Dirección de antígenos a receptores de células presentadoras de antígeno como estrategia para aumentar la inmunogenicidad de vacunas en la especie porcina

Tesis de la Universidad Complutense de Madrid, Facultad de Medicina, leída el 12-07-2011Depto. de FisiologíaFac. de MedicinaTRUEpu

Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells

Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-a. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-a treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC

Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

Monocyte subsets have been shown to differ in the pattern of chemokine receptor expression and their migratory properties, both in human and mouse. Previously we have characterized in the swine several monocyte subpopulations, based on the expression of CD163, Tük4 and SLA-II, which share features with the populations described in human and mouse. Here, we have analysed the expression of different chemokine receptors in the CD163−Tük4+SLA-II− and CD163+Tük4−SLA-II+ populations of porcine monocytes. CD163+Tük4−SLA-II+ monocytes expressed higher CX3CR1 but lower CCR2 and CXCR4 mRNA levels than CD163−Tük4+SLA-II− monocytes. Moreover, porcine CCL2 binding on Tük4+SLA-II− but not on Tük4−SLA-II+ cells was detected by using a CCL2-green fluorescence protein (pCCL2-GFP) fusion protein. Finally, flow cytometric analyses of monocytes recovered after chemotaxis assays show a clear increase in the proportion of Tük4+SLA-II− cells in the fraction migrating toward CCL2, consistent with the polarized CCR2 expression in this monocyte population. The pattern of expression of these chemokine receptors reinforces the similarities of these porcine subsets with their human and mouse counterparts

Pharmacological Elevation of Cellular Dihydrosphingomyelin Provides a Novel Antiviral Strategy against West Nile Virus Infection

The flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our collection of polyphenols against West Nile virus (WNV), a representative medically relevant flavivirus, led to the identification of N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (half-maximal effective concentrations [EC50s] of 2.2 and 0.24 μM, respectively, in Vero cells; EC50s of 2.2 and 1.9 μM, respectively, in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely, Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1), promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles in membrane properties. The addition of exogenous dhSM or Des1 blockage by using the reference inhibitor GT-11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide} confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.We thank Theodore C. Pierson (National Institutes of Health, USA) for the subgenomic replicon of WNV. This work was supported by the Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under grants PID2019-105117RR-C21 (to M.A.M.-A.), PID2019-105117RR-C22 (to M.-J.P.-P.), and PID2020-119195RJ-I00 (to N.J.d.O.) and by the AECSIC under grant PIE-201980E100 (to M.-J.P.-P. and A.S.-F.). This research work was also funded by the European Commission-NextGenerationEU (regulation EU 2020/2094) through CSIC’s Global Health Platform (PTI Salud Global). P.M.-C. was supported by an FPI fellowship (PRE2020-093374) from AEI/10.13039/501100011033. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewe

Porcine Macrophage Markers and Populations: An Update

Besides its importance as a livestock species, pig is increasingly being used as an animal model for biomedical research. Macrophages play critical roles in immunity to pathogens, tissue development, homeostasis and tissue repair. These cells are also primary targets for replication of viruses such as African swine fever virus, classical swine fever virus, and porcine respiratory and reproductive syndrome virus, which can cause huge economic losses to the pig industry. In this article, we review the current status of knowledge on porcine macrophages, starting by reviewing the markers available for their phenotypical characterization and following with the characteristics of the main macrophage populations described in different organs, as well as the effect of polarization conditions on their phenotype and function. We will also review available cell lines suitable for studies on the biology of porcine macrophages and their interaction with pathogens

Relevance of oxidative stress in inhibition of eIF2 alpha phosphorylation and stress granules formation during Usutu virus infection.

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile, Japanese encephalitis, Zika, and dengue viruses. USUV emerged in 1996 in Europe, where quickly spread across the continent causing a considerable number of bird deaths and varied neurological disorders in humans, including encephalitis, meningoencephalitis, or facial paralysis, thus warning about USUV as a potential health threat. USUV replication takes place on the endoplasmic reticulum (ER) of infected cells, inducing ER stress and resulting in the activation of stress-related cellular pathways collectively known as the integrated stress response (ISR). The alpha subunit of the eukaryotic initiation factor eIF2 (eIF2α), the core factor in this pathway, is phosphorylated by stress activated kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated inhibitor kinase (HRI), and general control non-repressed 2 kinase (GCN2). Its phosphorylation results, among others, in the downstream inhibition of translation with accumulation of discrete foci in the cytoplasm termed stress granules (SGs). Our results indicated that USUV infection evades cellular stress response impairing eIF2α phosphorylation and SGs assembly induced by treatment with the HRI activator ArsNa. This protective effect was related with oxidative stress responses in USUV-infected cells. Overall, these results provide new insights into the complex connections between the stress response and flavivirus infection in order to maintain an adequate cellular environment for viral replication

