Species specialization in cytokine biology: Is interleukin-4 central to the TH1–TH2 paradigm in swine? (2023)

Fetal and child’s immune systems differ from those of adults. Developing immune systems exhibit increased or decreased sensitivity to drugs, infection, or toxicants compared to adult immune systems. Understanding fetal and neonatal immune systems will help predict toxicity or the pathogenesis or prognosis of diseases. In this study, we evaluated whether the innate and adaptive immune system of fetal and young minipigs could respond to external stimuli compared to a medium-treated group and analyzed several immunological parameters for developmental immunotoxicity according to developmental stages. We performed a hematological analysis of fetal cord bloods and the bloods of neonatal and 4-week-old piglets. Splenocytes were isolated at each developmental stage and treated with lipopolysaccharide (LPS), R848, and concanavalin A (ConA). Various cytokines were measured in the cell supernatants. Total antibody production was also evaluated in serum. The percentage of lymphocytes was dominant in gestational weeks (GW) 10 and 12 and started to decrease from postnatal day (PND) 0. From PND0, the percentage of neutrophils increased. Interleukin (IL)-1β, IL-6, and interferon (IFN)-α were induced from GW10 in response to LPS and R848 stimulation. Th1 cytokine induction was detected from PND0 upon ConA stimulation, whereas Th2 cytokine release was observed from GW10. IgM and IgG production was sustained at low levels at fetal stages and was significantly increased after birth. This study reconfirmed that the fetal immune system could respond to external stimuli and that hematological analysis, cytokine evaluation, and antibody subclass measurement can be useful parameters for developmental immunotoxicity using minipigs.

The Northern Bolivian Altiplano is the human fascioliasis hyperendemic area where the highest prevalences and intensities in humans have been reported. Preventive chemotherapy was implemented in the last ten years. Surveillance showed high human infection and re-infection rates in between the annual triclabendazole monodose treatments. A complementary One Health control action was launched to decrease the infection risk. Among the multidisciplinary axes, there is the need to establish animal reservoir species priorities for a more efficient control. Laboratory and field studies were performed for the first time to assess the Fasciola hepatica transmission capacity of the pig and its potential reservoir role. The experimental follow-up of altiplanic pig isolates through altiplanic Galba truncatula snail vector isolates were performed at different miracidial doses and different day/night temperatures. Experiments included egg embryonation, miracidial infectivity, lymnaeid snail infection, intramolluscan larval development, cercarial production, chronobiology of the cercarial shedding, vector survival to infection, metacercarial infectivity of mammal host, and adult stage development. Surveys included the assessment of prevalence, intensity, egg measurements and egg shedding rates in nature. Pig contribution was evaluated by comparing with the main altiplanic reservoirs sheep and cattle. Results demonstrated that the pig assures the whole F. hepatica life cycle and participates in its transmission in this area. The fast egg embryonation, high cercarial production, long multi-wave shedding chronobiological pattern in monomiracidial infections at permanent 20°C temperature, and the high daily egg outputs per pig are worth mentioning. The high infection risk suggests early infection of freely running piglets and evolutionary long-term adaptation of the liver fluke to this omnivorous mammal, despite its previously evoked resistance or non-suitability. Genetic, physiological and immune similarities with humans may also underlie the parasite adaptation to humans in this area. The pig should be accordingly included for appropriate control measures within a One Health action against human fascioliasis. The pig should henceforth be considered in epidemiological studies and control initiatives not only in fascioliasis endemic areas with human infection risk on other Andean countries, but also in rural areas of Latin America, Africa and Asia where domestic pigs are allowed to run freely.

By definition no model is perfect, and this also holds for biology and health sciences. In medicine, murine models are, and will be indispensable for long, thanks to their reasonable cost and huge choice of transgenic strains and molecular tools. On the other side, non-human primates remain the best animal models although their use is limited because of financial and obvious ethical reasons. In the field of respiratory diseases, specific clinical models such as sheep and cotton rat for bronchiolitis, or ferret and Syrian hamster for influenza and Covid-19, have been successfully developed, however, in these species, the toolbox for biological analysis remains scarce. In this view the porcine medical model is appearing as the third, intermediate, choice, between murine and primate. Herein we would like to present the pros and cons of pig as a model for acquired respiratory conditions, through an immunological point of view. Indeed, important progresses have been made in pig immunology during the last decade that allowed the precise description of immune molecules and cell phenotypes and functions. These progresses might allow the use of pig as clinical model of human respiratory diseases but also as a species of interest to perform basic research explorations.

Swine dysentery (SD) is a global, production-limiting disease of pigs in commercial farms. It is associated with infection by Brachyspira hyodysenteriae and B. hampsonii, and characterized by mucohaemorrhagic diarrhea and colitis, SD prevention, treatment or control relies heavily on antimicrobials as no commercial vaccines are available. This is linked to our poor understanding of the disease pathogenesis. Our goal was to characterize the host-pathogen interactions during the early stage of infection. We employed dual RNA-seq to profile mRNA and miRNA following 1-h incubation of colonic explants with a pathogenic or a non-pathogenic B. hampsonii strain. Our results suggest that the pathogenic strain more efficiently interfered with the host's ability to activate and build a humoral response (through IL-4/CCR6/KLHL6 interactions), epithelial wound repair mechanisms (associated with LSECtin impairment of macrophages), induced mitochondrial dysfunction (linked to MDR1), and loss of microbiome homeostasis. The pathogenic strain also up-regulated the expression of stress-associated genes, when compared to the non-pathogenic strain. These results shed a light on the pathophysiological mechanisms that lead to SD and will contribute to the development of novel disease control tools.

We delineated the expression of DAP12 (DNAX-Activating Protein) and its associated receptors, TREM-1, TREM-2 and MDL-1 in pig alveolar monocyte/macrophages (AMM) that have attained M1 or M2 phenotypes. Pig AMM stimulated in vitro with IFN-γ and IL-4 induced the expression of M1 (TNFα and iNOS) and M2 (ARG1 and no MMR) phenotypic markers, respectively. In influenza virus infected pigs at seven days post-infection, in addition to substantial modulations in the M1 and M2 markers expression, DAP12, TREM-1 and MDL-1 were downregulated in AMM. Thus, DAP12 signaling promoted the anti-inflammatory pathway in AMM of influenza virus infected pigs.

We previously demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA), increased T helper 2-associated responses induced in pigs by infection with the parasitic nematode Ascaris suum We also showed that ATRA potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (CC motif) ligand 11 [(CCL11), CCL17, CCL22 and CCL26] associated with alternative activation (M2a) in porcine macrophages in vitro. Herein, several mechanisms whereby ATRA affects IL-4 signaling are profiled using large-scale real time PCR and RNA-Seq analysis. Twenty-three genes associated with M2a markers in other species were independently upregulated by both IL-4 and ATRA, including the adenosine receptor A2B (ADORA2B), cysteinyl leukotriene receptor 2 (CYSLTR2) and the vitamin D receptor (VDR). ATRA synergistically enhanced IL-4 up-regulation of Hepatitis A virus cellular receptor 2 (HAVCR2) and transglutaminase 2 (TGM2) and further repressed IL-4 down-regulated CD163 and Cytochrome b-245, beta polypeptide (CYBB) mRNA. Macrophages treated with ATRA exhibited a dose-dependent reduction in phagocytosis of opsonized Staphylococcus aureus. In addition, the combination of IL-4 and ATRA up-regulated the anti-inflammatory protein, IL-1R antagonist (IL1RN) and TGM2. These data indicate that ATRA induces a state of partial alternative activation in porcine macrophages, and amplifies certain aspects of M2a activation induced by IL-4. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.

Vaccine, Volume 35, Issue 8, 2017, pp. 1124-1131

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4⁺CD8αα⁺ T helper and CD8⁺ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Journal of Controlled Release, Volume 233, 2016, pp. 162-173

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.

