Ileal transcriptome analysis in obese rats induced by high-fat diets and an adenoviral infection.

BACKGROUND: Obesity has become a worldwide epidemic affecting millions of people. Obesity and associated health consequences tend to be complicated by diverse causes and multi-systemic involvement. Previous studies have investigated obesity induced by a single factor, such as a high-fat diet (HF) of typical energy-dense food and infection by an adipogenic virus, such as a widely studied human adenovirus serotype 36 (Ad-36). In this study, we hypothesized and investigated the synergistic effect of two causal factors, HF and Ad-36, in obesity induction. METHODS: The 7-week-old Wistar rats (n = 1214/group) were randomly divided into weight-matched groups and induced for obesity with mock-control, HF, Ad-36, or HF + Ad-36 for 8-30 weeks, and compared for obesity phenotype. A global transcriptomic RNA-Seq analysis was used to profile signature gene response pathways in ileal tissues from 8-week control and obese animals during this early phase of obesity induction. RESULTS: HF only and particularly co-administration of Ad-36 and HF (HF + Ad-36) induced significant obesity in rats (p < 0.05 or p < 0.005). Compared with either Ad-36 or HF alone, HF + Ad-36 treatment significantly aggravates obesity in rats regarding body weight (n = 12-14/group) and adiposity index (n = 6-7). Genome-wide transcriptomic analyses of intestinal tissues revealed signature genes on an inter-systemic scale, including many genes in the pathways of circadian rhythm and antiviral immunity focusing on IFN signaling. CONCLUSIONS: Ad-36 exacerbated the induction of obesity in rats compared with those treated with HF alone. Gene-responsive pathways involved in circadian rhythm and antiviral immunity in ileal tissues were significantly (p < 0.05, and FDR < 0.01) regulated during the early phase of obesity induction. This study provided a co-factorial model for obesity induction and profiled molecular targets for further validation and molecular manipulation.

Analyses of pig genomes provide insight into porcine demography and evolution.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Reduction of infection by inhibiting mTOR pathway is associated with reversed repression of type I interferon by porcine reproductive and respiratory syndrome virus.

Type I interferons (IFNs) are critical in animal antiviral regulation. IFN-mediated signalling regulates hundreds of genes that are directly associated with antiviral, immune and other physiological responses. The signalling pathway mediated by mechanistic target of rapamycin (mTOR), a serine/threonine kinase regulated by IFNs, is key in regulation of cellular metabolism and was recently implicated in host antiviral responses. However, little is known about how animal type I IFN signalling coordinates immunometabolic reactions during antiviral defence. Here, using porcine reproductive and respiratory syndrome virus (PRRSV), we found that the genes in the mTOR signalling pathway were differently regulated in PRRSV-infected porcine alveolar macrophages at different activation statuses. Moreover, mTOR signalling regulated PRRSV infection in MARC-145 and primary porcine cells, in part, through modulating the production and signalling of type I IFNs. Taken together, we determined that the mTOR signalling pathway involves PRRSV infection and regulates expression and signalling of type I IFNs against viral infection. These findings suggest that the mTOR signalling pathway has a bi-directional loop with the type I IFN system and imply that some components in the mTOR signalling pathway can be utilized as targets for studying antiviral immunity and for designing therapeutic reagents.

Antiviral regulation in porcine monocytic cells at different activation states.

UNLABELLED: Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity. IMPORTANCE: Activation statuses of monocytic cells, including monocytes, macrophages (Mϕs), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic, including our focus, PRRSV, which alone causes nearly $800 million economic loss annually in the U.S. swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine the intricate interaction of viral infection with activation statuses and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.

Structural and functional annotation of the porcine immunome.

BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Differential expression and activity of the porcine type I interferon family.

Type I interferons (IFNs) are central to innate and adaptive immunity, and many have unique developmental and physiological functions. However, in most species, only two subtypes, IFN-alpha and IFN-beta, have been well studied. Because of the increasing importance of zoonotic viral diseases and the use of pigs to address human research questions, it is important to know the complete repertoire and activity of porcine type I IFNs. Here we show that porcine type I IFNs comprise at least 39 functional genes distributed along draft genomic sequences of chromosomes 1 and 10. These functional IFN genes are classified into 17 IFN-alpha subtypes, 11 IFN-delta subtypes, 7 IFN-omega subtypes, and single-subtype subclasses of IFN-alphaomega, IFN-beta, IFN-epsilon, and IFN-kappa. We found that porcine type I IFNs have diverse expression profiles and antiviral activities against porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), with activity ranging from 0 to >10(5) U.ng(-1).ml(-1). Whereas most IFN-alpha subtypes retained the greatest antiviral activity against both PRRSV and VSV in porcine and MARC-145 cells, some IFN-delta and IFN-omega subtypes, IFN-beta, and IFN-alphaomega differed in their antiviral activity based on target cells and viruses. Several IFNs, including IFN-alpha7/11, IFN-delta2/7, and IFN-omega4, exhibited minimal or no antiviral activity in the tested target cell-virus systems. Thus comparative studies showed that antiviral activity of porcine type I IFNs is virus- and cell-dependent, and IFN-alphas are positively correlated with induction of MxA, an IFN-stimulated gene. Collectively, these data provide fundamental genomic information for porcine type I IFNs, information that is necessary for understanding porcine physiological and antiviral responses.

Cross-Species Genome-Wide Analysis Reveals Molecular and Functional Diversity of the Unconventional Interferon-ω Subtype.

Innate immune interferons (IFNs), particularly type I IFNs, are primary mediators regulating animal antiviral, antitumor, and cell-proliferative activity. These antiviral cytokines have evolved remarkable molecular and functional diversity to confront ever-evolving viral threats and physiological regulation. We have annotated IFN gene families across 110 animal genomes, and showed that IFN genes, after originating in jawed fishes, had several significant evolutionary surges in vertebrate species of amphibians, bats and ungulates, particularly pigs and cattle. For example, pigs have the largest but still expanding type I IFN family consisting of nearly 60 IFN-coding genes that encode seven IFN subtypes including multigene subtypes of IFN-α, -δ, and -ω. Whereas, subtypes such as IFN-α and -ß have been widely studied in many species, the unconventional subtypes such as IFN-ω have barely been investigated. We have cross-species defined the IFN evolution, and shown that unconventional IFN subtypes particularly the IFN-ω subtype have evolved several novel features including: (1) being a signature multi-gene subtype expanding primarily in mammals such as bats and ungulates, (2) emerging isoforms that have superior antiviral potency than typical IFN-α, (3) highly cross-species antiviral (but little anti-proliferative) activity exerted in cells of humans and other mammalian species, and (4) demonstrating potential novel molecular and functional properties. This study focused on IFN-ω to investigate the immunogenetic evolution and functional diversity of unconventional IFN subtypes, which may further IFN-based novel antiviral design pertinent to their cross-species high antiviral and novel activities.

Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7.

To investigate porcine Toll-like receptors (TLR) responding to viral pathogen associated molecular patterns, the full-length cDNA of porcine TLR3 and TLR7 were identified and characterized. Porcine TLR3 and TLR7 cDNA encode 904- and 1050-amnio-acid polypeptides, respectively. Both porcine TLR3 and TLR7 contain typical functional TLR domains and share about 80% sequence identity to other mammalian orthologues. Tissue expression profiles showed that TLR3 was highly expressed in kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin. Conversely, TLR7 was moderately and constitutively expressed in all tissues evaluated. Stimulation of mammalian cells transfected with porcine TLR3 and TLR7 constructs with TLR3 and TLR7 agonists [poly (I:C) and imiquimod (R837), respectively], and adenovirus elicited activation of interferon regulatory factors (IRFs). These data provide molecular and functional information for porcine TLR3 and TLR7, and implicate their role in mediating immune protection against porcine viral diseases.

Molecular cloning and characterization of equine Toll-like receptor 9.

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.

Antimicrobial peptides and bacteriocins: alternatives to traditional antibiotics.

Antimicrobial peptides (AMPs) are ubiquitous, gene-encoded natural antibiotics that have gained recent attention in the search for new antimicrobials to combat infectious disease. In multicellular organisms, AMPs, such as defensins and cathelicidins, provide a coordinated protective response against infection and are a principal component of innate immunity in vertebrates. In unicellular organisms, AMPs, such as bacteriocins, function to suppress competitor species. Because many AMPs kill bacteria by disruption of membrane integrity and are thus thought to be less likely to induce resistance, AMPs are being extensively evaluated as novel antimicrobial drugs. This review summarizes and discusses the antibiotic properties of AMPs highlighting their potential as alternatives to conventional antibiotics.

Central nervous system administration of interleukin-6 produces splenic sympathoexcitation.

Interleukin-6 (IL-6) is a multifunctional cytokine that has been shown to play a pivotal role in centrally-mediated physiological responses including activation of the hypothalamic-pituitary-adrenal axis. Cerebral spinal fluid (CSF) concentrations of IL-6 are elevated in multiple pathophysiological conditions including Alzheimer's disease, autoimmune disease, and meningitis. Despite this, the effect of IL-6 on central regulation of sympathetic nerve discharge (SND) remains unknown which limits understanding of sympathetic-immune interactions in health and disease. In the present study we determined the effect of intracerebroventricular (i.c.v, lateral ventricle) administration of IL-6 on splenic SND in urethane-chloralose-anesthetized rats. A second goal was to determine if icv injected IL-6 enters the brain parenchyma and acts as a volume transmission signal to access areas of the brain involved in regulation of sympathetic nerve outflow. i.c.v administration of IL-6 (10 ng, 100 ng, and 400 ng) significantly and progressively increased splenic SND from control levels in baroreceptor-denervated Sprague-Dawley rats. Administration of 100-ng and 400-ng IL-6 resulted in significantly higher SND responses when compared to those elicited with a 10-ng dose. Sixty minutes following icv administration, fluorescently labeled IL-6 was not distributed throughout the parenchyma of the brain but was localized to the periventricular areas of the ventricular system. Brain sections counter-stained for the IL-6 receptor (IL-6R) revealed that IL-6 and the IL-6R were co-localized in periventricular areas adjoining the third ventricle. These results demonstrate that icv IL-6 administration increases splenic SND, an effect likely achieved via signaling mechanisms originating in the periventricular cells.

Innate immunity and inflammation--New frontiers in comparative cardiovascular pathology.

Innate immunity and inflammation play key roles in a wide range of pathology - including heart disease and vasculopathies. Current thinking suggests "damage" rather than "foreignness" as the actual trigger of the immune system, which has caused a dramatic change in how we tend to view the etiopathology of most types of heart disease. The future potential of certain anti-inflammatory therapeutic strategies in addressing heart disease is intriguing. Still, the Janus face of immunity/inflammation cannot be over emphasized as adverse manipulation of these systems may prove ineffectual or worse, damaging. Knowledge on functional characteristics of individual immune mediators is undoubtedly a central theme, but in depth understanding of the multiple biological actions of these molecules, as well as their contextual function, is the corner stone in deciding on potential future targets for pharmacologic manipulation. Animal models of human heart disease are currently being investigated and clinical trials conducted to gain further knowledge in this essential area of cardiovascular research, but the scarcity of cardiovascular research focusing on signaling molecules and pathways of innate immunity is still evident. Genomic and proteomic research in heart disease is going through its formative years, and much is still unknown about the complex pathway dynamics utilized by the innate immune system. This review will provide an overview of the current literature focusing on innate immunity and the heart, and hopefully will spark an interest in further basic as well as clinical research. As more information on cardiovascular immunity becomes available, this will provide a better understanding and thus act as the foundation for potential development of new treatment strategies for treatment of cardiovascular disorders.

Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide.

