Activation of Epidermal Growth Factor Receptor Is Required for NTHi-Induced NF-κB-Dependent Inflammation

Inflammation is a hallmark of many serious human diseases. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor-kappa B (NF-κB)-dependent production of proinflammatory mediators. Epidermal growth factor receptor (EGFR) has been shown to play important roles in regulating diverse biological processes, including cell growth, differentiation, apoptosis, adhesion, and migration. Its role in regulating NF-κB activation and inflammation, however, remains largely unknown.In the present study, we demonstrate that EGFR plays a vital role in NTHi-induced NF-κB activation and the subsequent induction of proinflammatory mediators in human middle ear epithelial cells and other cell types. Importantly, we found that AG1478, a specific tyrosine kinase inhibitor of EGFR potently inhibited NTHi-induced inflammatory responses in the middle ears and lungs of mice in vivo. Moreover, we found that MKK3/6-p38 and PI3K/Akt signaling pathways are required for mediating EGFR-dependent NF-κB activation and inflammatory responses by NTHi.Here, we provide direct evidence that EGFR plays a critical role in mediating NTHi-induced NF-κB activation and inflammation in vitro and in vivo. Given that EGFR inhibitors have been approved in clinical use for the treatment of cancers, current studies will not only provide novel insights into the molecular mechanisms underlying the regulation of inflammation, but may also lead to the development of novel therapeutic strategies for the treatment of respiratory inflammatory diseases and other inflammatory diseases

The Escherichia coli O157:H7 carbon starvation-inducible lipoprotein Slp contributes to initial adherence in vitro via the human polymeric immunoglobulin receptor.

Escherichia coli O157:H7 is the most well-studied serotype of the enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 bacteria and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of the slp gene fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested and not the nonpathogenic control strain E. coli K12. Additionally, deletion of slp gene resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp gene complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor

Opposing roles for RhoH GTPase during T-cell migration and activation

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients (“go” signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition (“stop” signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1–GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1–GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals

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

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

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