Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the centre to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN M to their murine counterparts: subcapsular sinus M, medullary cord M and medullary sinus M. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of M, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (M). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN M populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning.
DNA vaccination is an attractive technology, based on its well-established manufacturing process, safety profile, adaptability to rapidly combat pandemic pathogens, and stability at ambient temperature; however an optimal delivery method of DNA remains to be determined. As pigs are a relevant model for humans, we comparatively evaluated the efficiency of vaccine DNA delivery in vivo to pigs using dissolvable microneedle patches, intradermal inoculation with needle (ID), surface electroporation (EP), with DNA associated or not to cationic poly-lactic-co-glycolic acid nanoparticles (NPs). We used a luciferase encoding plasmid (pLuc) as a reporter and vaccine plasmids encoding antigens from the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a clinically-significant swine arterivirus. Patches were successful at inducing luciferase expression in skin although at lower level than EP. EP induced the cutaneaous recruitment of granulocytes, of MHC2posCD172Apos myeloid cells and type 1 conventional dendritic cells, in association with local production of IL-1β, IL-8 and IL-17; these local responses were more limited with ID and undetectable with patches. The addition of NP to EP especially promoted the recruitment of the MHC2posCD172Apos CD163int and CD163neg myeloid subsets. Notably we obtained the strongest and broadest IFNγ T-cell response against a panel of PRRSV antigens with DNA + NPs delivered by EP, whereas patches and ID were ineffective. The anti-PRRSV IgG responses were the highest with EP administration independently of NPs, mild with ID, and undetectable with patches. These results contrast with the immunogenicity and efficacy previously induced in mice with patches. This study concludes that successful DNA vaccine administration in skin can be achieved in pigs with electroporation and patches, but only the former induces local inflammation, humoral and cellular immunity, with the highest potency when NPs were used. This finding shows the importance of evaluating the delivery and immunogenicity of DNA vaccines beyond the mouse model in a preclinical model relevant to human such as pig and reveals that EP with DNA combined to NP induces strong immunogenicity.
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces reproductive disorders in sows and respiratory illnesses in growing pigs and is considered as one of the main pathogenic agents responsible for economic losses in the porcine industry worldwide. Modified live PRRSV vaccines (MLVs) are very effective vaccine types against homologous strains but they present only partial protection against heterologous viral variants. With the goal to induce broad and cross-protective immunity, we generated DNA vaccines encoding B and T antigens derived from a European subtype 1 strain that include T-cell epitope sequences known to be conserved across strains. These antigens were expressed either in a native form or in the form of vaccibodies targeted to the endocytic receptor XCR1 and CD11c expressed by different types of antigen-presenting cells (APCs). When delivered in skin with cationic nanoparticles and surface electroporation, multiple DNA vaccinations as a stand-alone regimen induced substantial antibody and T-cell responses, which were not promoted by targeting antigens to APCs. Interestingly, a DNA-MLV prime-boost strategy strongly enhanced the antibody response and broadened the T-cell responses over the one induced by MLV or DNA-only. The anti-nucleoprotein antibody response induced by the DNA-MLV prime-boost was clearly promoted by targeting the antigen to CD11c and XCR1, indicating a benefit of APC-targeting on the B-cell response. In conclusion, a DNA-MLV prime-boost strategy, by enhancing the potency and breadth of MLV vaccines, stands as a promising vaccine strategy to improve the control of PRRSV in infected herds.
In human medicine, bronchoalveolar lavage is the main non-traumatic procedure allowing an insight into the respiratory Dendritic Cells (DC) and Macrophages populations. However, it has never been demonstrated in a relevant model that alveolar DC subpopulations were comparable to their interstitial counterparts. In a precedent work we observed that respiratory pig DC and Macrophages were more similar to the human ones than to the mouse ones. In the present work, thanks to our animal model, we were able to collect the rare bronchoalveolar DC and compare them to their interstitial counterparts. We observed that DC presented very similar gene-expression patterns in the alveolar and interstitial compartments, validating the study of human bronchoalveolar DC as surrogate of their interstitium counterparts.
Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two PRRSV genotypes have been identified: the genotype 1 from European origin and the genotype 2 from North American origin. Genotypes 1 have been further divided into four subtypes. Genotype 1 subtype 3 (type 1.3) such as Lena are more pathogenic than type 1.1 such as Lelystad or Flanders13. One main feature of all PRRSV strains is their capacity to delay the appearance of neutralizing antibodies, leading to a viremia that can last several months. The role of the cellular immune response in PRRSV clearance remains ill defined, with type 1.3 viruses triggering a higher Th1 response than type 1.1 without striking differences in the viremia time course. It has been proposed that the inability of pigs to achieve rapid sterilizing immunity upon PRRSV infection might rely on the virus capacity to infect and inhibit dendritic cells (DCs) functions, as it has been observed for in vitro differentiated monocytes derived DCs (moDCs). The higher pathogeny of type 1.3 viruses compared with type 1.1 may also result from their differential ability to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2 and moDCs have never been investigated. Here, the capacity of type 1 (Lelystad, Flanders13 and Lena) to infect DCs and to trigger differential T helper responses were compared. None of these 3 strains infected DCs neither in vivo nor in vitro. Moreover, Lena, but not LV and FL13, triggered cDC1 activation paralleled by a strong Th1 response. We thus propose that the ability of all PRRSV strains to alter the immune response is not due to the DCs infection or direct DCs inhibition.
Le virus du syndrome dysgenesique et respiratoire porcin (SDRPv) est un pathogene a l’origine de problemes respiratoires et de reproduction. La reponse immunitaire face au SDRPv est caracterisee par une viremie persistante et un retard dans la mise en place des anticorps neutralisants. Les macrophages alveolaires (AM) sont la cible principale du virus mais plusieurs etudes in vitro suggerent une infection des cellules dendritiques (DC). Dans ce manuscrit, de nouvelles cibles cellulaires du virus ont ete decouvertes dans le poumon et dans les ganglions tracheo-bronchiques. Les cellules du parenchyme pulmonaire AM-like sont infectes in vivo par le SDRPv de Type 1 alors que les cDC1, cDC2 et moDC du poumon ne sont pas infectees. La souche Lena Type 1.3 se distingue des autres souches par sa capacite a induire une polarisation vers une reponse Th1 in vitro. Une experience de transfert de lymphocytes T memoires in vivo suggerent une reponse cellulaire accrue et deletere lors de l’infection par la souche Lena. Trois populations de macrophages ont ete identifiees dans les ganglions et nommees : macrophages perifolliculaires (PFMacro), macrophages des cordons lymphatiques (cordMacro) et macrophages efferents (effMacro). Les effMacro et les PFMacro sont infectes par les souches europeennes. Quant a la reponse humorale, 5 stades de differenciation des LB dans les ganglions ont ete identifies et une etude de l’impact de l’infection sur la differenciation des lymphocytes B est en cours.
Increased levels of inflammatory markers such as tumour necrosis factor-α (TNFα) and interleukin- 6 (IL-6) have been associated with formation of new blood vessels, or angiogenesis, and linked to chronic inflammation in obesity. This study aimed to establish and use a versatile co-culture cell system to further investigate the role of TNFα and IL-6 in modulating (i) tubule formation and (ii) cell-cell interactions via matrix metalloproteinase (MMP) enzyme activity and secretion of vascular endothelial growth factor (VEGF), E-selectin and prostaglandin E2 (PGE2). Co-cultures of human endothelial cells and fibroblasts were incubated with TNFα (10 ng/mL) or IL-6 (10 ng/mL) added 2 and/or 7 days after co-culture establishment. Cell viability by enzymatic conversion was determined by MTT assay; tubule formation was detected by immunostaining; VEGF, E-selectin and PGE2 expression by ELISA analysis and MMP enzyme activity by gel zymography. Treatmentspecific and time dependent differences in tubule formation were observed: IL-6 significantly increased tubule formation, whilst TNFα significantly inhibited tubule formation. Treatment-specific differences in levels of MMP activities which correlate to tubule formation were also observed. This study showed inflammatory markers, typically associated with obese status, affect tubule formation differently in a heterogeneous cell environment similar to that observed in vivo. Keywords: Angiogenesis, co-culture, cytokines, fibroblasts, HUVEC, obesity.
Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.
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