The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity

Abstract

Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.

FIG. 1

Influence of OHHL and OdDHL on ConA mitogen-driven proliferation of murine spleen cells (+1 standard deviation). OdDHL inhibited [³H]thymidine uptake at concentrations over 5 × 10⁻⁵ M in two assays. OHHL (Inset) had no effect on the uptake of [³H]thymidine. DMSO at concentrations equivalent to those in AHL-containing samples had no effect on the assay (data not shown). Counts per minute (CPM) are in thousands. ∗∗, P < 0.01.

FIG. 2

Influence of OHHL and OdDHL on PTK activity. OHHL at 10⁻⁴ M inhibited phosphorylation of PKS-1 substrate by both p59^fyn (a) and p56^lck (b). OdDHL had no effect. ∗∗, P < 0.01. Error bars indicate standard deviations.

FIG. 3

Influence of OHHL and OdDHL on TNF-α and IL-12 production by LPS-stimulated adherent murine peritoneal macrophages. (a) OdDHL inhibited TNF-α release at 3.35 × 10⁻⁵ M. OHHL did not inhibit the production of TNF-α. CT, control level of TNF-α release. (b) OdDHL also inhibited IL-12 production by LPS-stimulated macrophages at all concentrations tested. DMSO at equivalent concentrations to those in AHL-containing samples had no effect on the assay (data not shown). Error bars indicate standard deviations. +ve, positive control (no OdDHL added). ∗∗, P < 0.01.

FIG. 4

Influence of OHHL and OdDHL on specific antibody production in the KAP model of a secondary antibody response. OdDHL inhibited the production of specific antibody by antigen-stimulated KLH-primed murine spleen cells at a concentration of 8 × 10⁻⁵ M (P < 0.01). Antibody production was significantly enhanced at lower concentrations of the compound. ∗, P < 0.05; ∗∗, P < 0.01. Error bars indicate standard deviations. The value of 1 represents the positive control level (CT). DMSO at equivalent concentrations to those in AHL-containing samples had no effect on the assay (data not shown).

FIG. 5

Influence of OdDHL on IgG1 production in the KAP model of a secondary antibody response. OdDHL, at 5 × 10⁻⁵ M, caused a significant increase in IgG1. ∗, P < 0.05. DMSO at equivalent concentrations to those in AHL-containing samples had no effect on the assay (data not shown). Error bars indicate standard deviations.

FIG. 6

Influence of OdDHL on IgE production by IL-4-stimulated human PBMCs. OdDHL caused an increase in IgE levels at 10⁻⁴ M. ∗, P < 0.05. DMSO at equivalent concentrations to those in AHL-containing samples had no effect on the assay (data not shown). Error bars indicate standard deviations.

FIG. 7

Influence of OdDHL on the immune system. The mammalian T-helper (Th) cell response can be subdivided into two distinct arms, Th-1 and Th-2; the development of T cells along these distinct lineages is stimulus dependent and results in the release of cytokines characteristic of each lineage. (a) bacterial pathogens induce a Th-1 response, characterized by the secretion of IL-12 and gamma interferon. This developmental pathway results in the generation of activated macrophage populations, which are bactericidal and proinflammatory. (b) TNF-α is a key player in the establishment of this inflammatory process. It causes neutrophil activation, adhesion and extravasation, and the secretion of further inflammatory cytokines and chemokines (IL-6 and IL-8). Conversely, environmental allergens and helminth parasites induce a Th-1 contrasuppressive Th-2 response, characterized by the secretion of IL-4, IL-5, and IL-13, which induce a state of immediate hypersensitivity. Given the demarcation of these developmental pathways into antibacterial and anthelminthic, it would be of evolutionary advantage to bacterial pathogens to suppress the Th-1 response. This would in turn be supportive of the Th-2 response. It can be envisaged that OdDHL, by influencing both T-cell and macrophage functions at the points indicated in panel a, modulates host inflammatory responses to a degree which may promote the growth and survival of the pathogen. Compounds with this type of immunological activity could have applications in disease states, such as toxic shock syndrome and cerebral malaria, which have a significant TNF-α component.