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J. Biol. Chem., Vol. 277, Issue 19, 17263-17270, May 10, 2002

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Glucocorticoids Synergistically Enhance Nontypeable Haemophilus influenzae-induced Toll-like Receptor 2 Expression via a Negative Cross-talk with p38 MAP Kinase^*

Tsuyoshi Shuto^§, Akira Imasato^§, Hirofumi Jono, Akihiro Sakai, Haidong Xu, Takahiro Watanabe, Davida D. Rixter, Hirofumi Kai^¶, Ali Andalibi, Fred Linthicum, Yue-Ling Guan, Jiahuai Han^**, Andrew C. B. Cato, David J. Lim, Shizuo Akira^§§, and Jian-Dong Li^§¶¶

From the  Gonda Department of Cell and Molecular Biology, House Ear Institute, and the Department of Otolaryngology, University of Southern California, Los Angeles, CA 90057, the ^¶ Department of Molecular Medicine, Kumamoto University, Kumamoto 862-0973, Japan, the  Department of Histopathology, House Ear Institute, Los Angeles, California 90057, the ^** Department of Immunology, the Scripps Research Institute, La Jolla, California 92037, the  Forschungszentrum Karlsruhe, Institute of Toxicology & Genetics, P. O. Box 3640, D-76021 Karlsruhe, Germany, and the ^§§ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, CREST of Japan Science and Technology Corporation, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan

Received for publication, December 20, 2001, and in revised form, February 18, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be up-regulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeable Hemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKK-IB-dependent NF-B pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38/ pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effective host defense against invading microorganisms requires the detection of foreign pathogens and the rapid deployment of an antimicrobial effector response (1). In the host innate immune system, the surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against pathogenic bacteria (2). They recognize the invading bacteria by directly interacting with pathogen-associated molecular patterns on a variety of bacteria via Toll-like receptors (TLRs)¹ expressed on the host (3). Activation of TLRs, in turn, leads to induction of direct antimicrobial activity that can result in elimination of the invading pathogen before a full adaptive immune response takes effect (4). In addition, activation of TLRs is also a prerequisite for the triggering of acquired immunity (5). TLRs are type I transmembrane receptors with leucine-rich repeats in the extracellular domains and cytoplasmic domains that resemble the mammalian IL-1 receptor (IL-1R) (6, 7). To date, 10 members of the human TLR family have been cloned. Of these, TLR2 and TLR4 have been well studied. While TLR4 seems to be mainly involved in Gram-negative bacteria lipopolysaccharide (LPS) signaling, TLR2 can respond to a variety of Gram-positive products, including peptidoglycan, lipoprotein, lipoteichoic acid, and lipoarabinomannan. The importance of TLR2 in host defense was further supported by the studies from knock-out mice showing decreased survival of TLR2-deficient mice after infection with Gram-positive Staphylococcus aureus (8). Moreover, our recent study demonstrated that TLR2 also plays an important role in activation of NF-B by lipoprotein from the Gram-negative bacterium nontypeable Hemophilus influenzae (NTHi), a major cause of otitis media and exacerbation of chronic obstructive pulmonary diseases (COPD) (9). Thus, it is clear that TLR2 plays a crucial role in host defense against both Gram-positive and -negative bacteria. In contrast to its relatively higher expression in lymphoid tissues, TLR2 is expressed at a low level in epithelial cells. Given the low expression of TLR2 in unstimulated human epithelial cells, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating NTHi-induced inflammatory response and whether TLR2 expression can be up-regulated by bacteria in human epithelial cells during bacterial infections. Here, we explore the possibility that NTHi up-regulates TLR2 in human epithelial cells via activation of specific signaling pathways. Our studies reveal that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by NTHi. Activation of IKK-IB-dependent NF-B pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38/ pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Moreover, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the inhibitory p38 MAP kinase pathway. These studies, although rather unexpected, may provide novel insights into the role of bacteria and glucocorticoids in regulating host defense and innate immune responses and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and COPD.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Caffeic acid phenethyl ester (CAPE), MG-132, and SB203580 were purchased from Calbiochem. Dexamethasone and RU486 were purchased from Sigma.

Bacterial Strains and Culture Conditions-- NTHi strain 12, a clinical isolate, was used in this study. 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 NAD (3.5 µg/ml). 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 phosphate-buffered saline and sonicated. Subsequently, the lysate was collected and stored at 70 °C. NTHi whole cell lysate was used in all the experiments, unless otherwise indicated.

