Resveratrol Impairs the Release of Steroid-Resistant Inflammatory Cytokines from Human Airway Smooth Muscle Cells in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) therapy is complicated by corticosteroid resistance of the interleukin 8 (IL-8)dependent and granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent chronic airway inflammation, for whose establishment human airway smooth muscle cells (HASMCs) might be crucial. It is unclear whether the release of inflammatory mediators from HASMCs is modulated by cigarette smoking and is refractory to corticosteroids in COPD. Resveratrol, an antiaging drug with protective effects against lung cancer, might be an alternative to corticosteroids in COPD therapy. Vascular endothelial growth factor (VEGF) might offer protection from developing emphysema. We tested the following hypotheses for HASMCs: 1) smoking with or without airway obstruction modulates IL-8, GM-CSF, and VEGF release; and 2) corticosteroids, but not resveratrol, fail to inhibit cytokine release in COPD. Cytokine release from HASMCs exposed to tumor necrosis factor alpha (TNF alpha), dexamethasone, and/or resveratrol was measured via enzyme-linked immunosorbent as-say and compared between nonsmokers (NS), smokers without COPD (S), and smokers with COPD (all n = 10). In response to TNF alpha, IL-8 release was increased, but GM-CSF and VEGF release was decreased in S and COPD compared with NS. Dexamethasone and resveratrol inhibited concentration-dependently TNF alpha-induced IL-8, GM-CSF, and VEGF release. For IL-8 and GM-CSF efficiency of dexamethasone was NS > S > COPD. That of resveratrol was NS = S = COPD for IL-8 and NS = S = COPD for GM-CSF. For VEGF the efficiency of dexamethasone was NS = S = COPD, and that of resveratrol was NS = S > COPD. All resveratrol effects were partially based on p38 mitogen-activated protein kinase blockade. In conclusion, smoking modulates cytokine release from HASMCs. Corticosteroid refractoriness of HASMCs in COPD is cytokine-dependent. Resveratrol might be superior to corticosteroids in COPD therapy, because it more efficiently reduces the release of inflammatory mediators and has limited effects on VEGF in COPD

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C.

BACKGROUND: In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C. METHODS: Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(-) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15). RESULTS: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis. CONCLUSION: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis

Frequency of CXCR3 expressing NK cells in chronic hepatitis C.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g004" target="_blank">Figure 4A</a> compares frequency of peripheral CXCR3(+) NK cells in HCV infected (n = 14) and healthy individuals (n = 16). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g004" target="_blank">Figure 4B:</a> Peripheral and liver-infiltrating NK cells from HCV-infected individuals were analyzed for expression of CXCR3 by flowcytometry Then frequency of circulating (left graph) and intra-hepatic (right graph) CXCR3(+)CD56Bright NK cells was compared between patients with progressive (F≥3; n = 6) and less advanced (F<3; n = 9) liver fibrosis. Results are given as box and whisker plots, with medians and 10th, 25th, 75th, and 90th percentiles. * indicates p<0.05.</p

Patient Characteristics.

a)<p>number of cases (number/total in %).</p>b)<p>mean (range).</p>c)<p>n.a. – not analyzed.</p

CXCR3 expression dissects phenotypically distinct NK cell subsets.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g001" target="_blank">Figure 1A:</a> PBMCs in whole blood specimen were stained with anti-CD3, anti-CD56, and anti-CXCR3. CD3⁽⁻⁾ CD56⁽⁺⁾ NK cells were then gated for quantification of CXCR3 surface expression. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g001" target="_blank">Figure 1B:</a> PBMCs from at least eight healthy donors were stained with anti-CD3, anti-CD56, and anti-CXCR3–conjugated mAb, as well as a mAb directed against the indicated maturation markers. CXCR3(+) and CXCR3(−) NK cell populations were then assessed for surface expression of the respective maturation marker. Results are given as box and whisker plots, with medians and 10th, 25th, 75th, and 90th percentiles. * indicates p<0.05; **indicates p<0.01; *** indicates p<0.001.</p

Role of NKG2D in anti-fibrotic activity of CXCR3-expressing NK cells.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g003" target="_blank">Figure 3A:</a> Circulating NK cells obtained from HCV(-) individuals (n = 5) were analyzed for co-expression of NKG2D and CXCR3. The figure compares frequency of NKG2D-positive cells (left graph) as well as density of NKG2D surface expression (relative fluorescence intensity, RFI) (right graph) between the four studied NK cell subsets. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g003" target="_blank">Figure 3B</a> shows the effect of NKG2D blockade on HSC-induced degranulation of CXCR3(+) and CXCR3(−) CD56Bright NK cell subsets obtained from healthy donors (n = 8). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g003" target="_blank">Figure 3C</a> compares HSC-induced degranulation of CXCR3(+) and CXCR3(−) CD56Bright NK cell subsets (n = 8) following pre-incubation with anti-NKG2D. Results are given as box and whisker plots, with medians and 10th, 25th, 75th, and 90th percentiles. * indicates p<0.05; **indicates p<0.01; *** indicates p<0.001.</p

CXCR3(+)CD56Bright NK cells have strong anti-fibrotic potential.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2A:</a> Purified peripheral NK cells from healthy individuals (n = 14) were co-incubated with primary human hepatic stellate cells (E:T ratio 1∶1) and then analyzed with respect to degranulation (CD107a) and expression of CXCR3 by flowcytometry. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2B</a> depicts NK cell degranulation following co-incubation with HSC at different effector : target (E:T) ratios as indicated (n = 5). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2C</a> shows CD107a expression of the four studied NK cell sub-populations obtained from healthy donors (n = 14) following co-incubation with primary hepatic stellate cells (E:T ratio 1∶1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2D</a> illustrates IFN-γ production of purified CXCR3(+) and CXCR3(−) NK cells obtained from HCV(-) individuals (n = 14) after co-culturing with primary HSC (E:T ratio 1∶1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2E</a> shows IFN-γ production of purified NK cells following co-incubation with hepatic stellate cells at different E:T ratios (n = 4). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038846#pone-0038846-g002" target="_blank">Figure 2F</a> displays IFN-γ production of the four studied NK cell sub-populations isolated from healthy donors (n = 14) following co-incubation with primary hepatic stellate cells (E:T ratio 1∶1).</p