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Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

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.

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.

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 (EomeshighT-betlow in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3−CD8αhigh cells, the porcine liver contains an abundant additional CD3−CD8αdim NK cell subpopulation. In the current study, we show that this porcine CD3−CD8αdim 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 EomeshighT-betlow expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e− and CXCR6+. Furthermore, the porcine EomeshighT-betlow liver NK cell population is able to produce IFN-γ 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 EomeshighT-betlow 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.