Identification of a porcine liver eomes-high-T-bet-low NK cell subset that resembles human liver resident NK cells

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (Eomes(high)T-bet(low) in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3-CD8 alpha(high) cells, the porcine liver contains an abundant additional CD3-CD8 alpha(dim) NK cell subpopulation. In the current study, we show that this porcine CD3-CD8 alpha(dim) liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an Eomes(high)T-bet(low) expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e(-) and CXCR6(+). Furthermore, the porcine Eomes(high)T-bet(low) liver NK cell population is able to produce IFN-gamma upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine Eomes(high)T-bet(low) NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease

The variable effect of polyploidization on the phenotype in Escallonia

To induce new variation within the Escallonia genus, chromosome doubling was performed in E. rubra, E. rosea, and E. illinita, three important species within this genus of mainly evergreen woody ornamental species. Obtained tetraploids and diploid controls were analyzed for rooting capacity, leaf and flower characteristics, and plant architecture using image analysis and cold tolerance. In the present study, a breeders' collection of 23 accessions was characterized cytogenetically and described morphologically. All analyzed species and cultivars were diploid (2n =2x =24), with exception of E. pendula, a tetraploid. Today, breeding in Escallonia is limited to lucky finds in seedling populations and few efforts in interspecific hybridization. Three selected Escallonia species underwent an in vitro chromosome doubling with both oryzalin and trifluralin applied as either a continuous or shock treatment. The treatments successfully induced polyploids in all three species. Image analysis revealed that tetraploid E. rosea had decreased shoot length (from 3.8 to 1.3 cm), higher circularity and more dense growth habit compared to diploids. No significant changes in cold tolerance were seen. Tetraploid E. illinita did not differ in shoot length, but an increased outgrowth of axillary buds on the main axis led to denser plants. For tetraploid E. rubra, an increase in plant height (from 4.9 to 5.5 cm) was observed together with a large decrease in circularity and density due to a more polar outgrowth of branches on the main axis. E. rubra tetraploids bore larger flowers than diploids and had an increased cold tolerance (from 7.7 to 11.8-C). Leaf width and area of tetraploids increased for both E. illinita and E. rubra, while a decrease was seen in E. rosea genotypes. For all three species, the rooting capacity of the tetraploids did not differ from the diploids. We conclude that the effect of polyploidization on Escallonia was highly variable and species dependent

Pseudorabies virus infection causes downregulation of Ligands for the activating NK cell receptor NKG2D

Herpesviruses display a complex and carefully balanced interaction with important players in the antiviral immune response of immunocompetent natural hosts, including natural killer (NK) cells. With regard to NK cells, this delicate balance is illustrated on the one hand by severe herpesvirus disease reported in individuals with NK cell deficiencies and on the other hand by several NK cell evasion strategies described for herpesviruses. In the current study, we report that porcine cells infected with the porcine alphaherpesvirus pseudorabies virus (PRV) display a rapid and progressive downregulation of ligands for the major activating NK cell receptor NKG2D. This downregulation consists both of a downregulation of NKG2D ligands that are already expressed on the cell surface of an infected cell and an inhibition of cell surface expression of newly expressed NKG2D ligands. Flow cytometry and RT-qPCR assays showed that PRV infection results in downregulation of the porcine NKG2D ligand pULBP1 from the cell surface and a very substantial suppression of mRNA expression of pULBP1 and of another potential NKG2D ligand, pMIC2. Furthermore, PRV-induced NKG2D ligand downregulation was found to be independent of late viral gene expression. In conclusion, we report that PRV infection of host cells results in a very pronounced downregulation of ligands for the activating NK cell receptor NKG2D, representing an additional NK evasion strategy of PRV

β-glucan-induced IL-10 secretion by monocytes triggers porcine NK cell cytotoxicity

Beta-glucans are naturally occurring polysaccharides present in cell walls of fungi, yeast, bacteria, cereals, seaweed, and algae. These microbe-associated molecular patterns (MAMPs) possess immunomodulatory properties. In human, it has been suggested that NK cells can be activated by β-glucans. Here, we aimed to elucidate whether β-glucans modulate porcine NK cell responses in vitro and if so, how these effects are mediated. We investigated the effect of two β-glucans, Macrogard and Curdlan, which differ in solubility and structure. Direct addition of β-glucans to purified porcine NK cells did not affect cytotoxicity of these cells against K562 target cells. However, when using PBMC instead of purified NK cells, β-glucan addition significantly increased NK cell-mediated cytotoxicity. This effect depended on factors secreted by CD14+ monocytes upon β-glucan priming. Further analysis showed that monocytes secrete TNF-α, IL-6, and IL-10 upon β-glucan addition. Of these, IL-10 turned out to play a critical role in β-glucan-triggered NK cell cytotoxicity, since depletion of IL-10 completely abrogated the β-glucan-induced increase in cytotoxicity. Furthermore, addition of recombinant IL-10 to purified NK cells was sufficient to enhance cytotoxicity. In conclusion, we show that β-glucans trigger IL-10 secretion by porcine monocytes, which in turn leads to increased NK cell cytotoxicity, and thereby identify IL-10 as a potent stimulus of porcine NK cell cytotoxicity

Paradoxical reaction in non HIV-tuberculosis, a rare case with pancreatic involvement

A 24-year-old young man presents a sudden hepatic cytolysis and cholestasis after two months of isoniazid, rifampin and pyrazinamide therapy for severe lung tuberculosis. Initial data included heroin consumption history, HIV-negative serology, drug sensitivity and a normal abdominal CT

Introgression of rol genes from rhizogenic Agrobacterium strains into Escallonia spp.

The introgression of rol-genes of rhizogenic Agrobacterium into the plant genome induces changes in plant phenotype and physiology. However, only limited experience with this technique is available for woody ornamentals. To induce new variation within the Escallonia genus, several species were co-cultivated with rhizogenic Agrobacterium strains. Co-cultivation of three rhizogenic Agrobacterium strains (Arqua1, LMG 63 and MAFF210266) with four Escallonia species (E. illinita, E. myrtoidea, E. rosea, and E. rubra), resulted in hairy roots production with a varying efficiency. Co-cultivation of E. rubra with MAFF210266, and E. myrtoidea with LMG63 did not yield hairy roots, while co-cultivation of E. rubra leaves with LMG63 was most successful for hairy root production (up to 80.6%). In addition, the efficiency of hairy root induction depended on the explant type (leaves or nodal sections). The presence of inserted rol-genes and aux-genes in hairy roots was molecularly confirmed using qPCR. Few shoots regenerated from hairy roots, but regeneration needs to be optimized for efficient implementation of rol-genes introgression in Escallonia breeding. Key Message: This research provides a protocol for the production of hairy roots with rol-genes inserted after co-cultivation of several species of Escallonia with rhizogenic Agrobacterium strains

Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells