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Published as doi: 10.1096/fj.07-104117.
 (The FASEB Journal. 2008;22:2168-2176.)
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Flagellin is the principal inducer of the antimicrobial peptide S100A7c (psoriasin) in human epidermal keratinocytes exposed to Escherichia coli

Arby Abtin*, Leopold Eckhart*, Michael Mildner*, Florian Gruber*, Jens-Michael Schröder{dagger} and Erwin Tschachler*,{ddagger},1

* Department of Dermatology, Medical University of Vienna, Vienna, Austria;

{dagger} Department of Dermatology, University Hospital Schleswig-Holstein, University of Kiel, Kiel, Germany; and

{ddagger} Centre de Recherches et d'Investigations Epidermiques et Sensorielles, Neuilly sur Seine, France

1Correspondence: Department of Dermatology, Medical University of Vienna, Waehringer Guertal 18–20, 1090 Vienna, Austria. E-mail: erwin.tschachler{at}meduniwien.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal keratinocytes (KCs) express antimicrobial peptides as a part of the innate immune response. It has recently been shown that the culture supernatant of Escherichia coli induces the expression of S100A7c (psoriasin) in KCs and that S100A7c efficiently kills E. coli. Here we have investigated which of the microbial components triggers the up-regulation of S100A7c expression. Exposure of human primary KCs to ligands of the human Toll-like receptors (TLRs) revealed that only the TLR5 ligand flagellin strongly induced the expression of S100A7c mRNA and protein, whereas all other TLR ligands had no significant effect. In contrast to the supernatant from flagellated wild-type (WT) E. coli, the supernatant of a flagellin-deficient E. coli strain ({Delta}FliC) did not induce S100A7c expression. Small interfering RNA-mediated knockdown of TLR5 expression suppressed the ability of KCs to up-regulate S100A7c expression in response to both flagellin and WT E. coli supernatant. Taken together, our data demonstrate that bacterial flagellin is essential and sufficient for the induction of S100A7c expression in KCs by E. coli.—Abtin, A., Eckhart, L., Mildner, M., Gruber, F., Schröder, J.-M., Tschachler, E. Flagellin is the principal inducer of the antimicrobial peptide S100A7c (psoriasin) in human epidermal keratinocytes exposed to Escherichia coli.


Key Words: innate immunity • Toll-like receptor • TLR • pathogen-associated molecular pattern • PAMP • antimicrobial defense


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUMAN SKIN IS AN EFFECTIVE BARRIER against invading microorganisms. This protective function is partly mediated by the presence of antimicrobial peptides (AMPs) in the skin (1 , 2) . Although known for a long time in other species (3 , 4) and in other organ systems of humans (5 , 6) , the presence and importance of AMPs in human skin has only recently been demonstrated (1 , 7) . AMPs of the skin comprise a heterogeneous group of molecules, such as human β-defensin (hBD) -1, hBD-2, hBD-3, RNase 7, cathelicidin, and S100A7c (2) . The antimicrobial activity described for AMPs is due to perforation and disruption of the bacterial membrane (e.g., hBD-2, RNase 7, and cathelicidin) (8 9 10) by deprivation of essential trace elements (e.g., S100A7c) (1) or by as yet unknown mechanisms (e.g., hBD-1 and hBD-3) (2) . Studies using mouse models with deficiencies in AMP genes have emphasized the importance of some of these peptides in the defense against invasive bacterial infections in epithelia of different organs (11 12 13) . For example, mice deficient in the expression of CRAMP (the mouse homolog to the human cathelicidin) were more susceptible to skin infections caused by group A Streptococcus (12) and urinary tract infections by Escherichia coli (11) .

