Altered NK cell function in obese healthy humans

Abstract

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 (<25 kg/m(2)) and obese (>30 kg/m(2)). 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.

Keywords: CD107a; Immunity; Leptin; Natural killer (NK) cells; Ob-R; Obesity; TRAIL.

Figure 1

Leukocyte subsets in healthy normal weight and obese subject . Isolated PBMCs from healthy normal weight and obese subjects were stained with antibodies and analyzed by flow cytometry. (A) NK (CD56⁺CD3⁻) cells as percentage of lymphocytes; (B) CD56^bright cells and (C) CD56^dim cells as percentage of NK cells. (D) Representative flow cytometry dot plots of CD56bright and CD56dim cells from a normal weight individual; insert in the dot plot show the prior gaiting for NK cells (CD56⁺CD3⁻) in the upper left quadrant. (E) CD3⁺CD56⁺ cells as percentage of lymphocytes; (F) T (CD3⁺CD56⁻) cells as percentage of lymphocytes. Data are expressed as mean + SEM. Significant effects of obese vs. normal weight subjects are indicated by an asterisk *p < 0.05.

Figure 2

Leptin receptor (Ob-R) expression and signaling pathway in PBMCs. PBMCs from healthy normal weight and obese subjects were stained with antibodies and analyzed by flow cytometry; (A) Numbers of Ob-R positive NK cells are shown in percentage of NK cells. Data are expressed as mean + SEM. Representative flow cytometry dot plots of ObR+ and ObR- NK cells from a normal weight (middle panel) and an obese individual (right panel) are shown. (B) Isolated PBMCs from normal weight and obese individuals (shown with body mass index (BMI)) were stimulated for 5 and 15 min with 50 nM human recombinant leptin. Phosphorylation of JAK2 (p-JAK2) was determined by Western blotting. Actin was used as control for equal loading. The experiment was repeated with 3 normal weight and 3 obese individuals, and a representative Western blot is shown. Quantitative analysis of the Western blots for p-JAK2. Values were normalized to actin and are shown as fold induction in arbitrary units (AU). Data are expressed as mean + SEM. Significant effects of obese vs. normal weight subjects are indicated by an asterisk *p < 0.05.

Figure 3

Expression of functional parameters of NK cells . PBMCs were stained with antibodies and analyzed by flow cytometry to evaluate the differences of functional parameters in normal weight and obese subjects. (A) TNF-related apoptosis-inducing ligand (TRAIL) positive NK cells, (B) CD178 positive NK cells, and (C) CD107a positive NK cells as percentage of NK cells. Representative flow cytometry dot plots of CD107a from a normal weight (middle panel) and an obese individual (right panel). Inserts in both dot plots show the prior gating for NK cells (CD56⁺CD3⁻) in the upper left quadrant. Data are expressed as mean + SEM. Significant effects of obese vs. normal weight subjects are indicated by an asterisk *p < 0.05.

Figure 4

Effects of a leptin stimulation on NK-cell-tumor-cell-conjugates and IFN-γ and Granzyme production of human NK cells. PBMCs from healthy normal weight and obese subjects were stimulated with 50 nM human recombinant leptin or treated with vehicle for 24 h. Thereafter, cells were stained with antibodies and analyzed by flow cytometry. (A) Numbers of NK-cell-tumor-cell-conjugates (K562 erythroleukemia line), (B) Granzyme positive NK, (C) IFN-γ positive NK cells are shown as percentage of NK cells. Data are expressed as mean + SEM. Significant effects of leptin stimulated IFN-γ positive vs. non-stimulated NK cells in the normal weight group are indicated by an asterisk *p < 0.05.

Figure 5

Effect of leptin stimulation on cell proliferation of human NK cells . PBMCs from healthy normal weight and obese subjects were stimulated with 50 nM human recombinant leptin or vehicle for 7 days, stained with antibodies and analyzed with flow cytometry. (A) Representative histograms of CSFE staining showing four proliferation peaks of NK cells from a normal weight (upper histogram) and an obese (lower histogram) subjects. The peaks, from P4 (parental generation) to P1, represent successive generations. (B) Numbers of proliferating NK cells as percentage of NK cells. Data are expressed as mean + SEM.