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Proc Natl Acad Sci U S A. Mar 9, 2004; 101(10): 3563–3568.
Published online Mar 1, 2004. doi:  10.1073/pnas.0400557101
PMCID: PMC373502
Medical Sciences

Synergistic activation of NF-κB by nontypeable Haemophilus influenzae and tumor necrosis factor α

Abstract

Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen causing otitis media in children and exacerbation of chronic obstructive pulmonary disease in adults. Like most other bacterial infections, NTHi infections are also characterized by inflammation, which is mainly mediated by cytokines and chemokines such as tumor necrosis factor α (TNF-α). Among a variety of transcription regulators, NF-κB has been shown to play a critical role in regulating the expression of large numbers of genes encoding inflammatory mediators. In review of the current studies on NF-κB regulation, most of them have focused on investigating how NF-κB is activated by a single inducer at a time. However, in bacteria-induced inflammation in vivo, multiple inducers including both exogenous and endogenous mediators are present simultaneously. A key issue that has yet to be addressed is whether the exogenous inducers such as NTHi and the endogenous factors such as TNF-α activate NF-κB in a synergistic manner. We show that NTHi and TNF-α, when present together, synergistically induce NF-κB activation via two distinct signaling pathways: NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves NF-κB-inducing kinase-IκB kinase β/γ-dependent phosphorylation and degradation of IκBα, whereas the NF-κB translocation-independent pathway involves mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 1-dependent activation of MAPK kinase 3/6-p38 MAPK pathway. In addition, the same signaling pathways are also involved in synergistic induction of TNF-α, IL-1β, and IL-8. These studies should deepen our understanding of the molecular mechanisms underlying the combinatorial regulation of inflammation and lead to development of therapeutic strategies for NTHi-induced infections.

Nontypeable Haemophilus influenzae (NTHi), a Gram-negative bacterium, is an important human pathogen (1). In children, it causes otitis media (OM), which is the most common childhood infection and the leading cause of conductive hearing loss (2, 3), whereas in adults, it exacerbates chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of death in the United States (4, 5). Like most bacterial infections, both OM and COPD are characterized also by inflammation, which is mediated mainly by inflammatory cytokines and chemokines, such as tumor necrosis factor α (TNF-α), IL-1β, and IL-8 (6-8). Among a variety of transcription regulators, NF-κB has been shown to play a critical role in regulating the expression of many genes encoding cytokines, chemokines, and other mediators involved in inflammatory responses (9).

Tremendous efforts have been made toward understanding how NF-κB is activated by various inducers, including bacteria, virus, and cytokines. However, current studies on NF-κB regulation have focused on investigating how NF-κB is activated by a single inducer at one time. Given the fact that, in bacteria-induced inflammation in vivo, multiple inflammation inducers, including both exogenous and endogenous mediators, are present simultaneously, a key issue is whether the exogenous inducers, such as NTHi, and the endogenous factors, such as TNF-α, activate NF-κB simultaneously in a synergistic manner.

NF-κB is a dimeric transcription factor that is composed of p50 (NF-κB1) and p65 (RelA) subunits (9). In resting cells, NF-κB is retained in the cytoplasm but enters the nucleus in response to various stimuli, including bacterial infections. Activation of NF-κB is controlled by an inhibitory subunit, IκB, which retains NF-κB in the cytoplasm. The activation of NF-κB requires sequential phosphorylation, ubiquitination, and degradation of IκB, as well as consequent exposure of a nuclear-localization signal on NF-κB molecule. Many kinases have been shown to phosphorylate IκB at specific N-terminal serine residues (10, 11). The most well studied kinases are the IκB kinases (IKKs) IKK-α, IKK-β, and IKK-γ (9). There is also strong evidence that IKK-β is phosphorylated and activated by one or more upstream activating kinases. One such upstream kinase, NF-κB-inducing kinase (NIK) has been identified (9). Phosphorylation of IκB by IKK pathway will lead eventually to the nuclear translocation of NF-κB, which, in turn, activates expression of target genes such as TNF-α, IL-1β, and IL-8, in the nucleus. In addition to the NF-κB nuclear translocation-dependent NIK-IKK-IκBα signaling pathway, our recent studies demonstrated that mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK pathway also mediates NTHi-induced NF-κB activation by an NF-κB nuclear translocation-independent mechanism (7).

TNF-α is produced by macrophages and epithelial cells. It plays a critical role in mediating inflammatory responses by up-regulation of genes encoding cell-adhesion molecules required for the recruitment of inflammatory cells and genes encoding important inflammatory cytokines, such as IFN-γ (12). Because the promoter regions of many of the TNF-α-regulated genes contain DNA binding sites for NF-κB, activation of NF-κB by TNF-α is essential to elicit effective immune and inflammatory responses. The interaction of TNF-α with its receptor has been shown to activate several signaling pathways, including an MAPK/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)-dependent MAPK signaling pathway and an NIK-IKK-IκBα pathway (13).

Based on our recent report that NTHi activates NF-κB by NIK-IKK-α/β-IκBα and MKK3/6-p38 MAPK signaling pathways and the essential involvement of TNF-α in mediating inflammatory responses, we hypothesized that NTHi and TNF-α may induce inflammatory responses by activation of NF-κB in a synergistic manner. Here, we show that NTHi and TNF-α, when present together, synergistically induce NF-κB activation by two distinct signaling pathways: NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves NIK-IKK-β/γ-dependent phosphorylation and degradation of IκBα, whereas the NF-κB translocation-independent pathway involves MEKK1-dependent activation of MKK3/6-p38 MAPK pathway. In addition, the same NIK-IKK-IκBα and MEKK1-MKK3/6-p38 signaling pathways are involved also in the synergistic induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α. Thus, the present studies provided direct evidence for the synergistic induction of NF-κB-dependent inflammatory responses by both exogenous and endogenous inducers and may lead to the development of therapeutic strategies for modulating the inflammation in otitis media and COPDs.

Materials and Methods

Reagents. MG-132 and SB203580 were purchased from Calbiochem. TNF-α was purchased from Life Technologies (Gaithersburg, MD).

Bacterial Strains and Culture Conditions. NTHi strain 12, which was used in the study, was a clinically isolated strain that was kindly provided by H. Faden (Children's Hospital of Buffalo, State University of New York, Buffalo, NY) (14). For making NTHi crude extract, NTHi were harvested from a plate of chocolate agar after overnight incubation and incubated in 30 ml of brain-heart infusion broth supplemented with (3.5 μg/ml nicotinamide adenine dinucleotide. After overnight incubation, NTHi were centrifuged at 10,000 × g for 10 min, and the supernatant was discarded. The resulting pellet of NTHi was suspended in 10 ml of PBS and sonicated. Subsequently, the lysate was collected and stored at -70°C. We chose to use NTHi lysates because of the following reasons. NTHi has been shown to be highly fragile, and it has the tendency to autolyse. Its autolysis can be triggered in vivo under various conditions, including antibiotic treatment. Therefore, the use of lysates of NTHi represents a common clinical condition in vivo, especially after antibiotic treatment.

Cell Culture. Human cervix epithelial cell line HeLa was maintained as described (7). Human middle-ear epithelial cell line (HMEEC-1) and primary normal human bronchial (NHBE) cells were maintained in bronchial epithelial basal medium purchased from Clonetics (San Diego) by following the manufacturer's instructions (16, 17). Wild-type rat Rat-1 cells and 5R cell line, which is derived from Rat-1 and lacks functional IKK-γ, were maintained in DMEM (17).

Real-Time Quantitative RT-PCR Analysis of TNF-α, IL-1β, and IL-8. Total RNA was isolated by using TRIzol reagent (Invitrogen) by following the manufacturer's instructions. For the reverse transcription reaction, TaqMan reverse transcription reagents (Applied Biosystems) were used. Briefly, the reverse transcription reaction was performed for 60 min at 37°C, followed by 60 min at 42°C by using oligo(dT) and random hexamers. PCR amplification was performed by using TaqMan Universal Master Mix. Predeveloped TaqMan assay reagents (probe and primer mixture of human TNF-α, IL-1β, and IL-8) were used to detect expression of each gene. In brief, reactions were performed in duplicate containing 2× Universal Master Mix, 2 μl of template cDNA, 200 nM primers, and 100 nM probe in a final volume of 25 μl, and they were analyzed in a 96-well optical-reaction plate (Applied Biosystems). Probes include a fluorescent reporter dye, 6-carboxyfluorescein (FAM), on the 5′ end and labeled with a fluorescent quencher dye, 6-carboxytetramethyl-rhodamine (TAMRA), on the 3′ end to allow direct detection of the PCR product. Reactions were amplified and quantified by using an ABI 7700 sequence detector and the manufacturer's corresponding software (Applied Biosystems). Relative quantity of TNF-α, IL-1β, and IL-8 mRNA was obtained by using the comparative Ct Method (for details, see User Bulletin 2 for the ABI PRISM 7700 sequence-detection system) and was normalized by using predeveloped TaqMan assay reagent human cyclophilin as an endogenous control (Applied Biosystems).

