Article

Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages Min Wan,* Anne M. van der Does,† Xiao Tang,* Lennart Lindbom,† Birgitta Agerberth,*,1,2 and Jesper Z. Haeggström*,1,2 Departments of *Medical Biochemistry and Biophysics, Division of Physiological Chemistry 2, and †Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden RECEIVED MAY 30, 2013; REVISED JANUARY 19, 2014; ACCEPTED JANUARY 27, 2014. DOI: 10.1189/jlb.0513304

ABSTRACT

Introduction

LL-37/hCAP-18 is the only human member of the cathelicidin family and plays an important role in killing various pathogens, as well as in immune modulation. In this study, we investigated the effect of LL-37 on bacterial phagocytosis by macrophages and demonstrate that LL-37 enhances phagocytosis of IgG-opsonized Gramnegative and Gram-positive bacteria in a dose- and time-dependent manner by dTHP-1 cells. In addition, LL-37 enhanced phagocytosis of nonopsonized Escherichia coli by human macrophages. Consistently, LL-37 elevated the expression of Fc␥Rs on macrophages but not the complement receptors CD11b and -c. Further studies revealed that the expression of TLR4 and CD14 is also increased on LL-37-treated macrophages. Several lines of evidence indicated that the FPR2/ALX receptor mediated LL-37-induced phagocytosis. However, TLR4 signaling was also coupled to the phagocytic response, as a specific TLR4 antibody significantly suppressed phagocytosis of IgG-opsonized E. coli and nonopsonized E. coli by dTHP-1 cells. Finally, macrophages from Cnlp⫺/⫺ mice exhibited diminished bacterial phagocytosis compared with macrophages from their WT littermates. In conclusion, we demonstrate a novel, immune-modulatory mechanism of LL-37, which may contribute to bacterial clearance. J. Leukoc. Biol. 95: 971–981; 2014.

Macrophages play a central role in host defense against infection via their recognition, phagocytic capacities, and killing of microbes. Efficient uptake and phagocytosis of bacteria by macrophages are achieved by opsonization of pathogens with Ig or complement proteins. Particles opsonized with IgG are recognized by Fc␥Rs, which are mainly expressed on phagocytic leukocytes [1, 2]. There are three major classes of Fc␥Rs, designated Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16) [1]. AMPs, or host-defense peptides, are important molecules in host defense against pathogenic microbes [3]. Mammals express a variety of small, amphipathic AMPs, including defensins and cathelicidins. The only endogenous cathelicidin in humans, LL-37/hCAP-18, is expressed by various cell types, such as different epithelial cells [4, 5], keratinocytes [6], and specific leukocyte subsets [7]. LL-37 is also present at high concentrations as the inactive proform hCAP-18 in the secondary granules of neutrophils [8]. Once hCAP-18 is secreted from neutrophils, it is processed into the active LL-37 peptide by proteinase 3 that is present in the primary granules of these cells [9, 10]. Besides a broad range of direct antimicrobial activities, LL-37 also exhibits functions important in immune responses and inflammation. For example, LL-37 serves as a chemoattractant for neutrophils, monocytes, and T cells [11] and promotes IL-8 and IL-1␤ release by monocytes [12, 13]. In addition, LL-37 modulates the differentiation of DCs [14] and macrophages [15]. Previously, we reported that LL-37 elevated bacterial phagocytosis by human neutrophils [16]. Moreover, it has been also demonstrated that neutrophil-derived HBP and HNP-1–3 boost bacterial phagocytosis by macrophages [17]. Here, we report that LL-37 stimulates bacterial phagocytosis by human macrophages and that this response is mediated via FPR2/ALX.

Abbreviations: ALX ⫽ Lipoxin A4 receptor, AMP⫽antimicrobial peptide, APC⫽allophycocyanin, CF⫽cystic fibrosis, Cnlp⫺/⫺⫽cathelicidin-deficient, CRAMP⫽cathelicidin-related antimicrobial peptide, dTHP-1⫽differentiated THP-1 cells, FPR2⫽formyl peptide receptor 2, Gr-1⫽granulocyte receptor-1, HBP⫽heparin-binding protein, hCAP-18⫽human cationic antimicrobial protein, HMDM⫽human monocyte-derived macrophage, HNP-1–3⫽human neutrophil peptide 1–3, LTB4⫽leukotriene B4, LXA4⫽lipid mediator lipoxin A4, mCRAMP⫽murine cathelicidin-related antimicrobial peptide, P2X7⫽P2X purinoceptor 7, PMN⫽polymorphonuclear leukocyte, RT⫽room temperature, sLL-37⫽sequence-scrambled LL-37 The online version of this paper, found at www.jleukbio.org, includes supplemental information.

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1. These authors contributed equally to this work. 2. Correspondence: Dept. of Medical Biochemistry and Biophysics, Division of Physiological Chemistry 2, Karolinska Institutet, S-171 77, Stockholm, Sweden. E-mail: [email protected] (B.A.) or [email protected] (J.Z.H.)

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MATERIALS AND METHODS

parallel wells were measured for each treatment, and the results were calculated from three to four independent experiments.

Reagents Fluorescein-conjugated Staphylococcus aureus and E. coli, opsonizing reagents, FITC-conjugated CD32, and FITC-conjugated CD64 antibodies were purchased from Life Technologies (Paisley, UK), and FITC-conjugated CD16, PE-conjugated CD11b, and APC-conjugated CD206 antibodies were from Miltenyi Biotec (Bergisch Gladbach, Germany). FITC-conjugated CD11c; FITC-conjugated antibodies against human CD14, CD54, CD68, and CD163 and against murine CD14; and Fc␥Rs (Fc␥RIII, CD16; Fc␥R II, CD32) were from BD Biosciences (Heidelberg, Germany). PMA, 2-ME, KN62, trypan blue, RIPA buffer, and RPMI medium were from Sigma (St. Louis, MO, USA); AG-1478 was from Cayman Chemical (Ann Arbor, MI, USA); TLR2 and TLR4 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and synthetic LL-37 (NH2-LLGDFFRKSKEKIGKEFKRIVQRIKDFFRNLVPRTES-COOH), synthetic HNP-1 (NH2-ACYCRIPACIAGERRYGTCIYQGRLWAFCC-COOH), specific FPR2 antagonist peptide WRW4 (WRWWWW-NH2), and specific FPR2 agonist peptide Wpeptide (WKYMVm-NH2) were obtained from Innovagen AB (Lund, Sweden). The endotoxin content in all peptides was detected by Limulus amoebocyte lysate assay (Charles River Laboratories, Wilmingon, MA, USA) and was found to contain ⬍0.0012 EU/mg endotoxin. HBP was a kind gift from Hans Flodgaard (Bartholin Instituttet XPU, Biocenter Copenhagen, Denmark). Murine rTNF-␣ was purchased from PeproTech (Rocky Hill, NJ, USA).

Intracellular killing assay dTHP-1 cells (1⫻106) were incubated with 1 or 10 ␮g/ml LL-37 for 24 h and then washed three times with PBS. Afterwards, cells were incubated with equal numbers of E. coli strain D21 or S. aureus strain B5381 without opsonization at 37°C under shaking. Immediately and after 2 h, macrophages were lysed using ice-cold water and vortexed for 30 s. Thereafter, the lysates were serially diluted and plated onto agar plates. The following day, viable bacteria were counted, and the percentage of surviving bacteria was determined.

Immunocytochemistry THP-1 cells were differentiated in 96-well black plates (BD Biosciences Heidelberg, Germany) or in the Lab-Tek II Chamber Slide system (Thermo Fisher scientific, Rochester, NY, USA). After stimulation with 1 or 10 ␮g/ml LL-37 for 8 or 24 h, cells were washed three times with PBS. Thereafter, the cells were incubated with FITC-CD16, FITC-CD32, FITC-CD64, or FITC-CD14 antibodies for 30 min at RT. After washing, cells were analyzed for fluorescence intensity by a fluorometer (BMG Labtech), or cells were mounted on slides using an antifading mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscopy (Olympus FV1000; Olympus, Tokyo, Japan).

Cell culture The THP-1 cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640, supplemented with 10% heat-inactivated FBS, HEPES (25 mM), 2-ME (0.05 mM), penicillin (100 U/ml), and streptomycin (100 ␮g/ml). THP-1 cell suspension was maintained in 0.3– 0.8 ⫻ 106 cells/ml, and cell differentiation was induced by PMA (10 ng/ml) for 48 h. Human mononuclear cells were isolated from freshly prepared buffy coats (Karolinska Hospital Blood Bank, Stockholm, Sweden) by gradient centrifugation on Ficoll-Paque Premium (GE Healthcare Bio-Sciences AB, Sweden). Differentiation of human monocytes to macrophages was achieved by plating mononuclear cells in cell-culture plates for 2 h, and then unbound cells were washed away with PBS, followed by cell culture over 7 days in RPMI 1640, supplemented with 10% autologous human serum (heat-inactivated), 25 mM HEPES, penicillin (100 U/ml), and streptomycin (100 ␮g/ml).

Phagocytosis assay Fluorescence-labeled dead S. aureus and E. coli were reconstituted in opsonizing reagents. IgG opsonization was performed, according to the manufacturer’s instructions (Life Technologies). Briefly, bacteria and the corresponding opsonization IgG were incubated at 37°C for 1 h. Before adding to cells, the mixture of bacteria and IgG antibodies was washed three times with PBS. Complement opsonization was attained by incubation of bacteria with fresh human serum for 1 h at 37°C. PMAdTHP-1 cells or human primary macrophages were incubated with LL-37 at different concentrations for several intervals, followed by washing three times with PBS. Thereafter, opsonized or nonopsonized bacteria in RPMI-1640 medium were added at a ratio of 20 bacteria/cell for 1 h at 37°C. Afterwards, fluorescence from redundant extracellular bacteria was quenched by trypan blue for 1 min, and the mean fluorescence inside the cells was measured by POLARstar fluorometer (BMG Labtech, Offenburg, Germany). The wells containing only fluorescent bacteria were set as negative controls, whereas the wells containing cells without LL-37 treatment were set as baseline controls. Phagocytosis (%) ⫽ (fluorescence in the experimental well⫺fluorescence in the negative control well)/(fluorescence in the baseline control well⫺fluorescence in the negative control well) ⫻ 100%. In each experiment, six

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Flow cytometric analysis dTHP-1 cells (1⫻106) were stimulated with 1 or 10 ␮g/ml LL-37 for 8 or 24 h. After washing three times with PBS, the cells were collected and incubated with PE-conjugated CD11b or APC-conjugated CD206 antibodies in PBS with 1% BSA for 15 min at 4°C or with FITC-CD11c antibody in PBS with 1% BSA for 30 min at RT. After washing, the fluorescence intensity of cells was measured by a FACSort (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using BD CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

SDS-PAGE and Western blot analysis After treatments with LL-37 at different concentrations for 8 or 24 h, dTHP-1 cells (2⫻106) were lysed with RIPA buffer together with a complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Samples with equal amounts of protein were resolved by SDSPAGE using NuPAGE Novex 4 –12% Bis-Tris gels (Life Technologies, