Oesophageal eosinophilia accompanies food allergy to hen egg white protein in young pigs.

BACKGROUND: Esophagitis with eosinophilia, inflammation, and fibrosis represent a chronic condition in humans with food allergies. OBJECTIVE: In this investigation, we asked whether esophagitis with an eosinophilic component is observed in young pigs rendered allergic to hen egg white protein (HEWP). METHODS: Food allergy was induced in young pigs using two protocols. In one protocol, sensitized pigs were challenged by gavage with a single dose of HEWP. Clinical signs were monitored for 24 hours, and then, gastrointestinal (GI) tissues were collected for histological examination. The phenotype of circulating, ovalbumin (OVA)-specific T cells also was examined in HEWP challenged animals. In the second protocol, sensitized animals were fed HEWP for 28 days. Animals were then examined by endoscopy and gastrointestinal tissues collected for histological examination. RESULTS: In pigs challenged by gavage with HEWP, clinical signs were noted in 5/6 pigs including diarrhoea, emesis, and skin rash. Clinical signs were not seen in any control group. Histological analysis revealed significant levels of oesophageal eosinophilic infiltration (P < .05) in 4/6 of these animals, with two also displaying eosinophilic infiltration in the stomach. Eosinophils were not increased in ileum or colon samples. Increased numbers of circulating, OVA-specific CD4+ T cells also were observed in pigs that received HEWP by gavage. In the group of animals fed HEWP, endoscopy revealed clinical signs of esophagitis including oedema, granularity, white spots, and furrowing, while histology revealed oedema, immune cell infiltration, and basal zone hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Food allergy in the pig can be associated with esophagitis based on histological and endoscopic findings, including eosinophilic infiltration. The young pig may, therefore, be a useful large animal model for the study of eosinophilic esophagitis in humans.

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-naïve regulatory T cells to proliferate.

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)(+) and N-protein(-) moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein(+) moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein(-) moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3(+) regulatory T cells present within CD4(+) T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4(+) T cells showed differences in regard to proliferation and frequency of Foxp3(+) T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.

Temporal Changes in Splenic Immune Cell Populations following Infection with a Very Virulent plus MDV in Commercial Meat-Type Chickens.

Marek's disease virus (MDV) can cause severe immunosuppression in chickens. Our previous study showed that infection with very virulent plus (vv+) MDV strains of one-day-old commercial meat-type chickens possessing maternal antibodies against MDV resulted in severe depletion of splenocytes at 28-30 days of age. In the present study, we have investigated the effect of vv+MDV strain 686 on splenic immunophenotypes at 6, 20, and 30 days post-infection (dpi). Both live and dead cells were analyzed, and the data were statistically compared to the uninfected control. The results revealed a decrease in the total live cell population starting on day 20, primarily affecting B cells, CD8ß+, and gamma delta (γδ) T cells, while the frequencies of both live and dead CD3+ and CD4+ T cells were increased. The MHC-I expression of CD3+ and CD4+ T cells was higher at 20 and 30 dpi, while the expression of MHC-II on these cells was downregulated at 6 dpi but was upregulated at 30 dpi. Collectively, these results suggest that maternal antibodies seem to delay the negative effects of vv+MDV on the splenic lymphoid populations, albeit being non-protective. Our results emphasize the importance of MD vaccination in vv+MDV endemic areas.

A TriAdj-Adjuvanted Chlamydia trachomatis CPAF Protein Vaccine Is Highly Immunogenic in Pigs.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.

Testable Candidate Immune Correlates of Protection for Porcine Reproductive and Respiratory Syndrome Virus Vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an on-going problem for the worldwide pig industry. Commercial and experimental vaccinations often demonstrate reduced pathology and improved growth performance; however, specific immune correlates of protection (CoP) for PRRSV vaccination have not been quantified or even definitively postulated: proposing CoP for evaluation during vaccination and challenge studies will benefit our collective efforts towards achieving protective immunity. Applying the breadth of work on human diseases and CoP to PRRSV research, we advocate four hypotheses for peer review and evaluation as appropriate testable CoP: (i) effective class-switching to systemic IgG and mucosal IgA neutralizing antibodies is required for protective immunity; (ii) vaccination should induce virus-specific peripheral blood CD4+ T-cell proliferation and IFN-γ production with central memory and effector memory phenotypes; cytotoxic T-lymphocytes (CTL) proliferation and IFN-γ production with a CCR7- phenotype that should migrate to the lung; (iii) nursery, finishing, and adult pigs will have different CoP; (iv) neutralizing antibodies provide protection and are rather strain specific; T cells confer disease prevention/reduction and possess greater heterologous recognition. We believe proposing these four CoP for PRRSV can direct future vaccine design and improve vaccine candidate evaluation.