Plasmids, Transfection, and Luciferase Assay. The expression plasmids IκBα(S32/36A), NIK(K429/430A), MEKK1(K432M), fp38α(AF), fp38β2(AF), MKK3β(A), MKK6β(A), IKK-β(K49A), and IKK-γ were described (7, 14-17). The reporter construct NF-κB luc was generated as described (7). It contains three copies of the NF-κB site from the IL-2 receptor α promoter. The following oligonucleotide primers were used: 5′-TCGAGACGGCAGGGGAATCTCCCTCTCCG-3′ and 3′-CTGCCGTCCCCTTAGAGGGAGAGGCAGT-5′. The reporter construct was sequenced to verify the number and orientation of inserted oligonucleotides. All transient transfections were performed in triplicate by using TransIT-LT1 reagent (Panvera, Madison, WI) by following the manufacturer's instructions.

Immunofluorescent Staining. Cells were cultured on four-chamber slides. After treatment with NTHi and/or TNF-α, the cells were fixed in 4% paraformaldehyde solution and incubated with mouse anti-p65 NF-κB Ab for 1 h (Santa Cruz Biotechnology). Primary Ab was detected with FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). Samples were examined and photographed by using an Axiophot microscope (Zeiss).

Western Blot Analysis. Abs against phospho-IκBα(Ser-32), IκBα, phospho-p38, p38, phospho-MKK3/6(Ser-189/207), and MKK3 were purchased from Cell Signaling Technology (Beverly, MA). Phosphorylation of IκBα, p38, and MKK3/6 was detected as described and by following the manufacturer's instructions (7, 14-17).

Results and Discussion

NTHi and TNF-α Synergistically Induce NF-κB-Dependent Transcription of TNF-α, IL-1β, and IL-8 in NHBE and HMEEC-1 Cells. To determine whether NTHi and TNF-α synergistically activate NF-κB in human epithelial cells, we first measured NF-κB-dependent promoter activity by using luciferase reporter plasmid in HeLa cells. As shown in Fig. 1A, simultaneous stimulation with NTHi and TNF-α resulted in a synergistic activation of NF-κB-dependent promoter activity in HeLa cells. Because NTHi is a major pathogen of airway and middle-ear infections (1-5), we assayed the HMEEC-1 cell line and NHBE cells also. Interestingly, the synergistic activation of NF-κB by NTHi and TNF-α was observed in all of these cells (Fig. 1 B and C). Because nuclear translocation is a key step for NF-κB to exert its transcriptional activity (9), we next sought to explore the possibility that NTHi and TNF-α synergistically activate NF-κB by inducing its nuclear translocation by using immunofluorescence staining of p65, a key subunit of NF-κB. As shown in Fig. 1D, p65 was localized in the cytoplasm of unstimulated HeLa cells. When the cells were stimulated with NTHi or TNF-α alone, p65 was partially translocated to the nucleus at 10 min, whereas simultaneous treatment with both NTHi and TNF-α resulted in synergistic induction of p65 translocation that was observed in all of the cells. Similar results were observed also in HMEEC-1 and NHBE cells (Fig. 1 E and F). These results suggest that NTHi synergizes with TNF-α to activate NF-κB by inducing its nuclear translocation in human epithelial cells, including primary cells.

Fig. 1.
NTHi and TNF-α synergistically activate NF-κB in human epithelial cells. Simultaneous stimulation with NTHi and TNF-α resulted in synergistic activation of NF-κB-dependent promoter activity in HeLa (A), HMEEC-1 (B), and ...

Because of the important role that NF-κB plays in regulating various key inflammatory mediators (10), we next sought to determine whether NTHi and TNF-α also synergistically induce several key NF-κB-dependent inflammatory mediators, including TNF-α, IL-1β, and IL-8, by performing real-time quantitative PCR analysis. As shown in Fig. 2, NTHi and TNF-α synergistically induced expression of TNF-α, IL-1β, and IL-8 in all of these three cell types, including HeLa (Fig. 2A), HMEEC-1 (Fig. 2B), and NHBE (Fig. 2C) cells.

Fig. 2.
NTHi and TNF-α synergistically induce expression of TNF-α, IL-1β, and IL-8 in HeLa (A), HMEEC-1 (B), and NHBE (C) cells, as assessed by performing real-time quantitative PCR analysis. Values are means ± SD. Data are representative ...

