Antimicrobial innate immune molecules produced by the epithelial cells provide the host with a constitutive or immediately inducible defense mechanism against invading pathogens. Under normal conditions, the middle ear of humans and laboratory animals remains sterile, although OM pathogens, which are part of the normal nasopharyngeal flora can reach the middle ear via the Eustachian tube causing infection . Furthermore, non-inflamed tubal and middle ear mucosa have been shown to contain relatively few immunocytes [46, 47]. All of these findings suggest that the components of the innate immune system may be important in the defense of the tubotympanum (middle ear and Eustachian tube). Furthermore, due to the premature and under-developed adaptive immune system in infants and young children, the role of AIIMs in protecting against OM pathogens becomes of paramount importance. Park and Lim  showed that the lysozyme positive cells in the epithelium and glandular structures of the Eustachian tube of adult and early post-natal animals were similar. Our previous studies also showed synergistic antimicrobial activities of AIIMs against OM pathogens [8, 14]. These results lend strong support to the hypothesis that AIIMs protect the tubotypmpanum during the neonatal and early postnatal periods when the adaptive immunity is not yet fully developed.
Among the AIIMs, β-defensin 2 plays a pivotal role against major OM pathogens and previous studies have shown that this molecule is inducible by a variety of agents, including cytokines, NTHi and its components/molecules [8, 14, 23, 27, 30]. Furthermore, we have previously shown that this molecule is expressed at high levels in the middle ear mucosa of patients with OM, but it is undetectable in the normal middle ear mucosa .
Some LOS mutants of NTHi exhibit reduced virulence and require higher doses in order to induce OM . Mutations in certain bacterial membrane components alter the susceptibility of the mutants towards AIIMs [49, 50]. However, these mutations did not significantly affect the inducibility of HBD-2 expression. To date, the exact nature and identity of the β-defensin 2 inducing molecule(s) remains elusive, but what is known is that it is maximally active and is present in the soluble WCL portion (Fig 1A and 1B) . Currently several NTHi macromolecules have been identified as vaccine candidates, which include adhesins, high molecular weight adhesins, pilus proteins, outer membrane proteins and LOS [52–55]. Identification of induction of the responsible ligand for beta-defensin 2 requires more research.
Treatment with NTHi WCL causes a substantial increase in HBD-2 mRNA expression in both HMEEC-1 and A549 cells. However, the kinetics of HBD-2 expression is a little different between the two cell lines (Fig 2A and 2B), which may be the result of the fact that different cells respond differently even to the same bacterial components . In vivo induction of mouse β defensin-2 with NTHi WCL treated mice middle ear epithelia exhibits a similar trend to that of HMEEC-1; the mRNA level peaked at 6 hour post-inoculation and dropped back down to basal levels by 12 hours (Fig 2C). This rapid increase in mouse β defensin-2 levels may indicate that the middle ear epithelial cells, but not inflammatory cells, were the sole source of the mouse β defensin-2 mRNA – β defensins are mainly produced by epithelial cells since monocytes would not have yet begun infiltrating the middle ear cavity during this period .
The Toll-like receptors (TLRs) have been shown to be critical for the recognition of bacteria and recent studies have demonstrated that TLR2 and TLR4 are involved in NTHi signaling [3, 57]. To test the involvement of either TLR2 or TLR4 in mediating NTHi WCL induced HBD-2 up-regulation, we used blocking antibodies, siRNA, and dominant negative mutants of TLR2 and TLR4 to inhibit the receptors' activities (Figs 3 – 5). All three methods revealed TLR2 as being the primary receptor for the recognition of NTHi WCL component.
Based on these results, we investigated the role and identity of other possible players downstream of the TLR2 pathway that may be involved in HBD-2 up-regulation by the NTHi WCL such as MyD88, which was previously shown to be the first molecule in the signaling cascade immediately downstream of the TLR2  and its associated proteins, interleukin-1 receptor-associated kinase 1 (IRAK1) [13, 19] and tumor necrosis factor receptor-associated factor 6 (TRAF6). MyD88 is recruited via its TIR death domain and promotes association and phosphorylation of the IRAK1. The phosphorylation of IRAK1 results in its dissociation from the complex and its interaction with TRAF6. This interaction results in the activation of downstream signaling in the majority of the epithelial cells studied [13, 19]. Our results using siRNA (Fig 4A, 4B &4E) and DN plasmids (Fig 5A &5B) indicate that MyD88, IRAK1 & TRAF6 are directly involved in regulating HBD-2 expression in the presence of NTHi WCL.
