The porcine immune system--differences compared to man and mouse and possible consequences for infections by Salmonella serovars

The porcine immune system differs in many aspects from that of humans and mice. Morphological differences in the lymphatic system (e.g. lymph nodes, Peyer's patches), and phenotypic differences in immune cells have been observed as well as functional differences in immune cell populations. Indeed, even the prenatal development of the embryonic piglet proceeds in a principally different way. However, it remains unclear to what extent these differences might contribute to the predisposition to and outcomes of bacterial infections, in particular those with zoonotic potential. In the following article we will review some of the peculiarities of the porcine immune system and consider possible implications for the course of infections in swine, with an emphasis on Salmonella serovars.     

Proline-arginine rich (PR-39) cathelicidin: Structure,expression and functional implication in intestinal health

Proline-Arginine-39 (PR-39) is a small cationic, proline and arginine rich, cathelicidin that plays an important role in the porcine innate immune system. Although PR-39 was first discovered in intestinal cell lysates of pigs, subsequent research has indicated that it is primarily expressed in bone marrow and other lymphoid tissues including the thymus and spleen, as well as in leukocytes. Mature PR-39 cathelicidin has anti-microbial activity against many gram-negative and some gram-positive bacteria. PR-39 is also a bridge between the innate and adaptive immune system with recognized immunomodulatory, wound healing, anti-apoptotic, and pro-angiogenic functions. The purpose of this review is to summarize our current knowledge about the structure, expression, and functions of PR-39 and its potential to promote intestinal homeostasis. This understanding is relevant in the search of alternative therapeutics against diarrheic enterocolitis, a major problem faced by pork producers both in terms of costs and risk of zoonosis.     

An overview of molecular and cellular mechanisms associated with porcine pregnancy success or failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.     

Porcine TLR3 characterization and expression in response to influenza virus and Bordetella bronchiseptica

We have provided a detailed structural analysis of porcine alveolar macrophage TLR3 extracellular domain (ECD). The porcine TLR3-ECD contains 18 leucine-rich repeats (LRRs) consisting of blocks of consensus motifs and non-consensus motifs containing insertions. Excluding the N-terminal and C-terminal LRRs, porcine TLR3 has two LRRs with insertions, resulting in one LRR of 39 amino acids and another LRR of 34 amino acids. Furthermore, we have conducted the first examination of the regulated expression of porcine alveolar macrophage TLR3 during in vivo co-infection with influenza virus and Bordetella bronchiseptica. There was a bi-phasic upregulation of porcine TLR3 during influenza virus infection (day 1 and day 10 post-infection). Co-infection resulted in an enhanced expression of porcine TLR3 only at day 1 post-infection. Interestingly, B. bronchiseptica induced an upregulation in alveolar macrophage TLR3 expression at day 10 post-infection. Based on our work and that of others, TLR3 likely plays a key role in the immune response of lung cells to influenza virus infection in several mammalian species.     

Equine beta-defensin-1: full-length cDNA sequence and tissue expression

beta-Defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immune defense. Although, previous investigations have identified beta-defensins in several mammalian species, no reports have identified equine beta-defensins. Using a strategy of database searching for expressed sequence tags (EST) we identified putative expression of equine beta-defensins in hepatic tissue. Based on this information, sequence specific primers were designed for the equine gene enabling the identification of the full-length cDNA sequence of equine beta-defensin-1. Comparative analyses showed that equine beta-defensin-1 has 46-52% amino-acid identity with other beta-defensins, sharing the greatest identity with porcine beta-defensin-1. Complete conservation of cysteine residues was maintained between the species evaluated, and RT-PCR analysis revealed diverse mRNA tissue expression for equine beta-defensin-1. These data extend the repertoire of equine antimicrobial peptides and expand our understanding of equine innate immunity.     