NIK-IKK-β/γ-Dependent IκBα Phosphorylation and Degradation Are Required for the Synergistic Activation of NF-κB by NTHi and TNF-α. NF-κB is normally present in the cytoplasm in an inactive state, and it is bound to members of the IκB inhibitor protein family, chiefly IκBα (9-11). In this complex, IκBα blocks the nuclear-localization signal, preventing nuclear translocation. To translocate NF-κB into the nucleus, the cytoplasmic NF-κB-IκBα complex needs to be disrupted (9). A common pathway to achieve disruption of this complex is based on specific phosphorylation of IκBα and the degradation of the phosphorylated IκBα protein by proteasomes. The inhibition of proteasome activity by specific inhibitors such as MG-132 prevents the degradation of IκBα and, thus, prevents the nuclear translocation of NF-κB (17-18). To determine the involvement of IκBα phosphorylation and degradation in the synergistic activation of NF-κB by NTHi and TNF-α, we first investigated the effect of proteasome inhibitor MG-132 in HeLa cells. As shown in Fig. 3A, MG-132 abrogated the synergistic activation of NF-κB. To confirm the involvement of IκBα further, we transfected the cells with a transdominant mutant (S32A,S36A) of IκBα in which two critical serine residues required for inducer-mediated phosphorylation were mutated (19). Overexpression of the transdominant mutant IκBα greatly inhibited the synergistic activation of NF-κB. Consistent with this finding, synergistic induction of phosphorylation and degradation of IκBα was observed also in cells treated with both NTHi and TNF-α, as assessed by performing Western blot analysis by using Abs against phosphorylated and total IκBα, respectively (Fig. 3B). These observations implicate the involvement of IκBα phosphorylation and degradation in the synergistic activation of NF-κB by NTHi and TNF-α.

Fig. 3.
NIK-IKK-β/γ-dependent IκBα phosphorylation and degradation are required for the synergistic activation of NF-κB by NTHi and TNF-α.(A) MG132 and overexpression of a transdominant mutant of IκBα ...

Recently the IκBα kinase IKK-α-IKK-β-IKK-γ complex has been shown to phosphorylate IκB at specific N-terminal serine residues (11). There is also strong evidence that IKK-β is phosphorylated and activated by upstream kinase NIK (9). On the basis of these studies, we investigated the role of NIK-IKK-β/γ pathway in the synergistic activation of NF-κB by NTHi and TNF-α. Cotransfection with a dominant-negative mutant form of IKK-β, IKK-β(K49A), markedly inhibited the NTHi-induced NF-κB activation (Fig. 3C). Consistent with these results, 5R, a cell line that is derived from Rat-1 cells and lacks functional IKK-γ, did not show an NTHi- and TNF-α-induced activation of NF-κB-driven promoter activity (17, 20). Their responsiveness to NTHi and TNF-α could be rescued fully by cotransfection of wild-type IKK-γ in 5R cells, similar to the response of wild-type Rat-1 cells (Fig. 3D). Likewise, overexpression of dominant-negative mutant form of NIK, NIK(K429/430A), also inhibited NTHi- and TNF-α-induced synergistic activation of NF-κB (Fig. 3E). Together, these findings clearly demonstrate that NTHi and TNF-α synergistically induce NF-κB by means of the activation of NIK-IKK-β/γ-dependent IκBα phosphorylation and degradation, which, in turn, leads to NF-κB nuclear translocation.

Activation of MKK3/6-p38 MAPK Pathway Is Required also for the Synergistic Activation of NF-κB by NTHi and TNF-α. Many cellular-stress stimuli can activate both NF-κB and p38 MAPK modules (7, 21-25). Because of this overlap, we explored the possibility that activation of p38 is involved also in the synergistic NF-κB activation. We first sought to determine whether activation of p38 MAPK is required for the synergistic NF-κB activation by assessing the effect of pyridinyl imidazole SB203580, a specific inhibitor for p38 MAPK (26). As shown in Fig. 4A, SB203580 abrogated the synergistic activation of NF-κB in response to NTHi and TNF-α. Moreover, overexpression of a dominant-negative mutant form of either p38α [fp38α(AF)] or p38β [fp38β2(AF)] inhibited the synergistic NF-κB activation also, confirming the involvement of p38 MAPK in the synergistic NF-κB activation. Similarly, the synergistic induction of p38 MAPK phosphorylation was observed also in cells treated with both NTHi and TNF-α, as evaluated by Western blot analysis by using antiphosphorylated-p38 MAPK (Fig. 4B). These results indicate that p38 MAPK is involved also in the synergistic activation of NF-κB induced by NTHi and TNF-α.

Fig. 4.
Activation of MKK3/6-p38 MAPK pathway is required for the synergistic activation of NF-κB by NTHi and TNF-α.(A) SB203580 and overexpression of dominant-negative mutants of p38α and p38β2 inhibited the synergistic activation ...

Two kinases of the MAPK superfamily, MKK3 and MKK6, have been identified as upstream activators of p38 MAPK. It has been demonstrated that MKK6 is a common activator of p38α and p38β, whereas MKK3 activated only p38α (22). To investigate further whether activation of MKK3/6 is involved also in NTHi- and TNF-α-induced synergistic NF-κB activation, a dominant-negative mutant form of either MKK3 [MKK3b(A)] or MKK6 [MKK6b(A)] was cotransfected into HeLa cells. The synergistic NF-κB activation was inhibited by both treatments (Fig. 4C). Consistent with these results, the synergistic induction of MKK3/6 phosphorylation was observed also in cells treated with both NTHi and TNF-α (Fig. 4D). Collectively, these data demonstrate that activation of MKK3/6-p38α/β MAPK pathway is also required for the synergistic activation of NF-κB activation by NTHi and TNF-α.

MEKK1 Is Involved in the NTHi- and TNF-α-Induced Synergistic NF-κB Activation by Acting as an Upstream Kinase for the p38 MAPK, but not for the IκBα, Pathway. Despite extensive analysis of both NIK-IKK-β/γ-IκBα and MKK3/6-p38 MAPK signal transduction pathways, the signaling events upstream of both pathways still remain poorly understood. Based on recent studies that MEKK1 is involved in NF-κB activation and that MEKK1 acts as an upstream activator for IKK-IκBα and p38 MAPK pathways (27-28), we next sought to determine whether MEKK1 acts upstream of both signaling pathways to mediate NTHi- and TNF-α-induced synergistic NF-κB activation. As shown in Fig. 5A, cotransfecting cells with a dominant-negative mutant of MEKK1 inhibited the synergistic activation of NF-κB. Interestingly, overexpressing the same dominant-negative mutant of MEKK1 attenuated the NTHi- and TNF-α-induced synergistic phosphorylation of p38 MAPK but not IκBα (Fig. 5 B and C). We conclude from these data that MEKK1 mediates NTHi- and TNF-α-induced synergistic activation of NF-κB by acting as a upstream kinase for a p38 MAPK, but not for an IKK-IκBα, signaling pathway.

Fig. 5.
MEKK1 is involved in the NTHi- and TNF-α-induced synergistic NF-κB activation by acting as an upstream kinase for the p38 MAPK, but not for the IκBα, pathway. (A) Overexpression of a dominant-negative mutant of MEKK1 inhibited ...

Requirement of NIK-IKK-IκBα and MEKK1-MKK3/6-p38 Pathways in NTHi- and TNF-α-Induced Synergistic NF-κB Activation Is Conserved in Multiple Human Epithelial Cells. Having identified the requirement of NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways in NTHi- and TNF-α-induced synergistic NF-κB activation in HeLa cells, we next sought to determine whether these signaling pathways are involved in mediating the synergistic activation of NF-κB in other human epithelial cells also, such as HMEEC-1 and NHBE cells. As shown in Fig. 6, the synergistic NF-κB activation by NTHi and TNF-α was abrogated by MG-132, SB203580, and overexpression of a dominant-negative mutant MEKK1 in both HMEEC-1 and NHBE cells. Thus, it is evident that the requirement of NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways in NTHi- and TNF-α-induced synergistic NF-κB activation is conserved in multiple human epithelial cells, including the primary human bronchial epithelial cells.

Fig. 6.
Requirement of NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways in NTHi- and TNF-α-induced synergistic NF-κB activation is well conserved in HMEEC-1 and NHBE cells. MG-132, SB203580, and coexpression of a dominant-negative mutant ...

NIK-IKK-IκBα and MEKK1-MKK3/6-p38 Pathways Are also Required for the Synergistic Induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α in Multiple Human Epithelial Cells. Because NF-κB plays an essential role in regulating various key inflammatory mediators (7, 10-11), we sought next to determine whether the same signaling pathways also mediate the synergistic induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α. As shown in Fig. 7, the same NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways are involved also in the synergistic induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α in multiple human epithelial cells, including HeLa (Fig. 7A), HMEEC-1 (Fig. 7B), and NHBE (Fig. 7C) cells, as assessed by performing real-time quantitative PCR analysis.