One arm of the antimicrobial defense of the skin is provided by members of the multifunctional S100 protein family. S100 proteins have a molecular mass of 9–13 kDa and are characterized by two calcium-binding EF-hand motifs (14) . Eleven proteins of the S100A family, i.e., S100A2, S100A3, S100A4, S100A6, S100A7c, S100A8, S100A9, S100A10, S100A11, S100A12, and S100A15, are expressed in the epidermis (15 16 17) . S100A7c, S100A12, and S100A15 have Gram-negative bacteria-killing activities (1 , 18 , 19) , and a heterodimer complex of S100A8/S100A9 suppresses the growth of the yeast Candida albicans (20) . S100A7c has been identified as a protein that is up-regulated in lesional psoriatic skin; hence, it was initially named psoriasin (21) . However, in the meantime it has been established that S100A7c is also expressed in normal skin (1 , 21) . S100A7c is encoded by a gene located on chromosome 1q21 within a cluster of five S100A7-like genes, S100A7a through S100A7e. S100A7a–S100A7c transcripts encode functional proteins whereas S100A7d and S100A7e are pseudogenes (22) . S100A7c shows 93 and 50% amino acid sequence similarity compared with S100A7a and S100A7b, respectively (22) . A recent report has provided evidence that the human S100A7 genes have arisen by repeated duplications of an ancient S100A7 gene whereas the mouse has lost the S100A7 gene (23) . Of all the proteins of the S100A7 family only S100A7c is present at high concentrations in human stratum corneum (2) . Gläser et al. (1) have shown that E. coli is effectively killed on human skin and that this killing activity was inhibited in vivo by a neutralizing antibody to S100A7c. Furthermore, this report demonstrated that S100A7c is up-regulated by exposing keratinocytes (KCs) to E. coli culture supernatants. Together, these data suggest that S100A7c has a critical role in the skin defense against E. coli and probably also against related bacteria.

The induction of antimicrobial molecules is a central mechanism of the innate immune defense. Components of different pathogens, i.e., pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors, such as the Toll-like receptors (TLRs) (24) , which are expressed on a wide variety of immune and nonimmune cells (25 26 27 28 29) . To date, 10 members of the TLR family of genes have been identified in humans (30 , 31) . Each TLR recognizes distinct PAMPs derived from various microorganisms. For instance, TLR3 recognizes viral double-stranded RNA (32) , TLR4 recognizes lipopolysaccharide (LPS) (33) , and TLR5 recognizes bacterial flagellin (34) . Engagement of TLRs by microbial components initiates signaling cascades that activate mitogen-activated protein kinases and induces nuclear translocation of NF-{kappa}B (35) , which consequently leads to the production of proinflammatory cytokines (35) and the induction of AMPs (such as hBD-2) in various epithelial cells (28 , 36 37 38) . In human KCs, functional expression of TLR1, 2, 3, 5, and 9 has been demonstrated (26 27 28) , whereas expression of TLR4 in KCs was detected by some but not other authors (27 , 39 , 40) . TLR2 has been shown to mediate the activation of NF-{kappa}B gene expression in response to Staphylococcus aureus (41) . The bacterial components by which E. coli induces the expression of S100A7c have not yet been identified.

Here we have investigated whether TLR ligands are able to up-regulate S100A7c expression in KCs and whether any of these ligands are involved in the induction of S100A7c by E. coli culture supernatants. The regulation of S100A7c was compared with that of the antimicrobial peptide hBD-2. Our results reveal that the induction by E. coli of both S100A7c and hBD-2 depends on the presence of bacterial flagellin and TLR5 on KCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human primary KCs prepared from neonatal foreskin were obtained from Clonetics (San Diego, CA, USA) and cultured in serum-free keratinocyte growth medium (KGM; Clonetics) as described previously (42) . For stimulation, third passage KCs were cultured in 12-well tissue culture plates (Corning Incorporated, Corning, NY, USA) and used at a confluence of 60–70%. Stimulation was performed in keratinocyte basal medium (KBM; Clonetics).