Porcine T-helper and regulatory T cells exhibit versatile mRNA expression capabilities for cytokines and co-stimulatory molecules.

T-helper (TH) and regulatory T cells (Tregs) are important modulators of immune responses. Aim of this study was to analyse their expression potential for cytokines and other immune-relevant molecules. Therefore, porcine PBMC, CD4(-) cells, CD4(+)CD25(-) resting, CD4(+)CD25(dim) activated TH cells, and CD4(+)CD25(high) Tregs were analysed on their mRNA expression potential ex vivo or after in vitro stimulation with CD3 and IL-2 by RT-qPCR. In vitro stimulation led to an increased production of pro-inflammatory (IL-6, TNFα) and TH (IL-2, IL-4, IL-17, IFN-γ) cytokines and a diverse production of immunosuppressive cytokines (IL-10 and TGF-ß) in PBMC, CD4(-), and CD4(+) cells. Resting and activated TH cells showed an increased expression of various immune-modulatory molecules indicating that porcine TH cells possess distinct immunological skills in order to react on the actual immune situation. In contrast, Tregs appear to fulfil mainly immunosuppressive functions characterized by increased production of IL-10, IL-35, CD40L, and CD25.

Heterologous vaccine immunogenicity, efficacy, and immune correlates of protection of a modified-live virus porcine reproductive and respiratory syndrome virus vaccine.

Although porcine reproductive and respiratory syndrome virus (PRRSV) vaccines have been available in North America for almost 30 years, many vaccines face a significant hurdle: they must provide cross-protection against the highly diverse PRRSV strains. This cross-protection, or heterologous vaccine efficacy, relies greatly on the vaccine's ability to induce a strong immune response against various strains-heterologous immunogenicity. Thus, this study investigated vaccine efficacy and immunogenicity of a modified live virus (MLV) against four heterologous type 2 PRRSV (PRRSV-2) strains. In this study, 60 pigs were divided into 10 groups. Half were MOCK-vaccinated, and the other half vaccinated with the Prevacent® PRRS MLV vaccine. Four weeks after vaccination, groups were challenged with either MOCK, or four PRRSV-2 strains from three different lineages-NC174 or NADC30 (both lineage 1), VR2332 (lineage 5), or NADC20 (lineage 8). Pre-and post-challenge, lung pathology, viral loads in both nasal swabs and sera, anti-PRRSV IgA/G, neutralizing antibodies, and the PRRSV-2 strain-specific T-cell response were evaluated. At necropsy, the lung samples were collected to assess viral loads, macroscopical and histopathological findings, and IgA levels in bronchoalveolar lavage. Lung lesions were only induced by NC174, NADC20, and NADC30; within these, vaccination resulted in lower gross and microscopic lung lesion scores of the NADC20 and NADC30 strains. All pigs became viremic and vaccinated pigs had decreased viremia upon challenge with NADC20, NADC30, and VR2332. Regarding vaccine immunogenicity, vaccination induced a strong systemic IgG response and boosted the post-challenge serum IgG levels for all strains. Furthermore, vaccination increased the number of animals with neutralizing antibodies against three of the four challenge strains-NADC20, NADC30, and VR2332. The heterologous T-cell response was also improved by vaccination: Not only did vaccination increase the induction of heterologous effector/memory CD4 T cells, but it also improved the heterologous CD4 and CD8 proliferative and/or IFN-γ response against all strains. Importantly, correlation analyses revealed that the (non-PRRSV strain-specific) serum IgG levels and the PRRSV strain-specific CD4 T-cell response were the best immune correlates of protection. Overall, the Prevacent elicited various degrees of efficacy and immunogenicity against four heterologous and phylogenetically distant strains of PRRSV-2.