Cell Culture-- Human cervix epithelial cell line HeLa was maintained as described previously (9, 14). Human middle ear epithelial cell line HMEEC-1 derived by human papillomavirus immortalization of primary human middle ear epithelial cells and primary human airway epithelial cells (NHBE) (Clonetics, Walkersville, MD) were maintained in bronchial epithelial basal medium (Clonetics) following manufacturer's instruction.

RT-PCR Analysis of TLR2-- Total RNA was isolated from human epithelial cells using a Qiagen kit (Valencia, CA) following the manufacturer's instruction. For the RT reaction, the Moloney murine leukemia virus preamplification system (Invitrogen) was used. PCR amplification was performed with Taq gold polymerase (PerkinElmer Life Sciences) for 32 cycles at 95 °C for 60 s, 63 °C for 60 s, and 72 °C for 60 s (for TLR2) and 32 cycles at 95 °C for 60 s, 50 °C for 60 s, and 72 °C for 60 s (for cyclophilin). The oligonucleotide primers were: TLR2, 5'-GCCAAAGTCTTGATTGATTGG-3' and 5'-TTGAAGTTCTCCAGCTCCTG-3'; cyclophilin, 5'-CCGTGTTCTTCGACATTGCC-3' and 5'-ACACCACATGCTTGCCATCC-3'.

Real-time Quantitative PCR Analysis of TLR2 and TLR4-- Total RNA was isolated from human epithelial cells as described above. For the RT reaction, TaqMan reverse transcription regents (Applied Biosystems, Foster City, CA) were used. Briefly, the RT reaction (final volume of total 50 µl) was conducted for 60 min at 37 °C followed by 60 min at 42 °C using oligo(dT) and random hexamers. PCR amplification was performed with TaqMan Universal Master Mix (Applied Biosystems). In brief, reactions were performed in duplicate containing 2× Universal PCR master mix, 2 µl of template cDNA, 200 nM of TLR2 primers (5'-GGCCAGCAAATTACCTGTGTG-3' and 5'-AGGCGGACATCCTGAACCT-3') and 100 nM of TLR2 probe (5'-TCCATCCCATGTGCGTGGCC-3') in a final volume of 25 µl and were analyzed in a 96-well optical reaction plate (Applied Biosystems). TLR2 primers and probes were synthesized by Applied Biosystems custom oligo synthesis service. Probes include a fluorescent reporter dye, FAM, on the 5' end and labeled with a fluorescent quencher dye, TAMRA, on the 3' end to allow direct detection of the PCR product. Reactions were amplified and quantified using an ABI 7700 sequence detector and manufacturer's software (Applied Biosystems). The relative quantity of TLR2 mRNA was obtained using the comparative CT method and was normalized using pre-developed TaqMan assay reagent human cyclophilin as an endogenous control (Applied Biosystems) (for details, see user Bulletin 2 for the ABI PRISM 7700 Sequence Detection System under www.appliedbiosystems.com/support/tutorials). Briefly, the TaqMan software (Applied Biosystem) was used to calculate a Ct value for each reaction, where the Ct value is the point in the extension phase of the PCR reaction that the product is distinguishable from the background. The Ct values were then normalized for TLR2 amplification by subtracting the Ct value calculated for cyclophilin, an endogenous control for the amount of mRNA from the same sample, to obtain a Ct using the following equation: Ct TLR2  Ct cyclophilin = Ct. The NTHi-dependent or other inducer-dependent fold induction of TLR2 was calculated relative to the Ct value obtained in the unstimulated cells. The normalized TLR2 expression was thus expressed as relative quantity of TLR2 mRNA (fold induction). TLR4 mRNA was measured using TLR4 primers (5'-CCAGTGAGGATGATGCCAGGAT-3' and 5'-GCCATGGCTGGGATCAGAGT-3') and a TLR4 probe (5'-TGTCTGCCTCGCGCCTGGC-3') and was then normalized similarly to TLR2.

