Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections

This information is current as Christa Maerki, Simone Meuter, Mark Liebi, Kathrin of September 24, 2021. Mühlemann, Mitchell J. Frederick, Nikhil Yawalkar, Bernhard Moser and Marlene Wolf J Immunol 2009; 182:507-514; ; doi: 10.4049/jimmunol.182.1.507

http://www.jimmunol.org/content/182/1/507 Downloaded from

References This article cites 40 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/182/1/507.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections1

Christa Maerki,* Simone Meuter,§ Mark Liebi,* Kathrin Mu¨hlemann,† Mitchell J. Frederick,¶ Nikhil Yawalkar,‡ Bernhard Moser,2§ and Marlene Wolf2,3*

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the CXCL14 is a broad-spectrum AMP with killing activity for cutaneous Gram-positive bacteria and Candida albicans as well as the Gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human ␤-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its Downloaded from AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity. The Journal of Immunology, 2009, 182: 507–514.

he skin is in constant contact with the environment and is (4, 5). Its selective ligand CCL1 is constitutively expressed in the http://www.jimmunol.org/ permanently challenged by a range of threats including dermal microvasculature and scattered cells within the epidermis. T potentially pathogenic microorganisms. Therefore, the This chemokine system likely represents a mechanism controlling existence of an elaborate local immune surveillance system is crit- the traffic and/or positioning of peripheral immune surveillance T ical for immune defense and skin integrity. The outermost layer of cells within healthy human skin. healthy skin consists of a stratified epidermis that is sealed on the By contrast to CCL1, a second chemokine, CXCL14, is much outside by the largely impermeable stratum corneum. In addition more highly expressed in healthy human skin, notably in keratin- to this physical barrier, it has been long recognized that the skin, ocytes and dermal fibroblasts, but is also present in other epithelial i.e., the epidermis and the underlying dermal tissue, harbors a va- tissues such as gut and kidney (6–8). Chemoattractant activity for by guest on September 24, 2021 riety of highly specialized immune cells, including dendritic cells peripheral blood led us to propose that CXCL14 con- 4 (DCs), , mast cells, and lymphocytes, each involved tributes to the homeostasis of cutaneous DCs, including dermal in the maintenance of tissue integrity and cutaneous immunity (1). DCs and Langerhans cells, by attracting DC precursors to distinct Our recent studies of the immune surveillance system in human niches within the skin where their final differentiation into bona skin are based on the paradigm that links the control of cell mi- fide APCs occurs (9). Indeed, in a tissue model with human epi- gration with immune cell function (2). Tissue-specific traffic of dermal equivalents, CXCL14-responsive monocytes were found to immune surveillance cells is dependent on the constitutive expres- develop into APCs with Langerhans cell characteristics (9). This sion of homeostatic (2, 3). Initial investigations led us chemokine was also shown to act on immature -derived to the CCR8, which we found preferentially DCs, indicating an additional role in DC localization within expressed on the majority of lymphocytes in healthy human skin CXCL14-producing tissues (10, 11). Mice deficient in CXCL14 do not show any gross abnormalities in the tissue distribution and

*Theodor Kocher Institute, †Institute for Infectious Diseases, and ‡Department of function of DCs, suggesting that murine CXCL14 is not involved Dermatology, University of Bern, Bern, Switzerland; §Department of Medical Bio- in the maintenance of the peripheral DC compartments, that chemistry and Immunology, Cardiff University School of Medicine, Cardiff, United CXCL14 is not the only chemokine involved in this process (func- Kingdom; and ¶Department of Head and Neck Surgery, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030 tional redundancy), or that this defect was neutralized through Received for publication July 16, 2008. Accepted for publication November 3, 2008. compensatory mechanism(s) (12). A detailed analysis is hampered The costs of publication of this article were defrayed in part by the payment of page by the striking breeding defect of homozygous CXCL14-null mice, charges. This article must therefore be hereby marked advertisement in accordance pointing to a role for CXCL14 in reproduction and/or embryogen- with 18 U.S.C. Section 1734 solely to indicate this fact. esis. It will be important to identify the CXCL14 receptor, because 1 This work was supported by Staatssekretariat fu¨r Bildung und Forschung Grant this information will lead us to the physiological target cells and, 03.04412 and European Framework Programme No. 6 Grant 518167. thus, to the function of this ill-defined chemokine in immune 2 B.M. and M.W. contributed equally to this work. processes. 3 Address correspondence and reprint requests to Dr. Marlene Wolf, Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3012 Bern, Switzerland. E-mail ad- The abundant expression of CXCL14 in normal human skin, dress: [email protected] notably in the epidermis, is unmatched by any other chemokine 4 Abbreviations used in this paper: DC, ; AMP, antimicrobial peptide; and suggested to us a chemokine-atypical function in local im- HBD, human ␤-defensin; MEC, minimal effective concentration; RDU, radial diffu- mune defense, which is the subject that we have investigated in the sion unit; S. coag.neg. spp., coagulase-negative Staphylococcus spp. present study. In addition to the physical barrier and the repertoire Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 of immune surveillance cells, the skin also harbors a variety of www.jimmunol.org 508 CXCL14 DISPLAYS POTENT ANTIMICROBIAL ACTIVITY

