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Airway epithelia regulate expression of human ß-defensin 2 through Toll-like receptor 2¹

XIAORONG WANG^*,2, ZHE ZHANG^*,, JEAN-PIERRE LOUBOUTIN^*,, CHRISTIAN MOSER^*,, DANIEL J. WEINER^*, and JAMES M. WILSON^*,

^* Department of Medicine, Medical Genetics Division, University of Pennsylvania School of Medicine,
The Wistar Institute, and the
Department of Pediatrics at Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

²Correspondence should be sent: 204 Wistar, 3601 Spruce St., Philadelphia, PA 19104, USA. E-mail: wilsonjm{at}mail.med.upenn.edu.

SPECIFIC AIMS

The goal of this study was to investigate whether Toll-like receptor 2 (TLR2) mediates induction of human ß-defensin 2 (hBD2) through the NF-B pathway in response to bacterial components in human airway epithelia.

PRINCIPAL FINDINGS

1. hTLR2 is expressed in airway epithelial cells
Human airway epithelial cells from non-CF and CF subjects were analyzed for expression of hTLR2 RNA by RT-PCR; transcripts were observed in all cell isolates (Fig. 1 A). Human lung tissue was analyzed for cell-specific expression of hTLR2 protein by immunohistochemistry using peptide-specific antibodies. Polyclonal sera made to COOH and NH₂-terminal peptides of hTLR2 were obtained commercially; we generated anti-sera in rabbits immunized with an internal peptide of hTLR2 distinct from the other known hTLRs. Specific staining of the apical surface of epithelial cells throughout the conducting airway was observed with all three antibodies (Fig. 1B ). Expression was localized to ciliated cells. Analysis of a number of irrelevant primary antibodies failed to reveal a similar pattern of staining (Fig. 1B and data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 1. Expression of hTLR2 in the human lung epithelium. A) Expression of hTLR2 in the cultured primary airway epithelial cells by RT-PCR. dCF: differentiated CF airway epithelial cells, dN: differentiated non-CF airway epithelial cells, uN: undifferentiated non CF airway epithelial cells, PC: positive control, NC: negative control. B) Immunohistochemical localization of hTLR2 in human lung. Cryostat sections (10 µm thick) of human lung were immunostained by antibodies against TLR2 (C: goat antibody against the carboxyl terminus, 1st column; N: goat antibody against the amino terminus, 2nd column; I: rabbit homemade antibody, 3rd column). Negative controls consisted of preincubation with PBS, incubation with nonimmune IgG diluted 1:100, or omission of the primary antibody. Absence of immunostaining was observed in the 3 types of controls. Results obtained with omission of the primary antibody are presented in the 4th column. First 2 rows: immunostaining of the apical surface of pseudo-stratified ciliated epithelial cells of the bronchus. Note the interruption of immunostaining in secretory cells. Third and fourth rows: immunostaining of the epithelial cells of the bronchioles and terminal bronchioles. The staining appears to be more diffuse in the cytoplasm than in the proximal airway. For each level of the airway, the intensity of immunostaining was comparable with the different antibodies considered.

2. hTLR2 mediates regulation of hBD2 through the NF-B pathway in 293 cells
The initial approach was to transfect into 293 cells (a human cell line not expressing TLRs) a vector constitutively expressing hTLR2 cDNA together with a construct in which a reporter gene is driven by 5' flanking regions of hBD2. Previous studies have shown that endogenous hBD2 is up-regulated in response to inflammatory mediators while other ß-defensins such as hBD1 are constitutively expressed. Reporter gene expression is evaluated in the presence or absence of inducers (i.e., components of bacteria). Deletion analysis mapped sequences that confer most of the hTLR2-dependent induction of hBD2 promoter activity to a region spanning –953 to +65 relative to TATA box (data not shown). 293 cells were transfected with an hTLR2 together with a luciferase gene driven by either the hBD1 or hBD2 promoter in the presence or absence of lipoteichoic acid (LTA, a component of gram-positive bacterial wall). The hBD1 promoter was unresponsive to LTA, consistent with the biology of the endogenous gene, while the hBD2 promoter was induced at least 10-fold in response to LTA (data not shown).

The regulating elements conferring responsiveness of the hBD2 gene to LTA were further mapped. Sequence analysis of 1018 bp of 5' flanking sequence of the hBD2 gene revealed three putative NF-B binding sites at –564, –175, and –165 relative to the TATA box (Fig. 2 A). Mutant promoter constructs were generated in which each Rel/NF-B site was individually ablated though several point mutations. Ablating site #1 had no effect on the induction profile, but similar mutations in sites #2 and #3 substantially inhibited peak induction (Fig. 2B ). LTA-stimulated firefly luciferase induction was significantly attenuated when a degradation-resistant IB mutant was introduced; on the other hand, when p50 and p65 were overexpressed, the firefly luciferase induction was greatly stimulated even in the absence of LTA stimulation (Fig. 2B ). This supported the notion that LTA-stimulated firefly induction was mediated by Rel/NF-B.

View larger version (37K): [in this window] [in a new window]   Figure 2. Putative NF-B binding sites are important for hBD2 transcription activation. A) Three putative NF-B binding sites exist in the proximal region (1 kb) of the hBD2 promoter located at –564, –175, and –165 relative to the TATA box. B) Three putative NF-B sites were mutated individually. Wt: the wild-type hBD2 promoter, Mt#1: the first NF-B site was mutated, Mt#2: the second NF-B site was mutated, Mt#3: the third NF-B site was mutated. C)Gel shift assay. Experiments were performed in different conditions. Cells in lanes 2–9 were stimulated with LTA. Lanes 10–12: cells were not stimulated with LTA. Lane 1: no nuclear extract was added. Lanes 2 and 10: nuclear extract was added, but no competitor. No antibodies were added. Lanes 3 and 11: a NF-B consensus oligo was mixed at 40-fold excess with the probe. Lanes 4 and 12: a scrambled oligo was mixed at 40-fold excess with the probe. Lanes 5–9: before adding the probe, nuclear extract was incubated with the polyclonal antibodies against indicated proteins. The probe for upper panel is oligo corresponded to NF-B site #1, the probe for middle panel is oligo corresponded to NF-B #2, and the probe for lower panel is oligo corresponded to NF-B site #3.

Interactions of the putative Rel/NF-B binding sites with the relevant transcription factors were characterized in gel shift experiments with cell extracts (Fig. 2C ). 293 cells were transfected with the hTLR2 plasmid and 18 h later incubated with the cell culture medium with or without LTA. Nuclear extracts were prepared 24 h later and incubated with double-stranded, end-labeled oligonucleotides representing the three putative Rel/NF-B sites. A shift by the labeled oligo was noted in extracts from LTA-treated cells for all three Rel/NF-B sites presumably representing binding to a nuclear protein. Binding was not noted in extracts from non-LTA-treated cells. For each Rel/NF-B site, binding was competed with excess cold oligo but not with a scrambled oligo. To further define the nature of the proteins binding to the oligos, incubations were performed with antibodies specific for Rel/NF-B family members including p50, p65, p52, RelB, and cRel. Binding of antibody to the protein/DNA complex results in a shift of the band to a slower migrating species. For Rel/NF-B sites #2 and #3, there is a complete supershift with antibody to p50 and p65, but not with the others. The results with Rel/NF-B site #1 are qualitatively similar except the p50 and p65 shifts were not complete. These data strongly support binding of Rel/NF-B proteins possibly in the form of p50/p65 heterodimers to Rel/NF-B sites #2 and #3. The lesser extent of involvement of site #1 might be due to reduced affinity of these proteins for the site and/or the binding competition of other transcription factors for this site in the airway epithelial cells.

3. hTLR2 mediate hBD2 up-regulation through NF-B pathway in primary airway epithelial cells
Experiments described thus far demonstrated the ability of transfected hTLR2 to activate the NF-B pathway and signal promoters of the innate immune response (i.e., hBD2) in 293 cells. Additional experiments were performed to confirm the role of TLR2 in airway epithelial cells. A dominant negative version of hTLR2 created by truncating the cytosolic domain was cotransfected with the hBD2-luciferase reporter gene. Expression of the dominant negative version of hTLR2 inhibited LTA-mediated induction of the hBD2 promoter. These experiments also confirmed that #2 and #3 NF-B sites are important for hBD2 regulation in the airway epithelia cells, because activity of the wild-type promoter and mutant promoter #1 was significantly increased when airway epithelia cells were stimulated with LTA, but mutant promoter #2 and #3 activities were not significantly increased upon stimulation with LTA; consistently, PT-PCR expression of hBD2 from the endogenous gene in human airway epithelial cells was significantly induced after stimulation with LTA (data not shown).

CONCLUSIONS AND SIGNIFICANCE

Our original hypothesis was that epithelia of the lung are capable of sensing infection and mobilizing an effective innate immune response through the secretion of effector molecules that have direct and indirect biological activities. We confirmed this hypothesis in the current study by demonstrating that the induction of an important antibacterial peptide (i.e., hBD2) is mediated by an epithelial expressed TLR that recognizes components of gram-positive bacteria leading to activation of the NF-B pathway.

Epithelia are not only passive physical barriers, but also play key roles in regulation of the innate immune response. This work contributes to the emerging concept that epithelia serve critical roles in the regulation and implementation of innate immune responses. Our working model is that TLR can sense bacterial infection on the airway surface; activation of the TLR can lead to up-regulation of antimicrobial peptides to facilitate elimination of the bacteria. At the same time, proinflammatory cytokines/chemokines can be induced and neutrophiles and microphages mobilized to the airway to help clear the infection (Fig. 3 ).

View larger version (51K): [in this window] [in a new window]   Figure 3. Our working model is that TLR can sense bacterial infection in the airway surface; activation of the TLR can lead to up-regulation of antimicrobial peptide to help to eliminate bacteria; at the same time, proinflammatory cytokines/chemokines are up-regulated and neutrophiles and microphages are mobilized to the airway to help clear the infection.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0616fje; doi: 10.1096/fj.02-0616fje

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