The effects of PCM and PCM CD154-depleted upon human hepatocyte ROS accumulation and cell death.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g005" target="_blank">Figures 5a</a> demonstrates representative flow cytometry plots to illustrate the effect of PCM (hatched red) and PCM CD154-depleted (solid grey) upon human hepatocyte ROS accumulation during H-R. The gate used to analyse primary human hepatocytes is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figures 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">2</a>. The area of interest within the flow cytometry plots is marked by the vertical ellipse. The area on the left of each ellipse again represents cell debris. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g005" target="_blank">Figure 5b</a>, human hepatocytes were treated with PCM or PCM CD154-depleted during H-R and the percentage of cells staining with both the ROS probe DCF and apoptotic marker, Annexin-V, were assessed by flow cytometry.. The percentage of human hepatocytes that stain for both DCF and Annexin-V are shown in parentheses. Data is representative 3 separate experiments (*p<0.05 relative to basal, **p<0.05 relative to PCM).</p

CD40 Expression on Primary Human Hepatocytes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a> demonstrates a representative flow cytometry plot of CD40 expression on primary human hepatocytes during normoxia, hypoxia and H-R. The plot on the left hand side represents a typical forward scatter (FS) versus side scatter (SS) plot of primary human hepatocytes. The FS versus SS plots shown is from the H-R sample of a liver preparation but similar plots were obtained during normoxia and hypoxia (data not shown). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1b</a> shows a bar chart with the pooled data of three separate experiments illustrating the level of CD40 expression on primary human hepatocytes. Data are expressed as MFI and readings are based upon values taken from cells within the gated region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a>.</p

JNK and p38 regulate CD40∶CD154 mediated human hepatocyte apoptosis but not necrosis.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g003" target="_blank">Figure 3a</a> show representative flow cytometry plots to illustrate the effect of the JNK inhibitor SP600125 upon CD40∶CD154 mediated apoptosis and necrosis during normoxia, hypoxia and H-R. The gate used to analyse primary human hepatocytes is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figures 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">2</a>. The area of interest within the flow cytometric plots are marked by the vertical ellipses. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g003" target="_blank">Figure 3b</a> illustrates the effects of the p38 inhibitor P169316 upon CD40∶CD154 mediated apoptosis and necrosis during normoxia, hypoxia and H-R. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g003" target="_blank">Figure 3c</a> shows a bar chart with the pooled data of three separate experiments illustrating the effects of both SP600125 and PD169316 upon CD40∶CD154 mediated human hepatocyte apoptosis and necrosis during normoxia, hypoxia and H-R. Data are expressed as increase/decrease relative to basal, where basal refers to the level of apoptosis or necrosis during normoxia alone. Data are expressed as mean ± S.E. (**p<0.01 relative to basal, *p<0.05 relative to basal, †p<0.05 relative to CD154, ††p<0.05 relative to basal).</p

FasL Expression on Primary Human Hepatocytes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4a</a> demonstrates a representative flow cytometry plot of FasL expression on primary human hepatocytes during normoxia, hypoxia and H-R. The gate used to analyse primary human hepatocytes is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figures 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">2</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4b</a>. demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte FasL expression during normoxia, hypoxia and H-R. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4c</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 upon human hepatocyte FasL expression during normoxia, hypoxia and H-R. Data are expressed as MFI and readings are based upon values taken from cells within the gated region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a>. Data are expressed as mean ± S.E. (*p<0.05 relative to basal, **p<0.05 relative to CD154).</p

CD40∶CD154 regulates ROS accumulation, apoptosis and necrosis within human hepatocytes in a NADPH Oxidase dependent manner during normoxia and H-R.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a> demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte ROS accumulation during normoxia, hypoxia and H-R. Typical FS versus SS plots of primary human hepatocytes during normoxia, hypoxia and H-R are shown to the left of each flow cytometric plot. The FS versus SS plots shown is from the H-R alone sample of a liver preparation but similar plots were obtained during normoxia and hypoxia (data not shown). The areas of interest on the flow cytometric plots are marked by the vertical ellipses. The area on the left of each ellipse represents cell debris. Cell debris is included within the plot as human hepatocytes vary considerably in size and therefore to include all viable human hepatocytes in the analysis a large gate is required on the flow cytometer, this by necessity includes the cell debris. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2b</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 and CD154+DPI upon human hepatocytes ROS accumulation. Data is expressed as MFI and readings are based upon values taken from cells within the gated region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a>. Data are expressed as mean ± S.E. (*p<0.01 relative to basal, **p<0.05 relative to basal, †p<0.001 relative to CD154). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2c</a> demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte apoptosis and necrosis during normoxia, hypoxia and H-R. Again, the area of interest within the flow cytometric plots is marked by the vertical ellipse. The same gate has been applied to primary human hepatocytes for these plots as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2d</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 and CD154+DPI upon human hepatocytes apoptosis and necrosis during normoxia, hypoxia and H-R. Data is expressed as increase/decrease relative to basal, where basal refers to the level of apoptosis or necrosis during normoxia alone. Data are expressed as mean ± S.E. (**p<0.01 relative to basal, *p<0.05 relative to basal, †p<0.05 relative to CD154, §p<0.05 relative to basal). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2e</a>. Human hepatocytes were treated with CD154 or CD154 following pre-treatment with rotenone (Rot) during normoxia, hypoxia and H-R. The percentage of cells staining with both the ROS probe DCF and apoptotic marker, Annexin-V, were assessed by flow cytometry. The percentage of human hepatocytes that stain for both DCF and Annexin-V are shown in parentheses. Data are representative of 3 separate experiments (†p<0.05 relative to basal, *p<0.05 relative to CD154).</p