CD200R family receptors are expressed on porcine monocytes and modulate the production of IL-8 and TNF-α triggered by TLR4 or TLR7 in these cells

The inhibitory receptor CD200R1 and its paired activating receptor CD200R1L are involved in the regulation of myeloid cell immune responses. The aim of this study was to analyze their distribution, regulation by cytokines, and function in porcine monocyte subsets. We had previously observed that CD200R1 and CD200R1L genes can generate different protein isoforms through alternative mRNA splicing, therefore in this study, we explored the diversity of transcripts in monocyte subsets, and described several new splicing variants of both CD200R1 and CD200R1L, some of which could be expressed on the porcine monocyte surface. A substantial proportion of CD163- SLAII+ and most CD163+SLAII+ monocytes expressed CD200R1 and CD200R1L receptors, while CD163- SLAII- monocytes did not. CD200R1 and CD200R1L expression was down-regulated in monocytes polarized by IFN-ɣ, a cytokine that induces classical activation of macrophages, while IL-10 which gives rise to regulatory macrophages, increased the expression of CD200R1. Finally, treatment of monocyte subsets with a monoclonal antibody specific for the inhibitory CD200R1 receptor and its splicing variants enhanced TNFα and IL-8 production, induced by TLR4 or TLR7 stimulation, suggesting a modulatory role for these receptors on porcine monocyte functions

CD200R1 and CD200R1L expression is regulated during B cell development in swine and modulates the Ig production in response to the TLR7 ligand imiquimoid

The CD200R family comprises a group of paired receptors that can modulate the activation of immune cells. They are expressed both on myeloid cells and lymphocyte subsets. Here we report that the expression of these receptors on porcine B cells is tightly regulated, being mainly expressed on mature cells. The expression of the inhibitory receptors CD200R1 and/or its splicing variant CD200R1X2, either in combination or not with the activating receptor CD200R1L, is upregulated in sIgM+ effector/memory cells, and tends to decline thereafter as these cells progress to plasmablasts or switch the Ig isotype. sIgM+ naïve and primed cells only express, by contrast, the CD200R1X2 receptor. B-1 like cells also express CD200R1 isoforms, either alone or in combination with CD200R1L. Treatment of peripheral blood mononuclear cells with a monoclonal antibody specific for inhibitory receptors, enhances the IgM and IgG production induced by TLR7 stimulation suggesting a modulatory role of B cell functions of these receptors.Funding: This work was supported by grants RTA2014-00003-00-00 from INIA and PID2019-10985RB-I00 from Agencia Estatal de Innovación (Ministerio de Ciencia e Innovación of Spain).Peer reviewe

Pathogenicity and virulence of West Nile virus revisited eight decades after its first isolation

30 Pág. Departamento de BiotecnologíaWest Nile virus (WNV) is a flavivirus which transmission cycle is maintained between mosquitoes and birds, although it occasionally causes sporadic outbreaks in horses and humans that can result in serious diseases and even death. Since its first isolation in Africa in 1937, WNV had been considered a neglected pathogen until its recent spread throughout Europe and the colonization of America, regions where it continues to cause outbreaks with severe neurological consequences in humans and horses. Although our knowledge about the characteristics and consequences of the virus has increased enormously lately, many questions remain to be resolved. Here, we thoroughly update our knowledge of different aspects of the WNV life cycle: virology and molecular classification, host cell interactions, transmission dynamics, host range, epidemiology and surveillance, immune response, clinical presentations, pathogenesis, diagnosis, prophylaxis (antivirals and vaccines), and prevention, and we highlight those aspects that are still unknown and that undoubtedly require further investigation.This work was supported by the Comunidad de Madrid [S2018/BAA-4370-ZOOVIR (PLATESA2-CM]; Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria [RTA2015-00009-00-00]; Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria [E-RTA2017-00003-C02-01]; Instituto de Salud Carlos III [COV20/00183]; Agencia Estatal de Investigación (ES) [PID2019-105117RR-C21/AEI/10.13039/501100011033].Peer reviewe