Reagents used for treatment of KCs
For in vitro assays, recombinant interleukin-1{alpha} (IL-1{alpha}; R&D Systems, Minneapolis, MN, USA), IL-8 (R&D Systems), and the following TLR ligands (InvivoGen, San Diego, CA, USA) were used: synthetic tripalmitoylated lipopeptide (Pam3CSK4; TLR1/2 ligand), heat-killed Listeria monocytogenes (HKLM; TLR2 ligand), poly(I:C), a synthetic analog of double-stranded RNA (TLR3 ligand), ultrapure LPS from E. coli K12 (TLR4 ligand), purified flagellin from Salmonella typhimurium (TLR5 ligand), synthetic lipoprotein of Mycoplasma salivarium (FSL1; TLR6/2 ligand), imiquimod (R837; TLR7 ligand), single-stranded RNA40 (TLR8 ligand), and ODN2006 (CpG oligonucleotide type B; TLR9 ligand). For IL-1{alpha} neutralizing experiments, KCs were used at a confluence of 60–70% in 12-well tissue culture plates. Before stimulation with IL-1{alpha} and flagellin, cells were preincubated for 90 min with a polyclonal goat IgG IL-1{alpha} neutralizing antibody (2 µg/ml; R&D Systems) or a polyclonal goat IgG isotype control (2 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For blocking IL-1 receptors, cells were incubated for 90 min with 100 ng/ml IL-1 receptor antagonist (IL-1Ra; Strathmann Biotec, Hamburg, Germany) before stimulation with flagellin or IL-1{alpha}.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)
After stimulation, cells were washed with PBS, and RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For cDNA synthesis RNA was reverse-transcribed with murine leukemia virus-reverse transcriptase using the Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA, USA) and oligo(dT) primers (Roche Diagnostics, Basel, Switzerland). cDNA sequences of the genes under investigation were obtained from GenBank. Primers were designed using PRIMER3 software from the Whitehead Institute for Biomedical Research (Cambridge, MA, USA). The following forward (F) and reverse (R) intron-spanning primers were used—for β-2-microglobulin: F, 5'-GATGAGTATGCCTGCCGTGTG-3'; R, 5'-CAATCCAAATGCGGCATCT-3'; for S100A7c: F, 5'-GGAGAACTTCCCCAACTTCCTT-3'; R, 5'-GGAGAAGACATTTTATTGTTCCT-3'; for S100A7a: F, 5'-AAATACACCGGACGTGATGG-3'; R, 5'-TCTTGTCCTTTTTCTCAAAGACAGT-3'; for S100A7b: F, 5'-GATGAATATCCCTCTAGGTGAGAAAGT-3'; R, 5'-GGTGCCACTCCATGCATTAT-3'; for hBD-2: F, 5'-ATCAGCCATGAGGGTCTTGT-3'; R, 5'-GAGACCACAGGTGCCATTTT-3'; for hBD-1: F, 5'-CCCAGTTCCTGAAATCCTGA-3'; R, 5'-CTTCTGGTCACTCCCAGCTC-3'; for hBD-3: F, 5'-AGCTGTGGCTGGAACCTTTA-3'; R, 5'-CGATCTGTTCCTCCTTTGGA-3'; for cathelicidin: F, 5'-GCTAACCTCTACCGCCTCCT-3'; R, 5'-GGTCACTGTCCCCATACACC-3'; for TLR5: F, 5'-CTAGAAGTCCCTTCTGCTAGGAC-3'; R, 5'-AAGGGGAAGGATGAAGCAGT-3'; and for IL-8: F, 5'-CTCTTGGCAGCCTTCCTGATT-3'; R, 5'-TATGCACTGACATCTAAGTTCTTTAGCA-3'. Quantitative real-time PCR was performed by LightCycler technology using the Fast Start SYBR Green I Kit for amplification and detection (Roche Diagnostics) as described previously (43) . Determination of relative quantification of target gene expression and amplification efficiencies was performed using a mathematical model by Pfaffl (44) . The expression of the target gene was normalized to the expression of β-2-microglobulin. All real-time PCRs were performed in triplicate. The specificity of the reactions was confirmed by sequencing of the PCR products.

