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

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America.
PLoS ONE (Impact Factor: 3.23). 11/2011; 6(11):e28216. DOI: 10.1371/journal.pone.0028216
Source: PubMed


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.

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    • "However, the NTHi lysate has been used for in vitro stimulation of the epithelial cells in this study because (1) live NTHi was found to up-regulate human β-defensin 2 with high variations (Fig. S4B); and (2) the NTHi lysate appeared to more significantly up-regulate human β-defensin 2 expression than the same CFUs of live NTHi as shown in Fig. S4C. Although bacterial lysates are not a perfect model, the NTHi lysate is commonly used for the study of host-pathogen interactions as shown in our prior studies [7], [36], [38], [42] and other studies [46], [66], [67] because live bacteria inhibit viability of mammalian cells. Furthermore, it is suggested that the NTHi lysate is able to up-regulate human β-defensin 2 expression more stably and significantly than live NTHi since a variety of bacterial molecules inside bacteria are able to directly access cytoplasmic PRRs after bacterial lysis. "
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    ABSTRACT: Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate β-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced β-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced β-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced β-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced β-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated β-defensin 2 regulation contributes to the protection against NTHi-induced otitis media.
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    • "Also, only lapatinib but not gefitinib can synergize the anti-tumor activity of MG-132 in TNBC cells (Figure 6B). Third, inhibition of either HER2 [46] or EGFR [47] by its specific siRNA has been reported to decrease but not increase NF-κB activity. Similar to this EGFR/HER2-independent role in NF-κB activation, lapatinib has also been reported to up-regulate the expression of pro-apoptotic TRAIL death receptors DR4 and DR5 through an off-target mechanism in colon cancer cells and, thus, sensitizes these cancer cells to TRAIL-induced apoptosis [21]. "
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    ABSTRACT: Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Our data showed that nuclear factor (NF)-kappaB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-kappaB involved Src family kinase (SFK)-dependent p65 and IkappaBalpha phosphorylations, and rendered these cells more vulnerable to NF-kappaB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-kappaB, and thus augment the anti-tumor activity of proteasome inhibitors. These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients.
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    ABSTRACT: Pneumonia, the most typical and frequent lower respiratory tract infection (LRTI), is a leading cause of health problems in the United States. Bacteria represent the most prevailing cause of pneumonia in both children and adults. Although pneumonia with a single bacterial infection is common, a significant portion of patients with pneumonia is polymicrobial. This infection is often complexed with other physiological factors such as cytokines and growth factors. Nontypeable Haemophilus influenzae (NTHi) is the most frequently recovered Gram-negative bacterial pathogen in the respiratory system and induces strong inflammatory responses. NTHi also synergizes with other respiratory pathogens, such as Streptococcus pneumoniae and respiratory viruses and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α). It is noteworthy that NTHi not only synergizes with growth factors such as transforming growth factor-beta (TGF-β), but also utilizes growth factor receptors such as TGF-β receptor and epidermal growth factor receptor (EGFR), to enhance inflammatory responses. Although appropriate inflammation is a protective response against invading pathogens, an uncontrolled inflammatory response is often detrimental to the host. Thus, inflammation must be tightly regulated. The human immune system has evolved strategies for controlling overactive inflammatory response. One such important mechanism is via regulation of negative feedback regulators for inflammation. CYLD, a multifunctional deubiquitinase, was originally reported as a tumor suppressor, but was recently identified as a negative regulator for nuclear factor-kappa B (NF-κB) signaling. It is induced by NTHi and TNF-α via a NF-κB-dependent mechanism, thereby serving as an inducible negative feedback regulator for tightly controlling inflammation in NTHi infection.
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