Cathelicidins, a group of cationic peptides found in leukocytes and epithelial cells, play a central role in the early innate immune defense against infection. Although these host defense peptides have been reported in several mammalian species, including primates, no cathelicidins have been identified in carnivores. Here we report the cloning, tissue expression and biological activity of a novel canine cathelicidin (K9CATH). The full-length cDNA sequence of K9CATH encodes a predicted 172 amino acid pre-propeptide that is 60-70% similar to other mammalian cathelicidins. Mass spectrometry analysis confirmed that the 38 aa mature K9CATH peptide was present in neutrophil granule contents. Synthetic K9CATH displayed broad antimicrobial activity against Gram-positive bacteria (Listeria monocytogenes, and Staphylococcus aureus; MICs (minimal inhibitory concentrations) 0.5 and 50 microM, respectively), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Pseudomonas aeruginosa, Proteus mirabilis; MICs 1.25 microM, Salmonella serotype Enteritidis; MIC 0.5 microM, and Neisseria gonorrhoeae; MIC 0.06 microM), and yeast (Candida albicans; MIC 12.5-50 microM). K9CATH demonstrated high antimicrobial activity against Ureaplasma canigenitalium, and lower activity against Ureaplasma urealyticum (MIC 0.06 and 50 microM, respectively). Similar to its ovine congener SMAP-29, K9CATH possesses salt-independent antimicrobial activity and LPS binding capacity. K9CATH displayed minimal hemolytic activity against human, dog and chicken erythrocytes. The potency and broad antimicrobial activity of K9CATH suggest that this peptide may act as a fundamental contributor to the innate immune responses in this carnivore species.

Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived cells to different stimuli.

Monocyte-derived DCs (mDCs) are major target cells in porcine reproductive and respiratory syndrome virus (PRRSV) pathogenesis; however, the plasticity of mDCs in response to activation stimuli and PRRSV infection remains unstudied. In this study, we polarized mDCs, and applied genome-wide transcriptomic analysis and predicted protein-protein interaction networks to compare signature genes involved in mDCs activation and response to PRRSV infection. Porcine mDCs were polarized with mediators for 30 hours, then mock-infected, infected with PRRSV strain VR2332, or a highly pathogenic PRRSV strain (rJXwn06), for 5 h. Total RNA was extracted and used to construct sequencing libraries for RNA-Seq. Comparisons were made between each polarized and unpolarized group (i.e. mediator vs. PBS), and between PRRSV-infected and uninfected cells stimulated with the same mediator. Differentially expressed genes (DEG) from the comparisons were used for prediction of interaction networks affected by the viruses and mediators. The results showed that PRRSV infection inhibited M1-prone immune activity, downregulated genes, predicted network interactions related to cellular integrity, and inflammatory signaling in favor of M2 activity. Additionally, the number of DEG and predicted network interactions stimulated in HP-PRRSV infected mDCs was superior to the VR-2332 infected mDCs and conformed with HP-PRRSV pathogenicity.

Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: cloning and induction by bacterial infection.

Hepcidin is a liver-expressed iron-regulating hormone that also is an antimicrobial peptide. Here we report the full-length cDNA sequences of porcine hepcidin and liver-expressed antimicrobial peptide-2 (LEAP-2). Porcine hepcidin and LEAP-2 cDNA sequences contain 411 and 525 bp, and encode predicted peptides of 82 and 77 amino acid residues, respectively. Both porcine hepcidin and LEAP-2 are highly expressed in liver and LEAP-2 also is expressed in intestinal tissues and kidney. Pigs infected with Salmonella enterica serovar Typhimurium showed inducible but differential expression of hepcidin and LEAP-2 in bone marrow and intestinal tissues. Conversely, although highly expressed in liver, expression of hepcidin mRNA in liver was not influenced by Salmonella infection. These findings provide fundamental comparative data showing the relationship of porcine hepcidin and LEAP-2 to other mammalian orthologs and indicate that bacterial infections influence their expression.

Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.

