Publications by authors named "Santiago F Gonzalez"

35 Publications

Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection.

NPJ Vaccines 2021 Apr 12;6(1):52. Epub 2021 Apr 12.

Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland.

Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.
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http://dx.doi.org/10.1038/s41541-021-00314-7DOI Listing
April 2021

Carboxymethyl-β-glucan/chitosan nanoparticles: new thermostable and efficient carriers for antigen delivery.

Drug Deliv Transl Res 2021 Apr 1. Epub 2021 Apr 1.

Center for Research in Molecular Medicine & Chronic Diseases (CIMUS), School of Pharmacy, Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, 15706, Spain.

In the last few decades, nanotechnology has emerged as an important tool aimed at enhancing the immune response against modern antigens. Nanocarriers designed specifically for this purpose have been shown to provide protection, stability, and controlled release properties to proteins, peptides, and polynucleotide-based antigens. Polysaccharides are particularly interesting biomaterials for the construction of these nanocarriers given their wide distribution among pathogens, which facilitates their recognition by antigen-presenting cells (APCs). In this work, we focused on an immunostimulant beta-glucan derivative, carboxymethyl-β-glucan, to prepare a novel nanocarrier in combination with chitosan. The resulting carboxymethyl-β-glucan/chitosan nanoparticles exhibited adequate physicochemical properties and an important protein association efficiency, with ovalbumin as a model compound. Moreover, thermostability was achieved through the optimization of a lyophilized form of the antigen-loaded nanoparticles, which remained stable for up to 1 month at 40 ºC. Biodistribution studies in mice showed an efficient drainage of the nanoparticles to the nearest lymph node following subcutaneous injection, and a significant co-localization with dendritic cells. Additionally, subcutaneous immunization of mice with a single dose of the ovalbumin-loaded nanoparticles led to induced T cell proliferation and antibody responses, comparable to those achieved with alum-adsorbed ovalbumin. These results illustrate the potential of these novel nanocarriers in vaccination.
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http://dx.doi.org/10.1007/s13346-021-00968-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015750PMC
April 2021

Targeting a scavenger receptor on tumor-associated macrophages activates tumor cell killing by natural killer cells.

Proc Natl Acad Sci U S A 2020 12 23;117(50):32005-32016. Epub 2020 Nov 23.

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17165 Stockholm, Sweden;

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.
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http://dx.doi.org/10.1073/pnas.2015343117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750482PMC
December 2020

Characterization of the Dynamic Behavior of Neutrophils Following Influenza Vaccination.

Front Immunol 2019 20;10:2621. Epub 2019 Nov 20.

Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.

Neutrophils are amongst the first cells to respond to inflammation and infection. Although they play a key role in limiting the dissemination of pathogens, the study of their dynamic behavior in immune organs remains elusive. In this work, we characterized the dynamic behavior of neutrophils in the mouse popliteal lymph node (PLN) after influenza vaccination with UV-inactivated virus. To achieve this, we used an image-based systems biology approach to detect the motility patterns of neutrophils and to associate them to distinct actions. We described a prominent and rapid recruitment of neutrophils to the PLN following vaccination, which was dependent on the secretion of the chemokine CXCL1 and the alarmin molecule IL-1α. In addition, we observed that the initial recruitment occurred mainly via high endothelial venules located in the paracortical and interfollicular regions of the PLN. The analysis of the spatial-temporal patterns of neutrophil migration demonstrated that, in the initial stage, the majority of neutrophils displayed a patrolling behavior, followed by the formation of swarms in the subcapsular sinus of the PLN, which were associated with macrophages in this compartment. Finally, we observed using multiple imaging techniques, that neutrophils phagocytize and transport influenza virus particles. These processes might have important implications in the capacity of these cells to present viral antigens.
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http://dx.doi.org/10.3389/fimmu.2019.02621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881817PMC
November 2020

Early production of IL-17A by γδ T cells in the trachea promotes viral clearance during influenza infection in mice.

Eur J Immunol 2020 01 4;50(1):97-109. Epub 2019 Dec 4.

Institute for Research in Biomedicine, Università della Svizzera italiana, via Vincenzo Vela 6, Bellinzona, Switzerland.

The innate immune response generated against influenza infection is critical for the inhibition of viral dissemination. The trachea contains different types of innate immune cells that protect the respiratory tract from pathogen invasion. Among them, γδ T cells have the ability to rapidly generate large amounts of pro-inflammatory cytokines to preserve mucosal barrier homeostasis during infection. However, little is known about their role during the early phase of influenza infection in the airways. In this study, we found that, early after infection, γδ T cells are recruited and activated in the trachea and outnumber αβ T cells during the course of the influenza infection that follows. We also showed that the majority of the recruited γδ T cells express the Vγ4 TCR chain and infiltrate in a process that involves the chemokine receptor CXCR3. In addition, we demonstrated that γδ T cells promote the recruitment of protective neutrophils and NK cells to the tracheal mucosa. Altogether, our results highlight the importance of the immune responses mediated by γδ T cells.
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http://dx.doi.org/10.1002/eji.201948157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003741PMC
January 2020

Design of polymeric nanocapsules to improve their lympho-targeting capacity.

Nanomedicine (Lond) 2019 12 7;14(23):3013-3033. Epub 2019 Nov 7.

Center for Research in Molecular Medicine & Chronic Diseases (CIMUS), Health Research Institute of Santiago de Compostela (IDIS), School of Pharmacy, Universidade de Santiago de Compostela, Campus Vida, 15706 Santiago de Compostela, Spain.

To design lympho-targeted nanocarriers with the capacity to enhance the activity of associated drugs/antigens whose target is within the lymphatic system. : Inulin (INU)-based nanocapsules (NCs), negatively charged and positively charged chitosan NCs were prepared by the solvent displacement techniques. The NCs were produced in two sizes: small (70 nm) and medium (170-250 nm). results indicated that small NCs interacted more efficiently with dendritic cells than the larger ones. The study of the NCs biodistribution in mice, using 3D reconstruction of the popliteal lymph node, showed that small INU NCs have the greatest access and uniform accumulation in different subsets of resident immune cells. Small and negatively charged INU NCs have a potential as lympho-targeted antigen/drug nanocarriers.
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http://dx.doi.org/10.2217/nnm-2019-0206DOI Listing
December 2019

Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1.

Nat Microbiol 2019 11 29;4(11):1930-1940. Epub 2019 Jul 29.

Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.

The early phase of influenza infection occurs in the upper respiratory tract and the trachea, but little is known about the initial events of virus recognition and control of viral dissemination by the immune system. Here, we report that inflammatory dendritic cells (IDCs) are recruited to the trachea shortly after influenza infection through type I interferon-mediated production of the chemokine CCL2. We further show that recruited IDCs express the C-type lectin receptor SIGN-R1, which mediates direct recognition of the virus by interacting with N-linked glycans present in glycoproteins of the virion envelope. Activation of IDCs via SIGN-R1 triggers the production of the chemokines CCL5, CXCL9 and CXCL10, which initiate the recruitment of protective natural killer (NK) cells in the infected trachea. In the absence of SIGN-R1, the recruitment and activation of NK cells is impaired, leading to uncontrolled viral proliferation. In sum, our results provide insight into the orchestration of the early cellular and molecular events involved in immune protection against influenza.
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http://dx.doi.org/10.1038/s41564-019-0506-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817362PMC
November 2019

Two-Photon Intravital Imaging of Leukocytes in the Trachea During Pneumococcal Infection.

Methods Mol Biol 2019 ;1968:183-194

Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.

Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluorescent protein (LysM-GFP) mouse, in which neutrophils express GFP. Additionally, we used a custom-designed platform, which allowed the intubation and fixation of the trachea after surgical exposition, and we injected intravenously a fluorescently labeled dextran solution to visualize the blood vessels.
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http://dx.doi.org/10.1007/978-1-4939-9199-0_15DOI Listing
August 2019

Influenza Vaccination Induces NK-Cell-Mediated Type-II IFN Response that Regulates Humoral Immunity in an IL-6-Dependent Manner.

Cell Rep 2019 02;26(9):2307-2315.e5

Institute for Research in Biomedicine (IRB), Università della Svizzera italiana, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. Electronic address:

The role of natural killer (NK) cells in the immune response against vaccines is not fully understood. Here, we examine the function of infiltrated NK cells in the initiation of the inflammatory response triggered by inactivated influenza virus vaccine in the draining lymph node (LN). We observed that, following vaccination, NK cells are recruited to the interfollicular and medullary areas of the LN and become activated by type I interferons (IFNs) produced by LN macrophages. The activation of NK cells leads to their early production of IFNγ, which in turn regulates the recruitment of IL-6+ CD11b+ dendritic cells. Finally, we demonstrate that the interleukin-6 (IL-6)-mediated inflammation is important for the development of an effective humoral response against influenza virus in the draining LN.
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http://dx.doi.org/10.1016/j.celrep.2019.01.104DOI Listing
February 2019

ATP released by intestinal bacteria limits the generation of protective IgA against enteropathogens.

