LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3kinase signalling
© Ngkelo et al; licensee BioMed Central Ltd. 2012
Received: 16 November 2011
Accepted: 12 January 2012
Published: 12 January 2012
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.
Bacterial infections are one of the dominant causes of acute exacerbations in chronic obstructive pulmonary disease (COPD). Lipopolysaccharide (LPS) is the most abundant component within the cell wall of Gram-negative bacteria. It can stimulate the release of interleukin 8 (IL-8, CXCL8, CXC ligand 8) and other inflammatory cytokines in various cell types, leading to an acute inflammatory response towards pathogens . These responses are initiated by the activation of the TLR signalling through adaptor proteins, and include induction of gene expression via the activation of the NF-κB and AP-1 signal transduction pathways . Bacterial LPS has been extensively used in models studying inflammation as it mimics many inflammatory effects of cytokines, such as TNF-α, IL-1β or IL-6. The cellular receptor transducing the LPS signal has been identified as Toll-like receptor 4 (TLR4) [3–5]. Binding of LPS to TLR4 leads to the activation of NF-κB through the recruitment and activation of MyD88, IL-1R kinase (IRAK), TNFR associated factor 6 (TRAF-6), as well as NADPH oxidase (Nox) [2, 6, 7]. NF-κB plays a crucial role in regulating the transcription of genes related to innate immunity and inflammation responses and several studies indicate its activation is controlled by reactive oxygen species (ROS) in immune modulation in the lungs and in monocytes [8–11].
Several studies searching for novel anti-inflammatory agents have led to the identification of a key role for phosphatidylinositol 3-kinase (PI3K) in transducing receptor-mediated signalling during inflammation in chronic inflammatory diseases, such as COPD . The PI3K family is divided into three classes (I, II and III) depending on their structure, substrate and function . The class I PI3Ks are further subdivided into class IA (p110α, p110β and p110δ) and class IB (p110γ). All class I PI3Ks mediate fundamental signalling pathways and cellular processes that orchestrate cell growth, proliferation, migration and survival . Class IA PI3Ks are activated via cell surface expressed receptor tyrosine kinases (RTKs) including insulin and growth factors whereas, class IB (p110γ) are activated by G-protein coupled receptors (both α and βγ subunits of G-proteins) [15, 16]. There is clear evidence that PI3Ks (PI3Kγ and PI3Kδ) play a crucial role in mediating both the innate and adaptive immune response by regulating leukocyte migration, activation and antigen response [17–19]. PI3K activation is important for neutrophil migration [20, 21] and this can play a significant role in disease exacerbations where neutrophilic influx is evident, as observed in COPD.
Several studies have shown that activation of PI3Ks by many microbial stimuli such as LPS, play a key role in regulating immune cell mechanisms, such as cytokine production [22, 23]. Moreover, global inhibition of the various PI3-kinase isoforms interferes with the TLR-mediated cellular signalling pathways and molecular responses such as B cell cytokine production and differentiation [24, 25]. However, the molecular mechanism by which LPS induces cytokine release in human monocytes is not fully understood and therefore warrants further investigation. In this study we demonstrate that LPS-stimulated release of proinflammatory cytokines from human monocytes is mediated through the activation of PI3K in both a ROS- and a G-protein-dependent manner, propagated through NOX4 activation.
Isolation of human peripheral blood mononuclear cells (PBMCs) and monocytes
PBMCs from healthy volunteers were isolated by centrifugation of whole blood on Histopaque®-1077 (Sigma; Poole, Dorset, UK) at 400 g for 30 min at room temperature. Cells collected from the interphase were washed with PBS then resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 2 ml L-glutamine. Human monocytes were purified by allowing the purified PBMCs resuspended in IMDM at 4 × 106 cells/ml to adhere to tissue culture plates for 1-2 hours at +37°C. Non-adherent cells were removed with warm HBSS twice before detaching adherent cells (> 90% monocytes) with cell dissociation buffer (Gibco BRL; Paisley, UK). The harvested monocytes were then resuspended in IMDM with 1% (w/v) BSA ready for the chemotaxis assay. The population of monocytes harvested by this method has been described previously  and the purity of the population was more than 95%. The study was approved by the local Ethics Committee with informed consent obtained from each participant.
Cell Culture and Treatments
Purified PBMCs were pre-treated with pharmacological agents as indicated in figure legends for 30 min before stimulation with LPS (100 ng/ml) for further 15 min prior to cell lysis and assessment of pAKT/Total AKT by either MSD assay (Mesoscale; Gaithersburg, MD) or immunoblotting. Alternatively, following the addition of LPS, the PBMCs were left to incubate for 16 hours at +37°C before culture supernatants were removed and assessed for both IL-6 and CXCL8 by sandwich enzyme-linked immunosorbent assay (R&D Systems; Abingdon, UK) according to the manufacturer's protocols.
