Perforin and granzyme A release as novel tool to measure NK cell activation in chickens.

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

A reverse vaccinology approach to design an mRNA-based vaccine to provoke a robust immune response against HIV-1.

There have been substantial advances in HIV research over the past three decades, but we are still far from our goal of eliminating HIV-1 infection entirely. Numerous ever-evolving antigens are produced as a result of HIV-1's genetic variability. Developing an effective vaccination is challenging because of the structural properties of the viral envelope glycoprotein that obscure conserved receptor-binding sites and the presence of carbohydrate moieties that prevent antibodies from reaching potential epitopes. To work on an HIV-specific vaccine, this study identified 5 HIV-surface proteins, from the literature, to screen potential epitopes and construct an mRNA vaccine. A wide range of immunological-informatics techniques were utilized to develop a construct that efficiently stimulated cellular and humoral immune responses. The vaccine was produced with 31 epitopes, a TLR4 agonist termed RpfE that acts as an adjuvant, secretion boosters, subcellular trafficking structures, and linkers. It was determined that this suggested vaccine would cover 98.9 percent of the population, making it widely available. We, furthermore, carried out an immunological simulation of the vaccine illustrating the active and stable responses from innate and adaptive immune cells, the memory cells remained active for up to 350 days after vaccine injection, whereas the antigen was excreted from the body within 24 hours. Docking performed with TLR-4 and TLR-3 showed significant interaction with -11.9kcal/mol and -18.2kcal/mol-1 respectively. Molecular dynamics simulations further validated the vaccine's stability, with a dissociation constant of 1.7E-11 for the TLR3-vaccine complex and 5.8E-11 for the TLR4-vaccine complex. Lastly, codon optimization was carried out to guarantee that the designed mRNA construct would be translated into the host successfully. This vaccine adaptation, if tested in-vitro, would be efficacious and potent as predicted.

Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures.

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian Wnt3a and Rspo1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. Isolated crypts from embryonic tissue of laying hens were growing in the presence of chicken WNT3 and RSPO1, whereas growth in the presence of mammalian Wnt3a and Rspo1 was limited. Moreover, the growth was increased by using Prostaglandin E2 (PGE2) and a Forkhead box O1-inhibitor (FOXO1-inhibitor), allowing to culture these organoids for 15 passages. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes and enteroendocrine cells in 2D structures. Overall, we show that chicken intestinal organoids can be cultured for multiple passages using chicken-derived WNT3 and RSPO1, PGE2, and FOXO1-inhibitor.

The Interplay between Salmonella and Intestinal Innate Immune Cells in Chickens.

Salmonellosis is a common infection in poultry, which results in huge economic losses in the poultry industry. At the same time, Salmonella infections are a threat to public health, since contaminated poultry products can lead to zoonotic infections. Antibiotics as feed additives have proven to be an effective prophylactic option to control Salmonella infections, but due to resistance issues in humans and animals, the use of antimicrobials in food animals has been banned in Europe. Hence, there is an urgent need to look for alternative strategies that can protect poultry against Salmonella infections. One such alternative could be to strengthen the innate immune system in young chickens in order to prevent early life infections. This can be achieved by administration of immune modulating molecules that target innate immune cells, for example via feed, or by in-ovo applications. We aimed to review the innate immune system in the chicken intestine; the main site of Salmonella entrance, and its responsiveness to Salmonella infection. Identifying the most important players in the innate immune response in the intestine is a first step in designing targeted approaches for immune modulation.