LPS-mediated production of pro/anti-inflammatory cytokines and eicosanoids in whole blood samples: Biological effects of +896A/G TLR4 polymorphism in a Sicilian population of healthy subjects

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Abstract

Toll-like receptors (TLRs) are the principal mediators of rapid microbial recognition: the lipopolysaccharide (LPS) receptor TLR4 seems to have a paradigmatic role. Single nucleotide polymorphisms (SNPs) in the TLR4 gene, such as +896A/G, known to attenuate receptor signaling, have been described. The +896A/G SNP is significantly less frequent in patients with myocardial infarction, Alzheimer's disease or prostate cancer, whereas it is overrepresented in centenarians. To clarify and confirm the biological effects of +896A/G SNP and its role in the pathophysiology of age-related diseases and longevity, we assessed the levels of IL-6, TNF-α, IL-10 and eicosanoids (LTB4 and PGE2) in LPS-stimulated whole blood samples in vitro of 50 young healthy Sicilians, screened for the presence of this SNP. To evaluate the possible influence of SNPs in PTGS2 and 5-Lo genes on eicosanoid production, the enrolled individuals were also genotyped for −765G/C PTGS2 and −1708G/A 5-Lo SNPs. Both pro-inflammatory cytokines and eicosanoids were significantly lower in carriers bearing the TLR4 mutation, whereas the anti-inflammatory IL-10 values were higher. On the basis of data reported herein, some suggestions can be drawn. First, pathogen load, by interacting with the host genotype, determines the type and intensity of inflammatory responses, according to the pro-inflammatory status and tissue injury, implicated in the pathophysiology of major age-related diseases. Second, adequate control of inflammatory response might reduce the risk of these diseases, and, reciprocally, might increase the chance of extended survival in an environment with reduced antigen (that is, pathogen) load.

Research highlights

▶ LSP mediated production of inflammatory mediators was induced in 50 WBA samples. ▶ Significantly different were their levels in the 50 samples screened for TLR4 SNP. ▶ Proinflammatory cytokines and eicosanoids were significantly lower in the carriers. ▶ In contrast, their antiinflammatory IL-10 values were higher. ▶ So, the biological effect of TLR4 SNP is an adequate control of inflammatory responses.

Introduction

Genetic variations in innate immune response are thought to influence the risk of several human diseases by altering host response to environmental stress (Lin et al., 2006, Goldstein, 2009, Hirschhorn, 2009, Kraft and Hunter, 2009). Good examples, under current examination, are innate immune pathways. Among these, TLR4, the key receptor for LPS, provides an ideal model. It permits the study of the consequences of its genetic variations and their relation to the function of the receptor pathway, and their implications in the risk of atherosclerosis, Alzheimer's disease (AD) and cancer (Balistreri et al., 2009, Beutler, 2002, Uematsu and Akira, 2008). A SNP, Asp299Gly or +896A/G (rs:4986790), has been identified in the TLR4 gene, encoding single amino-acid substitution in the extra-cellular receptor domain (Arbour et al., 2000). This SNP induces a blunted innate/inflammatory response to both foreign pathogens and endogenously generated inflammatory ligands (Arbour et al., 2000, Balistreri et al., 2009, Schröder and Schumann, 2005). In particular, it may influence inflammatory responses and the risk of major age-related diseases affecting the production of inflammatory mediators.