Bacterial strains and KC stimulation
E. coli NK 9373 and E. coli NK 9375 ({Delta}FliC; a flagellin-deficient strain having an in-frame deletion within the fliC gene) (45) were kindly provided by Dr. David Bates (Harvard University, Cambridge, MA, USA). Bacterial culture supernatants were prepared as described previously (1) with minor modifications. Briefly bacteria were grown in tryptic soy broth (TSB) (Fluka, Buchs, Switzerland) with agitation at 37°C until an optical density of 1.0 (OD600) was reached. Then 1 ml of this culture was combined with 9 ml of TSB and incubated overnight in 75-cm2 flasks (Cellstar, Frickenhausen, Germany) at 37°C without agitation. Optical density (OD600) of overnight cultures of E. coli strains was set with TSB to 1.7. Subsequently, bacteria were heat-killed in a water bath at 65°C for 60 min. Heat-killing was verified by a plating culture on Luria-Bertani agar. After heat-killing, cultures were centrifuged at 5000 g for 15 min. The resulting supernatants were diluted 1:100 in KBM and used to stimulate KCs from which the previous medium had been removed.

Cytokine measurement
Culture supernatants of stimulated KCs were depleted by centrifugation of detached cells or cell fragments and stored at –20°C until analysis. Concentrations of IL-1{alpha} and IL-8 were determined by ELISA (R&D Systems) according to the manufacturer’s instructions.

Small interfering (si) RNA transfections
Third-passage primary KCs were used at a confluence of 60–70% in 12-well tissue culture plates. KCs were transfected with the following Stealth siRNAs using Lipofectamine 2000 reagent (Invitrogen): TLR5-siRNA1, 5'-CAUCCUUCAUUUGGGAAGUUGAAUU-3'; TLR5-siRNA2, 5'-CCACCAGGAGUAUUUAGCCAUCUGA-3'; control siRNA1 (scrambled sequence of TLR5-siRNA1), 5'-CAUACUUGUUUAAGGGUUGACCAUU-3'; and control siRNA2 (scrambled sequence of TLR5-siRNA2), 5'-CCAGGAUGAUUAGAUACCCUCCUGA-3'. Lipofectamine 2000 (30 µl) was mixed with 60 µl of a 20 µM siRNA solution (1:1 mix of siRNA1 and -2) and 4 ml of OPTI-MEM medium (Gibco BRL, Gaithersburg, MD, USA). After 30 min at room temperature, 4 ml of KBM was added, and the solution was poured onto the KCs in 12-well tissue culture plates (600 µl/well) for 24 h. Afterward the medium was changed to KGM, and cells were left for another 24 h before stimulation.

Western blot analysis
For analysis of protein expression, KCs were lysed in SDS-PAGE loading buffer (50 mM Tris, pH 7.4, 2% SDS). After sonication, insoluble cell debris was removed by centrifugation, and protein concentration was measured by the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL, USA). Western blot analysis was performed as described previously (46) . The following first step antibodies were used: mouse monoclonal IgG1 anti-S100A7c clone 47C1068 (dilution 1:500; Abcam, Cambridge, UK) and mouse monoclonal IgG2b anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (dilution 1:400; Biogenesis, Poole, UK). The membranes were developed using the Chemiglow reagent (Alphainnotech, San Leandro, CA, USA) according to the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Flagellin but not other TLR ligands induces S100A7c expression in human primary KCs
To investigate the role of TLRs in the regulation of S100A7c expression in KCs, primary human epidermal KCs were stimulated with ligands for TLR1 through TLR9. The expression of S100A7c and other AMPs was determined by quantitative real-time PCR analysis. S100A7c mRNA was up-regulated by the TLR5 ligand flagellin but not by other TLR ligands (Fig. 1 A). The level of up-regulation of S100A7c induced by flagellin equalled that induced by IL-1{alpha} (Fig. 1A ). Among the other AMPs investigated, hBD-1, hBD-3, RNase7, and cathelicidin were not changed by stimulation with TLR ligands (data not shown). hBD-2 was up-regulated by flagellin, poly(I:C) (TLR3 ligand), and ODN2006 (TLR9 ligand) in a manner comparable with the results of previous studies (28 , 36) (Fig. 1B ). Similar to the results obtained with S100A7c, hBD-2 mRNA was almost as strongly induced by flagellin as by IL-1{alpha}. Because hBD-2 expression showed responsiveness to flagellin similar to that of S100A7c, it was used for comparison in all further experiments.


Figure 1
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  		Figure 1. Flagellin induces S100A7c and hBD-2 expression in human primary KCs. Normal human epidermal KCs were stimulated for 16 h with IL-1{alpha} (10 ng/ml) and the following TLR ligands: 0.5 µg/ml synthetic tripalmitoylated lipopeptide (Pam3CSK4; TLR1/2 ligand), 108 cells/ml heat-killed Listeria monocytogenes (HKLM; TLR2 ligand), 15 µg/ml poly(I:C) (a synthetic analog of double-stranded RNA; TLR3 ligand), 100 ng/ml ultrapure LPS from E. coli K12 (TLR4 ligand), 0.5 µg/ml purified flagellin from S. typhimurium (TLR5 ligand), 0.5 µg/ml FSL1 (a synthetic lipoprotein of M. salivarium; TLR6/2 ligand), 10 µg/ml imiquimod (R837; TLR7 ligand), 1 µg/ml single-stranded RNA40 (ssRNA; TLR8 ligand), and 2.5 µM ODN2006 (CpG oligonucleotide type B; TLR9 ligand). After stimulation, RNA was harvested and relative S100A7c (A) and hBD-2 (B) gene expression levels were quantified by real-time PCR and normalized to the expression of the housekeeping gene β-2-microglobulin. The mean values are displayed in relation to untreated controls. Data are means ± SD of one representative experiment of three, each done in triplicate. *P < 0.05 vs. untreated control; unpaired Student’s t test.

Flagellin induces S100A7c expression in an IL-1-independent manner
Next we determined the dose and time dependence of flagellin-induced AMP expression. S100A7c mRNA was significantly induced at a flagellin concentration of 5 ng/ml (Fig. 2 A), whereas 1 ng/ml flagellin sufficed to up-regulate hBD-2 mRNA expression (Fig. 2B ). Increasing concentrations of flagellin up to 10 ng/ml induced higher expression levels of both S100A7c and hBD-2 mRNA, whereas further elevation of the flagellin did not cause significant additional up-regulation (Fig. 2A, B ) and even reduced the inductive effect of flagellin (Fig. 2B ). The maximum up-regulation of both S100A7c and hBD-2 mRNA was observed at time points later than 8 h after addition of flagellin to the KC culture medium (Fig. 2C, D ). Essentially the same temporal expression pattern was induced by stimulation with IL-1{alpha} (Fig. 2C, D ), whereas IL-8 mRNA was up-regulated as early as 2 h after stimulation and decreased later (Fig. 2E ).


Figure 2
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  		Figure 2. Time- and dose-dependent effect of flagellin on S100A7c and hBD-2 expression. KCs were stimulated with the indicated concentrations of flagellin for 24 h. Thereafter, quantitative real-time PCR analysis of S100A7c (A) and hBD-2 (B) mRNA expression was performed. KCs were cultured for the indicated times in the presence of 10 ng/ml flagellin, and quantitative real-time PCR analysis of S100A7c (C), hBD-2 (D), and IL-8 (E) mRNA expression was performed. KCs were stimulated with IL-1{alpha} or IL-8 (both at 10 ng/ml) for 24 h; after stimulation, relative S100A7c and hBD-2 gene expression levels were determined by quantitative real-time PCR (F). All gene expression levels were normalized to the housekeeping gene β-2-microglobulin. The mean values are displayed in relation to untreated controls. Data are means ± SD of one representative experiment of three, each done in triplicate.

The time course of S100A7c or hBD-2 expression seemed to be compatible with an indirect mechanism of regulation involving flagellin-induced cytokine release and subsequent induction of S100A7c and hBD-2 by cytokines. We therefore investigated the role of two candidate cytokines, i.e., IL-1 and IL-8.

Because IL-1{alpha} is stored in KCs and can be released by various stress stimuli (47) , we determined by ELISA the IL-1{alpha} concentration in KC culture supernatant at 2 h after flagellin stimulation, a time point before the increase of S100A7c and hBD-2 expression (Fig. 2C, D ). However, IL-1{alpha} concentrations did not differ between supernatants of flagellin-stimulated and unstimulated KCs either at 2 h or 24 h after stimulation (Fig. 3 A).