Immunologic and pathologic characterization of a novel swine biomedical research model for eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergy-mediated condition with an increasing incidence in both children and adults. Despite EoE's strong impact on human health and welfare, there is a large unmet need for treatments with only one recently FDA-approved medication for EoE. The goal of this study was to establish swine as a relevant large animal model for translational biomedical research in EoE with the potential to facilitate development of therapeutics. We recently showed that after intraperitoneal sensitization and oral challenge with the food allergen hen egg white protein (HEWP), swine develop esophageal eosinophilia-a hallmark of human EoE. Herein, we used a similar sensitization and challenge treatment and evaluated immunological and pathological markers associated with human EoE. Our data demonstrate that the incorporated sensitization and challenge treatment induces (i) a systemic T-helper 2 and IgE response, (ii) a local expression of eotaxin-1 and other allergy-related immune markers, (iii) esophageal eosinophilia (>15 eosinophils/0.24 mm2), and (iv) esophageal endoscopic findings including linear furrows and white exudates. Thereby, we demonstrate that our sensitization and oral challenge protocol not only induces the underlying immune markers but also the micro- and macro-pathological hallmarks of human EoE. This swine model for EoE represents a novel relevant large animal model that can drive translational biomedical research to develop urgently needed treatment strategies for EoE.

Swine as biomedical animal model for T-cell research-Success and potential for transmittable and non-transmittable human diseases.

Swine is biologically one of the most relevant large animal models for biomedical research. With its use as food animal that can be exploited as a free cell and tissue source for research and its high susceptibility to human diseases, swine additionally represent an excellent option for both the 3R principle and One Health research. One of the previously most limiting factors of the pig model was its arguably limited immunological toolbox. Yet, in the last decade, this toolbox has vastly improved including the ability to study porcine T-cells. This review summarizes the swine model for biomedical research with focus on T cells. It first contrasts the swine model to the more commonly used mouse and non-human primate model before describing the current capabilities to characterize and extend our knowledge on porcine T cells. Thereafter, it not only reflects on previous biomedical T-cell research but also extends into areas in which more in-depth T-cell analyses could strongly benefit biomedical research. While the former should inform on the successes of biomedical T-cell research in swine, the latter shall inspire swine T-cell researchers to find collaborations with researchers working in other areas - such as nutrition, allergy, cancer, transplantation, infectious diseases, or vaccine development.

Host-Pathogen Interactions of Chlamydia trachomatis in Porcine Oviduct Epithelial Cells.

Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host-pathogen interactions in porcine oviduct epithelial cells (pOECs)-the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.

Systems Immunology Analyses Following Porcine Respiratory and Reproductive Syndrome Virus Infection and Vaccination.

This study was initiated to better understand the nature of innate immune responses and the relatively weak and delayed immune response against porcine reproductive and respiratory syndrome virus (PRRSV). Following modified live virus (MLV) vaccination or infection with two PRRSV-2 strains, we analyzed the transcriptome of peripheral blood mononuclear cells collected before and at three and seven days after vaccination or infection. We used blood transcriptional modules (BTMs)-based gene set enrichment analyses. BTMs related to innate immune processes were upregulated by PRRSV-2 strains but downregulated by MLV. In contrast, BTMs related to adaptive immune responses, in particular T cells and cell cycle, were downregulated by PRRSV-2 but upregulated by MLV. In addition, we found differences between the PRRSV strains. Only the more virulent strain induced a strong platelet activation, dendritic cell activation, interferon type I and plasma cell responses. We also calculated the correlations of BTM with the neutralizing antibody and the T-cell responses. Early downregulation (day 0-3) of dendritic cell and B-cell BTM correlated to both CD4 and CD8 T-cell responses. Furthermore, a late (day 3-7) upregulation of interferon type I modules strongly correlated to helper and regulatory T-cell responses, while inflammatory BTM upregulation correlated more to CD8 T-cell responses. BTM related to T cells had positive correlations at three days but negative associations at seven days post-infection. Taken together, this work contributes to resolve the complexity of the innate and adaptive immune responses against PRRSV and indicates a fundamentally different immune response to the less immunogenic MLV compared to field strains which induced robust adaptive immune responses. The identified correlates of T-cell responses will facilitate a rational approach to improve the immunogenicity of MLV.

The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV - the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains - one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28-35 dpi with sporadic rebounds thereafter. The local IgA response started 4-7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28-35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4-7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels - by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.

Maternal Autogenous Inactivated Virus Vaccination Boosts Immunity to PRRSV in Piglets.

Maternal-derived immunity is a critical component for the survival and success of offspring in pigs to protect from circulating pathogens such as Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2). The purpose of this study is to investigate the transfer of anti-PRRSV immunity to piglets from gilts that received modified-live virus (MLV) alone (treatment (TRT) 0), or in combination with one of two autogenous inactivated vaccines (AIVs, TRT 1+2). Piglets from these gilts were challenged with the autogenous PRRSV-2 strain at two weeks of age and their adaptive immune response (IR) was evaluated until 4 weeks post inoculation (wpi). The systemic humoral and cellular IR was analyzed in the pre-farrow gilts, and in piglets, pre-inoculation, and at 2 and 4 wpi. Both AIVs partially protected the piglets with reduced lung pathology and increased weight gain; TRT 1 also lowered piglet viremia, best explained by the AIV-induced production of neutralizing antibodies in gilts and their transfer to the piglets. In piglets, pre-inoculation, the main systemic IFN-γ producers were CD21α+ B cells. From 0 to 4 wpi, the role of these B cells declined and CD4 T cells became the primary systemic IFN-γ producers. In the lungs, CD8 T cells were the primary and CD4 T cells were the secondary IFN-γ producers, including a novel subset of porcine CD8α-CCR7- CD4 T cells, potentially terminally differentiated CD4 TEMRA cells. In summary, this study demonstrates that maternal AIV vaccination can improve protection of pre-weaning piglets against PRRSV-2; it shows the importance of transferring neutralizing antibodies to piglets, and it introduces two novel immune cell subsets in pigs-IFN-γ producing CD21α+ B cells and CD8α-CCR7- CD4 T cells.

In ovo vaccination with herpesvirus of turkey enhances innate and cellular responses in meat-type chickens: Effect of vaccine dose and strain.

In ovo vaccination with herpesvirus of turkey (HVT) or recombinant HVT (rHVT) is commonly used in meat-type chickens. Previous studies showed that in ovo vaccination with HVT enhances innate, cellular, and humoral immune responses in egg-type chicken embryos. This study evaluated if in ovo vaccination with HVT hastens immunocompetence of commercial meat-type chickens and optimized vaccination variables (dose and strain of HVT) to accelerate immunocompetence. A conventional HVT vaccine was given at recommended dose (RD), HVT-RD = 6080 plaque forming units (PFU), double-dose (2x), half-dose (1/2), or quarter-dose (1/4). Two rHVTs were given at RD: rHVT-A = 7380 PFU, rHVT-B = 8993 PFU. Most, if not all, treatments enhanced splenic lymphoproliferation with Concanavalin A and increased the percentage of granulocytes at day of age. Dose had an effect and HVT-RD was ideal. An increase of wing-web thickness after exposure to phytohemagglutinin-L was only detected after vaccination with HVT-RD. Furthermore, compared to sham-inoculated chickens, chickens in the HVT-RD had an increased percentage of CD3+ T cells and CD4+ T-helper cells, and increased expression of major histocompatibility complex (MHC)-II on most cell subsets (CD45+ cells, non-T leukocytes, T cells and the CD8+ and T cell receptor γδ T-cell subsets). Other treatments (HVT-1/2 and rHVT-B) share some of these features but differences were not as remarkable as in the HVT-RD group. Expression of MHC-I was reduced, compared to sham-inoculated chickens, in most of the cell phenotypes evaluated in the HVT-RD, HVT-2x and rHVT-A groups, while no effect was observed in other treatments. The effect of in ovo HVT on humoral immune responses (antibody responses to keyhole limpet hemocyanin and to a live infectious bronchitis/Newcastle disease vaccine) was minimal. Our study demonstrates in ovo vaccination with HVT in meat-type chickens can accelerate innate and adaptive immunity and we could optimize such effect by modifying the vaccine dose.

Mucosal Vaccination with UV-Inactivated Chlamydia suis in Pre-Exposed Outbred Pigs Decreases Pathogen Load and Induces CD4 T-Cell Maturation into IFN-γ+ Effector Memory Cells.

