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. 2012 Jan 12;9(1):1.
doi: 10.1186/1476-9255-9-1.

LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3kinase signalling

Anta Ngkelo  1 , Koremu Meja, Mike Yeadon, Ian Adcock, Paul A Kirkham
Affiliations

LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3kinase signalling

Anta Ngkelo et al. J Inflamm (Lond). .

Abstract

COPD is a disease of innate immunity and bacterial infections are a dominant cause of exacerbations in the later stages resulting in poor health and high mortality. The pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) is sensed by immune cells through activation of the toll-like receptor 4 (TLR4). This leads to the activation of NADPH oxidase (NOX) and NF-κB which together drive COPD inflammation. In this study we show in human PBMCs that LPS stimulated proinflammatory cytokine release (CXCL8 and IL6) was inhibited by approximately 50% by the broad specificity phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Our results also demonstrate that activation of PI3K following LPS stimulation is mediated by a NOX4 dependent mechanism releasing endogenous H2O2, as the NOX4 inhibitor apocynin blocked LPS induced AKT phosphorylation. Moreover, LPS-induced PI3K activation was inhibited by the anti-oxidant N-acetylcysteine in a concentration dependent manner (IC50 ~100 μM). In addition, our data demonstrated that inhibition of small G proteins, by pre-treatment with pertussis toxin, inhibited LPS-induced AKT phosphorylation. Furthermore, the G-protein inhibitors pertussis toxin and mastoparan both inhibited LPS-induced CXCL8 and IL-6 release by approximately 50%. Together, these data indicate there is a mechanism in human PBMCs where TLR4 activation by LPS leads to ROS generation through NOX4 and activation of the PI3K pathway. This effect is apparently mediated through small G proteins facilitating the release of pro-inflammatory cytokines.

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Figures

Figure 1
Figure 1
Impact of the PI3K inhibitor wortmannin on LPS-induced release of CXCL8 and IL-6. Purified human PBMCs were treated with 100 ng/ml LPS in the presence of wortmannin for 16 hrs. CXCL8 (a) and IL-6 (b) were assessed by ELISA and the data normalised to control (LPS stimulation only) for each experiment. The mean ± SEM for each data point from at least 3 independent experiments is shown. Sigmoidal dose response curves were plotted using Prism4 software. 100% of control (LPS-stimulated PBMCs only) represented 15 ng/ml CXCL8 and 30 ng/ml IL-6 respectively.
Figure 2
Figure 2
PI3K inhibition prevents C5a-induced chemotaxis in human monocytes. Human monocytes were pretreated with wortmannin for 30 min and then allowed to migrate in response to 3 nM C5a. Data was normalised relative to control, where control represents 100% chemotaxis in response to C5a only treatment. Results are displayed as mean ± SEM for 3 independent experiments and the sigmoidal dose response curve plotted using Prism4 software.
Figure 3
Figure 3
A time course of LPS-induced phosphorylation of Akt in human PBMCs. PBMCs were treated with 100 ng/ml LPS and at defined time points cells were harvested, lysed and assessed for the presence of Akt phosphorylation by Western blot. The blot is representative of the experiment repeated at least 3 times.
Figure 4
Figure 4
LPS-induced phosphorylation of AKT is inhibited by wortmannin and N-acettylcysteine in a concentration-dependent manner in human PBMCs. Human PBMCs were pre-treated with either (a) wortmanniun or (b) N-acetylcyteine for 30 minutes before stimulating with LPS for 15 minutes. For each data point, the % of phosphorylated AKT relative to total AKT present is plotted as the mean ± SEM from at least 3 independent experiments. Sigmoidal dose response curves were plotted using Prism4 software. As a negative control, cells were left untreated.
Figure 5
Figure 5
LPS-induced release of (a) CXCL8 and (b) IL-6 in human PBMCs. Cells were pre-treated with either the G-protein inhibitor pertussis toxin, the specific Giα inhibitor mastoparan (Mas+) or its inactive counterpart (Mas -). The data represents the mean ± SEM from 8 experiments. *P < 0.05 as determined by Kruskal Wallace non-parametric ANOVA followed by Dunnets post-test analysis.
Figure 6
Figure 6
Inhibition of LPS-induced pAkt by the G-protein inhibitor pertussis toxin and the NOX inhibitor Apocynin. PBMCs from healthy volunteers were pretreated with either Apocynin (300 μM) or pertussis toxin (100 ng/ml) for 30 min before LPS stimulation. Phosphorylated AKT and total AKT levels were visualised by Western blot and assessed semi-quantitatively by band densitometry. The data was normalised to total AKT for each treatment group and plotted as mean ± S.E.M of 7 independent experiments (**p < 0.01 as determined by Kruskal Wallace non-parametric ANOVA followed by Dunnets post-test analysis) showing the fold-increase in phospho-Akt compared to unstimulated cells.
Figure 7
Figure 7
Schematic representation of LPS-induced PI3K activity through apocynin sensitive NOX4 and pertusis toxin/mastoparan sensitive Gi protein activation. N-acetyl cysteine neutralises any NOX4 generated intracellular superoxide which can trigger activation of ROS sensitive Gi proteins releasing Gβγ subunits to activate PI3Kγ.
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