Inflammation is a hallmark of many serious human diseases. Appropriate inflammation is a protective host defense response to remove the injurious stimuli and initiate tissue healing and repair. However, overactive inflammation is detrimental to the host, leading to inflammatory diseases. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying tight regulation of inflammation remain largely unknown. Steroids and cyclooxygenase inhibitors have long been used as the main therapeutic anti-inflammatory agents, but they are frequently associated with significant detrimental effects in patients. In addition, inappropriate antibiotic treatment for bacterial infection contributes significantly to the worldwide emergence of antibiotic resistance. Thus, there is an urgent need for the development of novel anti-inflammatory agents.
Nontypeable Haemophilus influenzae
(NTHi), a gram-negative bacterium, is an important human pathogen in both children and adults 
. In children, it causes otitis media (OM), the most common childhood infection and the leading cause of conductive hearing loss 
. In adults, it exacerbates chronic obstructive pulmonary disease (COPD) 
, an important lung disease and the fourth leading cause of death in the United States 
. Like most bacterial infections, NTHi infection is characterized by inflammation, which is mainly mediated by nuclear factor-kappa B (NF-κB)-dependent production of proinflammatory mediators 
. NF-κB is a transcription factor consisting of homo- or heterodimers of Rel-related proteins 
. It has five members in mammalian cells: RelA (p65), RelB, c-Rel, p50/p105, and p52/p100. The heterodimer consisting of two subunits, p65 and p50, is most commonly involved in the regulation of a variety of physiologic processes, including inflammation, differentiation, proliferation, and survival, among others 
. In its inactive state, NF-κB resides in the cytoplasm and forms a multiprotein complex with an inhibitory subunit, inhibitor of NF-κB (IκB). Upon activation by external stimuli, the inflammatory signal converges on and activates a set of IκB kinases known as the IκB kinase (IKK) complex, which are composed of three subunits: IKKα, IKKβ, and IKKγ. IκBα is phosphorylated by IKKs and this phosphorylation results in the degradation and dissociation of IκBα from NF-κB. Once released from the complex involving IκBα, NF-κB translocates to the nucleus, where it binds to DNA and promotes the transcription of target genes. NF-κB is activated by inflammatory stimuli and involved in regulating expression of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thereby playing a critical role in mediating inflammatory responses 
Toll-like receptor 2 (TLR2) plays a crucial role in mediating NTHi-induced inflammatory response. However, directly blocking TLR2 signaling may result in some unwanted detrimental side effects because appropriate immune response mediated by TLR2 signaling is also required for host defense against invading bacterial pathogens. For instance, uncontrolled bacterial growth, decreased bacteria clearance and increased susceptibility to bacterial infection was observed in TLR2 KO mice 
and impairment of TLR2 signaling due to genetic mutations in human populations closely correlates with increased susceptibility to bacterial pathogens 
. Thus, identifying a non-TLR2 therapeutic target for NTHi infection is in high demand.
The epidermal growth factor receptor (EGFR) is a member of the HER family composed of four distinct receptors: EGFR/ErbB1, Her-2/ErbB2/c-neu, Her-3/ErbB3, and Her-4/ErbB4, which are predominantly located at the basolateral surface of polarized epithelial cells. EGFR is traditionally known as a growth factor receptor that mediates cell differentiation and proliferation. Elevated levels of EGFR and/or its cognate ligands have been shown to be involved in tumor growth 
. In addition, EGFR is activated by multiple TLRs to produce innate immune response in airway epithelium 
. Activation of EGFR plays an important role in recruiting leukocytes 
, inducing mucins and antimicrobial peptides to clear pathogens 
, and increasing wound repair 
. Recent studies from our group suggested that EGFR is at least in part activated by NTHi via NTHi-derived EGF-like growth factor and plays an important role in negatively regulating TLR2 induction during bacterial infections 
. In addition, exogenous EGF increases NTHi invasion of host epithelial cells, demonstrating the biological significance of TLR2 regulation by EGFR signaling 
. However, the role of EGFR in regulating NTHi-induced NF-κB signaling and inflammatory response in airway inflammatory diseases has yet to be fully explored.
Based on the essential role of TLR2 in NTHi-induced NF-κB signaling and inflammatory responses and the role of EGFR in controlling TLR2 induction, we hypothesized that EGFR may regulate NTHi-induced NF-κB activation and inflammation in the middle ear and lung. Here, we provide direct evidence for the critical role of EGFR signaling in regulating NTHi-induced inflammation in human middle ear and airway epithelial cells in vitro, and in mouse middle ear and lung in vivo. Our studies will not only provide novel insights into the molecular mechanisms underlying the regulation of inflammation, but will also facilitate translational research toward novel therapeutic strategies for the treatment of respiratory and other inflammatory diseases.