IL-10 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

Abstract

To determine to what extent lipopolysaccharide-induced IL-10 production capacity is determined by polymorphisms in toll-like receptor-4 (TLR4) and the IL-10 promoter region, we measured in vivo IL-10 and TNF-α production in patients undergoing elective cardiopulmonary bypass surgery, a major surgical trauma associated with ischemia-reperfusion injury that triggers an endotoxemia and profound inflammatory response in most patients. Ex vivo the IL-10 and TNF-α production was measured in a whole blood stimulation assay, using 3 LPS concentrations. Positive correlations were found between TNF-α and IL-10 production ex vivo, upon stimulation with each of the LPS concentrations. Also, the estimated TNF-α and IL-10 EC₅₀, and TNF-α_max and IL-10_max were positively correlated (r = 0.203; p = 0.023 and r = 0.287; p = 0.001, respectively), indicating that these parameters describing LPS sensitivity and maximal production capacity, respectively, can be estimated by measuring either TNF-α or IL-10. Interleukin-10 concentrations in patients experiencing endotoxemia in vivo negatively correlated with the IL-10 levels produced upon stimulation with 1000 ng/mL LPS as well as the estimated IL-10_max ex vivo. In vivo, a positive correlation between the TNF-α concentration at time-point 2 and the IL-10 concentration at time-point 3 was found, consistent with an important contribution of the magnitude of TNF-α release upon the subsequent IL-10 production. Carriers of the IL-10 promoter −1330G, −1082A, −819T, −592A (GATA) haplotype had lower IL-10 production ex vivo upon stimulation with 10 and 100 ng/mL LPS and higher EC₅₀ values (the estimated LPS concentration at which 50% of the maximal IL-10 response is reached) as compared to carriers of the other haplotypes combined, indicating decreased LPS sensitivity ex vivo. These individuals did not differ from the others in interleukin-10 production capacity upon stimulation with a high LPS concentration (i.e., 1000 ng/mL) and the estimated IL-10_max values, were similar, indicating unimpaired maximal IL-10 production capacity ex vivo. Carriers of the IL-10 promoter AGCC haplotype had lower EC₅₀ values as compared to carriers of the other haplotypes combined, indicating increased LPS sensitivity ex vivo. In accordance with this finding, carriers of the AGCC haplotype had higher circulating IL-10 levels in vivo. The common TLR4 polymorphisms (Asp299Gly and Thr399Ile) were associated with slightly higher IL-10 production capacity ex vivo and in vivo, however, this was not statistically significant. Our results indicate that polymorphisms in the proximal IL-10 promoter region are associated with in vivo and ex vivo LPS sensitivity. The contribution to the inter-individual variation, however, is limited since the variation between individuals in LPS sensitivity and IL-10 production capacity can only partly be attributed to these IL-10 promoter polymorphisms.

Introduction

Interleukin-10 (IL-10) is an important immunoregulatory cytokine. It is involved in the regulation of inflammatory responses through direct influence over tumor necrosis factor (TNF)-α production. Also, IL-10 plays an important role in the course of infectious diseases, for instance, the severity to which meningococcal meningitis progresses is associated with serum IL-10 concentrations, such that a high serum IL-10 level was observed in patients with a poor or fatal outcome, whereas patients with mild disease and a good prognosis had lower serum IL-10 levels [1], [2], [3]. In Gram-negative infection, the presence of lipopolysaccharide (LPS) in the circulation plays a pivotal role in the release of IL-10. Human monocytes, the main producers of IL-10, exhibit substantial inter-individual differences in IL-10 production upon LPS stimulation ex vivo [4]. A study investigating the inter-individual variation of IL-10 production following LPS stimulation of whole blood cultures ex vivo suggested that the differences in IL-10 production capacity can to a large extent be explained by differences in genetic background [5]. The inter-individual differences in produced IL-10 could not simply be explained by corresponding differences in TNF-α production, implicating that the differences in the individual's ability to produce IL-10 are not simply reflecting differences in the individual's ability to produce TNF-α [5]. It has been suggested that differential transcription is the principle mechanism of the inter-individual differences in IL-10 production [6]. Indeed, the IL-10 production is proportionally related to mRNA production and not to the half-life of IL-10 [7]. Given the fact that the differences in IL-10 production are most likely transcriptionally regulated, the IL-10 promoter region has been an important target for investigation [6]. In the IL-10 promoter, 11 single nucleotide polymorphisms (SNPs) have been described. Studies of SNPs in the proximal 1.1 kb have yielded the existence of a relative small number of haplotypes [8], [9], [10]. The dimorphic polymorphisms (G or A at position −1330, G or A at position −1082, C or T at position −819, C or A at position −592) are in preferential allelic association, namely AGCC, GACC and GATA haplotypes. It has been shown that the differences in the production capacity of IL-10 ex vivo are associated with IL-10 haplotypes [6]. Furthermore, the SNP in the IL-10 promoter at position −2849 was associated with differences in IL-10 production ex vivo upon stimulation with 1000 ng/mL LPS and transcriptional activity [11], [12], [13]. So far, other known SNPs in the IL-10 promoter have not been evaluated in this way.

Toll-like receptor-4 (TLR4) is part of a large family of transmembrane proteins and is believed to be crucial in mediating LPS effects. TLR4 is expressed on monocytes and macrophages, and to a lesser extent on lymphocytes and other cell types [14], [15]. The recently described association between the Asp299Gly polymorphism in TLR4 and Gram-negative septic shock suggests a functional defect in TLR4 leading to increased susceptibility to Gram-negative bacteremia [16].

Cardiopulmonary bypass surgery leads to perioperative endotoxemia in most patients, and this procedure may serve as a model to study the association of genetic polymorphism and endotoxin mediated IL-10 production in vivo. The most immunoreactive reactive component of endotoxin is lipid A, being a structural element of LPS.

The aims of the current study were to assess the inter-individual variation in IL-10 production upon whole blood stimulation with LPS, to determine the correlation between production rates and various SNPs in the IL-10 promoter region as well as the TLR4 coding region, and to determine how much of the inter-individual variation could be attributed to these genetic factors. We also studied the inter-individual variation in the in vivo IL-10 production following cardiopulmonary bypass surgery in the same way.