Plasmids, Transfections, and Luciferase Assays-- The expression plasmids hTLR₂, IB(S32/36A), IKK(K49A), fp38(AF) and wild-type (WT), fp382(AF) and WT, MKK3b(A), MKK6b(A), and phGR were described previously (9, 14, 15, 20). The expression plasmid of p65 was kindly provided by A. S. Baldwin (University of North Carolina, Chapel Hill, NC). The reporter construct NF-B luc was generated as described previously (9). It contains three copies of the NF-B site from the IL-2 receptor () promoter using the following oligonucleotides: 5'-TCGAGACGGCAGGGGAATCTCCCTCTCCG-3' and 3'-CTGCCGTCCCCTTAGAGGGAGAGGCAGCT-5'. The reporter construct was sequenced to verify number and orientation of inserted oligonucleotides. All transient transfections were carried out in duplicate for RT-PCR analysis and in triplicate for luciferase assays using TransIT-LT1 reagent (Panvera, Medison, WI) following the manufacturer's instruction, unless otherwise indicated. In all co-transfections with expression plasmids of signaling molecules, an empty vector was used as a control.

Immunofluorescent Staining-- Cells were cultured on four-chamber microscope slides. After NTHi treatment, the cells were fixed in paraformaldehyde solution (4%) and incubated with mouse anti-p65 NF-B monoclonal antibody for 1 h (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Primary antibody was detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.). Samples were viewed and photographed using a Zeiss Axiophot microscope.

Western Blot Analysis-- To detect TLR2 up-regulation at protein level, Western blot analysis was carried out using antibodies against human TLR2. To ensure the specificity, two antibodies against human TLR2 from different sources were used, including a polyclonal antibody to TLR2 (H-175) (Santa Cruz Biotechnology, Inc.) and a monoclonal antibody to TLR2 (IMG-319) (IMGENEX, San Diego, CA). HeLa cells transfected with human wild-type TLR2 expression plasmid served as a positive control for TLR2 expression. To assess phosphorylation of p38, Western blot analysis was carried out using antibodies against phospho-p38(Thr^180/182) and p38 (New England Biolabs, Beverly, MA). Phosphorylation of p38 was detected as described previously (9, 14).

Immunohistochemical Analysis of Human TLR2 Expression-- Celloidin-embedded sections of normal and diseased mucosa from archival temporal bone specimens were obtained from the House Ear Institute's Temporal Bone Collection. Following de-celloidination with ether and absolute ethanol, nonspecific binding sites on the slides were blocked using normal goat serum. The sections were then subjected to antigen retrieval (Zymed Laboratories Inc., South San Francisco, CA) and incubated with polyclonal antibody against human TLR2 (H-175) (Santa Cruz Biotechnology, Inc.). Signals were detected by the avidin/biotin complex method (Zymed Laboratories Inc.).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NTHi Up-regulates TLR2, but Not TLR4, in Human Epithelial Cells-- We first examined whether NTHi up-regulates TLR2 in human epithelial cells. We previously showed that TLR2 was expressed at a low level in a variety of human epithelial cells by RT-PCR analysis (9). As shown in Fig. 1A, the expression of TLR2 was very low in HeLa (human cervix epithelial) cells, but was up-regulated by NTHi. Similar results were also observed in HMEEC-1 (human middle ear epithelial) and primary human bronchial epithelial (NHBE) cells (data not shown). We next sought to confirm whether NTHi up-regulates TLR2 by performing real-time quantitative PCR analysis. NTHi indeed strongly up-regulates TLR2 mRNA in HeLa, HMEEC-1, and NHBE cells in a time-dependent manner (Fig. 1, B and C). Because TLR4 represents another important member of TLR family, we also evaluated the effect of NTHi on TLR4 expression in human epithelial cells. Interestingly, TLR4 did not appear to be strongly up-regulated (Fig. 1D). To determine whether up-regulation of TLR2 mRNA is accompanied by elevated TLR2 protein, Western blot analysis was carried out using antibodies against human TLR2. As shown in Fig. 1E, up-regulation of TLR2 was also observed at protein level. To further confirm whether TLR2 protein is elevated in diseased tissue in vivo, immunohistochemical analysis of TLR2 protein was performed in human middle ear tissues obtained from patients with chronic otitis media and normal individuals, respectively. In comparison with that from normal individuals, the expression of TLR2 protein is indeed higher in epithelial cells of middle ear tissue from patients with chronic otitis media (Fig. 1F). Taken together, these data demonstrate that TLR2, but not TLR4, is up-regulated in human epithelial cells by NTHi.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   NTHi up-regulates TLR2, but not TLR4, in human epithelial cells. A, the expression of TLR2 at mRNA level was measured by RT-PCR in NTHi-treated and untreated human cervix epithelial HeLa cells. HeLa cells transfected with human wild-type TLR2 expression plasmid (hTLR2) served as a positive control for TLR2 expression, whereas cyclophilin was used as a control for amount of RNA used in each reaction. RT was either included or not included in RT reactions to assure that the TLR2 PCR products result from cDNA, but not genomic DNA. RT-PCR analysis was carried out in duplicate. B, real-time quantitative PCR was performed to confirm up-regulation of TLR2 at mRNA level in HeLa, HMEEC-1 (human middle ear epithelial), and NHBE (primary human airway epithelial) cells. TLR2 mRNA levels were normalized to the level of cyclophilin that served as an internal control for the amount of RNA used in each reaction. C, NTHi up-regulates TLR2 mRNA in HeLa cells in a time-dependent manner. TLR2 mRNA was measured by real-time quantitative PCR. Similar results were also observed in NHBE cells. D, NTHi strongly up-regulates TLR2, but not TLR4 in HeLa cells, as assessed by real-time quantitative PCR. E, NTHi also up-regulates expression of TLR2 at protein level as assessed by Western blot analysis. HeLa cells were treated with and without NTHi for 5 h. Protein lysates were then analyzed by Western blotting using anti-hTLR2 antibody (H-175). Equal amounts of proteins were loaded. HeLa cells transfected with human wild-type TLR2 expression plasmid (TLR2) served as a positive control for TLR2 expression. Similar results were also observed in NHBE cells. F, the expression of human TLR2 is higher in mucosae from chronic otitis media patients in comparison with that from normal subjects. Immunohistochemical analysis of human TLR2 expression was performed in human middle ear mucosae from chronic otitis media patients and normal subjects. Archival human temporal bone sections were stained with a polyclonal anti-human TLR2 antibody (H-175). Note that the middle ear epithelium is thickened in the inflamed middle ear mucosa from chronic otitis media patients. Bar, 50 µm. CON, control.

IKK-IB-dependent Translocation and Activation of NF-B Is Required for NTHi-induced TLR2 Up-regulation-- Having demonstrated that TLR2 is up-regulated in human epithelial cells by NTHi, still unknown is which intracellular signaling pathways are involved. Based on the essential involvement of NF-B in regulating the expression of large numbers of genes involved in immunity and inflammation and our recent study showing the activation of NF-B by NTHi (9, 10), we determined the role of NF-B in NTHi-induced TLR2 up-regulation by using CAPE, a chemical inhibitor that is known to specifically block the translocation of p65 without affecting IB degradation (11). As shown in Fig. 2A, CAPE greatly reduced NTHi-induced TLR2 up-regulation, suggesting that activation of NF-B is involved in NTHi-induced TLR2 up-regulation. Because disruption of the NF-B·IB complex is required for NF-B nuclear translocation and activation, we next determined the requirement of IB degradation by assessing the effect of proteasome inhibitor MG-132 and overexpression of a transdominant mutant of IB on NTHi-induced TLR2 up-regulation (12, 13). As expected, MG-132 and overexpression of a transdominant mutant IB markedly inhibited TLR2 up-regulation (Fig. 2A). Concomitantly, NTHi-induced NF-B activation and translocation was also blocked by MG-132 in the same epithelial HeLa cells (Fig. 2, B and C). On the basis of a recent report that IB kinase  (IKK) acts as an immediate upstream kinase of IB, we investigated the role of IKK in NTHi-induced TLR2 up-regulation (10). Cotransfection with a dominant-negative mutant form of IKK (IKK(K44A)) abrogated NTHi-induced TLR2 up-regulation (Fig. 2D). We further confirmed the involvement of NF-B by transfecting the epithelial cells with an expression plasmid of wild-type p65, the major subunit of NF-B. Interestingly, expression of wild-type p65 induced TLR2 mRNA in a dose-dependent manner (Fig. 2E). As expected, expression of p65 also induced NF-B activation (Fig. 2F). Collectively, these findings clearly demonstrated that the IKK-IB-dependent translocation and activation of NF-B are required for NTHi-induced TLR2 up-regulation in human epithelial cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   IKK-IB-dependent translocation and activation of NF-B is required for NTHi-induced TLR2 up-regulation. A, CAPE, MG-132, and overexpression of a transdominant mutant of IB inhibit NTHi-induced TLR2 up-regulation in HeLa cells. Similar results were also observed in NHBE cells. B and C, concomitantly, NTHi-induced NF-B activation and translocation were also blocked by MG-132 in HeLa cells. Similar results were also observed in NHBE cells. D, overexpression of a dominant-negative mutant form of IKK (IKK(K44A)) abrogated NTHi-induced TLR2 up-regulation in HeLa cells. Similar results were also observed in NHBE cells. E and F, overexpression of wild-type p65 induces TLR2 mRNA and NF-B activation in a dose-dependent manner in HeLa cells. All real-time quantitative PCR reactions were carried out in duplicate. Values are the mean ± S.D.; n = 3. CON, control.