Activation of the TLR2-MyD88-IRAK1-TRAF6 can lead to the activation of many downstream signaling pathways, including members of the mitogen-activated protein kinase (MAPK) family [19, 35]. Previous studies have suggested that the p38 MAPK pathway is involved in HBD-2 induction in other cell systems [20, 58, 59]. We therefore sought to determine if p38 MAPK can act as a major signaling mediator for NTHi induced β-defensin 2 expression. Indeed, our results support data from previous work since such up-regulation was greatly inhibited by SB203580, a specific inhibitor of p38 MAPK but not by ERK inhibitors such as PD98059 and U0126. Also we used siRNA gene knock down to confirm the involvement of p38 MAPK and its associated upstream activators – MKK3 and MKK6. The results (Fig 4B &4E) support our hypothesis that NTHi WCL-specific HBD-2 up-regulation in HMEEC-1 takes place through the p38 MAPK cascade. The kinetics of this signaling was very rapid; phosphorylation of p38 MAPK started 15 minutes after stimulation and peaked at 45 minutes (Fig 5C). Near complete dephosphorylation was achieved by 60 minutes post-stimulation. Secretion of the β-defensin 2 was also reduced in cells treated with siRNAs targeted for down-regulating TLR2, IRAK1, TRAF6, and MKK(3/6) signaling molecules compared to a negative control. The importance of the role of TLR2 in NTHi WCL induced β-defensin 2 expression was also confirmed in vivo. The induction of mouse β-defensin 2 (mBD-2) mRNA by NTHi WCL in TLR2 KO mice middle ear was much lower than that of an age matched wild type control.
Beta-defensin exhibits not only innate immune activity such as direct killing of invading bacteria but also has been known to mediate adaptive immunity . The secreted β-defensin functions as an chemoattractant for CCR6 positive immature dendritic and memory T cells, facilitating the transition from innate immunity to adaptive immunity by producing inflammatory cytokines and chemokines such as IL-1 [19, 60–62]. Our previous studies showed that IL-1α is a potent inducer of β-defensin 2 and acts synergistically with NTHi WCL to up-regulate β-defensin 2 expression [27, 30]. These experiments showed that IL-1α-induced β-defensin 2 expression was mediated through the Src-dependent Raf-MEK1/2-ERK MAPK pathway and uses the same TIR domain of TLR2 that recognizes the unknown NTHi WCL component [27, 34]. The synergism between IL-1α and NTHi may be explained as follows: NTHi WCL stimulates HMEEC-1 through MyD88 dependent TLR2 signaling pathway inducing an early phase response with subsequent β-defensin 2 and proinflammatory cytokine (such as IL-1α) production [13, 34]. The secreted IL-1α may further stimulate the cells through MyD88 independent IL-1R pathway that initiates a late phase response [13, 34]. Therefore, even though active NTHi component/ligand and IL-1α utilize the same TIR domain and are working through the p38 and ERK MAPK signaling pathways respectively, they can act synergistically to amplify β-defensin 2 expression [13, 34, 63].
Recently a new group of receptors, Nacht-LRR, NOD-like Receptor, or CATERPILLAR (NLR), has been reported that resemble and function as intracellular receptors for bacterial recognition [19, 64, 65]. These receptors not only recognize invasive microbes and appropriate bacterial components/ligands, but also play pivotal roles in triggering inflammatory response by converting the inactive forms of pro-cytokines to active mature cytokines [66, 67]. Based on these facts, we speculate that these intracellular receptors may contribute to increasing the NTHi induced β-defensin 2 expression in HMEEC-1. Although NTHi is considered as a mucosal surface pathogen, there have been reports that NTHi can be internalized in the mucosal epithelium [68, 69]. While the significance of this bacterial internalization in the pathogenesis of otitis media is not yet known, it is possible that such internalized bacterial components can also induce β-defensin up-regulation. Studies are currently underway in our laboratory to test this possibility.