Chemotaxis assay: (plate based assay)
Chemotaxis was assessed as previously described . Briefly, monocytes were pre-loaded with Calcein-AM at 5 μg/107 cells for 20 min at +37°C, then resuspended in IMDM supplemented with 0.1% w/v BSA at 8 × 106 cells/ml and kept on ice in the dark until needed. Into the bottom well of a 96 well Neuroprobe 5 μM chemotaxis plate (Receptor Technologies Ltd; Adderbury, UK) was placed 29 μl of 3 nM C5a in medium (IMDM + 0.1% BSA), medium only (background) or cells in medium only (maximum signal). Into the upper chamber, 25 μl of calcein loaded cells were applied and the plate left to incubate for 90 min at +37°C. After 90 min unmigrated cells were washed away from the top chamber with PBS and the plate read using a Fluoroskan II plate reader (Labsystems, Cambridge, UK) in bottom reading mode (Ex 485 nm, Em 538 nm).
Measurement of AKT/pAKT by MSD assay
Phospho (Ser473) AKT and total AKT was evaluated using the MSD®MULTI-SPOT assay system from Mesoscale (Gaithersburg, MD. Cat # K15100D). PBMCs were aliquoted into a 96 well tissue culture plate at 3x106 cells/well then incubated with or without wortmannin or N-acetylcysteine for 30 min before treating the cells with 100 ng/ml LPS for a further 15 min. Cells were pelleted at 500 g for 3 minutes at +4°C and washed once with ice cold PBS before pelleting again and lysing the cell pellet with 100 μl ice cold lysis solution provided in the MSD assay kit. 20 μg of protein cell lysate was transferred to a 96 well MSD plate and phosphor-AKT and total AKT were assessed as per manufacturer's instructions.
Protein extraction and immunobloting
All experimental details are according to previous description (28). Whole Cell protein extracts were prepared using modified RIPA buffer (50 mM Tris HCL pH7.4. 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1% CHAPS, 1 mM EDTA with freshly added complete protease and phosphatase inhibitor cocktail II (Calbiochem; Feltham, UK). Protein concentration was determined using the Pierce BCA protein assay kit (Rockford, IL). Whole cell lysates were subjected to western blot after SDS PAGE using mouse monoclonal anti- phosphoAKT (Abcam; Cambridge, UK). Western Blots were stripped with Chemicon Re-Blot Plus western recycling kit (Chemicon International, Temecula, CA), blocked and reprobed with anti-AKT (Abcam). All other reagents were from Sigma (Poole, UK) unless otherwise stated.
Data were analysed by means of 1-way ANOVA to determine statistically significant variance between the groups for each end point assessed. Statistical significance between groups was then calculated by using the nonparametric Mann-Whitney t test with GraphPad Prism software (GraphPad Software, Inc, La Jolla, Calif). Data are expressed as means ± SEMs. Differences were considered significant at a P value of less than 0.01.
LPS-induced IL-6 and CXCL8 release is blocked by inhibition of PI3K
LPS-induced phosphorylation of AKT is inhibited by wortmannin and N-acetylcysteine
LPS induced inflammation is modulated through Gi-dependent signalling
Pertussis toxin and inhibition of NADPH oxidase prevents LPS-induced phosphorylation of AKT
We report here that stimulation of human PBMC by LPS, leading to the release of pro-inflammatory cytokines such as CXCL8 and IL-6, is dependent, in part, on PI3K signalling. Moreover, the activation of PI3K by LPS was G-protein dependent as the G protein inhibitor, pertussis toxin, clearly blocked PI3K signalling. However, neither pertussis toxin nor mastoparan could completely prevent LPS induced cytokine release, indicating multiple signalling pathways were at work. In contrast to pertussis toxin, however, the anti-oxidant N-acetylcysteine could only inhibit LPS-induced PI3K signalling by 50%.
ROS by their very nature are highly promiscuous and will target numerous redox sensitive molecules. Indeed, the protein tyrosine phosphatases are particularly susceptible to inhibition by ROS due to a key cysteine residue within the active site . This can lead to deregulated activation of PI3K and therefore increased phosphorylation of AKT . When ROS was inhibited by N-acetylcysteine in our experiments, we did not see complete inhibition of PI3K activity. The explanation for this may be two fold. Firstly, not all LPS-activated PI3K is driven through ROS and secondly the N-acetylcysteine concentrations used were not as high as that reported elsewhere, where up to 30 mM N-acetylcysteine was used. Moreover, in the experiments reported by Ashebourne et al., N-acetylcysteine could not completely inhibit LPS-induced pAKT expression unlike another anti-oxidant, α-tocopherol . This suggested that a compartmentalisation effect for the different anti-oxidants, whereby N-acetylcysteine, unlike tocopherol, could not penetrate the lipid compartment to neutralise the ROS located there.
In conclusion, we have shown that LPS can activate PI3Kγ activity through a ROS and pertussis toxin sensitive mechanism in human PBMC (Figure 7). Moreover, this PI3K dependent pathway is responsible for at least 50% of the functional response seen with respect to LPS-induced cytokine release from human PBMCs.
Anta Ngkelo was supported by a Pfizer/MRC-CASE PhD studentship award. Ian Adcock received support from the Wellcome Trust and Paul Kirkham received support from a Royal Society Industrial Fellowship.
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