HHQ and PQS, two Pseudomonas aeruginosa quorum-sensing molecules, down-regulate the innate immune responses through the nuclear factor-kappaB pathway

Abstract

To explore whether bacterial secreted 4-hydroxy-2-alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild-type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor-alpha and interleukin-6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild-type did not. The addition of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild-type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor-kappaB (NF-kappaB) binding to its binding sites and the expression of NF-kappaB target genes, and PQS delayed inhibitor of kappaB degradation, indicating that the effect of HHQ and PQS was mediated through the NF-kappaB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

Figure 1

Biosynthetic pathway of 4-hydroxy-2-alkylquinolines (HAQs) in Pseudomonas aeruginosa. Anthranilate is activated by PqsA to form anthraniloyl-CoA, and this is condensed with β-keto-fatty acid to produce 4-hydroxy-2-heptylquinoline (HHQ). The condensation is postulated to be catalysed by PqsB, PqsC and PqsD. Alternatively, the intermediates in this pathway can be converted to HAQ-N-oxides by PqsL. HHQ is hydroxylated by PqsH to produce 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal).

Figure 2

Stimulation of innate immune responses by the secreted molecules from pqsA mutant strain. (a,b) J774A.1 cells were treated with a vehicle control (0·5% dimethylsulphoxide) or extracts of culture supernatants from wild-type or two mutants (pqsA and pqsH) of Pseudomonas aeruginosa for 2 days. The concentrations of the extracts (1/16×, 1/64×, 1/128×) are presented as fold dilution compared with the original volume of the bacterial culture supernatants. The amount of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from the supernatants from the J774A.1 culture was measured by enzyme-linked immunosorbent assay. Data represent mean ± SD (n = 3). (c) Cell viability after treatment with various concentrations of the extracts for 2 days was measure by MTS assay. Data represent mean ± SD (n = 4). All data (a–c) are representative of three independent experiments with similar results.

Figure 3

Suppression of innate immune responses by 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal). (a,b) J774A.1 cells were treated with extracts of culture supernatant from the pqsA mutant at 1/16× concentration for 24 hr in the presence or absence of 0·28 μg/ml of HHQ or PQS. The amount of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from the culture supernatants was measured by enzyme-linked immunosorbent assay. Data represent mean ± SD (n = 8). Statistical differences among groups were analysed by analysis of variance. The dimethyl sulphoxide vehicle control showed no difference compared with no-treatment control in Fig. 2 so it was not included in the experiment shown here. (c) Cell viability after treatment with various concentrations of HHQ or PQS for 24 hr was measured by MTS assay. Data represent mean ± SD (n = 4). All data (a–c) are representative of three independent experiments with similar results.

Figure 4

Immune responses in the bronchoalveolar lavage (BAL) cells (a,b) and pathogenicity in the lung (c). (a,b) The cells in the BAL fluid from C57BL/6 mice were harvested and cultured in vitro with extracts of culture supernatants from PA14 (wild-type) or a pqsA mutant in the presence or absence of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) for 48 hr. The expression of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture supernatants was measured by enzyme-linked immunosorbent assay. Data represent mean ± SD (n = 3). Statistical differences among groups were analysed by a Kruskal–Walis test. (c) 1 × 10⁵ colony-forming units (CFUs) of Pseudomonas aeruginosa were injected into the lung of C57BL/6 mice intranasally (n = 5, each group). After 12 hr the mice were killed and lung lavage was performed. The lung was homogenized in 2 ml phosphate-buffered saline, and 100 μl of the lung homogenates were plated onto a Cetrimide agar (CA) plate to measure CFU. Statistical differences between groups were analysed by a Student’s t-test. All data (a–c) are representative of three independent experiments with similar results.

Figure 5

4-Hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) suppress the binding of nuclear factor-κB (NF-κB) to its recognition site. J774A.1 cells were pre-treated with 1·12 or 0·28 μg/ml of HHQ or PQS for 2 hr, and treated with the extracts from pqsA (or PA14) culture supernatants for 30 min. After the cells were harvested, nuclear extracts were prepared and mixed with oligomers containing the NF-κB binding sites or its mutated sequences. The binding of NF-κB to the oligomers was measured by the electrophoretic mobility shift assay. All data (a,c,e) are representative of three or four independent experiments with similar results. (b) Densitometric analysis of Fig. 5(a). Band intensity of each band in scanned films was calculated by ImageJ program and combined before statistical analysis by analysis of variance (anova). Data are presented as mean ± SD (n = 3). (d) Densitometric analysis of Fig. 5(c). Band intensity was calculated as for Fig. 5(b). Statistical analyses were performed by anova or Student’s t-test. Data are presented as mean ± SD (n = 4).

Figure 6

2-Heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) suppresses inhibitor of κB (IκB) degradation (a,b), and 4-hydroxy-2-heptylquinoline (HHQ) and PQS suppress the expression of its target genes (c). (a) J774A.1 cells were treated with the extract from pqsA culture supernatants in the presence or absence of 1·12 μg/ml of HHQ or PQS for various times. The cells were harvested and the IκB protein was detected by Western blotting with an anti-IκB antibody. Data are representative of five independent experiments with similar results. (b) Densitometric analysis of Fig. 6(a). Band intensity was calculated as Fig. 5(b). Statistical analyses were performed using Student’s t-test. Asterisks indicate the statistical difference (P < 0·05) between groups at the same time-point. Data are presented as mean ± SE (n = 5). (c) J774A.1 cells were pre-treated with 1·12 μg/ml or 0·28 μg/ml of HHQ or PQS for 2 hr, and treated with the extracts from pqsA (or PA14) culture supernatants for 90 min. After the cells were harvested, total RNA was isolated. Target gene expression was measure by reverse transcription–polymerase chain reaction. Data are representative of three independent experiments with similar results.