antimicrobial peptides (AMPs) that constitute the third type of weapon in the cutaneous arsenal against the constant onslaught of microbes (13). AMPs are a diverse family of small, mostly cationic polypeptides that have killing activity against a wide spectrum of bacteria, yeast, and some enveloped viruses (14–16). The major AMPs expressed in human skin are defensins, cathelicidin LL-37, and psoriasin (13, 17, 18), which are produced and released under either steady-state or inflammatory conditions by keratinocytes. Of interest, and in agreement with their role in acute infections, many AMPs also induce migration and effector responses in leukocytes (19, 20). Also, vice versa, a few chemokines were shown to have AMP activity (21–26). However, our understanding of their in- volvement in antimicrobial defense is rudimentary as compared with their primary role in leukocyte traffic (3) and with the vast literature describing nonchemokine AMPs (15, 27, 28). In this study, we describe CXCL14 as a highly active AMP with antimicrobial activity against Gram-positive and Gram-negative bacteria and also Candida albicans. Constitutive as opposed to

inflammation-dependent expression supports the view that Downloaded from CXCL14 fulfills a critical function in the maintenance of skin in- tegrity by keeping potentially infectious microbes at bay. This che- mokine may be less relevant in fighting microbes during estab- lished infections, where a range of inducible AMPs and other innate mechanisms are mobilized. http://www.jimmunol.org/ Materials and Methods Peptides, Abs, and microorganisms Chemokines were chemically synthesized as described (29). Human ␤-de- fensin (HBD) 2 was from Bachem, anti-human CXCL14 and CCL20 from R&D Systems, isotype controls from BD Biosciences and Sigma-Aldrich, and anti-HBD2 was provided by T. Ganz (University of California, Los Angeles, CA). Clinical isolates of oxacillin-susceptible and -resistant strains of coagulase-negative Staphylococcus spp. (S. coag.neg. spp.) and Staphylococcus aureus, Escherichia coli, Propionibacterium spp., and C. albicans were used. Bacteria and C. albicans were grown on agar plates at by guest on September 24, 2021 37°C in ambient air with the exception of Propionibacterium spp., which were cultured under anaerobic conditions. Cell lines Tu-138 is a human squamous cell carcinoma line (30), PamLy an immor- talized BALB/c keratinocyte cell line, and SCC7 a spontaneous squamous cell carcinoma line derived from C3H mice. DNA sequences correspond- ing to the complete coding regions (including signal peptides) from human or murine CXCL14 were generated by RT-PCR. Human CXCL14 was subcloned into the pDNR1 vector (Clontech) containing Lox P sites and FIGURE 1. CXCL14 expression is down-regulated in inflamed human transferred to the pLP-IRESneo expression vector using Cre recombinase. skin. A, Immunohistochemical analysis of tissue sections from normal skin Murine CXCL14 was cloned directly into the expression vector pIRESneo and skin affected by psoriasis and atopic dermatitis demonstrates differen- 3 (Clontech). Restriction site-modified primers used for PCR amplification were 5Ј-AGGATCCCCTCCCCATGTCCCTGC-3Ј (human sense); 5Ј- tial expression of CXCL14, CCL20, and HBD2. Insets, isotype control; ␮ GAAAGCTTCTATTCTTCGTAGACCCTGCG-3Ј (human antisense); 5Ј- scale bar, 50 m. B, Unlike many chemokines and AMPs, CXCL14 GTACCGGTCTCCTTGCCTCCCTGCTC-3Ј (mouse sense); and 5Ј- mRNA was significantly down-regulated in inflamed skin as measured by CCGAATTCATCGTCCACCCTATTCTTCGTA-3Ј (mouse antisense). Affymetrix GeneChip analysis. mRNA levels were compared between epi- Tu-138 cells were stably transfected with human CXCL14, whereas dermis of healthy (untreated) human skin and epidermis stimulated in vitro PamLy and SCC7 were stably transfected with murine CXCL14 using with TNF-␣ and LPS for 9 h. Expression changes of Ͼ2-fold with p Ͻ 0.05 Lipofectamine 2000 (Invitrogen). Clones were selected with 400 ␮g/ml (dotted lines) were taken as significant. G418 antibiotic. CXCL14 in culture supernatants was quantified using a commercially available ELISA kit (R&D Systems). Skin samples, RNA isolation, microarray hybridization 7.2 software (Silicon Genetics). Statistical comparison between stimulated Normal human skin samples from abdominal or mammary reduction sur- and normal skin was performed using the Welch t test with log transformed gery were digested with 1.25 U/ml Dispase II for 15–30 min at 37°C data. that passed this test with p Ͻ 0.05 and that showed changes in (Roche Diagnostics), before collecting the epidermis. The epidermis was expression Ͼ2-fold were assumed to be regulated. stimulated with 500 ng/ml LPS and 10 ng/ml TNF-␣ for 9h in RPMI 1640 (Invitrogen). RNA was extracted using the RNAzol B method (AMS Bio- Immunohistochemistry technology), purified using an RNeasy mini kit (Qiagen), and treated with DNase (Qiagen). RNA integrity was checked with a Bioanalyzer 2001 Skin biopsy procedure was approved by the local ethics committee and (Agilent Technologies). cRNA preparation and microarray hybridization study participants gave written informed consent before obtaining skin were conducted according to the supplier using GeneChip HG-U133A (Af- specimens. Skin samples were frozen in Tissue-Tek OCT compound fymetrix). Hybridized GeneChips were scanned with an Affymetrix mi- (Sakura Finetek) for the staining of CXCL14 or were formaldehyde fixed croarray scanner 3000 and fluorescence intensity raw data were collected and embedded in paraffin for CCL20 and HBD2. Immunohistochemistry using GeneChip MAS 5.0. Data analysis was performed with GeneSpring was performed as described (12) with the exception of a 10-min acetone The Journal of Immunology 509 Downloaded from http://www.jimmunol.org/

FIGURE 2. CXCL14 is a strong and broad-spectrum antimicrobial peptide against Gram-positive and Gram-negative bacteria. A, Antimicrobial activity of