Activation of MKK3/6-p38 / MAP Kinase Pathway Is Negatively Involved in NTHi-induced TLR2 Expression-- Many cellular stress stimuli can activate both NF-B and p38 MAP kinase modules. Because of this overlap as well as our recent report that NTHi strongly activates p38 MAP kinase (9, 14, 15), we investigated whether p38 MAP kinase is also involved in NTHi-induced TLR2 up-regulation. Surprisingly, the pyridinyl imidazole SB203580, a highly specific inhibitor for p38 MAP kinase greatly enhanced NTHi-induced TLR2 up-regulation in a dose-dependent manner (16), suggesting that activation of p38 MAP kinase may be negatively involved in NTHi-induced TLR2 up-regulation (Fig. 3A). Moreover, overexpression of a dominant-negative mutant form of either p38 (fp38(AF)) or p38 (fp38(AF)) also enhanced the NTHi-induced TLR2 expression, whereas overexpression of a wild-type p38 or p38 reduced it, thereby further supporting the negative involvement of p38 MAP kinase (Fig. 3B). As immediate upstream kinases of p38  and , two MAP kinase kinases (MKK3 and MKK6) have been identified (15). To further investigate whether activation of MKK3/6 is also negatively involved in NTHi-induced TLR2 up-regulation, a dominant-negative mutant form of either MKK3 (MKK3b(A)) or MKK6 (MKK6b(A)) was transfected into HeLa cells. The NTHi-induced TLR2 up-regulation was enhanced (Fig. 3C). Thus, we concluded from this data that activation of MKK3/6-p38 / MAP kinase pathway may be negatively involved in NTHi-induced TLR2 up-regulation.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Activation of MKK3/6-p38 / MAP kinase pathway is negatively involved in NTHi-induced TLR2 expression. A, SB203580 greatly enhances NTHi-induced TLR2 up-regulation in HeLa cells in a dose-dependent manner. B, overexpression of a dominant-negative mutant form of either p38 (p38(AF)) or p38 (p38(AF)) also enhances, whereas overexpression of a wild-type p38 or p38 reduces, the NTHi-induced TLR2 expression in HeLa cells. C, overexpression of a dominant-negative mutant form of either MKK3 (MKK3b(A)) or MKK6 (MKK6b(A)) enhances NTHi-induced TLR2 up-regulation in HeLa cells. All real-time quantitative PCR reactions were carried out in duplicate. Values are the mean ± SD; n = 3. Similar results were also observed in NHBE cells. CON, control.

Glucocorticoids Synergistically Enhance NTHi-induced TLR2 Up-regulation via a Likely Negative Cross-talk with p38 MAP Kinase Pathway-- Glucocorticoids have been used widely as anti-inflammatory agents. They exert their anti-inflammatory effects by down-regulating NF-B-dependent transcription of a variety of genes involved in the inflammatory response (17). Since we have shown that activation of NF-B is required for NTHi-induced TLR2 up-regulation, it is therefore logical that glucocorticoids may attenuate NTHi-induced TLR2 up-regulation. To test this, we first evaluated the effect of dexamethasone (DEX), a glucocorticoid analog in human epithelial cells, on NTHi-induced up-regulation of TLR2 mRNA. Surprisingly, DEX synergistically enhanced NTHi-induced TLR2 up-regulation at mRNA level in a dose-dependent manner (Fig. 4A). Concomitantly, Western blot analysis showed that DEX also synergistically enhanced NTHi-induced TLR2 up-regulation at protein level (Fig. 4B). These results are rather unexpected, because they were in sharp contrast to the inhibitory effect of DEX on cytokine production (18). Additionally, RU486, a synthetic antiglucocorticoid that acts as a competitor against binding to the glucocorticoid receptor (GR), counteracted the enhancing effect of DEX on NTHi-induced TLR2 up-regulation, suggesting the involvement of GR (Fig. 4C) (19). Moreover, overexpression of wild-type GR in HeLa cells greatly enhanced the synergistic effect of DEX on NTHi-induced TLR2 up-regulation, confirming the involvement of GR (Fig. 4D) (20). Note that a maximal response was observed with 1 µM DEX. Together, these results indicate that DEX synergistically enhanced NTHi-induced TLR2 up-regulation at both mRNA and protein levels via a GR-dependent mechanism.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Glucocorticoids synergistically enhance NTHi-induced TLR2 up-regulation via a likely negative cross-talk with p38 MAP kinase pathway. A, DEX synergistically enhances NTHi-induced TLR2 up-regulation at the mRNA level in HeLa cells in a dose-dependent manner. Similar results were also observed in NHBE and HMEEC-1 cells. B, DEX (1 µM) also synergistically enhances NTHi-induced TLR2 up-regulation at protein level in HeLa cells as assessed by Western blot analysis using anti-hTLR2 antibody (IMG-319). Equal amounts of proteins were loaded. C, RU486 (1 µM) counteracts the enhancing effect of DEX (1 µM) on NTHi-induced TLR2 up-regulation at mRNA level in HeLa. D, overexpression of wild-type human GR (phGR) in HeLa cells further greatly enhances the synergistic effect of DEX on NTHi-induced TLR2 up-regulation. E, effects of DEX on NTHi-induced TLR2 up-regulation in HeLa cells that were pretreated with SB203580. F, NTHi-induced phosphorylation of p38 MAP kinase is inhibited by DEX (1 µM) pretreatment in HeLa cells, which can be counteracted by RU486 (1 µM). Similar results were also observed in NHBE cells. All real-time quantitative PCR reactions were carried out in duplicate. Values are the mean ± S.D.; n = 3. CON, control.

One key issue that has yet to be addressed is how DEX synergistically enhances NTHi-induced TLR2 up-regulation. Because our results in Fig. 3, A and B, indicated that inhibition of p38 MAP kinase also synergistically enhanced NTHi-induced TLR2 up-regulation and glucocorticoids have been shown to inhibit p38 MAP kinase activity (21), we therefore postulated that glucocorticoids may enhance NTHi-induced TLR2 up-regulation via a negative cross-talk with p38 MAP kinase pathway. Our hypothesis was first supported by the result shown in Fig. 4E (upper panel) that DEX no longer enhanced NTHi-induced TLR2 up-regulation if the cells were already pretreated with SB203580, suggesting that DEX and SB203580 may target the same p38 MAP kinase signaling pathway. However, considering that the result was observed with 5 µM SB203580 and 1 µM DEX, a dose shown to elicit a maximal response when used alone, we cannot rule out the possibility that the system was already working at its maximal level. We therefore studied the effect of low doses of both SB203580 and DEX on NTHi-induced TLR2 expression. Interestingly, when 0.1 µM DEX was added to the cells that were already pretreated with 0.1 µM SB203580, NTHi-induced TLR2 up-regulation was further enhanced (Fig. 4E, lower panel), thereby suggesting that their ability to enhance TLR2 up-regulation is likely additive to each other. However, it is still unclear whether or not DEX indeed additively enhances TLR2 up-regulation via inhibition of p38 MAP kinase. We therefore investigated the direct effect of DEX on NTHi-induced phosphorylation of p38 MAP kinase. As shown in Fig. 4F, NTHi-induced phosphorylation of p38 was attenuated by DEX pretreatment. Thus, our data demonstrated that glucocorticoids synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with p38 MAP kinase pathway, although our data do not preclude the possibility that a direct protein-DNA interaction between GR and glucocorticoid response element in the regulatory region of the TLR2 gene may also contribute to this synergistic regulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

From what we have shown above, it is evident that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by NTHi via a positive IKK-IB-dependent NF-B pathway and a likely negative MKK3/6-p38 / pathway. The up-regulation of TLR2 by NTHi appears to be consistent with the function of a Drosophila Toll protein whose expression is also up-regulated by a pathogen challenge (22). Our finding may have several important implications in host defense and immune response against bacteria. First, the very low expression of TLR2 we observed in unstimulated epithelial cells is likely to be an important aspect of TLR2 function, because under limiting conditions, cellular responses to pathogen-associated molecular patterns could be more stringently regulated by controlling the amounts of TLR protein produced (23). Second, the increased TLR2 expression will probably contribute to the accelerated immune response by epithelial cells as well as resensitization of epithelial cells to invading pathogens. If so, regulation of TLR2 expression may be one of the immune regulatory mechanisms commonly involved in host defense against many bacterial strains. Finally, the observation that TLR2 expression is up-regulated by NTHi suggests that invading bacteria cannot only initiate the host immune response, but can also modulate the eventual responsiveness of epithelial cells to the invading bacteria by regulating the TLR2 expression level (24). Thus, these observations offer a new insight for fully understanding the important role of TLR2 in epithelial cell defense and immune response against invading bacteria.