Nat Commun 2019 01 16;10(1):250. Epub 2019 Jan 16.

Institute for Research in Biomedicine, Università della Svizzera Italiana, Via Vincenzo Vela 6, 6500, Bellinzona, Switzerland.

T cell dependent secretory IgA (SIgA) generated in the Peyer's patches (PPs) of the small intestine shapes a broadly diverse microbiota that is crucial for host physiology. The mutualistic co-evolution of host and microbes led to the relative tolerance of host's immune system towards commensal microorganisms. The ATP-gated ionotropic P2X7 receptor limits T follicular helper (Tfh) cells expansion and germinal center (GC) reaction in the PPs. Here we show that transient depletion of intestinal ATP can dramatically improve high-affinity IgA response against both live and inactivated oral vaccines. Ectopic expression of Shigella flexneri periplasmic ATP-diphosphohydrolase (apyrase) abolishes ATP release by bacteria and improves the specific IgA response against live oral vaccines. Antibody responses primed in the absence of intestinal extracellular ATP (eATP) also provide superior protection from enteropathogenic infection. Thus, modulation of eATP in the small intestine can affect high-affinity IgA response against gut colonizing bacteria.
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http://dx.doi.org/10.1038/s41467-018-08156-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335424PMC
January 2019

Engineering polymeric nanocapsules for an efficient drainage and biodistribution in the lymphatic system.

J Drug Target 2019 Jun - Jul;27(5-6):646-658. Epub 2019 Jan 9.

a Center for Research in Molecular Medicine & Chronic Diseases (CIMUS) Health Research Institute of Santiago de Compostela (IDIS), School of Pharmacy , Universidade de Santiago de Compostela , Campus Vida, Santiago de Compostela , Spain.

Polymer-based nanocarriers have shown potential for enhancing the immunological response of antigens. However, the key drivers for this response have not been fully elucidated. The objective of this work was to evaluate the influence of particle size (≈100 versus 200 nm) and surface composition of polymeric nanocapsules (chitosan, polyarginine and carboxymethyl-β-glucan) on their ability to target specific immune cells in the lymphatics. For this purpose, we used a powerful imaging technique, two-photon intravital microscopy, which minimises tissue damage in the visualisation of biological processes at cellular/subcellular levels. As expected, particle size was critical in the distribution and lymph node accumulation of all nanocapsules. Chitosan particles with a mean size below 100 nm accumulated significantly more in the popliteal lymph node than those with a larger size. Additionally, a comparative analysis of 100 nm nanocapsules with different polymeric shells indicated that cationic nanocapsules (chitosan and polyarginine) show higher accumulation in the popliteal lymph node than the anionic ones (carboxymethyl-β-glucan). In contrast, these anionic nanocapsules showed significant accumulation in the lumbar lymph node. In conclusion, tuning the physicochemical properties and composition of the nanocapsules allows the modulation of their lymphatic uptake and biodistribution, which may have important implications in the immune response.
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http://dx.doi.org/10.1080/1061186X.2018.1561886DOI Listing
July 2020

A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity.

PLoS Pathog 2018 10 1;14(10):e1007335. Epub 2018 Oct 1.

Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.

Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.
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http://dx.doi.org/10.1371/journal.ppat.1007335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181439PMC
October 2018

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy.

J Vis Exp 2018 08 17(138). Epub 2018 Aug 17.

Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI);

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal. Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.
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http://dx.doi.org/10.3791/58355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128112PMC
August 2018

Epithelial-mesenchymal transition in cancer metastasis through the lymphatic system.

Mol Oncol 2017 07 26;11(7):781-791. Epub 2017 Jun 26.

Department of Microbiology, Tumor Biology and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden.

