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Am J Physiol Lung Cell Mol Physiol 290: L730-L737, 2006. First published November 18, 2005; doi:10.1152/ajplung.00271.2005
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Pneumococci induced TLR- and Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter in lung epithelial cells

Bernd Schmeck,1 Sylvia Huber,1 Kerstin Moog,1 Janine Zahlten,1,2 Andreas C. Hocke,1 Bastian Opitz,1 Sven Hammerschmidt,3 Tim J. Mitchell,4 Michael Kracht,5 Simone Rosseau,1 Norbert Suttorp,1 and Stefan Hippenstiel1

Departments of 1Internal Medicine/Infectious Diseases and 2Paradontology, Charité—Universitätsmedizin Berlin, Berlin; 3Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany; 4Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom; and 5Institute of Pharmacology, Medizinische Hochschule Hannover, Hannover, Germany

Submitted 23 June 2005 ; accepted in final form 11 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-{kappa}B-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-{kappa}B subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter.

phosphatidylinositol 3-kinase; Rac1; p65/RelA; BEAS-2B cells; Toll-like receptor; interleukin-8; nuclear factor-{kappa}B

PNEUMONIA IS THE MOST COMMON cause of death due to infectious diseases in industrialized countries (14). Over 40% of all cases are due to Streptococcus pneumoniae, which is the most common etiologic agent of community-acquired pneumonia (5, 31). Activation of lung epithelial cells by pneumococci entering the host and interacting with specific receptors may be an important early step in initiating effective antipneumococci immune response (23, 32, 42).

Immune reaction against pneumococci seems to depend on components from the cell wall rather than from the capsule (44, 53, 54). Pneumococcal lipoteichoic acid (LTA) in particular was found to activate innate immunoreaction (16, 24, 43). The innate immune system recognizes pathogens by pattern recognition receptors, e.g., the Toll-like receptor (TLR) family (1). TLR2, which is thought to detect mainly surface patterns of gram-positive bacteria, was found to recognize cell wall preparations of S. pneumoniae synergistically with CD14 (58). Pneumococcal LTA was detected by TLR2, lipopolysaccharide-binding protein (LBP), and CD14, but not by MD-2 or TLR4 (43), which is considered to recognize mainly gram-negative surface pattern like lipopolysaccharide (LPS) (1). Peptidoglycan preparations from pneumococci were also described to stimulate TLR2 (55). TLR2 activation by S. pneumoniae was even enhanced by addition of penicillin (30). Malley et al. (29) showed that the important exotoxin pneumolysin was, in contrast, recognized by TLR4. However, TLR2 seems to synergize with other TLRs in pathogen recognition by heterodimerization (33). For example, TLR1/TLR2 dimers were activated by triacylated lipoproteins, whereas TLR2/TLR6 recognized diacylated lipoproteins (49). Whether dimerization also plays a role in recognition of pneumococci is not known. In addition to identification of extracellular pneumococci by TLRs, stimulation of nucleotide-binding oligomerization domain proteins, in particular by Nod2, by invading pneumococci may also contribute significantly to host cell activation (32).

Recently, Rho-GTPases were considered as part of the TLR2 (3, 52) and TLR4 (20) signaling pathways. The Rho-GTPases RhoA, Rac1, and Cdc42 belong to the Ras superfamily of small GTP-binding proteins (7), are well-characterized regulators of the cytoskeleton (36, 37), and participate in signal transduction (19, 34, 40) and apoptosis regulation (18).

In this study we analyzed activation of and signal transduction in lung epithelial cells by pneumococci. We found that S. pneumoniae-stimulated IL-8 release was reduced by Rac1-inhibition. Characterizing cellular activation in more detail, we found that pneumococci enhanced TLR1 and TLR2 mRNA levels in human bronchial epithelial cells, and viable pneumococci were synergistically recognized by TLR1 and TLR2. Pneumococci-induced signaling involved myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), Rac1, and Cdc42. Recruitment of NF-{kappa}B-subunit p65/RelA to the IL-8 promoter was regulated by Rac1-dependent phosphorylation of serine 276, whereas Rac1 did not regulate pneumococci-induced binding of phosphorylated c-Jun. Together, S. pneumoniae activated epithelial cells by TLR1/2 and PI3K-Rac1-NF-{kappa}B pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM, fetal calf serum (FCS), trypsin-EDTA solution, CA-650, and antibiotics were obtained from Life Technologies (Karlsruhe, Germany). Nsc23766 (Nsc) was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD. Drs. R. R. Schumann and N. Schröder (Charité—Universitätsmedizin Berlin, Germany) kindly supplied recombinant human LBP. All other chemicals used were of analytical grade and obtained from commercial sources.

Cell lines. Bronchial epithelial cell line BEAS-2B was a kind gift of Dr. Curtis Harris (National Institutes of Health, Bethesda, MD) (12, 35). Human embryonic kidney cells HEK-293 were purchased from ATCC (Rockville, MD). Chinese hamster ovarian cells (CHO-TLR4/CD14) stably expressing CD14, human (h) TLR4, and an NF-{kappa}B-dependent reporter gene (CD25) vector were kindly provided by Dr. Douglas Golenbock (University of Massachusetts Medical School, Worcester, MA) (17, 28).

Bacterial strains and isogenic mutants. S. pneumoniae R6x, the unencapsulated derivative of D39, was used in this study (11, 41, 42). S. pneumoniae were cultured in Todd-Hewitt broth (Oxoid, Basingstoke, UK) supplemented with 0.5% yeast extract (THY) to mid-log phase or grown on blood agar.

Pneumolysin. Recombinant pneumolysin was expressed in Escherichia coli and was purified from cell extracts as described elsewhere (39). Protein homogeneity was confirmed by SDS-PAGE. The stock toxin was essentially free (<2 pg/ml) of contaminating bacterial endotoxin. Our stock concentration was 0.21 mg/ml, which corresponds to 1.3 x 106 hemolytic units/ml (9). The determination of hemolytic units results from a hemolysis test with sheep erythrocytes. In short, erythrocytes were exposed to dilution series of purified pneumolysin, and hemolysis was determined; 1 hemolytic unit results in 50% hemolysis (41).

Plasmids and transient transfection procedures. HEK-293 cells were cultured in 12-well plates with DMEM supplemented with 10% FCS. Subconfluent cells were cotransfected by the calcium phosphate precipitation method according to the manufacturer's instructions (Clonetech, Palo Alto, CA) with 0.2 µg of NF-{kappa}B-dependent luciferase reporter (27), 0.2 µg of respiratory syncytial virus (RSV) beta-galactosidase plasmid, 0.2 µg human (h) TLR4 expression vector or 0.05 µg hTLR2, 0.6 µg hTLR1, 0.02 µg CD14 [all generously provided by Carsten Kirschnig, Munich, Germany, and Tularik, San Francisco, CA (25, 46)], or 0.2 µg hTLR6 (kindly provided by Dr. Shizou Akira, University of Osaka, Osaka, Japan) and a dominant-negative (dn) mutant of MyD88, or RhoN19, RacN17, and Cdc42 (generous gifts of Drs. Marta Muzio and Anne Ridley, respectively) expression vectors. We kept DNA concentration constant by adding an appropriate amount of control vector. All transfections were performed in duplicate. Cells were incubated with R6x for 6 h on the following day. Luciferase activity was measured by using Luciferase Reporter-Gene Assay (Promega, Mannheim, Germany), and results were normalized for transfection efficiency with values obtained by RSV-beta-galactosidase as described previously (32). Morphological changes in HEK-293 cells transfected with dn RhoA/Rac1/Cdc42 were analyzed by phase-contrast microscopy before bacteria stimulation.

IL-8, IL-1beta, TNF-{alpha} ELISA. Confluent BEAS-2B or transfected HEK-293 cells were stimulated as indicated in a humidified atmosphere. After incubation supernatants were collected and processed for cytokine quantification by sandwich ELISA as described previously (20).

RT-PCR. For analysis of TLR1, 2, 4, 6, and GAPDH gene expression in BEAS-2B cells, total RNA was isolated with RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed using AMV reverse transcriptase (Promega, Heidelberg, Germany). Generated cDNA was amplified by PCR with specific primers for TLR1 (5'-CGG GAT CCA CAC TGA GAG TCT GCA CA-3', 5'-CGA ATT CCA GCT GGA GGA TCC TAA-3'), TLR2 (5'-GCC AAA GTC TTG ATT GAT TGG-3', 5'-TTG AAG TTC TCC AGC TCC TG-3'), TLR4 (5'-TAC AGA AGC TGG TGG CTG TG-3', 5'-CCA GAA CCA AAC GAT GGA CT-3'), TLR6 (5'-AGA ACT CAC CAG AGG TCC AAC C-3', 5'-GAA GGC ATA TCC TTC GTC ATG AG-3'), and GAPDH (5'-CCA CCC ATG GCA AAT TCC ATG GCA-3', 5'-TCT AGA CGG CAG GTC AGG TCC-3'). After 32 amplification cycles, PCR products were analyzed on 1.5% agarose gels, stained with ethidium bromide, and subsequently visualized. To confirm use of equal amounts of RNA in each experiment, all samples were checked for GAPDH mRNA expression.

FACS analysis of CHO cells. CHO-TLR4/CD14 cells were plated at a density of 2 x 105 cells/well in six-well dishes. The following day, cells were stimulated as indicated in Ham's F-12 medium containing 10% FBS (total volume of 2 ml/well). After 18 h, the cells were harvested with trypsin-EDTA and labeled with allophycocyanin anti-CD25 in PBS/1% FBS for 30 min on ice. After labeling, the cells were washed once and resuspended in PBS/1% FBS. The cells were analyzed by flow cytometry using a FACScalibur (Becton Dickinson) (28).

Chromatin immunoprecipitation. BEAS-2B cells were stimulated with 107 cfu/ml S. pneumoniae R6x for 2.5 h, culture medium was removed, and 1% formaldehyde was added. After 1 min, cells were washed in ice-cold 0.125 M glycin in PBS and then rapidly collected in ice-cold PBS, centrifuged, and washed twice with ice-cold PBS. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [10 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P (NP)-40, 1% desoxycholic acid, 0.1% SDS, 1 mM EDTA, and 1% aprotinin], and the chromatin was sheared by sonication. Lysates were cleared by centrifugation, and supernatants were stored in aliquots at –80°C until further use. Antibodies used for immunoprecipitation were purchased from Cell Signaling, Beverly, MA (P-c-Jun and P-Ser276-p65) and Santa Cruz Biotechnology, Santa Cruz, CA (p65/RelA). Immunoprecipitations from soluble chromatin were carried out overnight at 4°C. Immune complexes were collected with protein A/G agarose for 60 min and washed twice with RIPA buffer and once with high-salt buffer (2 M NaCl, 10 mM Tris, pH 7.5, 1% NP-40, 0.5% desoxycholic acid, and 1 mM EDTA) followed by another wash in RIPA buffer and one wash with TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA). We extracted immune complexes in elution buffer (TE buffer containing 1% SDS) by shaking the lysates for 15 min at 30°C. Then, they were digested with RNase (1 µg/20 µl) for 30 min at 37°C. After proteinase K digestion (1 µg/ 5 µl for 6 h at 37°C and 6 h at 65°) DNA was extracted with a PCR purification kit (Qiagen, Hilden, Germany). IL-8 promoter DNA was amplified by PCR using Hotstart Taq (Qiagen) polymerase. The PCR conditions were 95°C for 15 min, 33–35 cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 20 s. PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining of gels. Equal amounts of input DNA was controlled by RT-PCR and gel electrophoresis (42).

The following promoter-specific primers were used: IL-8 sense 5'-AAG AAA ACT TTC GTC ATA CTC CG-3', antisense 5'-TGG CTT TTT ATA TCA TCA CCC TAC-3'.

Statistical methods. Data are shown as means ± SE of at least three independent experiments. A one-way ANOVA was used for data of Figs. 1, A–D; 2, B–E; 3, A–C; and 4, A–D. Main effects were then compared by a Newman-Keuls posttest. P < 0.01 was considered to be significant and indicated by asterisks.


