Preben Boysen

The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen‐free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free‐living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ‐free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.

NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics

Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46+ cells comprised 1–10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46+ cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46+ cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46+ cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL‐2 for a limited number of cell divisions. IL‐2‐activated NKp46+ cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL‐2‐activated NKp46+ cells killed the FcγR+ target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is anactivating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

ABSTRACT Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-γ) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-γ by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-γ production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-γ production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-γ production in young cattle provides an explanation for the nonspecific IFN-γ response frequently encountered in young cattle when using the IFN-γ test in diagnosis of mycobacterial infections.

The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

ABSTRACT Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection.

Induction of gut IgA production through T cell‐dependent and T cell‐independent pathways

The gut immune system protects against mucosal pathogens, maintains a mutualistic relationship with the microbiota, and establishes tolerance against food antigens. This requires a balance between immune effector responses and induction of tolerance. Disturbances of this strictly regulated balance can lead to infections or the development inflammatory diseases and allergies. Production of secretory IgA is a unique effector function at mucosal surfaces, and basal mechanisms regulating IgA production have been the focus of much recent research. These investigations have aimed at understanding how long‐term IgA‐mediated mucosal immunity can best be achieved by oral or sublingual vaccination, or at analyzing the relationship between IgA production, the composition of the gut microbiota, and protection from allergies and autoimmunity. This research has lead to a better understanding of the IgA system; but at the same time seemingly conflicting data have been generated. Here, we discuss how gut IgA production is controlled, with special focus on how differences between T cell‐dependent and T cell‐independent IgA production may explain some of these discrepancies.

Bovine natural killer cells

Natural killer (NK) cells have received much attention due to their cytotoxic abilities, often with a focus on their implications for cancer and transplantation. But despite their name, NK cells are also potent producers of cytokines like interferon-gamma. Recent discoveries of their interplay with dendritic cells and T-cells have shown that NK cells participate significantly in the onset and shaping of adaptive cellular immune responses, and increasingly these cells have become associated with protection from viral, bacterial and parasitic infections. Furthermore, they are substantially present in the placenta, apparently participating in the establishment of normal pregnancy. Consequently, NK cells have entered arenas of particular relevance in veterinary immunology. Limited data still exist on these cells in domestic animal species, much due to the lack of specific markers. However, bovine NK cells can be identified as NKp46 (CD335) expressing, CD3(-) lymphocytes. Recent studies have indicated a role for NK cells in important infectious diseases of cattle, and identified important bovine NK receptor families, including multiple KIRs and a single Ly49. In this review we will briefly summarize the current understanding of general NK cell biology, and then present the knowledge obtained thus far in the bovine species.

Bovine CD2 - /NKp46 + cells are fully functional natural killer cells with a high activation status

BackgroundNatural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2.ResultsBovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity.ConclusionThese results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation.

Natural killer cells in free‐living Mus musculus have a primed phenotype

Recent reports have shown that natural killer (NK) cells may be long‐lived, possess memory‐like features and may need microbial priming to become fully reactive. Thus, the notion that these cells are typically innate, nonadaptive lymphocytes has been challenged. If microbial priming is essential for functional maturity, it is necessary to raise the question whether NK cells of laboratory mice, kept under strict hygienic conditions, represent these cells adequately. In their natural habitat, mice will encounter microbes to a greater extent, and we here investigated whether NK cells of feral mice showed signs of being primed. In comparison with C57BL/6 mice raised under specific pathogen‐free conditions, NK cells from feral mice had high expression of CD69, KLRG1, granzyme B and NKp46 and a higher proportion of CD27+ cells, mostly CD11b−, as well as a higher presence in peripheral lymph nodes. Following cytokine stimulation, feral mouse NK cells had quickly inducible CD25 expression and a stronger interferon‐gamma response. These findings indicate a high degree of pre‐activation of NK cells of free‐living mice, indicating a strong environmental impact on NK cells, which may be highly relevant for interpretation of studies in the mouse model.

Natural killer cells in lymph nodes of healthy calves express CD16 and show both cytotoxic and cytokine-producing properties.

Natural killer (NK) cells were recently shown to play an important immunomodulatory role in lymph nodes. We here report the presence, phenotype and function of NK cells resident in lymph nodes of several anatomical sites of healthy calves. NKp46+/CD3-lymphocytes, recently demonstrated to precisely identify NK cells in all tested species, were present in the paracortex and the medulla of bovine lymph nodes. Most lymph node-derived NK cells expressed CD16 and perforin, and a lytic capacity was demonstrated, while a well-developed interferon-gamma response to interleukin-2 and interleukin-12 stimulation was also seen. Lymph node-derived NK cells differed from those in blood by a higher expression of the activation markers CD44 and CD25, as well as CD8. L-selectin (CD62L) was expressed by the majority of lymph node-derived NK cells, consistent with a dependency of this molecule for migration to lymph nodes. Unlike in blood, the majority of lymph node NK cells had little or no CD2 expression. Compared to available literature, calf lymph nodes contained NK cells in numbers equal to or higher than reported in humans, and clearly higher than in mice. These findings suggest a cytotoxic role of lymph node residing NK cells, beyond the predominantly cytokine-producing role previously inferred from studies on human NK cells.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age.

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3(-)CD2(+) and NKp46(+) largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3(-)CD2(+)NKp46(+). The remainder of the NK-like cells comprised two minor populations, CD3(-)CD2(+)NKp46(-) and CD3(-)CD2(-)NKp46(+); the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3(-)CD2(+) and NKp46(+) NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8alphaalpha and CD8alphabeta and did not express CD21, WC1, CD14 or gammadelta TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3(-)CD2(-)NKp46(+) population expressed CD8alpha compared to CD3(-)CD2(+)NKp46(+) cells. Furthermore, a significantly greater proportion of the CD3(-)CD2(+)NKp46(-) population expressed CD8 compared to total CD3(-)CD2(+) cells. Adult cattle had a significantly higher proportion of perforin(+) cells compared to calves aged </=6 weeks. In this age group, the majority of perforin(+) cells expressed NKp46, while in adults the majority of perforin(+) cells were NKp46(-). However, the proportion of NKp46(+) and CD3(-)CD2(+) cells that expressed perforin was not significantly different in any age group tested.