CXCL14 was compared with selected chemokines and HBD2 against Gram-negative E. coli, Gram-positive S. coag.neg. spp., Gram-positive S. aureus, Gram- by guest on September 24, 2021 positive Propionibacterium spp., and the yeast C. albicans. Data from two to four independent experiments (ϮSD) represent inhibition of microbial growth. The

MEC corresponds to the x-intercept of a linear least mean square regression of the relationship between zone diameters vs log10 peptide concentration. Peptide refers to the type of AMP tested (see legend to right of E. coli graph). B, Bacteriostatic capacity of CXCL14, compared with CCL20 and HBD2, was analyzed with the colony-forming assay against E. coli and S. coag.neg. spp. The percentage of growth inhibition is expressed as the ratio of colonies counted in the presence of a peptide to the number of colonies on a control plate as described in Materials and Methods (ϮSD of triplicate experiments).

fixation for CXCL14 and epitope retrieval for 40 min in boiling 1 mM Results EDTA (pH 8) for CCL20. Inflammatory conditions inhibit CXCL14 expression in human skin Antimicrobial assays Constitutive expression in healthy human skin has been taken to Antimicrobial activities were evaluated by the radial diffusion assay (21) or suggest a role for CXCL14 in local immune homeostasis, notably the colony-forming assay. Radial diffusion units (RDUs) were defined as in the steady-state turnover of epidermal Langerhans cells (9). follows: [diameter of clear zone in millimeters Ϫ diameter of the well] ϫ However, production under homeostatic conditions does not a pri- 10. This assay was also used to determine the antimicrobial activity of cell ori exclude a contribution of this ill-defined chemokine in inflam- culture supernatants from CXCL14-transfected cells cultured in antibiotic- matory processes that may include acute infections and chronic free medium for 24 h. The relationship between the net zone diameters and skin diseases. To approach this question, we performed immuno- the log10 of the concentration of peptide that was introduced into the well was calculated by the method of least mean squares. The minimal effective histochemical analysis with healthy and diseased human skin (Fig. concentration (MEC) was defined as the x-intercept specified by this func- 1A). As expected, CXCL14 protein was highly present in healthy tion (21). The colony-forming assay was performed with exponentially human skin, notably in the epidermis and scattered cells of the growing bacteria (OD ϭ 0.55) in Luria broth medium. Bacteria were 620 dermis, but was only produced in isolated patches in skin affected washed twice with 10 mM potassium phosphate buffer (pH 7.4) supple- mented with 1% (v/v) Luria broth medium and then diluted to a final by either psoriasis or atopic dermatitis (large CXCL14-negative concentration of 1 ϫ 105 CFU/ml in the same buffer. One hundred micro- areas are not shown in Fig. 1A). CXCL14 expression is regulated liters of this bacterial suspension was incubated with peptide or culture differently than two other skin AMPs, the chemokine CCL20 and supernatant for3hat37°C under rotation (250 rpm). Growth inhibition HBD2, which showed just marginal staining in healthy skin but was analyzed after plating serial dilutions of the bacterial suspensions on were significantly up-regulated in psoriatic and atopic dermatitis Luria broth agar plates and culturing for 16–18 h as described (31) and is expressed as the ratio of colonies counted to the number of colonies on a lesions (32, 33). HBD2 expression in atopic dermatitis, however, is control plate [1 Ϫ (CFU after peptide incubation)/(CFU after control in- less prominent, and insufficient HBD2 production is believed to be cubation)] ϫ 100. the reason why these patients are more prone to infections (34). 510 CXCL14 DISPLAYS POTENT ANTIMICROBIAL ACTIVITY

Table I. CXCL14 is a potent AMP ods, the radial diffusion assay measuring the distance of the clear- ing zone from the antibiotic deposition to the edge of bacterial MECsa (␮M) growth (Fig. 2A) and the colony-forming assay measuring growth

E. S. coag.neg. Propionibacterium S. C. inhibition in liquid culture as a function of antibiotic concentration coli spp spp. aureus albicans (Fig. 2B). The selected microbes are generally benign but become

CXCL14 0.32 1.02 1.16 5.17 0.56 pathogenic as a result of epithelial injury. CXCL14 exhibited CCL20 0.46 1.58 5.23 5.54 0.09 strong antimicrobial activity toward Gram-negative E. coli, Gram- CCL27 0.67 Ͼ80 Ͼ80 Ͼ80 Ͼ80 CXCL8 9.9 Ͼ150 Ͼ150 Ͼ150 Ͼ150 positive species S. coag.neg. spp., Gram-positive S. aureus, and HBD2 2.54 5.83 Gram-positive Propionibacterium spp., as well as the fungus C. a MEC was calculated by least mean squares regression at the x-intercept of the albicans. We quantified and compared the potency of CXCL14 by relationship between zone diameters vs log10 peptide concentration. determining MECs that were calculated by least mean squares re- gression at the x-intercept of the relationship between zone diam-

eters vs log10 peptide concentration (21) (Fig. 2A and Table I). The Next, we conducted a global expression analysis with RNA antimicrobial activity of CXCL14 showed a certain degree of spe- from whole skin (not shown) and isolated epidermis tissue from cies variation, being most effective against E. coli, C. albicans, and healthy individuals as well as TNF-␣- and LPS-treated epidermis S. coag.neg. spp. (MECs of 0.32, 0.56, and 1.02 ␮M, respectively). ␣ tissue to mimic acute infection (Fig. 1B). Of note, the TNF- /LPS We next included inducible skin peptides such as HBD2, CCL20, treatment reduced the expression of CXCL14 by 31-fold. This is in

and CCL27 and the acute phase chemokine CXCL8 in the radial Downloaded from sharp contrast to other chemokines or AMPs, which were either diffusion assay to gauge the level of antimicrobial activity obtained markedly up-regulated like CCL20 (356-fold), CXCL8 (180-fold), with CXCL14. CCL20 had similar antimicrobial properties as CXCL2 (8-fold), S100A7/psoriasin (15-fold), and HBD2 (3-fold), CXCL14 whereas CCL27 and CXCL8 were considerably less po- or remained unchanged like CXCL9, CXCL12, and HBD1 (Fig. tent (against E. coli,) or even inactive (against Gram-positive bac- 1B). LL-37, another well-characterized human AMP in skin, was teria and C. albicans). HBD2 only inhibited growth of E. coli and absent and not affected by the inflammatory stimuli, consistent

S. coag.neg. spp. The colony-forming assay confirmed these find- http://www.jimmunol.org/ with a previous report (35). Together, these results strongly argue ings by showing strong growth inhibition of CXCL14 and CCL20 for a function of CXCL14 in healthy human skin and underscore E. coli S. coag.neg. B the distinct expression profile that separates CXCL14 from other against and spp. (Fig. 2 ). HBD2 was bacte- skin chemokines and AMPs. riostatic against E. coli, but not against S. coag.neg. spp. (Fig. 2B; data not shown). CXCL14 kills Gram-negative and Gram-positive bacteria Next, we examined the effect of CXCL14 on the growth of and C. albicans primary isolates of both oxacillin-resistant and oxacillin-sensitive CXCL14 is not only highly expressed in skin but also in other strains of S. coag.neg. spp. and S. aureus. Radial diffusion assays using two different chemokine concentrations (15 and 30 ␮M) epithelial tissues with barrier functions such as gut or kidney (7). by guest on September 24, 2021 This substantial and epithelial tissue-selective expression raised demonstrated that growth inhibition by CXCL14 was largely in- the question whether CXCL14 may display antimicrobial activity dependent of the state of antibiotic resistance (Fig. 3). We noted a and thereby contribute to the prevention of local bacterial infec- slightly lower (although statistically insignificant) sensitivity in tions. The antimicrobial effect of CXCL14 was investigated with oxacillin-resistant isolates. Collectively, our findings identify Gram-negative and Gram-positive bacterial species including typ- CXCL14 as a highly potent AMP with broad-spectrum activity ical commensals of the skin and pathogens colonizing the skin and that includes clinical isolates of Gram-negative and Gram-positive the mucosa as well as the fungus C. albicans by two distinct meth- bacteria.

FIGURE 3. Antimicrobial activity of CXCL14 against oxacillin-susceptible and oxacillin-resistant S. coag.neg. spp. and S. aureus. The antimicrobial activity of 30 ␮M (filled column) and 15 ␮M (open column) CXCL14 against oxacillin-sensitive (s1–s5) and oxacillin-resistant (r1–r5) strains of S. coag.neg. spp. (left panel) and S. aureus isolates (right panel) was tested by the RDU assay. Representative results for each bacterial strain of two experiments are shown. The Journal of Immunology 511 Downloaded from http://www.jimmunol.org/

FIGURE 4. Robust antimicrobial activity of CXCL14 in increased so- dium chloride concentrations. Bacteriostatic activities of 0.53 ␮M CXCL14, 0.62 ␮M CCL20, and 2.33 ␮M HBD2 (corresponding to 5 ␮g/ml CXCL14 and CCL20 or 10 ␮g/ml HBD2) in the presence of 10, 50, and 100 mM sodium chloride were determined with the colony-forming p Ͻ 0.05 by Student’s t ,ء .assay as described in Materials and Methods test. by guest on September 24, 2021

CXCL14 shows antimicrobial activity under physiological conditions Human skin harbors a slightly acidic pH (Ͻ7.0) and a variable salt milieu (36). The antimicrobial activity of CCL20 (23, 25) and HBD2 (37) is greatly reduced or absent at physiological salt con- FIGURE 5. CXCL14 in culture supernatants of epidermal cell lines ex- centrations (150 mM NaCl). We then compared the salt sensitivity hibits strong antimicrobial activity. A, Culture supernatants of CXCL14-trans- of CXCL14 with CCL20 and HBD2 at increased NaCl concentra- fected Tu138 cells reveal strong antimicrobial activity against E. coli (filled columns) and S. coag.neg. spp. (open columns) as determined by RDU assay. tions but at levels where antimicrobial activity was not completely p Ͻ 0.001). B, Antimicrobial ,ءءء ;Data Ϯ SEM of triplicate experiments abrogated. Using the CFU assay as described for Fig. 2B, our activity of synthetic CXCL14 (5 ␮M) for E. coli was neutralized by anti- results show that CXCL14 inhibited the growth of E. coli by 65% CXCL14 Ab (Mab866) but not by IgG isotype control Ab. C, The antimicro- at 100 mM NaCl (Fig. 4). Growth of S. coag.neg. spp. was less bial activity of diluted culture supernatants of CXCL14-transfected Tu138 inhibited, but still 70 and 45% inhibition were seen at 50 and 100 cells was mainly due to CXCL14 as assessed for E. coli (filled columns) and mM NaCl, respectively. These experiments show that CXCL14 is S. coag.neg. spp. (open columns) with either Mab866 or control Ab. Respec- less salt sensitive than CCL20 and HBD2. We also tested the in- tive antimicrobial activity of synthetic CXCL14 (B) and CXCL14-Tu138 cul- hibition of E. coli growth by CXCL14, CCL20, and HBD2 at pH ture supernatants (C) was taken as 100%, and inhibition by anti-CXCL14 or 5.5, but no significant effect was observed (data not shown). IgG isotype Abs was calculated using the log-linear relationship between zone Thus far, all antimicrobial experiments were conducted with diameter and peptide concentration (B) or the difference between zone diam- ;p Ͻ 0.05 ,ء .(eters (C) (mean percentage Ϯ SEM of triplicate experiments synthetic CXCL14. To clarify whether natural, cell-derived .p Ͻ 0.001; revealed by Student’s t test ,ءءء CXCL14 also had AMP activity, we investigated the culture su- pernatants of squamous cell lines that were transfected with CXCL14-encoding plasmids. Although keratinocytes in human displayed strong antimicrobial activity against E. coli and S. co- skin are a rich source of CXCL14 (Fig. 1A), production of this ag.neg. spp., whereas supernatants from control cultures (parental chemokine in primary cultures of keratinocytes (or related cell cells or vector-only transfected cells) were considerably less active lines) is moderate to nondetectable (6–9). A CXCL14-transfected (Fig. 5A). The residual activity was probably due to alternative human squamous carcinoma-derived cell line (Tu-138) secreted AMPs. To assign the observed inhibition of bacterial growth to 4–5 ng/ml CXCL14 into the culture medium, whereas this che- CXCL14, we included CXCL14 neutralizing and control Abs in mokine was below detection in supernatants of parental cells. Of our assays. In control experiments with synthetic CXCL14, addi- note, culture supernatants of CXCL14-transfected Tu-138 cells tion of anti-CXCL14 Abs reduced the antimicrobial activity by 512 CXCL14 DISPLAYS POTENT ANTIMICROBIAL ACTIVITY Downloaded from

FIGURE 6. Potent antimicrobial activity of CXCL14 produced by murine epidermis-derived cell lines. A, The antimicrobial activity of culture super- natants from the murine CXCL14-transfected or the vector-transfected parental BALB/c keratinocyte cell line PamLy were tested in the RDU assay (left

panel). Data are representative of three experiments. The contribution of secreted CXCL14 to the antimicrobial activity was determined by the addition of http://www.jimmunol.org/ either anti-CXCL14 Ab (Mab866) or isotype control Ab (right panel); antimicrobial activity of diluted CXCL14-transfected PamLy culture supernatants was assumed as 100% and inhibition by Ab or IgG isotype control calculated as described in Fig. 5C. A representative experiment is shown. ***, p Ͻ 0.001. B, Exactly as in A except that culture supernatants of the murine CXCL14-transfected squamous carcinoma cell line SCC7 were examined. *, p Ͻ 0.05.

88%, whereas the control Ab had no effect (Fig. 5B). Importantly, study. In the present study we report that the antimicrobial potency the same anti-CXCL14 treatment reduced RDU values in culture of CXCL14 compares favorably to that of known AMPs such as supernatants of CXCL14-transfected Tu-138 cells to the level of HBD2 or the chemokine CCL20. We further show that CXCL14 is the background activity seen with supernatants of control cells a constitutive chemokine with exceptionally high-level expression by guest on September 24, 2021 (Fig. 5C). Due to considerable variation in the RDU values ob- in healthy human skin, an organ that is constantly exposed to en- tained with cell culture supernatants from different experiments, vironmental microbes (18). In fact, this abundance allowed us to the antimicrobial activity was expressed as the percentage of re- isolate the natural form of CXCL14 and to confirm that its struc- duction in the RDU values in the presence of anti-CXCL14 or ture and function are identical with those of the synthetic form we control Abs as compared with untreated cell culture supernatants routinely use in our experiments (9). We also find that the expres- (100% antimicrobial activity). Similar results were obtained with sion of CXCL14 mRNA and protein within human skin is sub- CXCL14 DNA-transfected murine squamous carcinoma (SCC7) stantially inhibited by inflammatory stimuli. This finding is re- or immortalized BALB/c keratinocytes (PamLy) cell lines (Fig. 6). markable because inflamed skin (like other diseased tissues) is a This is not surprising, because human and murine CXCL14 display rich source of both chemokines that participate in controlling the very close structural homology (12). Collectively, our data dem- recruitment and localization of immune cells (3) and AMPs that onstrate that native CXCL14 works as well as synthetic CXCL14 support the innate immune system in its fight against infectious in growth inhibition of bacteria. Resistance to neutralization by microbes. Inhibition of chemokine expression by inflammatory several factors, including cell-associated and serum proteases, che- stimuli has not been reported for any other chemokine and points mokine-binding proteins, and exposure to prolonged tissue culture to a role for CXCL14 in homeostatic as opposed to inflammation- suggest that natural CXCL14 in healthy skin may exist long driven immune processes and may include bacterial killing at early enough to provide protection against infection by local microbes stages of infection (before the establishment of inflammatory con- under steady-state conditions. ditions; see below). Previously characterized chemokines with bactericidal activity Discussion include CCL20, which is abundantly expressed in inflamed human Our study provides compelling evidence of a role for CXCL14 in epidermis and has broad-spectrum antimicrobial activity for both antimicrobial immunity. This conclusion is drawn from data show- Gram-positive and Gram-negative bacteria (23, 25). Interestingly, ing that synthetic and cell culture supernatant-derived CXCL14 is CCL20 shares with ␤-defensins its selectivity for CCR6 (20). highly potent in inhibiting the growth of a variety of microbes that However, it is not clear how the targeting of CCR6ϩ T cells and include species typically associated with the human skin micro- CCR6ϩ DCs is linked with the AMP activity of the CCR6 ligand, flora such as Staphylococci species, Propionibacteria, and the fun- especially so because a preference for CCR6ϩ immune cells in gus C. albicans as well as the prototype enterobacterium E. coli. response to infections has not been demonstrated. Many AMP-type Antimicrobial activity of CXCL14 has been reported in a previous chemokines require extremely high concentrations to kill bacteria publication (23) where numerous chemokines including CXCL14 in in vitro assays. These concentrations greatly exceed their opti- were screened for antimicrobial activity against E. coli. The re- mal concentrations for the induction of leukocyte recruitment and ported activity was moderate and was not further examined in that may not be generated in peripheral tissues, thus arguing against The Journal of Immunology 513

FIGURE 7. Model of CXCL14 participation in acute skin infection. CXCL14 is constitutively expressed by keratinocytes (K) in the epidermis and fibroblasts (F) in the dermis. CXCL14 is not found in the healthy stratum corneum (SC or S. corneum) and, therefore, is not involved in the homeostasis of the commensal skin microflora. Instead, this model predicts that CXCL14 kills bacteria that have penetrated into the live part of the skin, for instance as a result of microinjuries. At late stages of infections, after an inflammatory milieu has been established, antimicrobial defense is taken over by inducible AMPs, chemokines, and recruited effector cells. M␾, ; T, T cell; N, neutrophil. Downloaded from a major role in bacterial killing. Collectively, currently known che- flora by favoring skin-typical bacteria and yeast over potentially mokines with AMP activity are formidable chemoattractants for pathogenic species. Similar to the commensals in the gut micro- target cells irrespective of their relevance to infections and are flora, the host may benefit from the cutaneous microflora due to its produced under inflammatory settings, i.e., under conditions where secretion of antibiotics and factors supporting tissue regeneration the sites of infections are dominated by an inflammatory infiltrate. and control of inflammation (40). CXCL14 is abundantly ex-

Defensins are prominent AMPs in human skin and include pressed in the epidermis and dermis of healthy human skin (9); http://www.jimmunol.org/ HBD1, which is constitutively expressed, and HBD2 and HBD3, however, this chemokine is not described in skin wash fluids and which are absent in healthy skin but highly up-regulated under extracts from stratum corneum tissue samples (38) and, thus, is inflammatory conditions (18). Chemokines display structural sim- unlikely to play a major role in the homeostasis of the skin mi- ilarities with defensins, including abundance of cationic residues, croflora. Instead, we propose that CXCL14 fulfills its antimicrobial intramolecular disulfide bonds, and tertiary structure. A prominent function in the very first moment of skin injury when microbes feature of chemokines and defensins is the formation of large, pass the stratum corneum and reach the live part of the epidermis positively charged patches on the surface of the molecule, and it is (or the upper dermis) (Fig. 7). Such microinjuries may result from suggested that the positive charges interact with negatively frequent skin abrasions and superficial cuts that generally remain charged bacterial membrane components such as lipopolysaccha- unnoticed. Due to the location in the skin and the broad-range by guest on September 24, 2021 ride or teichoic acid, leading to permeabilization and subsequent AMP activity, CXCL14 is an ideal candidate for immediate in- death of the bacteria (23, 28). CXCL14 is a highly cationic protein volvement in antimicrobial defense against cutaneous and foreign with an estimated isoelectric point (pI) of 9.9 and a net charge of microbes. In this role, CXCL14 may be supported by HBD1, ϩ13 at pH 7 and displays an amphipathic character, thereby re- which is also produced in the skin under steady-state conditions vealing all of the important physicochemical properties of an (15). HBD1 is also active against E. coli but, in contrast to AMP. However, the most abundant AMP in human skin is pso- CXCL14, appears to be less effective against members of the cu- riasin (38), also known as Ca2ϩ-binding protein S100A7. Keratin- taneous microflora that are also known to cause disease upon tissue ocytes in the outermost layer of human skin, in proximity to the penetration (40). We propose that CXCL14 kills bacteria before stratum corneum and apical parts of hair follicles, are responsible they manage to establish macroscopic foci of infection and inflam- for the strikingly focal expression of this AMP, a characteristic mation. In fact, the expression of CXCL14 is drastically reduced fitting with a role in skin homeostasis as opposed to infection (see under inflammatory conditions, ruling out a role for this AMP in below). The killing activity of psoriasin is attributed in part to its late-stage infections characterized by the participation of innate ability to sequester Zn2ϩ, one of several cofactors that bacteria immune cells, including neutrophils and monocytes (Fig. 5). This require for combating oxidative stress. Two other abundant AMPs stage features the production of a new wave of inducible AMPs, in the stratum corneum, which are proposed to control the local including several defensins, cathelicidins, and chemokines that microflora under homeostatic conditions, are RNase 7 and ly- synergize in the killing of local microbes and in the recruitment of sozyme (18). Our data support a model that features CXCL14 as a antimicrobial immune cells. In our model, CXCL14 plays a unique novel AMP with a distinct function at an early stage of infection part in the antimicrobial defense by acting on microbes at the site (Fig. 7). The microflora in the outermost layer of human skin is of entry well before the mobilization of inflammatory cells and controlled by several factors, including a physical barrier made up mediators. Finally, our model may not be restricted to the skin, by the corneocytes and extracellular matrix of the stratum cor- because CXCL14 is also present in human intestinal and colonic neum, harsh physiochemical conditions (acidic pH and salt and tissue (7, 8). It will be essential to investigate the mechanism(s) by temperature fluctuations), and water deprivation as well as antimi- which CXCL14 is inhibiting bacterial growth. Such studies may crobial proteins (18, 39). The combination of these factors ensures inform about the feasibility of transforming this information into the maintenance of a healthy microflora and, at the same time, much needed alternative antibiotics. provides a defense against skin-extraneous microbes. For instance, psoriasin does not harm the numerous Gram-positive bacterial spe- cies present in healthy human skin but instead is highly efficient in Acknowledgments killing the enterobacterium E. coli (38). In this regard, the antimi- We thank Ursula Ackermann for helping with the microbe cultures and crobial “climate” in healthy human skin supports the local micro- Tomas Ganz for providing anti-HBD2 Abs. 514 CXCL14 DISPLAYS POTENT ANTIMICROBIAL ACTIVITY

Disclosures 21. Cole, A. M., T. Ganz, A. M. Liese, M. D. Burdick, L. Liu, and R. M. Strieter. 2001. Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like The authors have no financial conflict of interest. antimicrobial activity. J. Immunol. 167: 623–627. 22. Krijgsveld, J., S. A. Zaat, J. Meeldijk, P. A. van Veelen, G. Fang, B. Poolman, E. Brandt, J. E. Ehlert, A. J. Kuijpers, G. H. Engbers, et al. 2000. Thrombocidins, References microbicidal proteins from human blood platelets, are C-terminal deletion prod- 1. Bos, J. D., and M. L. Kapsenberg. 1993. The skin immune system: progress in ucts of CXC chemokines. J. Biol. Chem. 275: 20374–20381. cutaneous biology. Immunol. Today 14: 75–78. 23. Yang, D., Q. Chen, D. M. Hoover, P. Staley, K. D. Tucker, J. Lubkowski, and 2. Schaerli, P., and B. Moser. 2005. Chemokines: control of primary and memory J. J. Oppenheim. 2003. Many chemokines including CCL20/MIP-3␣ display an- T-cell traffic. Immunol. Res. 31: 57–74. timicrobial activity. J. Leukocyte Biol. 74: 448–455. 3. Moser, B., M. Wolf, A. Walz, and P. Loetscher. 2004. Chemokines: multiple 24. Hieshima, K., H. Ohtani, M. Shibano, D. Izawa, T. Nakayama, Y. Kawasaki, levels of leukocyte migration control. Trends Immunol. 25: 75–84. F. Shiba, M. Shiota, F. Katou, T. Saito, and O. Yoshie. 2003. CCL28 has dual 4. Schaerli, P., L. Ebert, K. Willimann, A. Blaser, R. S. Roos, P. Loetscher, and roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial B. Moser. 2004. A skin-selective homing mechanism for human immune sur- activity. J. Immunol. 170: 1452–1461. veillance T cells. J. Exp. Med. 199: 1265–1275. 25. Starner, T. D., C. K. Barker, H. P. Jia, Y. Kang, and P. B. McCray, Jr. 2003. 5. Ebert, L. M., S. Meuter, and B. Moser. 2006. Homing and function of human skin CCL20 is an inducible product of human airway epithelia with innate immune ␥␦ T cells and NK cells: relevance for tumor surveillance. J. Immunol. 176: properties. Am. J. Respir. Cell Mol. Biol. 29: 627–633. 4331–4336. 26. Linge, H. M., M. Collin, P. Nordenfelt, M. Morgelin, M. Malmsten, and 6. Sleeman, M. A., J. K. Fraser, J. G. Murison, S. L. Kelly, R. L. Prestidge, A. Egesten. 2008. The human CXC-chemokine chemotactic protein D. J. Palmer, J. D. Watson, and K. D. Kumble. 2000. B cell- and monocyte- 2 (GCP-2)/CXCL6 possesses membrane disrupting properties and is antibacte- activating chemokine (BMAC), a novel non-ELR ␣-chemokine. Int. Immunol. rial. Antimicrob. Agents Chemother. 52: 2599–2607. 12: 677–689. 27. Gallo, R. L., M. Murakami, T. Ohtake, and M. Zaiou. 2002. Biology and clinical 7. Kurth, I., K. Willimann, P. Schaerli, T. Hunziker, I. Clark-Lewis, and B. Moser. relevance of naturally occurring antimicrobial peptides. J. Allergy Clin. Immunol. 2001. Monocyte selectivity and tissue localization suggests a role for breast and 110: 823–831. kidney-expressed chemokine (BRAK) in macrophage development. J. Exp. Med. 28. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415: 194: 855–861. 389–395. Downloaded from 8. Frederick, M. J., Y. Henderson, X. Xu, M. T. Deavers, A. A. Sahin, H. Wu, 29. Clark-Lewis, I., L. Vo, P. Owen, and J. Anderson. 1997. Chemical synthesis, D. E. Lewis, A. K. El-Naggar, and G. L. Clayman. 2000. In vivo expression of purification, and folding of C-X-C and C-C chemokines. Methods Enzymol. 287: the novel CXC chemokine BRAK in normal and cancerous human tissue. 233–250. Am. J. Pathol. 156: 1937–1950. 30. Liu, T. J., W. W. Zhang, D. L. Taylor, J. A. Roth, H. Goepfert, and 9. Schaerli, P., K. Willimann, L. M. Ebert, A. Walz, and B. Moser. 2005. Cutaneous G. L. Clayman. 1994. Growth suppression of human head and neck cancer cells CXCL14 targets blood precursors to epidermal niches for Langerhans cell dif- by the introduction of a wild-type p53 gene via a recombinant adenovirus. Cancer ferentiation. Immunity 23: 331–342. Res. 54: 3662–3667.