Cytokines and eicosanoids represent the key regulators of innate/inflammatory response. High serum or plasma levels of these molecules have been correlated with the major age-related diseases, confirming their role in atherosclerosis, AD and cancer pathophysiology (Akiyama et al., 2000, Boyce, 2008, Bruunsgaard et al., 2001, Candore et al., 2007a, Candore et al., 2007b, Caruso et al., 2009, Krabbe et al., 2004, Leon, 2007, Rogers, 2008, Sheu et al., 2008). However, the magnitude of cytokine and eicosanoid production has been shown to vary individually and is likely based on genetic heterogeneity (Bucova et al., 2008, Candore et al., 2007a, Candore et al., 2007b, Caruso et al., 2009, Grimble, 2003, Rea et al., 2006, Reyes-Gibby et al., 2008). This might determine changes in the innate/inflammatory response and, consequently, in tissue injury and the processes involved in the development and progression of age-related diseases (Bucova et al., 2008, Candore et al., 2007a, Candore et al., 2007b, Caruso et al., 2009, Grimble, 2003, Rea et al., 2006, Reyes-Gibby et al., 2008). Hence, certain people develop diseases and others remain healthy. One or more functional SNPs in one or more genes might be responsible. Accordingly, recent studies have suggested the role of +896A/G TLR4 SNP in cytokine and eicosanoid production (Arbour et al., 2000, Ferwerda et al., 2007, Ferwerda et al., 2008, Hoshino et al., 1999, Michel et al., 2003, Norata et al., 2005, Poltorak et al., 1998, von Aulock et al., 2003, Werner et al., 2003). On the other hand, an association of this TLR4 SNP with an increased risk of Gram negative infections and septic shock has been demonstrated (Agnese et al., 2002, Lorenz et al., 2002, Schröder and Schumann, 2005). However, contrasting data have been obtained in other studies (Child et al., 2003, Erridge et al., 2003, Read et al., 2001, Heesen et al., 2003, Imahara et al., 2005, Schippers et al., 2005, von Aulock et al., 2003, Yang et al., 2004).

Changes in eicosanoid levels are also correlated with SNPs in the promoter region of PTGS2 and 5-Lo genes, respectively codifying the cyclooxygenase-(Cox)-2 and 5-lipoxygenase (5-Lo), enzymes involved in arachidonic acid metabolism. Two functional (−765G/C PTGS2 and −1708G/A 5-Lo) SNPs have recently been identified and associated with the major age-related diseases (Candore et al., 2007a, Candore et al., 2007b, Caruso et al., 2009, Cipollone et al., 2004, In et al., 1997, Orbe et al., 2006).

To clarify and confirm the possible pathophysiological effects of +896A/G TLR4 SNP (Balistreri et al., 2004, Balistreri et al., 2008, Balistreri et al., 2010), we analysed the levels of IL-6, TNF-α, IL-10 and eicosanoids (LTB4 and PGE2) in LPS-stimulated whole blood samples in vitro of 50 young healthy Sicilians, screened for the presence of +896A/G TLR4 SNP. To evaluate the possible influences of SNPs in PTGS2 and 5-Lo genes on eicosanoid production, we also screened the enrolled individuals for the −765G/C PTGS2 and −1708G/A 5-Lo SNPs. Hence, our idea was to test whether the three SNPs have a major influence on the production of inflammatory mediators, when they operate in combination to create a “risk profile,” as suggested in our previous studies (Candore et al., 2007a, Candore et al., 2007b, Caruso et al., 2009).

Section snippets

Population studied and TLR4, PTGS2 and 5-Lo genotyping

The study included 50 Sicilian Caucasoids from Palermo and neighbourhood (age range: 25–50 years; 24 females and 26 males), in good health according to their clinical history and blood tests (complete blood cell count, erythrocyte sedimentation rate, glucose, urea nitrogen, creatinine, electrolytes, C reactive protein, liver function tests, iron, proteins). They have previously been genotyped for the following SNPs: +896A/G TLR4 SNP, −765G/C PTGS2 SNP (rs:20417) and −1708 G/A 5-Lo SNP (no

LPS contamination

LPS contamination in supernatants of cell cultures at baseline conditions from both low (+896G+) and high (+896A+) responder subjects was detected at all times. LPS levels were 0.142 pg/ml (0.102–0.208). A positive correlation was observed between LPS and IL-6 and TNF-α levels in supernatants of cell cultures from subjects with high (+896A+) responder SNP (n = 40, r = 0.433, p < 0.001 and r = 0.33, p < 0.013, respectively, data not shown). Furthermore, a negative correlation was found between LPS and

Discussion

Current evidence supports the involvement of innate immunity and inflammation in the pathophysiology of major age-related diseases (Caruso et al., 2005, Licastro et al., 2005, Vasto et al., 2007). Different mediators, i.e. pro-inflammatory cytokines and eicosanoids, display a crucial role in atherosclerosis, neurodegeneration and carcinogenesis. Their production and release have prevalently been associated with the activation of TLR4 receptor (Balistreri et al., 2009, Candore et al., 2007a,

Acknowledgements

This work was supported by grants from the Italian Ministry of Education, University and Research to CC and GC.

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