported by grant Nº RTI2018-093647-B-I00 to AC-A from Ministerio de Ciencia, Innovación e Universidades (MCIU), Agenda Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional (FEDER). AV-N is a recipient of an FPI fellowship (PRE2019-090341) from the Spanish Ministry of Science, Innovation, and Universities

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses

Bacterial phagocytosis and antigen cross-presentation to activate CD8 T cells are principal functions of professional antigen presenting cells. However, conventional CD4 T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 T cells, which proliferate and become cytotoxic in response. CD4 T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 T cells with low PD-1 expression. Moreover, transphagocytic CD4 T cells induce protective anti-tumour immune responses by priming CD8 T cells, highlighting the potential of CD4 T cells as a tool for cancer immunotherapy.Spanish Ministries of Science and Technology (MICINN; BFU2011-29450 to E.V.) and of Economy and Competitiveness (MINECO; SAF2014-56716-REDT and BFU2014-59585-R to E.V., SAF2014-55579-R to F.S.M., SAF2013-47975-R to B.A., SAF2014-58895-JIN to A.C.-A.), the ISCIII (PI14/00526; CP11/00145; CPII16/00022 to J.M.G.-G.), the Fundación Ramón Areces (to J.M.G.-G.), the Madrid regional government (INDISNET-S2011/BMD-2332 to F.S.M.) and the European Research Council (ERC-2011-AdG 294340-GENTRIS to F.S.M.; ERC 2013-AdG 334763 NOVARIPP to B.A.)Peer Reviewe

Biocompatible Probes Based on Rare-Earth Doped Strontium Aluminates with Long-Lasting Phosphorescent Properties for In Vitro Optical IMAGING

(This article belongs to the Special Issue Metals and Metal Oxides for Biomedical Applications: Current Challenges and Opportunities)In recent decades, the demand for biomedical imaging tools has grown very rapidly as a key feature for biomedical research and diagnostic applications. Particularly, fluorescence imaging has gained increased attention as a non-invasive, inexpensive technique that allows real-time imaging. However, tissue auto-fluorescence under external illumination, together with a weak tissue penetration of low wavelength excitation light, largely restricts the application of the technique. Accordingly, new types of fluorescent labels are currently being investigated and, in this search, phosphorescent nanoparticles promise great potential, as they combine the interesting size-dependent properties of nanoscale materials with a long-lasting phosphorescence-type emission that allows optical imaging well after excitation (so avoiding autofluorescence). In this work, core-shell structures consisting of SrAlO:Eu,Dy luminescent cores encapsulated within a biocompatible silica shell were prepared, showing a green persistent phosphorescence with an afterglow time of more than 1000 s. A high-energy ball milling procedure was used to reduce the size of the starting phosphors to a size suitable for cellular uptake, while the silica coating was produced by a reverse micelle methodology that eventually allows the excitation and emission light to pass efficiently through the shell. Confocal fluorescence microscopy using HeLa cancer cells confirmed the potential of the all-ceramic composites produced as feasible labels for in vitro optical imaging.This work was supported by the Agencia Estatal de Investigación through PID2019-104118RB-C21 project and the Ministerio de Economía, Industria y Competitividad, Agencia Estatal de Investigación, through MAT2016-80182-R project. Dr T. Jardiel acknowledges the European Science Foundation (ESF) and the Ramon y Cajal Program of MICINN for financial support. D.G. Calatayud also acknowledges the Fundación General CSIC (ComFuturo Program) for financial support. Dr. A Cruz-Adalia wishes to thank the Confocal Microscopy Core Facility of the Centro Nacional de Biotecnología (CNB) for its technical support and acknowledges the Spanish Ministry of Science, Innovation and Universities (MICINN), Agenda Estatal de Investigación, and Fondo Europeo de Desarrollo Regional (FEDER) for the Ramon y Cajal Program and financial support (RTI2018-093647-B-I00).Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23063410/s

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells

We have investigated the expression and role of galectin-1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT-PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin-1, -3 and -9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin-1 and galectin-9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non-lesional as well as lesional skin samples from psoriasis patients had low levels of galectin-1 at the mRNA and protein levels, in parallel with low levels of IL-10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin-1 expression showed that this protein was down-regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c + DCs from psoriasis patients. Expression of galectin-1 correlated with IL-17 and IL-10 expression and with the psoriasis area and index activity. Addition of galectin-1 to co-cultures of human monocyte-derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL-10 secretion in co-cultures of monocyte-derived dendritic cells with CD4 + T cells. Our results suggest a model in which galectin-1 down-regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients.Spanish Ministry of Science and Innovation (PI080946,SAF-2008-02635,SAF-2011-25834); EU–Mexico FONCICYT (Grant No. C002-2009-1 ALA/127249); INSINET (Grant No. 01592006); MEICA (Genoma Espana)Peer Reviewe