Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-toshoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5# end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

A clear-cut delineation of bovine bona fide dendritic cells (DC) from monocytes has proved challenging, given the high phenotypic and functional plasticity of these innate immune cells and the marked phenotypic differences between species. Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 (cDC1, cDC2), plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4−, cDC2 being Flt3+CD172a+CD13−CD4−, pDC being Flt3+CD172adimCD13−CD4+, and monocytes being Flt3−CD172ahighCD13−CD4−. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16−) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for “marker molecules” that—when targeted by flow cytometry—will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.

No abstract

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine–rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide–DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid–complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

Clostridium perfringens β-toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but without obvious thrombosis of affected vessels, suggesting altered hemostasis in the early phase of the disease. The objective of the present study was to investigate the effect of CPB on platelets, with respect to primary hemostasis. Our results demonstrate that CPB binds to porcine and human platelets and forms oligomers resulting in a time- and dose-dependent cell death. Platelets showed rapid ultrastructural changes, significantly decreased aggregation and could no longer be activated by thrombin. This indicates that CPB affects the physiological function of platelets and counteracts primary hemostasis. Our results add platelets to the list of target cells of CPB and extend the current hypothesis of its role in the pathogenesis of C. perfringens type C enteritis.

18Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell

Cathelicidins encompass a family of cationic peptides characterized by antimicrobial activity and other functions, such as the ability to enhance the sensing of nucleic acids by the innate immune system. The present study aimed to investigate the ability of the bovine cathelicidins indolicidin, bactenecin (Bac)1, Bac5, bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, and BMAP-34 to inhibit the growth of bacteria and to enhance the sensing of nucleic acid by the host’s immune system. BMAP-27 was the most effective at killing Staphylococcus aureus, Streptococcus uberis, and Escherichia coli, and this was dependent on its amphipathic structure and cationic charge. Although most cathelicidins possessed DNA complexing activity, only the alpha-helical BMAP cathelicidins and the cysteine-rich disulfide-bridged Bac1 were able to enhance the sensing of nucleic acids by primary epithelial cells. We also compared these responses with those mediated by neutrophils. Activation of neutrophils with phorbol myristate acetate resulted in degranulation and release of cathelicidins as well as bactericidal activity in the supernatants. However, only supernatants from unstimulated neutrophils were able to promote nucleic acid sensing in epithelial cells. Collectively, the present data support a role for certain bovine cathelicidins in helping the innate immune system to sense nucleic acids. The latter effect is observed at concentrations clearly below those required for direct antimicrobial functions. These findings are relevant in development of future strategies to promote protection at mucosal surfaces against pathogen invasion.