New technologies for studying immune regulation in ruminants

There remains a great need for the development of reagents and techniques to study immune regulation in ruminants. These tools are fundamental to our understanding of the mechanisms of immune modulation and underpin the rational design of disease control strategies in livestock. Technological advances in molecular biology have resulted in the development of a new range of techniques to measure and quantify gene expression in normal and disease states. However, the ability to measure protein expression, produce proteins (usually in recombinant form) and to block receptor-ligand interactions remains essential for elucidating the function of the molecules of interest. The aims of this workshop were to look at the current status of reagents for studying ruminant immunology, the prospects for future developments, and also to discuss reagents availability between different groups worldwide.     

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

ABSTRACT: This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.     

Molecular analyses of disease pathogenesis: application of bovine microarrays

The molecular analysis of disease pathogenesis in cattle has been limited by the lack of availability of tools to analyze both host and pathogen responses. These limitations are disappearing with the advent of methodologies such as microarrays that facilitate rapid characterization of global gene expression at the level of individual cells and tissues. The present review focuses on the use of microarray technologies to investigate the functional pathogenomics of infectious disease in cattle. We discuss a number of unique issues that must be addressed when designing both in vitro and in vivo model systems to analyze host responses to a specific pathogen. Furthermore, comparative functional genomic strategies are discussed that can be used to address questions regarding host responses that are either common to a variety of pathogens or unique to individual pathogens. These strategies can also be applied to investigations of cell signaling pathways and the analyses of innate immune responses. Microarray analyses of both host and pathogen responses hold substantial promise for the generation of databases that can be used in the future to address a wide variety of questions. A critical component limiting these comparative analyses will be the quality of the databases and the complete functional annotation of the bovine genome. These limitations are discussed with an indication of future developments that will accelerate the validation of data generated when completing a molecular characterization of disease pathogenesis in cattle.     

Interaction between innate immunity and porcine reproductive and respiratory syndrome virus

Innate immunity provides frontline antiviral protection and bridges adaptive immunity against virus infections. However, viruses can evade innate immune surveillance potentially causing chronic infections that may lead to pandemic diseases. Porcine reproductive and respiratory syndrome virus (PRRSV) is an example of an animal virus that has developed diverse mechanisms to evade porcine antiviral immune responses. Two decades after its discovery, PRRSV is still one of the most globally devastating viruses threatening the swine industry. In this review, we discuss the molecular and cellular composition of the mammalian innate antiviral immune system with emphasis on the porcine system. In particular, we focus on the interaction between PRRSV and porcine innate immunity at cellular and molecular levels. Strategies for targeting innate immune components and other host metabolic factors to induce ideal anti-PRRSV protection are also discussed.     

Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies

With the growing emergence of antibiotic resistance and rising consumer demands concerning food safety, vaccination to prevent bacterial infections is of increasing relevance. Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. Despite all the research and trials that were performed with A. pleuropneumoniae vaccination in the past, a safe vaccine that offers complete protection against all serotypes has yet not reached the market. However, recent advances made in the identification of new potential vaccine candidates and in the targeting of specific immune responses, give encouraging vaccination perspectives. Here, we review past and current knowledge on A. pleuropneumoniae vaccines as well as the newly available genomic tools and vaccination strategies that could be useful in the design of an efficient vaccine against A. pleuropneumoniae infection.     

Characterization of the cytokine pattern of porcine bone marrow-derived cells treated with 1alpha,25(OH)D

The biologically active form of vitamine D(3) [1alpha,25(OH)(2)D(3)] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1alpha,25(OH)(2)D(3) on the cytokine pattern of porcine bone marrow-derived cells from piglets aged 1-3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell-derived mRNA was subjected to semiquantitative RT-PCR specific for a panel of porcine cytokines (IL-1alpha, IL-6, IL-8, IL-10, and TNF-alpha). In addition, an immunofluorescence analysis using anti-porcine mAbs specific for IL-1beta, IL-2, IL-4, IL-6, IL-12, TNF-alpha, and IFN-gamma was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF-kappaB ligand (RANKL) bone marrow cell- as well as porcine white blood cell-derived mRNA was investigated by RT-PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT-PCR, expression of IL-1alpha, IL-6, IL-8, IL-10 and TNF-alpha mRNA could be found in cells cultured with and without 1alpha,25(OH)(2)D(3). Immunofluorescence analysis revealed that IL-1, IL-6, and TNF-alpha were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1alpha,25(OH)(2)D(3) tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL-specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.     