Proposed mechanism of CD40-CD154 mediated apoptosis and necrosis in human hepatocytes during normoxia, hypoxia and H-R.

<p>During normoxia (blue arrows) activation of CD40 expressed upon human hepatocyte by CD154 results in the translocation of the TRAF adaptor molecules to the cell membrane. The TRAF molecules are then responsible for the recruitment of the flavoenzyme NADPH Oxidase to the CD40∶TRAF complex. Here NADPH Oxidase can induce the production of ROS, primarily in the form of hydrogen peroxide. This process can be inhibited by the NADPH Oxidase inhibitor DPI. The resultant accumulation of ROS can result directly in necrotic cell death or it can activate the MAPK members, JNK and p38, to induce human hepatocyte apoptosis. However, during hypoxia (red arrows) CD40 activation on human hepatocytes does not result in ROS accumulation but can induce FasL expression via mechanisms that NADPH Oxidase-dependent and ROS-independent (dotted black line). This increased FasL expression can result in autocrine (green arrow) and/or paracrine (orange arrows) Fas-mediated apoptosis. Due to the increases in intracellular antioxidants levels during hypoxia CD154 does not increase apoptosis. However, mitochondrial ROS does contribute to apoptosis during hypoxia. During H-R, CD40 activation can lead to NADPH Oxidase-ROS-JNK/p38 dependent apoptosis, NADPH Oxidase-ROS-dependent necrosis and NADPH Oxidase∶ROS∶FasL expression with resultant Fas-mediated apoptosis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g006" target="_blank">Figure 6</a> highlights the importance of local microenvironment in shaping the effects of CD40∶CD154.</p

Study design.

<p>Series 1: Isolated primary human hepatocytes (PHH) were left in standard media for 2 days and then received media supplemented with fatty acids. After 2 days of fat loading the fatty PHH were allocated to the defatting treatment group where the media was supplemented with the defatting cocktail of drugs, and the control groups, the standard control group and the vehicle control group that received vehicle only. Lean hepatocytes were kept in standard culture conditions throughout the experimental period. The experimentation period lasted for two days thereafter. Series 2: Human intra-hepatic endothelial cells (HIEC) and cholangiocytes were immuno-magnetically separated with Dynabeads conjugated with cell-specific monoclonal antibody. The cells were left in culture for 2 days in standard media to reach confluence and then were allocated to the intervention group that received the defatting cocktail and the control groups, the standard control group and the vehicle control group that had the media supplemented with the vehicle only. The experimentation period lasts for two days thereafter.</p

Defatting of fat loaded primary human hepatocytes (PHH).

<p>Panel A: The positive are of oil red O of the defatting treatment group was reduced by 28% in comparison with the vehicle control group over 24 hours and 54% over 48 hours. Panel B: Intracellular triglyceride levels of the defatting treatment group were reduced by 32% within 24 hours of treatment and 35% within 48 hours, in comparison with the fatty vehicle control group. Panel C: Oil red O staining picture of PHH of the defatting group at the end of the 48 hours of treatment. There is a predominance of small lipid droplets in the cytoplasm of the cells and the nucleus is in its usual position. Series 2: shows a series of oil red O staining pictures from PHH in culture at different time points of the experiments. Panel D shows lean cells in culture, after the incubation with fatty acids they become loaded with fat (panel E). Those fat loaded PHH were then incubated with only the vehicle of the drugs for 48 hours and the lipid content decreased over time (panel F) or had the defatting treatment that showed the significant higher decrease in the area of oil red O (panel G). Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Release of total ketones in the supernatants.

<p>Fat loaded primary human hepatocytes that had the defatting treatment showed an increase in cell culture supernatant levels of total ketone bodies of 1.22-fold over 24 hours and 1.40-fold over 48 hours. Data reports the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Results of fat loading of primary human hepatocytes.

<p>Panel A: The supplementation of the media with the combination of fatty acids resulted in a cell viability rate of 81% after 48 hours of incubation. Panel B: Oil red O staining image of PHH at the end of the fat loading period. There is predominance of large lipid droplets displacing the nucleus of the cells to the periphery (black arrow). Panel C: At the end of 48 hours of fatting load there was a significant increase of 14-fold of the positive area of oil red O. Panel D: Intracellular triglycerides increased 8-fold within 48 hours of incubation with fatty acids. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p