Another major interesting finding in this study is the synergistic enhancement of NTHi-induced TLR2 up-regulation by glucocorticoids via a negative cross-talk with the inhibitory p38 MAP kinase signaling pathway. This result, although rather unexpected, may provide a novel insight into the role of glucocorticoids in host defense and inflammatory responses in the pathogenesis of infectious diseases. Recently, it has been shown that TLR2-deficient mice are highly susceptible to bacterial infection, demonstrating a critical role of TLR2 in host defense against invading bacteria (8). Therefore, the synergistic enhancement of NTHi-induced TLR2 expression by glucocorticoids would undoubtedly contribute to the host defense against bacteria. Since we also provided direct evidence that activation of NF-B is involved in NTHi-induced TLR2 up-regulation and glucocorticoids are widely known to inhibit NF-B activity (17, 18), a critical question that has yet to be answered is whether glucocorticoids also inhibit NTHi-induced NF-B activation, and if so, what mechanisms account for this synergistic enhancement of TLR2 expression. To determine whether glucocorticoids inhibit NF-B activity, we directly evaluated the effect of glucocorticoids on NTHi-induced NF-B-dependent promoter activity by using luciferase reporter plasmid in HeLa cells. As expected, glucocorticoids reduced NTHi-induced NF-B activation by 70-80% (Fig. 5). Therefore, it may well be that additional positive signaling pathways that cannot be inhibited by DEX, together with the direct induction of TLR2 by DEX as well as the inhibition of the negative p38 MAP kinase pathway by DEX, are responsible for the synergistic up-regulation of TLR2 by NTHi and DEX. Thus, the model of the synergistic up-regulation of TLR2 by NTHi and glucocorticoids has become more complex (Fig. 6). Future studies will focus on identifying other positive signaling pathways involved in TLR2 induction. Cloning of the regulatory region of human TLR2 will help to elucidate the transcriptional regulatory mechanisms involved in TLR2 up-regulation. These studies may lead to novel therapeutic intervention for modulating host defense and innate immune and inflammatory responses for otitis media and COPD.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Glucocorticoids attenuated NTHi-induced NF-B activation. HeLa cells were transiently transfected with an NF-B-regulated luciferase reporter construct and were then pretreated with 1 µM DEX for 2 h before being stimulated with NTHi for 3 h. Luciferase activity was then assessed in NTHi-treated and untreated cells. Values are the means ± SD; n = 3. CON, control.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Schematic representation of the signaling pathways involved in TLR2 up-regulation by NTHi and glucocorticoids in human epithelial cells. As indicated, NTHi up-regulates TLR2 expression via a positive IKK-IB-dependent NF-B pathway and a likely negative MKK3/6-p38 / pathway. Glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the inhibitory p38 MAP kinase pathway. The up-regulated TLR2 leads to enhanced immune and inflammatory responses.

	ACKNOWLEDGEMENT

We thank A. S. Baldwin for p65 expression plasmid.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant RO-1 DC04562 (to J.-D. L.) and by a grant from the Henry L. Guenther Foundation (to D. J. L. and J.-D. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^¶¶ To whom correspondence should be addressed: The Gonda Dept. of Cell and Molecular Biology, House Ear Inst., University of Southern California, 2100 West Third St., Los Angeles, CA 90057. Tel.: 213-273-8083; Fax: 213-273-8088; E-mail: jdli@hei.org.

Published, JBC Papers in Press, February 26, 2002, DOI 10.1074/jbc.M112190200

	ABBREVIATIONS

The abbreviations used are: TLR, Toll-like receptor; CAPE, caffeic acid phenethyl ester; COPD, chronic obstructive pulmonary disease; DEX, dexamethasone; GR, glucocorticoid receptor; IKK, IB kinase; NTHi, nontypeable H. influenzae; MAP kinase, mitogen-activated protein kinase; MKK, MAP kinase kinase; NHBE, normal human bronchial epithelial cells; RT, reverse transcriptase.

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