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A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease.

Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Skerrett SJ, Beutler B, Schroeder L, Nachman A, Ozinsky A, Smith KD, Aderem A - J. Exp. Med. (2003)

Bottom Line: Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood.We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells.Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, 1441 N. 34th St., Seattle, WA 98103, USA. thawn@u.washington.edu

ABSTRACT
Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5392STOP) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila, a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

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FliC stimulation of cytokine production. (A–E) Cells were stimulated with LPS (•), purified S. typhimurium FliC (○), heat-killed wild-type L. pneumophila (⋄), or FlaA− (FliC mutant) L. pneumophila (×). (A and B) A549 cells; (C and D) Calu-3 cells; and (E) whole blood. (F–G) PBMCs were harvested from individuals with homozygous wild-type TLR5 (392RR; n = 6) or stop codon TLR5 heterozygotes (392R*; n = 4). Cells were stimulated with FliC at 30 ng/ml−1 (F) or LPS at 10 ng/ml−1 (G). (A–G) Cells were stimulated for 18 h and supernatants were assayed for cytokine production by ELISA. Data are representative of experiments performed at least twice in triplicate with standard deviations indicated. (F and G) The mean level and standard deviation of IL-6 were derived from averaging the responses of different individual's cells stimulated in triplicate.
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fig3: FliC stimulation of cytokine production. (A–E) Cells were stimulated with LPS (•), purified S. typhimurium FliC (○), heat-killed wild-type L. pneumophila (⋄), or FlaA− (FliC mutant) L. pneumophila (×). (A and B) A549 cells; (C and D) Calu-3 cells; and (E) whole blood. (F–G) PBMCs were harvested from individuals with homozygous wild-type TLR5 (392RR; n = 6) or stop codon TLR5 heterozygotes (392R*; n = 4). Cells were stimulated with FliC at 30 ng/ml−1 (F) or LPS at 10 ng/ml−1 (G). (A–G) Cells were stimulated for 18 h and supernatants were assayed for cytokine production by ELISA. Data are representative of experiments performed at least twice in triplicate with standard deviations indicated. (F and G) The mean level and standard deviation of IL-6 were derived from averaging the responses of different individual's cells stimulated in triplicate.

Mentions: These genetic findings raise the question of why a deficiency in TLR5 would be deleterious to the host because there are other PAMP receptors, including TLR4, which could presumably still recognize L. pneumophila and mediate an inflammatory response. To understand TLR5 regulation of signaling in pulmonary epithelia, we stimulated A549 lung cell lines and measured culture supernatant IL-8 levels. As seen in previous analyses, LPS was unable to stimulate IL-8 production, even at doses of 1,000 ng ml−1 (Fig. 3 A, not depicted) (31). In contrast, flagellin potently stimulated IL-8 in a dose-dependent fashion, starting at concentrations of 1 ng ml−1. Furthermore, although heat-killed wild-type L. pneumophila induced IL-8 production, a flagellin mutant strain (FlaA−) was nonstimulatory (Fig. 3 B). Similar results were obtained for IL-6 and IL-8 production in Calu-3 lung epithelial cells (Fig. 3, C and D; and not depicted). In contrast to epithelial cells, both wild-type and FlaA− strains were similarly able to induce TNF-α production in a whole blood cytokine assay (Fig. 3 E). This control assay indicates that the FlaA− mutant did not have a generalized loss of PAMPs that compromised its overall stimulatory capacity. Overall, these results suggest a dominant role for flagellin and TLR5 in regulating the pulmonary epithelial innate immune response to L. pneumophila.


A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease.

Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Skerrett SJ, Beutler B, Schroeder L, Nachman A, Ozinsky A, Smith KD, Aderem A - J. Exp. Med. (2003)

FliC stimulation of cytokine production. (A–E) Cells were stimulated with LPS (•), purified S. typhimurium FliC (○), heat-killed wild-type L. pneumophila (⋄), or FlaA− (FliC mutant) L. pneumophila (×). (A and B) A549 cells; (C and D) Calu-3 cells; and (E) whole blood. (F–G) PBMCs were harvested from individuals with homozygous wild-type TLR5 (392RR; n = 6) or stop codon TLR5 heterozygotes (392R*; n = 4). Cells were stimulated with FliC at 30 ng/ml−1 (F) or LPS at 10 ng/ml−1 (G). (A–G) Cells were stimulated for 18 h and supernatants were assayed for cytokine production by ELISA. Data are representative of experiments performed at least twice in triplicate with standard deviations indicated. (F and G) The mean level and standard deviation of IL-6 were derived from averaging the responses of different individual's cells stimulated in triplicate.
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Related In: Results  -  Collection

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fig3: FliC stimulation of cytokine production. (A–E) Cells were stimulated with LPS (•), purified S. typhimurium FliC (○), heat-killed wild-type L. pneumophila (⋄), or FlaA− (FliC mutant) L. pneumophila (×). (A and B) A549 cells; (C and D) Calu-3 cells; and (E) whole blood. (F–G) PBMCs were harvested from individuals with homozygous wild-type TLR5 (392RR; n = 6) or stop codon TLR5 heterozygotes (392R*; n = 4). Cells were stimulated with FliC at 30 ng/ml−1 (F) or LPS at 10 ng/ml−1 (G). (A–G) Cells were stimulated for 18 h and supernatants were assayed for cytokine production by ELISA. Data are representative of experiments performed at least twice in triplicate with standard deviations indicated. (F and G) The mean level and standard deviation of IL-6 were derived from averaging the responses of different individual's cells stimulated in triplicate.
Mentions: These genetic findings raise the question of why a deficiency in TLR5 would be deleterious to the host because there are other PAMP receptors, including TLR4, which could presumably still recognize L. pneumophila and mediate an inflammatory response. To understand TLR5 regulation of signaling in pulmonary epithelia, we stimulated A549 lung cell lines and measured culture supernatant IL-8 levels. As seen in previous analyses, LPS was unable to stimulate IL-8 production, even at doses of 1,000 ng ml−1 (Fig. 3 A, not depicted) (31). In contrast, flagellin potently stimulated IL-8 in a dose-dependent fashion, starting at concentrations of 1 ng ml−1. Furthermore, although heat-killed wild-type L. pneumophila induced IL-8 production, a flagellin mutant strain (FlaA−) was nonstimulatory (Fig. 3 B). Similar results were obtained for IL-6 and IL-8 production in Calu-3 lung epithelial cells (Fig. 3, C and D; and not depicted). In contrast to epithelial cells, both wild-type and FlaA− strains were similarly able to induce TNF-α production in a whole blood cytokine assay (Fig. 3 E). This control assay indicates that the FlaA− mutant did not have a generalized loss of PAMPs that compromised its overall stimulatory capacity. Overall, these results suggest a dominant role for flagellin and TLR5 in regulating the pulmonary epithelial innate immune response to L. pneumophila.

Bottom Line: Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood.We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells.Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, 1441 N. 34th St., Seattle, WA 98103, USA. thawn@u.washington.edu

ABSTRACT
Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5392STOP) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila, a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

Show MeSH
Related in: MedlinePlus