Human NK Cell Subset Functions Are Differentially Affected by Adipokines

Background: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Adjuvant Effect of Orally Applied Preparations Containing Non-Digestible Polysaccharides on Influenza Vaccination in Healthy Seniors: A Double-Blind, Randomised, Controlled Pilot Trial.

Senior individuals can suffer from immunosenescence and novel strategies to bolster the immune response could contribute to healthy ageing. In this double-blind, randomised, controlled pilot trial, we investigated the ability of non-digestible polysaccharide (NPS) preparations to enhance the immune response in a human vaccination model. In total, 239 subjects (aged 50-79 years) were randomised to consume one of five different NPS (yeast β-glucan (YBG), shiitake β-glucan (SBG), oat β-glucan (OBG), arabinoxylan (AX), bacterial exopolysaccharide (EPS)) or control (CTRL) product daily for five weeks. After two weeks of intervention, subjects were vaccinated with seasonal influenza vaccine. The post-vaccination increases in haemagglutination inhibition antibody titres and seroprotection rate against the influenza strains were non-significantly enhanced in the NPS intervention groups compared to CTRL. Specifically, a trend towards a higher mean log2 fold increase was observed in the AX group (uncorrected p = 0.074) combined with a trend for an increased seroprotection rate, AX group (48.7%) compared to CTRL (25.6%) (uncorrected p = 0.057), for the influenza A H1N1 strain. Subjects consuming AX also had a reduced incidence of common colds compared to CTRL (1 vs. 8; p = 0.029 in Fisher exact test). No adverse effects of NPS consumption were reported. The findings of this pilot study warrant further research to study AX as an oral adjuvant to support vaccine efficacy

Human NK cell subset functions are differentially affected by adipokines.

BACKGROUND: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host's primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. METHODS: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). RESULTS: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56(dim) NK cells. The production of GzmA in CD56(bright) NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. CONCLUSION: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Altered NK cell function in obese healthy humans

Background: Obesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (30 kg/m2). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme+ and IFN-γ+ NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined. Results: As opposed to similar NK cell counts, the number of CD3+CD56+ cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells. Conclusions: NK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge

Lymphocyte numbers after 24 hours of stimulation with ACM (adipocte-conditioned medium).

<p>Human peripheral blood mononuclear cells (PBMCs) were isolated from six leukocyte filters and cultured for 24 hours with R10-medium conditioned with 0.1%, 1% or 10% of SGBS ACM. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the materials and methods section. NK cells, T cells and NKT cells are shown as percent of lymphocytes. CD56^dim and CD56^bright NK cells are shown as percent of all NK cells.</p

Ob-R (leptin receptor) expression after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) isolated from six leukocyte filters in each case were cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 or day 11 after induction of adipogenic differentiation. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) Ob-R-expressing human blood NK cells as percentage of human blood NK cells; B) Ob-R-expressing human blood CD56^dim NK cells as percentage of human blood CD56^dim NK cells; C) Ob-R-expressing human blood CD56^bright NK cells as percentage of human blood CD56^bright NK cells. D) Ob-R-expressing human blood NK cells as percentage of human blood NK cells from lean (nw, BMI <25, n = 3) and overweight (ow, BMI ≥25, n = 3) humans after 24 hours of stimulation with 1% SGBS ACM harvested on day 7 after induction of adipogenesis, controls were incubated for 24 hours with medium only. All data represent means+SEM.</p

Ability of human blood NK cells to form conjugates with cells of the K562 erythroleukemia line after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) isolated from six leukocyte filters in each case were cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 or day 11 after induction of adipogenic differentiation. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) About 30% of the NK cells (CD3⁻CD56⁺) were able to form conjugates with K562 target cells (upper right quadrant) irrespective of the cultivation conditions. B) Conjugate forming human blood NK cells as percentage of human blood NK cells. All data represent means+SEM.</p

IFN-γ and GzmA expression after ACM stimulation.

<p>Human peripheral blood mononuclear cells (PBMCs) isolated from six leukocyte filters in each case were cultured for 24 hours with R10-medium conditioned with 1% of SGBS ACM harvested on day 7 (d7) or day 11 (d11) after induction of adipogenesis. PBMCs incubated for 24 hours with medium only served as controls. A detailed description of the incubation procedure can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075703#s2" target="_blank">materials and methods</a> section. A) IFN-γ-expressing human blood CD56^dim and CD56^bright NK cells as percentage of human blood CD56^dim and CD56^bright NK cells treated with 1% of the ACM harvested on day 7 after induction of adipogenic differentiation; B) GzmA-expressing human blood CD56^dim NK cells as percentage of human blood CD56^dim NK cells treated with 1% of the ACM harvested on day 7 or 11 after induction of adipogenic differentiation; C) GzmA-expressing human blood CD56^bright NK cells as percentage of human blood CD56^bright NK cells treated with 1% of the ACM harvested on day 7 or 11 after induction of adipogenic differentiation; Statistically significant differences between the stimulation with SGBS ACM and the medium control are depicted with an asterisk (*). Statistically significant differences between the stimulation with SGBS ACM harvested on day 7 after induction of adipocyte differentiation and the same dosage of SGBS ACM harvested on day 11 after induction of adipogenic differentiation are depicted with a rhomb (#). Statistically significant differences between the numbers of IFN-γ expressing CD56^dim and CD56^bright NK cells are depicted with a paragraph sign (§). All data represent means+SEM.</p