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

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

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

CXCL1 serum levels in patients with alcoholic cirrhosis and healthy controls with distinct <i>CXCL1</i>

<div><p><b>rs4074 genotypes</b>. </p> <p>This figure shows CXCL1 serum concentrations in 66 healthy controls (grey columns) and in 66 patients with alcoholic cirrhosis (black columns) stratified with respect to rs4074 variants G/G, G/A and A/A. Results are shown as means ± standard errors. Groups were compared with the Mann-Whitney U test. </p></div

Frequency of the rs4074 A allele in the study groups.

<p>This figure illustrates that carriers of the <i>CXCL1 </i><i>rs4074</i> A allele were equally frequent in patients with alcohol related cirrhosis without and with HCC, but overrepresented in comparison to patients with alcohol abuse without liver damage and to healthy controls. Statistical significances refer to Fisher´s exact test.</p

Upregulation of fibrosis markers in human HSC after stimulation with CXCL1.

<p>After incubation with and without 250pg/ml recombinant CXCL1 for 16 hours, human HSC were stained for α-SMA and Collagen type I and analysed by flow cytometry. This representative set of histograms shows that α-SMA and Collagen type I expression increases after CXCL1 stimulation (solid lines) as compared to the unstimlated controls (dotted line).</p

In Vitro CXCL1 induction in TLR2- and TLR4-transfected HEK293 cells.

<p>HEK293 cells stably transfected with human TLR2 (HEK/TLR2) and human TLR4 (HEK/TLR4) as well as untransfected HEK293 cells (HEK -) were incubated with sera from patients with alcoholic cirrhosis (box plots on the right side) and sera from healthy controls (box plots on the left side). Stimulation with sera from cirrhotic patients showed significantly enhanced CXCL1 secretion in the TLR2- and TLR4-transfected cells but not in the un-transfected control cells. In contrast, sera from healthy controls did not up-regulate CXCL1 secretion in the TLR2- and TLR4-transfected HEK293 cells. P values were calculated using Mann-Whitney U test.</p