Chlamydia trachomatis (Ct) infections are the most frequent bacterial sexually transmitted disease, and they can lead to ectopic pregnancy and infertility. Despite these detrimental long-term sequelae, a vaccine is not available. Success in preclinical animal studies is essential for vaccines to move to human clinical trials. Pigs are the natural host to Chlamydia suis (Cs)-a chlamydia species closely related to Ct, and are susceptible to Ct, making them a valuable animal model for Ct vaccine development. Before making it onto market, Ct vaccine candidates must show efficacy in a high-risk human population. The high prevalence of human Ct infection combined with the fact that natural infection does not result in sterilizing immunity, results in people at risk likely having been pre-exposed, and thus having some level of underlying non-protective immunity. Like human Ct, Cs is highly prevalent in outbred pigs. Therefore, the goal of this study was to model a trial in pre-exposed humans, and to determine the immunogenicity and efficacy of intranasal Cs vaccination in pre-exposed outbred pigs. The vaccine candidates consisted of UV-inactivated Cs particles in the presence or absence of an adjuvant (TriAdj). In this study, both groups of vaccinated pigs had a lower Cs burden compared to the non-vaccinated group; especially the TriAdj group induced the differentiation of CD4+ cells into tissue-trafficking CCR7- IFN-γ-producing effector memory T cells. These results indicate that Cs vaccination of pre-exposed pigs effectively boosts a non-protective immune response induced by natural infection; moreover, they suggest that a similar approach could be applied to human vaccine trials.

Dietary arachidonate in milk replacer triggers dual benefits of PGE2 signaling in LPS-challenged piglet alveolar macrophages.

BACKGROUND: Respiratory infections challenge the swine industry, despite common medicinal practices. The dual signaling nature of PGE2 (supporting both inflammation and resolution) makes it a potent regulator of immune cell function. Therefore, the use of dietary long chain n-6 PUFA to enhance PGE2 effects merits investigation. METHODS: Day-old pigs (n = 60) were allotted to one of three dietary groups for 21 d (n = 20/diet), and received either a control diet (CON, arachidonate = 0.5% of total fatty acids), an arachidonate (ARA)-enriched diet (LC n-6, ARA = 2.2%), or an eicosapentaenoic (EPA)-enriched diet (LC n-3, EPA = 3.0%). Alveolar macrophages and lung parenchymal tissue were collected for fatty acid analysis. Isolated alveolar macrophages were stimulated with LPS in situ for 24 h, and mRNA was isolated to assess markers associated with inflammation and eicosanoid production. Culture media were collected to assess PGE2 secretion. Oxidative burst in macrophages was measured by: 1) oxygen consumption and extracellular acidification (via Seahorse), 2) cytoplasmic oxidation and 3) nitric oxide production following 4, 18, and 24 h of LPS stimulation. RESULTS: Concentration of ARA (% of fatty acids, w/w) in macrophages from pigs fed LC n-6 was 86% higher than CON and 18% lower in pigs fed LC n-3 (P < 0.01). Following LPS stimulation, abundance of COX-2 and TNF-α mRNA (P <  0.0001), and PGE2 secretion (P < 0. 01) were higher in LC n-6 PAM vs. CON. However, ALOX5 abundance was 1.6-fold lower than CON. Macrophages from CON and LC n-6 groups were 4-fold higher in ALOX12/15 abundance (P < 0.0001) compared to LC n-3. Oxygen consumption and extracellular acidification rates increased over 4 h following LPS stimulation (P < 0.05) regardless of treatment. Similarly, increases in cytoplasmic oxidation (P < 0.001) and nitric oxide production (P <  0.002) were observed after 18 h of LPS stimulation but were unaffected by diet. CONCLUSIONS: We infer that enriching diets with arachidonic acid may be an effective means to enhance a stronger innate immunologic response to respiratory challenges in neonatal pigs. However, further work is needed to examine long-term safety, clinical efficacy and economic viability.

The T-Cell Response to Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause severe reproductive and respiratory pathologies resulting in immense monetary and welfare costs for the swine industry. The vaccines against PRRSV are available; but they struggle with providing protection against the plethora of heterologous PRRSV strains. To improve PRRSV vaccine development, the aim of this study was to provide an in-depth analysis of the crucial heterologous T-cell response to type-2 PRRSV. Following PRRSV modified live virus (MLV) vaccination or infection using one high- or one low-pathogenic PRRSV-strain, this nine-week study evaluated the T-cell response to different PRRSV strains. Our results demonstrate an important role for T cells in this homo- and heterologous response. Specifically, the T-helper cells were the main responders during viremia. Their peak response at 28 dpi correlated with a reduction in viremia, and their homing receptor expression indicated the additional importance for the anti-PRRSV response in the lymphatic and lung tissue. The cytocoxic T lymphocyte (CTL) response was the strongest at the site of infection-the lung and bronchoalveolar lavage. The TCR-γδ T cells were the main responders post viremia and PRRSV induced their expression of the lymph node homing the chemokine receptor, CCR7: This indicates a crucial role for TCR-γδ T cells in the anti-PRRSV response in the lymphatic system.

Expression of T-Bet, Eomesodermin, and GATA-3 Correlates With Distinct Phenotypes and Functional Properties in Porcine γδ T Cells.

Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2- γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α-/dimCD27+perforin- phenotype. T-bet expression was mainly found in a subset of CD2+ γδ T cells with an opposing CD8αhighCD27dim/-perforin+ phenotype. Eomes+ γδ T cells were also found within CD2+ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes+ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2-GATA-3+ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2+T-bet+ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2- γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2-CD8α-/dimCD27+perforin- γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2+ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2- γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described.

Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents.

The staining of intracellular antigens is a standard method in flow cytometry but still only occasionally used for immunologic studies in veterinary species. In order to improve information about suitable fixation/permeabilisation protocols in porcine lymphocytes we tested six fixation/permeabilisation variants for the staining of different intracellular antigens: CD79alpha, perforin, interferon-gamma and the cell cycle associated protein Ki-67. For the fixation/permeabilisation procedure, four commercial kits from BD Biosciences, ADG Bio Research, Dako and AbD Serotec were tested in comparison to non-commercial fixation solutions of formaldehyde together with either saponin or Tween-20 for permeabilisation. Perforin and Ki-67 expressing cells were optimally stained only with permeabilisation reagents containing saponin, which includes the BD kit. In contrast, labelling of CD79alpha and IFN-gamma was possible with all investigated fixation/permeabilisation variants; however, here saponin and Tween protocols induced a higher degree of unspecific binding. Also, scatter properties of cells treated with saponin were worse compared to samples prepared with the kits from ADG, Dako and Serotec. Simultaneous staining of cell surface antigens was not negatively affected by any of the fixation/permeabilisation variants. A universal recommendation for a single fixation/permeabilisation strategy could not be deduced from our data but our work provides a useful guideline for optimal staining of common intracellular antigens analyzed in swine lymphocytes.

Detection of Foxp3 protein expression in porcine T lymphocytes.

Regulatory T cells (Tregs) are potent regulators of various immune reactions. Due to the lack of Treg-specific markers their analysis had often been challenging until the discovery of the transcription factor Forkhead-box p3 (Foxp3) which serves as this highly demanded marker. So far, antibodies staining human and murine Foxp3 have been developed. This study describes the analysis of four commercially available anti-Foxp3 antibodies for reactivity with their specific antigen in cells derived from porcine lymphoid tissues. One out of the four antibodies showed selective reactivity with porcine CD25(+) T lymphocytes. The intracellular antigen was expressed on a small subset of CD25(dim) cells and the majority of the CD25(high) positive T-cell subpopulation. Intracellular antigen positive cells showed a heterogeneous expression of other leukocyte differentiation antigens. The majority belonged to the CD4(+)CD8(+) T-lymphocyte subpopulation, but were also found in the CD4(+)CD8(-) subpopulation. Another small minority was included in the CD4(-)CD8(+) T-lymphocyte subpopulation. Additionally, a small fraction of the putative Foxp3(+) cells showed an expression of MHC-II molecules. These staining patterns in three and four colour flow cytometry analyses indicated that the cells detected by a rat anti-mouse/rat-Foxp3 antibody expressed the porcine Foxp3. The expression of the putative Foxp3 protein in distinct leukocyte subsets was confirmed by molecular analysis of Foxp3 mRNA expression. Using Western blot analysis specific protein bands could only be detected in fractions that also exhibited the corresponding Foxp3 mRNA expression. These experiments also revealed that the antibody recognized a single chain protein with a molecular mass of about 45kDA similar to Foxp3 described for other species. In summary, these data strongly indicate the reactivity of this antibody with porcine Foxp3. Thereby, this rat anti-mouse/rat Foxp3 antibody presents a powerful tool for the identification of porcine regulatory T cells.