Toll-like receptor (TLR) 2 and TLR4 gene expression in canine heart

Toll-like receptors (TLRs) are archetypal pattern recognition receptors of immediate importance for an efficacious innate immune response. TLRs exhibit marked differential tissue activity and their levels within a discrete cell type can be highly dynamic. Of 13 known mammalian paralogues, three TLRs have been identified in the dog. Although cardiac TLR expression has been reported in other species, this study is the first to present evidence that these innate immune receptors are expressed in the canine heart. Heart tissue samples from all four chambers were collected from healthy dogs immediately after euthanasia and stored at -80°C until analysis. Total RNA was extracted with TRI Regent. Specific primers were designed for amplification of canine TLR2 and TLR4 based on previously reported sequences for these genes. Reverse transcription was performed with M-MLV reverse transcriptase. PCR amplification was performed and PCR products analyzed by agarose gel electrophoresis. Bands were excised from the gel and the DNA isolated and cloned using the TA Cloning® Kit. The correct sequence for each product was verified by nucleotide sequencing. TLR4 expression was detected in the left ventricle and right atrium; TLR2 was detectable at low levels in the right atrium only. Identity of the RT-PCR products was confirmed by sequencing. Our findings show that at least two TLR paralogues- namely TLR2 and TLR4 - are expressed in the canine heart. Additional studies are warranted to determine these immune receptors' potential implication in the development of naturally occurring heart disease in the dog

Molecular evolution of the porcine type I interferon family: subtype-specific expression and antiviral activity

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ~60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family

Antiviral regulation in porcine monocytic cells at different activation states

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

Porcine type I interferons: polymorphic sequences and activity against PRRSV

Background: Type I interferons (IFN) are a heterogeneous group of cytokines central to innate and adaptive antiviral immune responses. We have recently reported that porcine type I IFNs comprise at least 39 functional genes with diverse antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). Here we report that potential cytokine polymorphisms exist in several genes of porcine type I IFNs. Results: We have detected more than 100 potential polymorphic mutations, which include nucleotide substitutions and deletions, within the coding regions of porcine type I IFNs. Approximately 50% of the nucleotide changes were mutations that resulted in non-conserved amino acid substitution, as well as deletions that produced frame shifts in the open reading frames (ORFs). We have identified more than 20 polymorphic mutants that showed alterations in anti-PRRSV and anti-vesicular stomatitis virus (VSV) activity in vitro. In particular, some mutations in IFN-α2, IFN-α3, IFN-α8, IFN-α12 and IFN-ω5 significantly altered the antiviral activity of expressed proteins in comparison to the wild-type or variant with more similarity to the wild-type. Conclusions: Multiple polymorphic isoforms potentially exist within subtypes of the porcine type I IFN family. Polymorphic mutations are more common in multiple-member subtypes than single-member subtypes, and most are found within the IFN-α subclass. Some polymorphic isoforms have altered amino acid composition and shifted ORFs, which show significantly different antiviral activity in vitro

Effect of regrouping unfamiliar pigs at weaning on immune function

Pigs weaned at 3 weeks of age and regrouped with unfamiliar individuals had a 4-fold increase in plasma cortisol when compared to pigs that remained in a litter group. However, cellular measures of immune function were not altered by regrouping.; Swine Day, Manhattan, KS, November 10, 198

Effect of feeding streptococcus faecium to artificially reared pigs

Two trials were conducted with a total of 112 artificially reared pigs to evaluate the effect feeding Streptococcus faecium. The areas studied were growth and feed efficiency, mortality rate, daily scour score, blood parameters (total leukocyte numbers and differentials), and in vivo determination of cell-mediated immunity. The results of the trial indicate that there was no significant advantage to feeding Streptococcus faecium to artificially reared pigs.; Swine Day, Manhattan, KS, November 10, 198

Cellular immune responses in artificially reared pigs

An experiment was conducted to determine the influence of artificial rearing on the cellular immune response of young pigs. Artificially reared pigs had lower cellular immune reactivity than sow-reared controls. These results indicate that artificial rearing may result in immunosuppression in young pigs.; Swine Day, Manhattan, KS, November 15, 198

Neutrophil and lymphocyte response to vitamins C and E supplementation in young calves

Calves were bottle-fed milk replacers at 10% of weekly adjusted body weight for 8 wk. Treatments were 1) no supplements (control), 2) .16 oz vitamin C, or 3) .16 oz vitamin C plus 125 IU/lb vitamin E. Lymphocytes and neutrophils isolated from day 14 and day 28 blood samples were assayed for neutrophil-mediated S. aureus phagocytosis and antibody-dependent cellular cytotoxicity, and for mitogen induced lymphocyte proliferation. Eye and nasal discharges of calves supplemented with vitamin C and vitamins C plus E were less than those of control calves for wk 1 to 8. Lymphocyte proliferation with the mitogens showed a trend for higher responses at wk 2 in vitamin C plus E supplemented calves. Neutrophils of calves supplemented with vitamin C showed decreased phagocytosis and lysis functions compared to those of control calves at wk 2 and 4. Neutrophil function of calves supplemented with vitamins C plus E was near or slightly higher than that of controls at wk 2 and 4, suggesting that the addition of vitamin E negated the adverse effects that vitamin C alone had on neutrophil functions.; Dairy Day, 1989, Kansas State University, Manhattan, KS, 1989; The 1989 Annual KSU Dairy Day is known as Dairy Day, 198

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

Citation: Sang, Y. M., Liu, Q. F., Lee, J., Ma, W. J., McVey, D. S., & Blecha, F. (2016). Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity. Scientific Reports, 6, 17. doi:10.1038/srep29072Interferons (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