MKK3/6-p38 mediates EGFR-dependent inflammation induced by NTHi in middle ear epithelial cells.

<p>(<i>A</i>) Cells were incubated with NTHi with or without SB203580, and NTHi-induced NF-κB activation was measured by performing luciferase assay. (<i>B</i>) Cells were incubated with NTHi with or without SB203580, and mRNA expression of TNF-α, IL-1β and IL-8 was measured by performing Q-PCR analysis. (<i>C</i>) Cells were incubated with NTHi with or without AG1478, and whole cell protein was collected and blotted against total- and phospho-p38 and MKK3/6. Data represent the mean±SD of at least three independent experiments, and each experiment was performed in triplicate. *<i>p</i><0.05 vs. control; #<i>p</i><0.05 vs. NTHi alone.</p

PI3K/Akt mediates EGFR-dependent inflammation induced by NTHi in middle ear epithelial cells.

<p><i>(A)</i> Cells were incubated with NTHi with or without wortmannin, and NTHi-induced NF-κB activation was measured by performing luciferase assay. <i>(B)</i> Cells were incubated with NTHi with or without wortmannin, and whole cell protein was collected and blotted against total- and phospho-IKKα/β and IκBα. <i>(C)</i> Cells were incubated with NTHi with or without wortmannin, and mRNA expression of TNF-α, IL-1β and IL-8 was measured by performing Q-PCR analysis. (<i>D</i>) Cells were incubated with NTHi with or without AG1478, and whole cell protein was collected and blotted against total- and phospho-Akt. Data represent the mean±SD of at least three independent experiments, and each experiment was performed in triplicate. *<i>p</i><0.05 vs. control; #<i>p</i><0.05 vs. NTHi alone.</p

EGFR mediates NTHi-induced NF-κB activation by inducing IKKα/β and IκBα activation in middle ear epithelial cells.

<p>(<i>A</i>) Cells were treated with NTHi at various time points as indicated in the figure, and whole cell protein was collected and blotted against total- and phospho-IKKα/β and IκBα. (<i>B</i>) Cells were treated with NTHi with or without AG1478, and whole cell protein was collected and blotted against total- and phospho-IKKα/β and IκBα. Data are representative of three or more independent experiments.</p

EGFR mediates NTHi-induced upregulation of proinflammatory mediators in middle ear epithelial cells.

<p>(<i>A</i>–<i>C</i>) Cells were treated with EGFR inhibitor AG1478 for 1 hour followed by 5 hour NTHi treatment. The mRNA expression of TNF-α, IL-1β and IL-8 was measured by performing Q-PCR analysis. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. *<i>p</i><0.05 vs. control; #<i>p</i><0.05 vs. NTHi alone.</p

EGFR is activated by NTHi in middle ear epithelial cells.

<p>HMEEC-1 cells were treated with NTHi for the time indicated, then were lysed and blotted with anti-phospho-EGFR and EGFR antibody. Data are representative of three or more independent experiments.</p

Schematic representation of EGFR-mediated inflammation.

<p>As indicated, EGFR is activated by NTHi, and mediates NTHi-induced NF-κB activation and inflammation via MKK3/6-p38 and PI3K/Akt signaling pathways.</p

Inhibition of EGFR inhibited NTHi-induced inflammation in the middle ear and lung tissues of mice <i>in vivo</i>.

<p><i>(A & B)</i> Animals were intraperitoneally inoculated with AG1478 or vehicle control. Two hours after AG1478 inoculation, NTHi was inoculated into middle ear via tympanic membrane (A) or into trachea (B). The mRNA expression of TNF-α, IL-1β, and MIP-2 was measured in the middle ear (A) or lung tissues (B) of mice inoculated with NTHi or saline as a control. *<i>p</i><0.05 vs. untreated group; #<i>p</i><0.05 vs. NTHi alone. (<i>C & D</i>) Middle ear (C) and lung tissues (D) of mice inoculated with NTHi with or without AG1478 were stained with Hematoxylin and Eosin for histological analysis (H&E stain, magnification ×200). <i>(E & F</i>) Bronchoalveolar lavage (BAL) was performed in NTHi-inoculated mice with or without AG1478, and total and polymorphonuclear (PMN) neutrophils were counted (E) and cytocentrifuged to stain with Hemacolor (F).</p