Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin

Abstract

Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-alpha and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-alpha production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-alpha. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.

FIG. 1

FliC-induced activation of HeNC2 cells. HeNC2 cells were cultured with increasing concentrations of FliC. (A) TNF-α in culture supernatants was measured after 24 h by ELISA. The amount of TNF-α produced in unstimulated control cultures was subtracted from each value. Similar results were obtained in three independent experiments. (B) NO production was assessed after 48 h by measuring the amount of nitrite, a stable byproduct of NO. The values represent the mean of duplicate determinations (standard deviation, <5%) from two independent experiments.

FIG. 2

Anti-TLR4 monoclonal antibody does not block the stimulatory effect of FliC. HeNC2 cells were cultured in the presence of 3 × 10⁻¹¹ M FliC with no antibody added (open bars), with 20 μg of anti-IL-1β antibody 3ZD/ml (gray bars), or with 20 μg of anti-TLR4 antibody MTS510/ml (black bars). (A) TNF-α production after 24 h. (B) NO production after 48 h. The values represent the mean of duplicate determinations (standard deviation, <5%). Similar results were obtained in three independent experiments. The percent inhibition relative to controls in which no antibody was added is indicated in parentheses.

FIG. 3

FliC induces TNF-α production in LPS-hyporesponsive GG2EE cells. GG2EE cells were cultured with the indicated concentrations of FliC for 24 h. The amount of TNF-α in culture supernatants was measured by ELISA. Similar results were obtained in three independent experiments.

FIG. 4

FliC activates IRAK in HeNC2 and GG2EE cells. HeNC2 and GG2EE cells (10⁷ cells per sample) were cultured in the absence (lanes 1 and 4) or presence of 10⁻⁹ M FliC (lanes 2 and 5) or 1 ng of LPS/ml (lanes 3 and 6) for 15 min at 37°C. (A) IRAK was immunoprecipitated (IP) from cell lysates using anti-IRAK antibody, and the protein was visualized by Western blotting using the same antibody. (B) IRAK kinase activity in the same cell lysates was measured by in vitro kinase assay using myelin basic protein as a substrate (MBP-P). The fold induction of IRAK activity relative to the unstimulated control is indicated below each lane. (C) TNF-α production in culture supernatants after 24 h. Similar results were obtained in three independent experiments.

FIG. 5

FliC activates IRAK in the human promonocytic THP-1 cell line. THP-1 cells (5 × 10⁶ cells per sample) were cultured for 1 h in the absence (lane 1) or presence of 10⁻¹¹ M FliC (lane 2), 10⁻¹⁰ M FliC (lane 3), or 10⁻⁹ M FliC (lane 4). (A) Western blot of IRAK expression. (B) IRAK kinase activity. Similar results were obtained in three independent experiments.

FIG. 6

Time course of FliC-induced IRAK activation in HeNC2 cells. (A) HeNC2 cells (10⁷ cells per sample) were cultured in the absence or presence of 10⁻⁹ M FliC. Cultures with FliC added were prepared in duplicate. Cells were lysed at the indicated time points, and IRAK activity was measured by in vitro kinase assay. (B) Fold induction of IRAK activity relative to unstimulated controls.