Veterinary Immunology and Immunopathology, Volume 248, 2022, Article 110418

As maternal passive immunity wanes at 6–8 weeks, young calves must rely on their own naïve and developing immune system for protection against pathogens. Typically, an infection in the young induces a T cell-mediated response, which skews towards a Th2 phenotype and results in a reduced effector response. Our study examines the implications this transitional period of immunocompetency has on cellular metabolism in young calves, focusing on effector function of CD4⁺ T cells in comparison to those from adult cows. Results from sorted CD4⁺ T cells from young calves and adult cows activated by α-CD3:α-CD28, show that young calves exhibit a significantly greater propensity to produce the Th2 cytokine, IL-4, in comparison to IFN-γ. Concomitantly, cells from young calves and adult cows exhibit no statistical difference in cell surface marker expression induced by α-CD3:α-CD28 stimulation. Metabolically, activated CD4⁺ T cells from young calves show significantly greater utilization of mitochondrial respiration, measured by oxygen consumption rate (OCR), and greater glycolytic reserve, measured by extracellular acidification rate (ECAR). However, adult cows have a significantly higher change in glycolytic rate after α-CD3:α-CD28 stimulation compared to young calves. Further, CD4⁺ T cells from young calves have an increased mRNA expression signature associated with glycolytic metabolism (GAPDH, HK2, FBP1, HIF1A) and Th2-associated metabolic signaling (RPTOR) in comparison to adult cows. The distinct metabolic phenotype and associated gene expression in activated CD4⁺ T cells may be intrinsic drivers of the Th2-biased response by young calves. Additionally, CD4⁺ recent thymic emigrant cells (RTEs) may further contribute to altered effector function, as they are preferential precursors to Tregs, and based on the microenvironment, have the propensity to polarize toward Th2. Evaluation of T cell master transcription regulators, as well as measuring signal joint T cell receptor excision circles between young calves and adult cows, we observed a significantly increased proportion of RTEs from sorted CD4⁺ T cells. In this study, we show a unique metabolic profile exhibited by activated CD4⁺ T cells from young calves in which mitochondrial respiration and glycolytic capacity is significantly increased compared to adult cows.

Vaccine, Volume 34, Issue 18, 2016, pp. 2121-2128

There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage (“nicking”) required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines.

American Journal of Transplantation, Volume 18, Issue 2, 2018, pp. 328-340

Transplant tolerance induction makes it possible to preserve functional grafts for a lifetime without immunosuppressants. One powerful method is to generate mixed hematopoietic chimeras in recipients by adoptive transfer of donor-derived bone marrow cells (BMCs). In our murine transplantation model, we established a novel method for mixed chimera generation using sublethal irradiation, CD40-CD40L blockade, and invariant natural killer T-cell activation. However, numerous BMCs that are required to achieve stable chimerism makes it difficult to apply this model for human transplantation. Here, we show that donor-derived splenic T cells could contribute to not only the reduction of BMC usage but also the establishment of complete chimerism in model mice. By cotransfer of T cells together even with one-fourth of the BMCs used in our original method, the recipient mice yielded complete chimerism and could acquire donor-specific skin-allograft tolerance. The complete chimeric mice did not show any remarks of graft versus host reaction in vivo and in vitro. Inhibition of the apoptotic signal resulted in increase in host-derived CD8⁺ T cells and chimerism brake. These results suggest that donor-derived splenic T cells having veto activity play a role in the depletion of host-derived CD8⁺ T cells and the facilitation of complete chimerism.

Developmental & Comparative Immunology, Volume 58, 2016, pp. 1-17

The adaptive immune system of higher vertebrates is believed to have evolved to counter the ability of pathogens to avoid expulsion because their high rate of germline mutations. Vertebrates developed this adaptive immune response through the evolution of lymphocytes capable of somatic generation of a diverse repertoire of their antigenic receptors without the need to increase the frequency of germline mutation. The focus of our research and this article is on the ontogenetic development of the lymphocytes, and the repertoires they generate in swine. Several features are discussed including (a) the “closed” porcine placenta means that de novo fetal development can be studied for 114 days without passive influence from the mother, (b) newborn piglets are precocial permitting them to be reared without their mothers in germ-free isolators, (c) swine are members of the γδ−high group of mammals and thus provides a greater opportunity to characterize the role of γδ T cells and (d) because swine have a simplified variable heavy and light chain genome they offer a convenient system to study antibody repertoire development.