Elsevier

Tuberculosis

Volume 84, Issue 6, 2004, Pages 347-352
Tuberculosis

The toll-like receptor 4 Asp299Gly variant: no influence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia

https://doi.org/10.1016/j.tube.2004.02.001Get rights and content

Abstract

Setting: Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Studies in a murine model of pulmonary TB have identified a role for Toll-like receptor 4 (TLR4) in the development of chronic lung infection with Mycobacterium tuberculosis. The Asp299Gly polymorphism in the human TLR4 gene is associated with in vivo hyporesponsiveness to lipopolysaccharide (LPS) in Caucasians.

Objective: To determine whether TLR4 Asp299Gly influences LPS responses or susceptibility to pulmonary TB in humans in a Gambian population sample.

Design: We compared whole blood monokine responses to LPS in 245 healthy blood donors stratified by TLR4 Asp299Gly genotype to assess whether this polymorphism was functional in this population. A case-control study of 640 subjects was conducted to investigate whether TLR4 Asp299Gly was associated with TB.

Results: LPS-induced tumour necrosis factor, interleukin-1β and interleukin-10 production was not influenced by TLR4 Asp299Gly genotype. There was no association between TLR4 Asp299Gly and TB.

Conclusion: Our data suggest that TLR4 Asp299Gly has no influence on monocyte LPS responses or susceptibility to TB in Gambians and could be an ancient neutral polymorphism.

Introduction

Mycobacterium tuberculosis infects 2 billion people globally, yet only 10% develop clinical disease.1 Even so, tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Prolonged treatment with multiple drugs is required and compliance is poor. This has contributed to the global rise in multi-drug-resistant TB.2 Furthermore, BCG vaccination does not protect against TB in countries where disease is most prevalent.3 The identification of factors that predispose to (or protect against) disease could aid the development of new therapies and vaccines.

The increased rate of TB in immunodeficient individuals, particularly those co-infected with the human immunodeficiency virus, highlights the importance of the host-immune response in the containment of disease. However, most people who develop TB are not immunosuppressed, and genetic variation in the host immune response is an important determinant of susceptibility to TB.4 Most of the genes identified to date are involved in innate immune responses, including solute carrier family 11 member 1, SLC11A1 (formerly natural resistance associated macrophage protein 1, NRAMP1), the vitamin D receptor gene and genes within the interleukin-12/interferon-γ (IL-12/IFN-γ) pathway.4., 5., 6., 7. The innate response is activated by the interaction of microbial components with receptor complexes expressed on the surface of host cells including macrophages and dendritic cells.8 This interaction induces macrophage antimicrobial functions, the production of inflammatory cytokines such as tumour necrosis factor (TNF), and primes specific immune responses by the production of IL-12. Several receptors are involved in the recognition of mycobacterial cell wall components. More recently, the importance of Toll-like receptors (TLRs) in cellular activation by M. tuberculosis has been described.9., 10. TLR2 interacts with M. tuberculosis cell wall components to induce cellular activation, killing of intracellular microbes and apoptosis.11 TLR4, initially identified as the mediator of lipopolysaccharide (LPS) inflammatory responses,12 can also interact with both heat labile soluble mycobacterial factor and whole viable M. tuberculosis to initiate innate responses.13., 14. M. tuberculosis-induced TNF and nitric oxide production can be blocked by the lipopolysaccharide (LPS) lipid A antagonist E5531.15 In a recent study, Tlr4-deficient mice developed chronic lung infection resembling human disease when exposed to aerosolized M. tuberculosis.16 This was associated with uncontrolled M. tuberculosis growth within the lung tissue, reduced macrophage recruitment to lung tissue, and reduced amounts of IL-12p40, TNF and monocyte chemoattractant protein-1 in lung homogenates.

Two mis-sense mutations have been identified in the extracellular domain of the human homologue, TLR4, (Asp229Gly and Thr399Ile) that are associated with hyporesponsiveness to LPS in alveolar macrophages and epithelial cells, and peripheral blood mononuclear cells.17., 18. We have investigated the role of the Asp229Gly polymorphism in susceptibility to TB in an outbred Gambian population sample: the Thr399Ile polymorphism was absent in a different Gambian population sample.19

Section snippets

Methods

This study had the approval of the MRC/Gambia Government Ethics Committee. The study cohort has been described elsewhere.20 Briefly, 320 men with pulmonary TB (mean age 36 years, range 19–58) were recruited from the TB clinic at Serrekunda Health Centre, The Gambia. All cases had microscopically proven, smear-positive TB in the context of clinical and radiological findings consistent with pulmonary TB, in the absence of other chronic illness, HIV infection, or treatment with corticosteroids.

Results

Allele and genotype frequencies for the TLR4 Asp299Gly polymorphism in the TB cases and matched control subjects are shown in Table 1. No differences were observed in either allele or genotype frequency between the TB cases and the controls, and no deviation from Hardy-Weinberg equilibrium was detected. Allele frequency differences were assessed in an additional population sample of 148 individuals, representing five Gambian ethnic groups (Table 2). The genotype frequencies in each sample were

Discussion

We have found no association between the Asp299Gly variant of TLR4 and pulmonary TB, suggesting that it has no major role in susceptibility to TB in this population. Furthermore, there is no association between carriage of the Asp299Gly G allele and LPS hyporesponsiveness using a whole blood assay in this Gambian population. Our negative findings have a number of possible interpretations. Firstly, the Asp299Gly polymorphism may influence susceptibility to TB, but if the effect were small (i.e.

Acknowledgements

We thank Professors Keith McAdam and Jenefer Blackwell for their support. This study was supported by the MRC (UK). MJ Newport holds a Wellcome Trust Advanced Clinical Fellowship. A Awomoyi received financial support from the Sir Halley Stewart Trust. GS thanks Mathurin Diatta for help with data collection.

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    1

    Co-corresponding author. Laboratory of Human Genetics, MRC The Gambia, PO Box 273, Banjul, The Gambia. Tel.: +220-4-494069; fax: +220-4-495919/496513.

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