Fig. 7.
NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways are required also for the synergistic induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α in many human epithelial cells, including primary cells. MG-132, SB203580, and ...

In conclusion, our studies demonstrate that NTHi, a major human pathogen of otitis media and COPD, synergizes with TNF-α, a key proinflammatory cytokine, to activate NF-κB strongly in a variety of human epithelial cells, including the primary bronchial epithelial cells (Fig. 8). NTHi- and TNF-α-induced synergistic NF-κB activation is mediated by two distinct signaling pathways: NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves NIK-IKK-β/γ-dependent phosphorylation and degradation of IκBα, whereas the NF-κB translocation-independent pathway involves MEKK1-dependent activation of MKK3/6-p38 MAPK pathway. In addition, the same NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways are involved also in the synergistic induction of TNF-α, IL-1β, and IL-8 by NTHi and TNF-α. Moreover, the requirement of NIK-IKK-IκBα and MEKK1-MKK3/6-p38 pathways in NTHi- and TNF-α-induced synergistic NF-κB activation and cytokine induction is well conserved in a variety of human epithelial cells, including the primary human bronchial epithelial cells.

Fig. 8.
Schematic representation depicting how NTHi and TNF-α synergistically activate NF-κB as well as the induction of TNF-α, IL-1β, and IL-8 in various human epithelial cells, including NHBE cells. As indicated, NTHi- and TNF-α-induced ...

Of particular interest in the present studies is the observation that NTHi and the key inflammatory mediator TNF-α synergize with each other to induce NF-κB activation and consequently the NF-κB-dependent up-regulation of several key inflammatory mediators including IL-1β and IL-8 in addition to TNF-α itself. In review of the current studies on NF-κB regulation in bacterial infections, most studies have fully focused on investigating how NF-κB is activated by a single inducer at a time (8-11). Although these studies are indeed critical for our understanding of the molecular basis underlying bacteria-induced inflammation, the information derived from these studies may be insufficient for fully understanding how NF-κB is induced in vivo where both exogenous and endogenous mediators are present simultaneously. In the pathogenesis of NTHi infections, NTHi first interacts with its receptors sitting on the surface of host cells, such as Toll-like receptor 2 (TLR2) (7), which, in turn, leads to the activation of multiple signaling cascades, chiefly the NIK-IKK-IκBα-NF-κB pathway and the MKK-p38 MAPK pathway. One of the consequence of the activation of these signaling pathways is the up-regulation of genes encoding important inflammatory cytokines such as TNF-α and the genes encoding cell-adhesion molecules required for the recruitment of inflammatory cells such as E-selectin and VCAM (vascular cell-adhesion molecule 1). The up-regulated TNF-α in response to NTHi will in turn act on the host cells and synergize with NTHi to induce NF-κB by means of activation of two distinct signaling pathways, the NIK-IKK-IκBα and the MEKK1-MKK3/6-p38 MAPK pathways. Therefore, the signaling mechanisms underlying bacteria-induced inflammation in the presence of both exogenous and endogenous inducers appear to be more complicated than the signaling mechanisms underlying bacteria-induced inflammation in the presence of a single inducer. Future studies could possibly focus on investigating comparing other inflammatory mediators such as IL-1β with the effect of TNF-α on the NTHi-induced NF-κB-dependent inflammatory responses. Understanding the molecular mechanisms underlying the combinatorial regulation of inflammation in bacterial infections is pivotal to the development of therapeutic strategies for the modulation of bacteria-induced inflammation.

Acknowledgments

We thank Dr. Shoji Yamaoka for kindly providing rat Rat-1 and 5R cell lines. This work was supported by National Institutes of Health Grants DC005843, DC004562, and HL070293 (to J.-D.L.).

Notes

Abbreviations: COPD, chronic obstructive pulmonary disease; HMEEC-1, human middle-ear epithelial cell line; IKK, IκB kinase; MAPK, mitogen-activated protein kinase; MEKK1, MAPK/extracellular signal-regulated kinase kinase kinase 1; MKK, MAPK kinase; NIK, NF-κB-inducing kinase; NHBE cells, primary normal human bronchial cells; NTHi, nontypeable Haemophilus influenzae; TNF-α, tumor necrosis factor α.

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