Figure 3
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  		Figure 3. Flagellin induction of S100A7c and hBD-2 is IL-1{alpha}-independent. A) KCs were stimulated with flagellin (10 ng/ml) for the indicated times, and culture medium was measured for IL-1{alpha} secretion by ELISA. Data are means ± SD of one representative experiment of two, each done in triplicate. B, C) KCs were preincubated for 90 min with anti-IL-1{alpha} (2 µg/ml), with isotype control (2 µg/ml) or with IL-1Ra (100 ng/ml), as indicated. Afterwards, cells were treated with IL-1{alpha} or flagellin (both at 10 ng/ml) or left untreated for 24 h in the continued presence of the reagents used for preincubation. Relative gene expressions of S100A7c (B) and hBD-2 (C) were quantitated by real-time PCR and normalized to the housekeeping gene β-2-microglobulin. The mean values are displayed in relation to untreated controls. Data are means of triplicates ± SD. Similar results were obtained in a second experiment. *P < 0.05, determined by unpaired Student’s t test. n.s., not significant.

To exclude the theoretical possibility that released IL-1{alpha} is captured by cellular receptors and exerts its effects without increasing in concentration in the bulk supernatant, KCs were preincubated with an IL-1{alpha} neutralizing antibody or its corresponding isotype control, and/or with an IL-1Ra that neutralizes both IL-1{alpha} and IL-1β. Neither treatment interfered with S100A7c or hBD-2 induction by flagellin (Fig. 3B, C ), demonstrating that extracellular IL-1 is not required for the observed effects of flagellin. Consistent with previous reports (48 , 49) , addition of IL-8 did not alter the mRNA expression levels of S100A7c and hBD-2 (Fig. 2F ).

Flagellin-deficient E. coli does not induce S100A7c expression
To determine the contribution of flagellin in the induction of S100A7c expression by E. coli culture supernatant, we compared a wild-type (WT) flagellated E. coli strain (NK 9373) and a flagellin-deficient ({Delta}FliC) E. coli strain (NK 9375) with regard to their capacity to induce S100A7c expression. Culture supernatants of E. coli strains were diluted 1:100 in KBM and applied to KC cultures for 16 and 48 h. Quantitative real-time PCR revealed that WT E. coli induced S100A7c and hBD-2 mRNA expression, whereas {Delta}FliC E. coli did not (Fig. 4 A, B). Neither WT nor {Delta}FliC E. coli strains were able to induce the expression of hBD-1, hBD-3, RNase7, and cathelicidin (Supplemental Fig. 1). Western blot analysis of cell lysates showed that the increase of S100A7c mRNA in response to flagellin or WT E. coli also resulted in a strong increase of S100A7c protein (Fig. 4C ). In contrast, {Delta}FliC E. coli lacked the ability to induce S100A7c protein production (Fig. 4C ). Addition of flagellin (10 ng/ml) to the {Delta}FliC E. coli culture supernatant restored the induction of S100A7c protein expression (Fig. 4C ), demonstrating that the failure of this strain to induce S100A7c is not due to the presence of an inhibitor. Notably, supernatants of {Delta}FliC E. coli were also able to induce secretion of IL-8 by KCs, although less than WT E. coli (Fig. 4D ). These findings demonstrate that flagellin is essential for E. coli-mediated induction of S100A7c and hBD-2 but only partially contributes to E. coli-mediated cytokine secretion from KCs.


Figure 4
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  		Figure 4. {Delta}FliC does not induce S100A7c or hBD-2 in human primary KCs. A, B) Culture supernatants of WT and {Delta}FliC E. coli strains and tryptic soy broth [control (ctrl.)] were diluted 1:100 in KBM and added to KCs from which the previous culture medium had been removed. Culture was continued for 16 h. After stimulation, relative S100A7c (A) and hBD-2 (B) gene expression levels were determined by quantitative real-time PCR. The results were normalized to the expression of the housekeeping gene β-2-microglobulin. Mean values are displayed in relation to the control. Data are means ± SD of triplicate samples from one representative of four independent experiments. C) S100A7c and GAPDH Western blot analysis of cell lysates after 48 h of stimulation with IL-1{alpha}, flagellin (both at 10 ng/ml), and E. coli culture supernatants (1:100 in KBM). The Western blot is representative of at least four independent experiments. D) Culture medium was collected after 16 h of stimulation, and the concentration of IL-8 was determined by ELISA. Data are means ± SD of triplicate samples from one representative of three independent experiments. *P < 0.05, unpaired Student’s t test.