Differential regulation of porcine beta-defensins 1 and 2 upon Salmonella infection in the intestinal epithelial cell line IPI-2I

Intestinal epithelial cells represent the first line of defence against pathogenic bacteria in the lumen of the gut. Besides acting as a physical barrier, epithelial cells orchestrate the immune response through the production of several innate immune mediator molecules including beta-defensins. Here, we establish the porcine intestinal cell line IPI-2I as a new model system to test the regulation of porcine beta-defensins 1 and 2. Gene expression of both defensins was highly upregulated by foetal calf serum components in normal growth medium. In serum-free medium, baseline expression remained low, but pBD-2 gene expression was increased 10-fold upon infection with Salmonella Typhimurium. Arcobacter cryaerophilus and Salmonella Enteritidis, pathogenic bacteria with comparable adhesion and invasion characteristics, failed to increase pBD-2 mRNA levels. Heat killed or colistin-treated Salmonella Typhimurium had no effect, showing that the upregulation of pBD-2 was dependent on the viability of the Salmonella Typhimurium. Gene expression of pBD-1 was regulated differently since an increase in pBD-1 mRNA was observed by Salmonella Enteritidis infection. We conclude that the IPI-2I cells can serve as a new model to study porcine beta-defensin regulation and that pBD-1 and pBD-2 are differentially regulated in this cell line.     

Fc receptors in livestock species

Many of the receptors for the Fc domain of immunoglobulins in cattle, sheep, pigs and horses have been cloned and characterized recently. This review summarises recent developments and relates them to the current understanding of the primary structure, cellular specificity and binding properties of Fc receptors (FcRs). Although there is an obvious overall similarity to their human and mouse counterparts, some Fc receptors in domestic animals are unusual, perhaps most notably the bovine Fcgamma2R, which although related to other mammalian FcgammaRs, belongs to a novel gene family and the porcine FcgammaRIIIA, which associates with a molecule that contains significant homology to the cathelin family of antimicrobial proteins. Accumulating data suggest the possibility of a different role for the FcRn in ruminants, which may secrete IgG onto the mucosal surfaces, rather than absorbing it, as was suggested by mouse studies. These differences may be linked to the diversity of immunoglobulin classes in different mammalian species, and may contribute to different immune functions. The observations made so far emphasize the importance of elucidating and analyzing the roles of these molecules within the immune system of each host animal, rather than inferring roles from conclusions made in human and mouse studies. A better understanding of Fc receptor expression on immune effector cells should help in developing new immunization protocols, while knowledge of the Fc receptors involved in immunoglobulin transport, especially in the mammary gland, may help to develop new products which could be used not only for veterinary purposes but perhaps also for human therapy.     

Ocular immunology in equine recurrent uveitis

Equine recurrent uveitis (ERU) is a disease with high prevalence and relevance for the equine population, since it results in blindness. Over the last decade, important advancements have been made in our understanding of the underlying immune responses in this disease. ERU is mediated by an autoaggressive Th1 response directed against several retinal proteins. Interphotoreceptor-retinoid binding protein (IRBP) and cellular retinaldehyde-binding protein (CRALBP) are capable to induce ERU-like disease in experimental horses, with the unique possibility to activate relapses in a well-defined manner. Further, proteomic evidence now suggests that retinal Mueller glial cells (RMG) may play a fatal role in uveitic disease progression by directly triggering inflammation processes through the expression and secretion of interferon-γ. Ongoing relapses in blind eyes can be associated with stable expression of the major autoantigens in ERU retinas. This review briefly summarizes the most significant developments in uveitis immune response research.