It was already in the 18th century when the French surgeon LeDran first noted that breast cancer patients with spread of tumor cells to their axillary lymph nodes had a drastically worse prognosis than patients without spread (LeDran et al., ). Since then, metastatic spread of cancer cells to regional lymph nodes has been established as the most important prognostic factor in many types of cancer (Carter et al., ; Elston and Ellis, ). However, despite its clinical importance, lymph metastasis remains an underexplored area of tumor biology. Fundamental questions, such as when, how, and perhaps most importantly, why tumor cells disseminate through the lymphatic system, remain largely unanswered. Accordingly, no treatment strategies exist that specifically target lymph metastasis. The identification of epithelial-mesenchymal transition (EMT) as a mechanism, which allows cancer cells to dedifferentiate and acquire enhanced migratory and invasive properties, has been a game changer in cancer research. Conceptually, EMT provides an explanation for why epithelial cancers with poor differentiation status are generally more aggressive and prone to metastasize than more differentiated cancers. Inflammatory cytokines, such as TGF-β, which are produced and secreted by tumor-infiltrating immune cells, are potent inducers of EMT. Thus, reactivation of EMT also links cancer-related inflammation to invasive and metastatic disease. Recently, we found that breast cancer cells undergoing TGF-β-induced EMT acquire properties of immune cells allowing them to disseminate in a targeted fashion through the lymphatic system similar to activated dendritic cells during inflammation. Here, we review our current understanding of the mechanisms by which cancer cells spread through the lymphatic system and the links to inflammation and the immune system. We also emphasize how imaging techniques have the potential to further expand our knowledge of the mechanisms of lymph metastasis, and how lymph nodes serve as an interface between cancer and the immune system.
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http://dx.doi.org/10.1002/1878-0261.12092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496496PMC
July 2017

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node.

Cell Rep 2017 03;18(10):2427-2440

Institute for Research in Biomedicine, Università della Svizzera Italiana, via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. Electronic address:

The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-β (IFN-β) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.
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http://dx.doi.org/10.1016/j.celrep.2017.02.026DOI Listing
March 2017

Dynamic intravital imaging of cell-cell interactions in the lymph node.

J Allergy Clin Immunol 2017 01;139(1):12-20

Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland. Electronic address:

In the last decade, the application of 2-photon intravital microscopy as a tool to study cell interactions in different areas of the immune system has offered an unprecedented opportunity to understand the complexity of cell behavior in relation to immune functions. In this review we describe the latest advances in the field of live imaging in the lymph nodes, grouping the different cell populations in 2 compartments according to their motility: the sessile compartment, which is formed by resident cells of stromal origin, macrophages, and resident dendritic cells, and the motile compartment, which is mainly formed by T and B lymphocytes. Here we review how the use of in vivo imaging has contributed to our understanding of the role of these cells in the initiation of the immune response in the draining lymph nodes.
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http://dx.doi.org/10.1016/j.jaci.2016.11.008DOI Listing
January 2017

Development of a SYBR green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida.

Appl Microbiol Biotechnol 2016 Dec 12;100(24):10585-10595. Epub 2016 Nov 12.

Departamento de Microbiología y Parasitología, Edificio CIBUS Facultad de Biología and Instituto de Investigación y Análisis Alimentario, Universidad de Santiago de Compostela, 15782, Santiago de Compostela, Spain.

A SYBR Green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 ± 0.35 °C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The SYBR Green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.
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http://dx.doi.org/10.1007/s00253-016-7929-2DOI Listing
December 2016

Endocytosis and recycling of immune complexes by follicular dendritic cells enhances B cell antigen binding and activation.

Immunity 2013 Jun 13;38(6):1164-75. Epub 2013 Jun 13.

The Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.

Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.
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http://dx.doi.org/10.1016/j.immuni.2013.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773956PMC
June 2013

Cochlin produced by follicular dendritic cells promotes antibacterial innate immunity.

Immunity 2013 May 16;38(5):1063-72. Epub 2013 May 16.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Cochlin, an extracellular matrix protein, shares homologies with the Factor C, a serine protease found in horseshoe crabs, which is critical for antibacterial responses. Mutations in the COCH gene are responsible for human DFNA9 syndrome, a disorder characterized by neurodegeneration of the inner ear that leads to hearing loss and vestibular impairments. The physiological function of cochlin, however, is unknown. Here, we report that cochlin is specifically expressed by follicular dendritic cells and selectively localized in the fine extracellular network of conduits in the spleen and lymph nodes. During inflammation, cochlin was cleaved by aggrecanases and secreted into blood circulation. In models of lung infection with Pseudomonas aeruginosa and Staphylococcus aureus, Coch(-/-) mice show reduced survival linked to defects in local cytokine production, recruitment of immune effector cells, and bacterial clearance. By producing cochlin, FDCs thus contribute to the innate immune response in defense against bacteria.
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http://dx.doi.org/10.1016/j.immuni.2013.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758559PMC
May 2013