Because it has been reported that S100A7a, misleadingly named human S100A15 (50) , is also up-regulated by E. coli (18) , we compared the potential biological significance of S100A7a and S100A7c in the defense against E. coli. Like S100A7c, S100A7a was up-regulated by IL-1{alpha}, flagellin, and WT E. coli but not by {Delta}FliC E. coli (Supplemental Fig. 2A). In contrast with the conclusions of another report (18) , we did not observe an increase in S100A7a in response to the TLR4 ligand LPS (data not shown). However, when we compared the absolute expression levels of both S100A7a and S100A7c by quantitative real-time PCR we found that S100A7c was expressed at a 650-fold higher level than S100A7a (12285±836 vs. 19±3 mRNA copies per 1 µg of total RNA) (Supplemental Fig. 2B). Transcripts of the third potentially functional S100A7 isoform, S100A7b, could not be detected in KCs (Supplemental Fig. 2B). These data suggest that S100A7a and S100A7c are regulated by E. coli components in a similar manner; however, S100A7c is expressed at a much higher level.

Knockdown of TLR5 abolishes S100A7c and hBD-2 induction by E. coli
Flagellin is known to induce antimicrobial defense via two pathways, namely activation of the intracellular receptor ICE-protease-activating factor (Ipaf) followed by caspase-1-mediated processing of IL-1β (51) and/or binding to TLR5 and MyD88-dependent intracellular signaling (24) . Because our results obtained with the IL-1Ra argued against a role of IL-1β and, accordingly, also against a role of the Ipaf pathway (51) , we focused our further investigations on the putative role of TLR5 in the response of KCs toward E. coli. siRNA technology was used to knock down TLR5 expression in KCs. Transfection of KCs with TLR5 siRNA led to a down-regulation of TLR5 mRNA by 70% compared with siRNA with a scrambled sequence (Fig. 5 A). Knockdown of TLR5 strongly inhibited the induction of S100A7c and hBD-2 mRNA by flagellin and WT E. coli supernatant (Fig. 5B, C ). Western blot analysis of cell lysates showed that knockdown of TLR5 also suppressed the increase of S100A7c protein production by flagellin and WT E. coli supernatant (Fig. 5D ).


Figure 5
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  		Figure 5. Knockdown of TLR5 abolishes the induction of S100A7c and hBD-2 by E. coli supernatant in human primary KCs. KCs were transfected either with scrambled (scr; {square}) or TLR5 ({blacksquare}) siRNA. Forty-eight hours after transfection, cells were stimulated with flagellin (10 ng/ml), culture supernatants of WT and {Delta}FliC E. coli strains or tryptic soy broth [control (ctrl.)] diluted (1:100) in KBM. Sixteen or 48 h later mRNA and protein, respectively, were prepared and analyzed. A–C) Relative expression of TLR5 (A), S100A7c (B), and hBD-2 (C) was quantified by real-time PCR. The results were normalized to the expression of the housekeeping gene β-2-microglobulin. Mean values are displayed in relation to untreated controls. Data are means ± SD of triplicate samples from one representative of three independent experiments. D) S100A7c and GAPDH Western blot analysis of TLR5 knockdown KC lysate stimulated for 48 h as indicated. The data are representative of three independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
S100A7c is one of the most potent antimicrobial agents of the skin surface and it appears to be relatively specific for E. coli (1) . The fact that S100A7c is expressed in a focal pattern in the epidermis and the finding that E. coli culture supernatants are able to increase S100A7c abundance on human skin have indicated that S100A7c is up-regulated by bacterial substances entering the epidermis through microwounds (1 , 2) . However, distinct exogenous molecular triggers of S100A7c have remained elusive. In the present study, we demonstrate that S100A7c is induced by flagellin, a component of flagellated bacteria, including E. coli.

A key result of this study was the demonstration that flagellin deficiency of E. coli is associated with a lack of induction of S100A7c. Because we compared two E. coli strains that differed exclusively at the locus of the fliC gene, which was destroyed by targeted deletion in E. coli NK9375, mutations affecting other gene products can be excluded as causative factors for the observed difference. Together with our demonstration that purified flagellin is sufficient to induce S100A7c up-regulation, the data obtained with E. coli supernatants strongly suggest that flagellin is the only relevant E. coli component that stimulates S100A7c expression in KCs. However, there remains the theoretical possibility that an as yet unknown factor attaches to flagellin and contributes to its effects. The ultimate proof may involve the use of ultrapure flagellin or flagellin fragments produced by peptide synthesis in vitro instead of purification from flagellated bacteria or bacteria expressing recombinant flagellin.

Complementary to our findings regarding the role of bacterial flagellin in S100A7c regulation, we have identified the flagellin receptor TLR5 as an essential element of the sensory and signaling system for E. coli in KCs. Furthermore, our data also demonstrate that TLR5 is critical for the induction of hBD-2. Compared with the results of a previous report (52) that showed ~7-fold up-regulation of hBD-2 promoter activity in response to bacterial muramyl dipeptide (MDP), the 50- to 100-fold up-regulation of hBD-2 mRNA expression by flagellin observed in our study appears to be more relevant in a physiological context. However, it is conceivable that TLR5 on the KC membrane and intracellular NOD2, the receptor of MDP, cooperate in sensing E. coli and triggering hBD-2 expression.

S100A7c is up-regulated by flagellin more than 8 h after stimulation of KCs. The response time from addition of flagellin to the production of S100A7c mRNA was longer than that for the induction of IL-8 but was comparable to the kinetics of S100A7c expression in KCs observed by others after stimulation with various cytokines (1 , 49) . The exploration of the cause for this relatively slow response exceeds the scope of our present study. However, our data argue against a role of autocrine activation of S100A7c production by IL-1 and IL-8, because blockade of the former did not inhibit flagellin-induced S100A7c expression and addition of the latter did not induce the expression of S100A7c when added to KCs.

Our data on the regulation of S100A7c extend the range of inducers of S100A7c in KCs. Previously, the cytokines IL-1β, tumor necrosis factor-{alpha}, IFN-{gamma} (1) , IL-6, IL-17, IL-20, IL-22, IL-24, and oncostatin M (49) , as well as late states of KC differentiation (53) , have been shown to enhance the expression level of S100A7c. The role of flagellin in the control of S100A7c therefore needs to be further evaluated in combination with other factors, many of which may be highly relevant in the context of cutaneous wounds. Unfortunately, the mouse is not a suitable model system for the investigation of S100A7 regulation because the mouse has lost the S100A7 genes during evolution (50) . The murine gene most closely related to S100A7 is S100A15, the ortholog of which has been lost during human evolution (50) . Although S100A15 may fulfill a role in the mouse equivalent to that of S100A7 in humans, the regulatory mechanisms of both genes are likely to differ (18) .

It is remarkable that the presence of the inducer of S100A7c, flagellin, distinguishes E. coli from the nonflagellated bacteria of the commensal skin microflora (54 , 55) . The specific response of KCs to a flagellated bacterium by induction of S100A7c and hBD-2 expression may exemplify a previously unappreciated ability of the innate immune system to discriminate between potentially harmful microbes and commensal bacteria. In line with this concept, S100A7c has been shown to exert virtually no bactericidal activity against the skin-resident bacterium Staphylococcus epidermidis (1) . Further studies are necessary to determine whether the antimicrobial defense of the skin is adapted to specifically respond to and attack opportunistic bacteria while preserving the resident microflora.


   ACKNOWLEDGMENTS
 
The authors are grateful to David Bates (Harvard University, Cambridge, MA, USA) for providing E. coli strains. We thank Veronika Mlitz, Martina Schmidt, Christine Poitschek, Awaz Uthman, and Heidemarie Rossiter for helpful discussions. J.-M.S. was supported by Deutsche Forschungsgemeinschaft, SFB 617.

Received for publication December 10, 2007. Accepted for publication January 17, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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