Figure 1
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  		Fig. 1. Pneumococci induced Rac1-dependent IL-8 release. BEAS-2B cells were stimulated with the indicated concentrations of Streptococcus pneumoniae (S.p.) R6x (A) for 16 h, and IL-8 concentration was measured in the supernatant. *P < 0.01 compared with control. BEAS-2B cells were preincubated with indicated concentrations (µM) of Rac1 inhibitor Nsc23766, stimulated with 107 cfu/ml S.p. R6x (B), 100 ng/ml TNF-{alpha} (C), or 50 ng/ml IL-1beta (D) for 16 h, and IL-8 release was measured in the supernatant. *P < 0.01 compared with stimulation without Nsc23766.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
S. pneumoniae induced Rac1-dependent expression of IL-8 in human bronchial epithelial cells. Recognition of pneumococci by tracheobronchial epithelium and subsequent expression of proinflammatory cytokines may contribute significantly to the prominent leukocyte recruitment observed in pneumococci pneumonia. Therefore, we assessed the potential of pneumococci-induced IL-8 expression in human bronchial epithelial cells. Bronchial epithelial BEAS-2B cells stimulated with S. pneumoniae R6x displayed dose dependently enhanced IL-8-expression (Fig. 1A). In terms of BEAS-2B cell stimulation, S. pneumoniae turned out to be as effective as 100 ng/ml TNF-{alpha}, which resulted in an IL-8 release of ~20 ng/ml in the cell supernatant (Fig. 1C). To rule out the possibility that pneumococci-related IL-8 liberation was due to secondary autocrine cytokine stimulation via epithelially generated TNF-{alpha} or IL-1beta, TNF-{alpha} and IL-1beta release was also analyzed in S. pneumoniae-infected BEAS-2B cells. Although TNF-{alpha} increased in parallel to IL-8, significant IL-1beta release was detected only after 10 h of cell infection (data not shown), suggesting that secondary effects of TNF-{alpha} and IL-1beta are not important in the experimental system used. To test the hypothesis that Rho-GTPases might be involved, we used the specific Rac1 inhibitor Nsc. The compound was shown to specifically block activation of Rac1, but not RhoA or Cdc42, at concentrations between 50 and 200 µM (13). Nsc dose dependently reduced IL-8 release in pneumococci-stimulated BEAS-2B cells (Fig. 1B). Nsc also reduced TNF-{alpha}- or IL-1beta-related IL-8 generation (Fig. 1, C and D, respectively). Thus Rac1 seems to be critically involved in pneumococci-related IL-8 release by cultured human bronchial epithelial BEAS-2B cells.

S. pneumoniae induced NF-{kappa}B-dependent gene transcription by TLR2, and pneumolysin by TLR4. To analyze epithelial recognition of S. pneumoniae in more detail, we studied TLR mRNA expression. Unstimulated BEAS-2B cells expressed TLR4 and TLR6, whereas TLR1 and TLR2 mRNA was barely detectable (Fig. 2A). Infection of BEAS-2B cells with pneumococci time dependently induced expression of TLR2 mRNA after 6 h and TLR1 mRNA after 12 h. In contrast, TLR4 and TLR6 mRNA expression was constitutively high in control and infected cells.


Figure 2
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  		Fig. 2. S.p. and pneumolysin (Ply) activated NF-{kappa}B-dependent gene transcription via Toll-like receptor (TLR) 2 and TLR4, respectively. A: BEAS-2B cells were infected with 106 cfu/ml R6x as indicated, and mRNA for TLR1, TLR2, TLR4, TLR6, and GAPDH was detected by RT-PCR. One representative gel out of 3 is shown. HEK-293 cells were transfected with plasmids encoding an NF-{kappa}B-dependent luciferase reporter, RSV beta-galactosidase, and as indicated with TLR2 (B), TLR4 (D), or not transfected with a TLR receptor (C). Cells were exposed to R6x S.p. (106 cfu/ml) or Ply (0.1–1 µg/ml) for 6 h, and luciferase activity was determined and normalized on beta-galactosidase activity. *P < 0.01 compared with control. E: HEK-293 cells were transfected with plasmids encoding an NF-{kappa}B-dependent luciferase reporter gene, RSV beta-galactosidase, and TLR4. Ply (1 µg/ml) or LPS (100 ng/ml) was preincubated with polymyxin B (PMB, 10 µg/ml for 1 h) and then given to TLR4-transfected HEK-293 cells for 6 h. Luciferase activity was determined and normalized on beta-galactosidase activity. F: CHO-TLR4/CD14 cells were stimulated with Ply (1 µg/ml) or LPS (100 ng/ml) for 18 h, and CD25 reporter gene expression was measured by FACS analysis. C, control; PGN, peptidoglycan; RLU, relative light units. Graphs from 1 representative experiment out of 3 are shown. Numbers represent fluorescence median of 3 experiments (fluorescence means ± SE).