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

Immunity 2012 Aug 9;37(2):276-89. Epub 2012 Aug 9.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.
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http://dx.doi.org/10.1016/j.immuni.2012.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556784PMC
August 2012

Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Nat Immunol 2012 Apr 1;13(5):499-510. Epub 2012 Apr 1.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31(-) LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α(7). Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.
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http://dx.doi.org/10.1038/ni.2262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366863PMC
April 2012

Molecular cloning and expression of two β-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after β-glucan feeding.

Fish Shellfish Immunol 2012 Mar 2;32(3):494-501. Epub 2012 Jan 2.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.

In this study, we described the partial structure, mRNA tissue distribution and regulation of two carp mucin and two β-defensin genes. This is the first description of these genes in fish. The genes might provide relevant tools to monitor feed-related improvements of fish health under aquaculture conditions. Carp mucin 2 and mucin 5B genes show a high similarity to their mammalian and avian counterparts. The carp β-defensin 1 and β-defensin 2 genes cluster together well with their piscine family members. The influence of a β-glucan immunomodulant on the expression of these genes in mucosal tissues could be confirmed for the first time. Muc5B expression was significantly increased in the skin. For Muc2 no significant up- or down-regulation could be observed. Significantly higher expression levels of β-defensin 2 in gills and both β-defensin genes in skin were found. Thus, the mucosal system can be influenced by the addition of β-glucans to the food.
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http://dx.doi.org/10.1016/j.fsi.2011.12.008DOI Listing
March 2012

Trafficking of B cell antigen in lymph nodes.

Annu Rev Immunol 2011 ;29:215-33

The Immune Disease Institute and Program in Molecular and Cellular Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.
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http://dx.doi.org/10.1146/annurev-immunol-031210-101255DOI Listing
June 2011

The role of innate immunity in B cell acquisition of antigen within LNs.

Adv Immunol 2010 ;106:1-19

Departments of Pediatrics and Pathology, Children's Hospital, The Immune Disease Institute, Program in Cellular and Molecular Medicine, and Harvard Medical School, Boston, Massachusetts, USA.

Over the past decade, it has become apparent that B cells acquire antigens primarily from membrane surfaces and that uptake is an active process involving a synapse between the B cell receptor, coreceptor, and the antigen surface. However, understanding how antigens are delivered to follicular dendritic cells (FDC), which are the primary depot for B cell antigen within the lymph node follicles, is only recently beginning to be dissected. The application of fluorescent-based imaging techniques such as multiphoton intravital microscopy to visualize trafficking of B cells and antigens into draining lymph nodes has provide insights that would not otherwise be made. At least three novel pathways for transport of lymph-borne antigens to the B cell compartment have been identified. Based on these studies, a new paradigm of how lymphocytes and antigens traffic within the peripheral lymph nodes is evolving. Understanding how the physical properties of the antigen influences its uptake and processing could be relevant in the design of new vaccines.
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http://dx.doi.org/10.1016/S0065-2776(10)06001-3DOI Listing
November 2010

Complement-dependent transport of antigen into B cell follicles.

J Immunol 2010 Sep;185(5):2659-64

The Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, MA 02115, USA.

Since the original proposal by Fearon and Locksley (Fearon and Locksley. 1996. Science 272: 50-53) that the complement system linked innate and adaptive immunity, there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers revealed an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of Ag to the B cell compartment. In this review, we discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine, such as mannose-binding lectin opsonization of influenza and uptake by macrophages, and the capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes.
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http://dx.doi.org/10.4049/jimmunol.1000522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3477863PMC
September 2010

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

Nat Immunol 2010 May 21;11(5):427-34. Epub 2010 Mar 21.

The Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.
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http://dx.doi.org/10.1038/ni.1856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424101PMC
May 2010

B cell acquisition of antigen in vivo.

Curr Opin Immunol 2009 Jun 8;21(3):251-7. Epub 2009 Jun 8.

Immune Disease Institute, Program in Cellular and Molecular Medicine, Children's Hospital, Boston, MA 02115, USA.

