Relationship between a toll-like receptor-4 gene polymorphism, bacterial vaginosis-related flora and vaginal cytokine responses in pregnant women

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Abstract

Objective: The relationship between a single nucleotide polymorphism (TLR4 896 A>G) in the toll-like receptor-4 (TLR4) gene, qualitative and quantitative changes in vaginal micro-flora and vaginal interleukin (IL)-1β and IL-1 receptor antagonist (IL-1ra) concentrations in pregnant women were evaluated. Study design: Qualitative and quantitative microbial methods were used to characterize vaginal micro-flora of 238 women at 18–22 weeks gestation. Polymerase chain reaction was used to determine TLR4 genotype. IL-1β and IL-1ra concentrations in vaginal lavage samples were measured by ELISA. Results: The TLR4 variant was identified in 10.3% of women. Carriage of this variant was associated with a median increase in vaginal pH (P=0.05), a greater than 10-fold increase in vaginal Gardnerella vaginalis levels (P<0.0001) and a 10-fold increase in the vaginal concentration of three species of anaerobic Gram-negative rods, Prevotella, Bacteroides, and Porphyromonas (P=0.08). Colonization with G. vaginalis and/or the anaerobic Gram-negative rods resulted in elevated vaginal IL-1 (P=0.01) and IL-1ra (P<0.0002) concentrations in women who were TLR4 896A homozygotes, but not in TLR4 896G carriers. Conclusion: The TLR4 896 A>G polymorphism contributes to inter-individual differences in the vaginal immune defense against G. vaginalis and anaerobic Gram-negative rods.

Introduction

The term “bacterial vaginosis” (BV) is used to describe the clinical condition characterized by increased quantities of malodorous vaginal discharge, a vaginal pH of >4.7, and a shift away from a Lactobacilli-dominant vaginal flora and towards a predominance of Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis [1], [2]. The immune response to BV is characterized by the release of pro-inflammatory cytokines, mainly interleukin-1β (IL-1β) [3], [4], [5]. This is often accompanied by an increase in the concentration of IL-1 receptor antagonist (IL-1ra) [3], which exerts its effects by competing with IL-1β for binding to IL-1 receptors on target cells [6]. In this way, IL-1ra regulates the pro-inflammatory action of IL-1 so that the invading pathogenic micro-organisms are destroyed while normal tissue architecture and function are maintained. Among the organisms isolated from the vagina, the degree of colonization with anaerobic Gram-negative rods and G. vaginalis strongly correlates with the concentrations of IL-1β and IL-1ra in the vaginal fluid, suggesting that these micro-organisms in the lower genital tract are potent stimulators of local IL-1β and IL-1ra production [7].

The innate component of the immune system has been highly conserved throughout evolution and is the first line of defense against microbial pathogens. Specific pattern recognition receptors, present on epithelial cells at mucosal surfaces as well as on macrophages and dendritic cells, recognize invariant structures, called pathogen-associated molecular patterns that are present on microbial cell surfaces. The binding of pathogen-associated molecular patterns to pattern recognition receptors stimulates the activation of a pro-inflammatory immune response and activation of components of adaptive immunity [8]. One of the most widely studied pattern recognition receptors is called toll-like receptor-4 (TLR4). This receptor resembles the toll protein identified in Drosophila [9] and specifically interacts with the lipopolysaccharide (LPS) of Gram-negative bacteria [10]. The specific mechanism involves LPS first binding to LPS-binding protein and membrane-associated CD14. TLR4 then binds to this complex and triggers a cascade of intracellular events leading to transcription of cytokine genes [11], [12].

The gene coding for TLR4 in man is polymorphic and the TLR4 896 A>G polymorphism results in the replacement of aspartic acid with glycine at amino acid 299 in the final protein [13]. This substitution leads to a marked reduction in responsiveness to LPS both in vitro and in vivo [13], [14]. Individuals carrying at least one copy of the TLR4 896G variant exhibited a reduced airway responsiveness to inhaled LPS, as compared to TLR4 896A homozygotes [13]. A recent study demonstrated an increased incidence of Gram-negative bacterial infections in critically ill patients who carried a TLR4 896G variant [15]. Conversely, there appeared to be no relation between TLR4 alleles and human meningococcal [16], chlamydial [17], or vaginal candidal [18] infections.

We hypothesize that the TLR4 receptor may be involved in the regulation of vaginal colonization with anaerobic Gram-negative rods and G. vaginalis, and that the TLR4 896 A>G polymorphism may influence the magnitude of vaginal colonization with these micro-organisms as well as the extent of the cytokine response. This study was performed to test this hypothesis.

Section snippets

Subjects

All women presenting for their first prenatal visit to the Brigham and Women’s Hospital, Boston, from January 1, 2000 to March 1, 2002 were given an enrolment package containing a description of the study. Eligible subjects were identified by the study nurse. Exclusion criteria were <15 years old, antimicrobial treatment within 4 weeks of sample collection, multiple gestation, an obstetric or chronic medical condition that increased the likelihood of a preterm birth, placenta previa, Rh

Results

Of 231 women enrolled in the study, 28 subjects were subsequently excluded from the analysis for the following reasons: in utero fetal demise at 19 weeks gestation due to fetal cytomegalovirus infection (n=1), lost to follow-up (n=1), vaginal lavage samples unavailable for analysis (n=14), failure to amplify a DNA sequence by PCR (n=4), induction of labor before 37 weeks due to pregnancy-induced hypertension (n=5), and elective cesarean delivery prior to 37 weeks due to idiopathic fetal hydrops

Discussion

LPS is a potent stimulant for IL-1 and IL-1ra production as part of the host immune response to invading micro-organisms. Inter-individual differences have been reported in the release and synthesis of cytokines by human monocytes stimulated by LPS in vitro [21]. Based on studies in a murine model, such differences in cytokine production in response to LPS have been in part attributed to polymorphisms in the gene encoding TLR4 [14]. In accordance with such observations, the results of the

Acknowledgements

This study was supported by grants RO1 HD35667 and RO1 HD41676 from the National Institute of Child Health and Disease.

References (24)

  • R. Medzhitov et al.

    A human homologue of the Drosophila toll protein signals activation of adaptive immunity

    Nature

    (1997)
  • M.J. Sweet et al.

    Endotoxin signal transduction in macrophages

    J. Leukoc. Biol.

    (1996)
  • Cited by (0)

    1

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