 
Next we analyzed the role of TLR receptors for pneumococci recognition by transfecting HEK-293 cells with TLR2 or TLR4. Expression of a NF-{kappa}B-dependent reporter gene in S. pneumoniae (106 cfu/ml)-, pneumolysin (0.1 and 1 µg/ml)-, or positive control (peptidoglycan or LPS, respectively)-exposed cells was measured. Neither pneumococci nor pneumolysin activated NF-{kappa}B-dependent gene transcription in HEK-293 cells without transfected TLR2 or TLR4 (Fig. 2C), but reporter gene expression was strongly activated by 10 ng/ml TNF-{alpha} (data not shown). S. pneumoniae-induced NF-{kappa}B activity occurred exclusively in TLR2-expressing cells (Fig. 2B) and pneumolysin-induced NF-{kappa}B activity occurred exclusively in TLR4-expressing HEK-293 cells (Fig. 2D).

We verified recognition of pneumolysin by TLR4 using CD14- and hTLR4-positive CHO cells expressing an NF-{kappa}B-dependent reporter gene. These cells are unresponsive to TLR2 activation due to a single base-pair deletion in the tlr2 gene (25). NF-{kappa}B-dependent reporter gene expression was noted in CHO-TLR4/CD14 cells upon stimulation with LPS (100 ng/ml) or pneumolysin (1 µg/ml) (Fig. 2F).

To exclude effects of possibly contaminating LPS in the pneumolysin preparation we made use of polymyxin B (PMB). Preincubation of LPS (100 ng/ml) with 10 µg/ml PMB blocked activation of NF-{kappa}B in hTLR4-transfected HEK-293 cells almost completely. In contrast, PMB had no inhibitory effect on pneumolysin-induced activation of NF-{kappa}B in these cells (Fig. 2E). Thus relevant LPS contamination seems to be unlikely in the pneumolysin preparation used.

TLR1 and TLR2 synergistically induced S. pneumoniae-dependent NF-{kappa}B-dependent gene transcription, but TLR4, CD14, or LBP did not enhance recognition of S. pneumoniae. Binding of bacteria to TLR homo- or heterodimers is considered to activate host cells (1). We noted expression of a reporter gene activated by the important proinflammatory transcription factor NF-{kappa}B in pneumococci-stimulated HEK-293 cells overexpressing TLR2, but not TLR1 or TLR6 alone (Fig. 3A). TLR2 is reported to recognize pathogens in cooperation with other TLR receptors. TLR2-dependent NF-{kappa}B activation by pneumococci was synergistically enhanced by coexpression of TLR1. TLR6 did not boost pneumococci-induced reporter gene levels in context with TLR2. Thus TLR1 and TLR2 seem to be synergistically involved in pneumococci-induced cell activation.


Figure 3
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  		Fig. 3. Pneumococci activated NF-{kappa}B-dependent gene transcription synergistically by TLR1 and TLR2, but independently of CD14 and LBP. A: HEK-293 cells were transfected with plasmids encoding an NF-{kappa}B-dependent luciferase reporter gene, RSV beta-galactosidase, and combinations of TLR1, TLR2, and TLR6. Cells were exposed to viable R6x (106 cfu/ml) for 6 h, and luciferase activity was determined and normalized on beta-galactosidase activity. *P < 0.01 compared with TLR2-expressing cells alone. B: HEK-293 cells were transfected with plasmids encoding an NF-{kappa}B-dependent luciferase reporter gene, RSV beta-galactosidase, and control vector or combinations of TLR2 and TLR4. Cells were exposed to viable R6x (106 cfu/ml) for 6 h, and luciferase activity was determined and normalized on beta-galactosidase activity. *P < 0.01 compared with TLR2-expressing cells alone. C: HEK-293 cells were transfected with plasmids for an NF-{kappa}B-dependent luciferase reporter, RSV beta-galactosidase, TLR2, and when indicated CD14. They were exposed to 106 cfu/ml R6x for 6 h in the presence or absence of 10% FCS or LPS-binding protein (LBP, 1 or 2 µg/ml), and luciferase activity was determined and normalized on beta-galactosidase activity. n.s., Not significant.

 
Pneumococci did not induce NF-{kappa}B-dependent gene transcription in HEK-293 by TLR4 alone (Fig. 2D). We performed cotransfection experiments with TLR2 and TLR4 to see whether they act synergistically with respect to pneumococci-dependent cell activation. However, HEK-293 cells cotransfected with TLR2 and TLR4 displayed no significant change in pneumococci-induced NF-{kappa}B-activation above effects seen in TLR2-carrying cells (Fig. 3B).

The recognition of LPS by TLR4 and some other bacterial surface molecules, e.g., LTA by TLR2, is assisted by LBP and CD14 (43). To test whether this is also the case for living pneumococci and human TLR2, we stimulated TLR2-expressing HEK-293 cells with S. pneumoniae in the presence of FCS or recombinant LBP and transfection of CD14, respectively, and measured NF-{kappa}B-dependent reporter gene expression (Fig. 3C). Although there was a trend for enhanced NF-{kappa}B activity when FCS or a combination of CD14 and LBP were added, no significant effect of serum, CD14, or LBP on the recognition of viable pneumococci by TLR2 was found within the dose and time frame tested.

S. pneumoniae induced MyD88-, PI3K- and Rac1-dependent gene transcription and recruitment of phosphorylated p65 to the IL-8 gene promoter. Next, we analyzed pneumococci-induced signal transduction. MyD88, PI3K, and Rho-GTPases were considered as part of the TLR2 pathway (22). Therefore, hTLR2-expressing HEK-293 cells were transfected with dn mutants of MyD88, RhoA, Rac1, and Cdc42, stimulated with S. pneumoniae, and NF-{kappa}B-dependent reporter gene expression was measured. dn-MyD88 (Fig. 4A), dn-Rac, and dn-Cdc42 (Fig. 4B) reduced pneumococci-induced NF-{kappa}B activity, whereas dn-RhoA had no inhibitory effect, although it induced specific morphological changes in transfected HEK-293 cells (data not shown). In addition, we measured IL-8 release by transfected HEK-293 cells and similarly found that Rac1 and Cdc42 but not RhoA were necessary for induction of IL-8 release by S. pneumoniae-infected cells (Fig. 4C).


Figure 4
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  		Fig. 4. S.p. induced myeloid differentiation factor 88 (MyD88)-, phosphatidylinositol 3-kinase (PI3K)- and Rac1-dependent gene transcription and recruitment of phosphorylated p65 to the IL-8 gene promoter. HEK-293 cells were transfected with plasmids encoding an NF-{kappa}B-dependent luciferase reporter gene, RSV beta-galactosidase, TLR2, and dominant negative (dn) mutants of MyD88 (A), RhoA, Rac1, or Cdc42 (B, C). They were exposed to pneumococci (R6x, 106 cfu/ml) for 6 h and luciferase activity (A, B) or IL-8-release (C) was determined. *P < 0.01 compared with cells without dominant mutants. BEAS-2B cells were preincubated with indicated concentrations (µM) of PI3K inhibitor LY-294002 (D) stimulated with 106 cfu/ml S.p. R6x for 16 h, and IL-8-release was measured in the supernatant. *P < 0.01 compared with control. E: BEAS-2B cells were infected with 106 cfu/ml S.p. R6x (120 min) in presence or absence of Nsc23766 (Nsc, 200 µM) and binding of phosphorylated c-Jun (Ser63) and p65 (phosphorylated at Ser276 or unphosphorylated) was detected at the IL-8 gene promoter by chromatin immunoprecipitation. One representative experiment out of 3 is shown.

 
As PI3K is considered as an upstream activator of Rho proteins, we verified the involvement of this pathway in pneumococci-infected human bronchial epithelial cells: Blocking of PI3K in BEAS-2B cells by specific inhibitor LY-294002 dose dependently reduced IL-8 release (Fig. 4D).

To understand Rac1 involvement in IL-8 expression on a more molecular level, we measured transcription factor recruitment to the native IL-8 gene promoter in human bronchial epithelial cells by chromatin immunoprecipitation (Fig. 4E). S. pneumoniae infection of BEAS-2B cells induced binding of activator protein (AP)-1 subunit c-Jun phosphorylated on serine 63, and NF-{kappa}B subunit p65/RelA phosphorylated on serine 276 (Fig. 4F). Recruitment of serine 276-phosphorylated p65/RelA was blocked, and p65/RelA binding was reduced by inhibition of Rac1, whereas binding of phosphorylated c-Jun remained unaffected by Nsc in pneumococci-infected BEAS-2B cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we found activation of pneumococci-infected lung epithelial cells by TLR1/2-induced PI3K and Rac1 signal transduction. S. pneumoniae stimulated Rac1-dependent IL-8-release by human bronchial epithelial cells. Pneumococci enhanced TLR1 and TLR2 expression in these cells. Viable pneumococci were synergistically recognized by TLR1 and TLR2, whereas pneumolysin was only recognized by TLR4. TLR4, LBP, or CD14 displayed no significant effect on pneumococci-induced TLR2-dependent cell activation. Pneumococci-induced signaling involved MyD88, PI3K, Rac1, and Cdc42. Recruitment of NF-{kappa}B subunit p65/RelA to the IL-8 promoter was regulated by Rac1-dependent phosphorylation of serine 276, whereas Rac1 did not influence pneumococci-induced binding of phosphorylated c-Jun.

Tracheobronchial epithelium is part of the important mechanic barrier of the lung against invading pathogens and represents the first line of defense of the innate immune system in the lung. We analyzed pneumococci-induced activation of the well-established human bronchial epithelial cell line BEAS-2B, addressing the chemotactic IL-8 as a model cytokine, which was suggested to play an important role in lung inflammation (48).

Rho-GTPases have been shown to signal downstream of TLRs, e.g., TLR2 (3) in HEK-293 and THP-1 cells and TLR4 (20) in human endothelial cells. These pathways seem to include MyD88 and PI3K (3, 22). In accordance, dn-MyD88 and chemical inhibition of PI3K reduced S. pneumoniae-induced gene transcription in pneumococci-exposed epithelium. Transfection of dn Rac1 and Cdc42 as well as the specific Rac1 inhibitor Nsc (13) blocked NF-{kappa}B activity and IL-8 release, whereas RhoA was not involved. Interestingly, Rac1-dependent IL-8 release was also observed in intestinal epithelial cells infected with gram-negative Yersinia enterocolitica (15). Moreover, contribution of Rac1 in NF-{kappa}B-activation has been demonstrated after stimulation with heat-killed Staphylococcus aureus (3). In these experiments Cdc42 was also activated but without effect on NF-{kappa}B activation (3). Because Rho proteins, in particular Rac1, also participated in IL-1beta (45)- and TNF-{alpha} (19)-dependent NF-{kappa}B activation in various cells (and also in BEAS-2B, as shown here) these GTPases must be considered as important signaling mediators in inflammatory reactions. However, dn mutants as well as chemical inhibitors have their technical limitations, and further studies employing Rac1-defective cells are needed.

Different mechanisms have been suggested for Rho-GTPase-dependent activation of NF-{kappa}B: Arbibe et al. (3) implicated TLR2-/PI3K-/Rac1-dependent phosphorylation of NF-{kappa}B. p65/RelA subunit of NF-{kappa}B was indeed phosphorylated PI3K dependently at serine 536 after TLR2 stimulation (47), but involvement of Rac1 was not addressed in that study. We show herein that Rac1 inhibition reduced pneumococci-induced binding of p65 to the IL-8 promoter and blocked phosphorylation of p65 on serine 276. This phosphorylation site has been implicated in DNA binding of NF-{kappa}B (8). In contrast, the AP-1 subunit c-Jun, which was phosphorylated on serine 63 in pneumococci-stimulated epithelial cells, was recruited to the IL-8 promoter independently of Rac1, indicating an NF-{kappa}B-specific mechanism. In other studies, RhoA was shown to be involved in TLR2-dependent phosphorylation of p65/RelA on serine 311 by PKC{zeta} (52) and thrombin-induced phosphorylation of p65/RelA on serine 536 by IKKbeta (2) in different cells and stimulation conditions.

In unstimulated epithelial cells, strong mRNA expression of TLR4 and TLR6 was noted, whereas TLR1 and TLR2 expression seemed to be low. Experiments with pneumococci-stimulated BEAS-2B cells demonstrated increased TLR1 and 2 expression and unchanged levels of TLR4 and TLR6 mRNA. Armstrong et al. (4) recently reported modulation of TLR2 and TLR4 expression by LTA and LPS in human type II alveolar epithelial cells.

TLR2 seemed to be involved in sensing of pneumococci (43), and mice lacking TLR2 were highly susceptible to pneumococcal meningitis due to lower bacterial clearance and enhanced inflammation (10). However, although TLR2 contributed significantly to the early inflammatory response in murine pneumococcal pneumonia, it did not contribute to antibacterial defense per se (26). Recent reports have given evidence that TLR2 synergizes with TLR1 (57) or TLR6 (50) to activate cells. In our model, pneumococci-induced NF-{kappa}B activation was much stronger in cells expressing TLR1 and TLR2 compared with cells transfected with TLR2 alone or together with TLR6. TLR1 and TLR2 were found to interact physically (51) and form heterodimers for signal transduction (38). Thus subsequent upregulation of these pattern recognition receptors in pneumococci-infected epithelial cells may sensitize the epithelial barrier to pneumococci infection, thereby strengthening innate immune response. Pneumolysin activated TLR4-expressing cells, an observation in accordance with Malley et al. (29), who demonstrated an impact of TLR4 on pneumococcal clearance in mouse infection. Interestingly, viable pneumococci were not recognized by TLR4 in our model, nor did TLR4 synergize with TLR2 in S. pneumoniae recognition, most likely because pneumolysin is localized in the bacterial cytosol and only released by autolysin action (21). In addition to pathogen recognition receptors localized at the surface, intracellular detection of invading pneumococci by Nod2/CARD15 proteins may participate in target cell activation as shown recently (32).

LBP and CD14 are important cofactors for LPS sensing by the immune system and also facilitate recognition of pneumococcal LTA (43, 55). Moreover, LBP has become a very actively investigated molecule in sepsis, as well as other infectious diseases and is considered as therapeutical target (59). Weber et al. (55) found, in a mouse model of pneumococcal meningitis, involvement of LBP in pathogenesis. Therefore, we analyzed the impact of LBP and CD14 on recognition of viable pneumococci by TLR2-expressing cells. However, in our model, we did not find significantly increased TLR2-dependent NF-{kappa}B activation by pneumococci in the presence of CD14 or LBP. LBP knockout mice accordingly displayed increased susceptibility to gram-negative Haemophilus influenzae infection, but similarly sufficient immune reaction against S. pneumoniae (6) or TLR2 stimulus nonmannose-capped lipoarabinomannan (56). Therefore, the function of LBP in cell activation seems to be stimulus and cell dependent.

In summary, S. pneumoniae activated human bronchial epithelial cells in vitro by TLR1/2 and a PI3K-Rac1-NF-{kappa}B-dependent pathway, whereas pneumolysin stimulated TLR4 signaling. Further studies on Rho protein involvement in pneumococcal pneumonia are required and may pave the way to the development of new therapeutic strategies.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by Bundesministerium für Bildung und Forschung Grants BMBF Competence Network CAPNETZ C15 (to B. Schmeck), BMBF Competence Network CAPNETZ C15 (to S. Hippenstiel), BMBF Competence Network CAPNETZ C8 (to S. Hammerschmidt), BMBF Competence Network CAPNETZ C4 (to N. Suttorp and S. Rosseau), and Deutsche Forschungsgemeinschaft Grants DFG SFB479 TP A7 (to S. Hammerschmidt), DFG Kr1143/4-1 (to M. Kracht), and GRK325 (to J. Zahlten).


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Kerstin Möhr and Sylvia Schapke (Department of Internal Medicine/Infectious Diseases, Charité—Universitätsmedizin Berlin) and Christa Albert (Research Center for Infectious Diseases, Würzburg) is greatly appreciated.

Part of this work will be included in the doctoral theses of S. Huber and K. Moog.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Hippenstiel, Dept. of Internal Medicine/Infectious Diseases Charité—Univ. Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: stefan.hippenstiel{at}charite.de)

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.


   REFERENCES
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 ABSTRACT
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 RESULTS
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 REFERENCES
 

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