The fate of B lymphocytes is dictated in large part by cognate antigen and the environment in which it is encountered. Yet we are only now beginning to understand where and how B cells acquire antigen. Recent studies identify multiple pathways by which lymph-borne antigens enter the B cell follicles of LNs. Size is a major factor as particulate antigens and large IC are bound by subcapsular sinus macrophages. By contrast, small antigens (under 70kDa) are rapidly channeled into follicles via conduits secreted by fibroblastic reticular cells (FRC). Interestingly, the conduits not only deliver antigen to follicular dendritic cells (FDC) but also provide a rich source of B cell chemokine, that is, CXCL-13. Thus, the follicular conduits provide an 'antigen highway' for B cells trafficking within the LN. These new findings provide an important discovery in understanding how B cells acquire cognate antigen.
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http://dx.doi.org/10.1016/j.coi.2009.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717615PMC
June 2009

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

Immunity 2009 Feb 29;30(2):264-76. Epub 2009 Jan 29.

Departments of Pediatrics and Pathology, Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.
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http://dx.doi.org/10.1016/j.immuni.2008.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699624PMC
February 2009

Differential transcription of multiple forms of alpha-2-macroglobulin in carp (Cyprinus carpio) infected with parasites.

Dev Comp Immunol 2008 26;32(4):339-47. Epub 2007 Jul 26.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland.

Alpha-2-macroglobulin (a2M) is a non-specific protease inhibitor involved in host defense mechanisms, inhibiting both endogenous and exogenous proteases. It is unique among the plasma anti-proteases with respect to the diversity of proteases that it can inactivate. Carp a2M consists of an alpha and beta chain of which the first includes the bioactive regions. Previously, three a2M alpha chain sequences were reported for East-Asian common carp. We studied a2M alpha chain variability in European common carp and report the cloning of a fourth a2M alpha chain with distinct sequence diversity in the bait region. The role of a2M in the immune response to parasites was studied in the liver of carp infected with Trypanoplasma borreli or with Ichthyophthirius multifiliis. Quantitative gene transcription analysis showed a differential regulation of the four isoforms, most clearly seen in infections with I. multifiliis. A2M3 was the only a2M isoform with a highly upregulated transcription during infection, suggesting that this particular isoform is of foremost biological importance.
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http://dx.doi.org/10.1016/j.dci.2007.06.007DOI Listing
May 2008

Complement expression in common carp (Cyprinus carpio L.) during infection with Ichthyophthirius multifiliis.

Dev Comp Immunol 2007 6;31(6):576-86. Epub 2006 Oct 6.

Department of Veterinary Pathobiology, Laboratory of Fish Diseases, The Royal Veterinary and Agricultural University, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark.

A real-time PCR assay for determination of the complement response to infection with the ectoparasite Ichthyophthirius multifiliis in carp is presented. Specific primers were designed for selected genes representing the three pathways of the carp complement system. The investigated complement molecules were C1r/s, C3, C4, C5, factor I, factor B/C2-A (Bf/C2-A), mannose-binding lectin (MBL) and MBL-associated serine protease (MASP). The expression of the selected genes was analyzed on RNA extracts from skin, liver, and whole blood from carp at 3, 12, 24, 36, and 48 h post-infection (pi) with I. multifiliis. A pronounced up-regulation of Bf/C2-A, in skin, blood, and liver (250-, 60-, and 4-fold respectively), was observed at later sampling points pi (24-48 h). In addition, an intermediate (from 5 to 13-fold) down-regulation of MASP was observed in skin and liver samples at 36 and 48 h pi with respect to control fish. MBL was expressed only in liver and no variation in the transcription level of this lectin was observed. Complement factor C3 was significantly up-regulated in liver (4-fold up-regulation, 24 h pi). The presented results indicate that infection with the parasite I. multifiliis in carp to a large extent stimulates the expression of complement molecules. Moreover, the dramatic and early up-regulation of Bf/C2-A in skin indicates a role of this molecule as an acute-phase reactant. Furthermore, our study confirms the role of fish skin as an important extra-hepatic site of expression of complement molecules as well as an active regulator of complement expression. Expression of some of the components of the complement system in blood suggests that leukocytes in carp act as an important extra-hepatic source of complement molecules.
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http://dx.doi.org/10.1016/j.dci.2006.08.010DOI Listing
May 2007