Interferons (IFNs) are key cytokines identified in vertebrates and evolutionary dominance of intronless IFN genes in amniotes is a signature event in IFN evolution. For the first time, we show that the emergence and expansion of intronless IFN genes is evident in amphibians, shown by 24-37 intronless IFN genes in each frog species. Amphibian IFNs represent a molecular complex more complicated than those in other vertebrate species, which revises the established model of IFN evolution to facilitate re-inspection of IFN molecular and functional diversity. We identified these intronless amphibian IFNs and their intron-containing progenitors, and functionally characterized constitutive and inductive expression and antimicrobial roles in infections caused by zoonotic pathogens, such as influenza viruses and Listeria monocytogenes. Amphibians, therefore, may serve as overlooked vectors/hosts for zoonotic pathogens, and the amphibian IFN system provides a model to study IFN evolution in molecular and functional diversity in coping with dramatic environmental changes during terrestrial adaption.

Cross-species analysis of the mammalian beta-defensin gene family: presence of syntenic gene clusters and preferential expression in the male reproductive tract.

Mammalian beta-defensins are an important family of innate host defense peptides with pleiotropic activities. As a first step to study the evolutionary relationship and biological role of the beta-defensin family, we identified their complete repertoires in the human, chimpanzee, mouse, rat, and dog following systemic, genome-wide computational searches. Although most beta-defensin genes are composed of two exons separated by an intron of variable length, some contain an additional one or two exons encoding an internal pro-sequence, a segment of carboxy-terminal mature sequences or untranslated regions. Alternatively, spliced isoforms have also been found with several beta-defensins. Furthermore, all beta-defensin genes are densely clustered in four to five syntenic chromosomal regions, with each cluster spanning <1.2 Mb across the five species. Phylogenetic analysis indicated that, although the majority of beta-defensins are evolutionarily conserved across species, subgroups of gene lineages exist that are specific in certain species, implying that some beta-defensins originated after divergence of these mammals from each other, while most others arose before the last common ancestor of mammals. Surprisingly, RT-PCR revealed that all but one rat beta-defensin transcript are preferentially expressed in the male reproductive tract, particularly in epididymis and testis, except that Defb4, a human beta-defensin-2 ortholog, is more restricted to the respiratory and upper gastrointestinal tracts. Moreover, most beta-defensins expressed in the reproductive tract are developmentally regulated, with enhanced expression during sexual maturation. Existence of such a vast array of beta-defensins in the male reproductive tract suggests that these genes may play a dual role in both fertility and host defense.

PU.1-mediated transcriptional regulation of prophenin-2 in primary bone marrow cells.

Prophenin-2 (PF-2) is a cathelicidin, 97-amino-acid antimicrobial protein stored in neutrophil secondary granules. PF-2 is expressed specifically in porcine immature myeloid cells; however, little is known about its regulation. In this study, we characterized the 5' regulatory regions of the PF-2 gene to understand the molecular mechanisms regulating its expression. Using bioinformatic approaches, site-directed mutagenesis, and transactivation experiments, we found that the PF-2 gene was regulated by transcription factor PU.1. In addition, PF-2 expression also is regulated by the cytokines GM-CSF and IL-3. Taken together, these results identify cis- and trans-acting factors involved in the regulation of PF-2 and clarify mechanisms of cathelidicin gene regulation.

Cloning of porcine triggering receptor expressed on myeloid cells-1 (TREM-1) and its induction by lipopolysaccharide, peptidoglycan, and Salmonella enterica serovar Typhimurium infection.

Triggering receptors expressed on myeloid cells (TREM) are a family of activating receptors expressed on neutrophils and monocytes. These receptors are involved in regulation of immunity by inducing the expression of inflammatory cytokines and adhesion molecules, augmenting dendritic cell maturation, and are implicated in septic shock. Here we report the cloning of full-length TREM cDNA from porcine bone marrow cells, which predicts a 238 amino-acid peptide. Treating porcine bone marrow cells with lipopolysaccharide or peptidoglycan caused an increase in TREM-1 expression. Moreover, bone marrow cells derived from pigs that were orally challenged with Salmonella enterica serovar Typhimurium showed increases in TREM-1 at 8 and 24 h post-infection, respectively. Complete down-regulation of TREM-1 expression was observed at 48 h post-infection. These findings provide fundamental comparative data indicating that bacterial infection induces TREM-1 expression.