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. 2005 Apr;73(4):2344-50.
doi: 10.1128/IAI.73.4.2344-2350.2005.

Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium

Muhammad Iqbal  1 , Victoria J Philbin, G S K Withanage, Paul Wigley, Richard K Beal, Marianne J Goodchild, Paul Barrow, Ian McConnell, Duncan J Maskell, John Young, Nat Bumstead, Yvonne Boyd, Adrian L Smith
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Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium (V体育ios版)

Muhammad Iqbal et al. Infect Immun. 2005 Apr.

Abstract

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5+ cells to flagellin induced elevated levels of chicken interleukin-1beta (chIL-1beta) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1beta mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken VSports手机版. .

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Figures

FIG. 1.
FIG. 1.
Amino acid sequence of chTLR5 protein and alignment with huTLR5 and moTLR5 proteins. Accession numbers were AJ626848 for chTLR5, NM003268 for huTLR5, and AF186107 for mTLR7. Alignment was performed using the ClustalX program and edited in the Genedoc program; shading was performed using the conserved mode (black shading for conserved residues and light grey shading for similar residues). The double continuous lines denote leucine-rich repeats, the single continuous line indicates transmembrane domain, and the dotted line represents the TIR domain.
FIG. 2.
FIG. 2.
Expression of chTLR5 mRNA in tissues (A), immune cell populations (B), and cultured cells (C) by RT-PCR from 8-week-old line 72 chickens. Products were resolved by agarose gel electrophoresis and visualized with ethidium bromide. Data are representative of three PCR amplifications with independent samples. Small Int., small intestine.
FIG. 3.
FIG. 3.
Exposure of chicken cell lines to flagellin induces upregulation of chIL-1β mRNA but not chIL-6 mRNA. Three adherent cell cultures (CEF, CKC, and HD11) plated in 24-well plates (4 wells/treatment) were exposed to 10, 100, or 200 ng of purified Salmonella-derived flagellin/ml or were mock treated for 6 h. Real-time RT-PCR (Taqman) was used to determine the levels of cytokine and 28S mRNA, with the latter used to normalize the cytokine data, depicted as the mean relative fold increase ± standard error, compared with mock-treated wells. An asterisk indicates a statistically significant difference (P < 0.05) between flagellin-treated and mock-treated wells. The data are representative of three independent experiments.
FIG. 4.
FIG. 4.
Numbers of aflagellar (fliM mutant) and wild-type (WT) S. enterica serovar Typhimurium F98 cells in cecal contents and livers (A) and heterophil infiltration in the cecal wall (B) after oral infection of 3-day-old chicks (4/group). (A) Bars represent mean numbers of Salmonella CFU/g ± standard error on a logarithmic scale. Asterisks indicate statistically significant differences between flagellin-treated and mock-treated wells (P < 0.05). ND, not detected. (B) Heterophil infiltration was assessed microscopically in hematoxylin-eosin-stained sections of cecal wall at 9, 24, and 48 h p.i. +/−, relative degree of infiltration; -, level of infiltration in the uninfected chickens; +++, heavy infiltrate with numerous heterophils in the tissue.
FIG. 5.
FIG. 5.
The levels of chIL-1β and chIL-6 mRNA in CT and small intestine (Small int.) of chicks challenged with aflagellar (fliM mutant) or wild-type (wt) S. enterica serovar Typhimurium F98. Bars represent the mean fold increases ± standard errors (4 birds/group) of cytokine mRNA by real-time PCR (Taqman) using uninfected birds as the fold denominator. *, statistically significant difference between infected and uninfected chickens (P < 0.05); **, statistically significant difference between chickens infected with wild-type and fliM mutant Salmonella (P < 0.05).
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