10. Shellenberger, T. D., M. Wang, M. Gujrati, A. Jayakumar, R. M. Strieter, 31. Wu, Z., D. M. Hoover, D. Yang, C. Boulegue, F. Santamaria, J. J. Oppenheim, http://www.jimmunol.org/ M. D. Burdick, C. G. Ioannides, C. L. Efferson, A. K. El-Naggar, D. Roberts, et J. Lubkowski, and W. Lu. 2003. Engineering disulfide bridges to dissect antimi- al. 2004. BRAK/CXCL14 is a potent inhibitor of and a chemotactic crobial and chemotactic activities of human ␤-defensin 3. Proc. Natl. Acad. Sci. factor for immature dendritic cells. Cancer Res. 64: 8262–8270. USA 100: 8880–8885. 11. Shurin, G. V., R. L. Ferris, I. L. Tourkova, L. Perez, A. Lokshin, L. Balkir, 32. Ali, R. S., A. Falconer, M. Ikram, C. E. Bissett, R. Cerio, and A. G. Quinn. 2001. B. Collins, G. S. Chatta, and M. R. Shurin. 2005. Loss of new chemokine Expression of the peptide antibiotics human ␤ defensin-1 and human ␤ defen- CXCL14 in tumor tissue is associated with low infiltration by dendritic cells sin-2 in normal human skin. J. Invest. Dermatol. 117: 106–111. (DC), while restoration of human CXCL14 expression in tumor cells causes 33. Nakayama, T., R. Fujisawa, H. Yamada, T. Horikawa, H. Kawasaki, attraction of DC both in vitro and in vivo. J. Immunol. 174: 5490–5498. K. Hieshima, D. Izawa, S. Fujiie, T. Tezuka, and O. Yoshie. 2001. Inducible 12. Meuter, S., P. Schaerli, R. S. Roos, O. Brandau, M. R. Bosl, U. H. von Andrian, expression of a CC chemokine liver- and activation-regulated chemokine and B. Moser. 2007. Murine CXCL14 is dispensable for dendritic cell function (LARC)/macrophage inflammatory protein (MIP)-3 ␣/CCL20 by epidermal ker- and localization within peripheral tissues. Mol. Cell. Biol. 27: 983–992. atinocytes and its role in atopic dermatitis. Int. Immunol. 13: 95–103.

13. Braff, M. H., A. Bardan, V. Nizet, and R. L. Gallo. 2005. Cutaneous defense 34. Ong, P. Y., T. Ohtake, C. Brandt, I. Strickland, M. Boguniewicz, T. Ganz, by guest on September 24, 2021 mechanisms by antimicrobial peptides. J. Invest. Dermatol. 125: 9–13. R. L. Gallo, and D. Y. Leung. 2002. Endogenous antimicrobial peptides and skin 14. Brown, K. L., and R. E. Hancock. 2006. Cationic host defense (antimicrobial) infections in atopic dermatitis. N. Engl. J. Med. 347: 1151–1160. peptides. Curr. Opin. Immunol. 18: 24–30. 35. Schauber, J., R. A. Dorschner, K. Yamasaki, B. Brouha, and R. L. Gallo. 2006. 15. Ganz, T. 2003. Defensins: antimicrobial peptides of innate immunity. Nat. Rev. Control of the innate epithelial antimicrobial response is cell-type specific and Immunol. 3: 710–720. dependent on relevant microenvironmental stimuli. Immunology 118: 509–519. 16. Izadpanah, A., and R. L. Gallo. 2005. Antimicrobial peptides. J. Am. Acad. Der- 36. Schittek, B., R. Hipfel, B. Sauer, J. Bauer, H. Kalbacher, S. Stevanovic, matol. 52: 381–390. M. Schirle, K. Schroeder, N. Blin, F. Meier, et al. 2001. Dermcidin: a novel 17. Niyonsaba, F., and H. Ogawa. 2005. Protective roles of the skin against infection: human antibiotic peptide secreted by sweat glands. Nat. Immunol. 2: 1133–1137. implication of naturally occurring human antimicrobial agents ␤-defensins, 37. Singh, P. K., H. P. Jia, K. Wiles, J. Hesselberth, L. Liu, B. A. Conway, cathelicidin LL-37 and lysozyme. J. Dermatol. Sci. 40: 157–168. E. P. Greenberg, E. V. Valore, M. J. Welsh, T. Ganz, et al. 1998. Production of 18. Schroder, J. M., and J. Harder. 2006. Antimicrobial skin peptides and proteins. ␤-defensins by human airway epithelia. Proc. Natl. Acad. Sci. USA 95: Cell Mol. Life Sci. 63: 469–486. 14961–14966. 19. Yang, D., A. Biragyn, L. W. Kwak, and J. J. Oppenheim. 2002. Mammalian 38. Glaser, R., J. Harder, H. Lange, J. Bartels, E. Christophers, and J. M. Schroder. defensins in immunity: more than just microbicidal. Trends Immunol. 23: 2005. Antimicrobial psoriasin (S100A7) protects human skin from Escherichia 291–296. coli infection. Nat. Immunol. 6: 57–64. 20. Hoover, D. M., C. Boulegue, D. Yang, J. J. Oppenheim, K. Tucker, W. Lu, and 39. Elias, P. M. 2007. The skin barrier as an innate immune element. Semin. Immu- J. Lubkowski. 2002. The structure of human macrophage inflammatory pro- nopathol. 29: 3–14. tein-3␣ /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding 40. Cogen, A. L., V. Nizet, and R. L. Gallo. 2008. Skin microbiota: a source of activities with human ␤-defensins. J. Biol. Chem. 277: 37647–37654. disease or defense? Br. J. Dermatol. 158: 442–455.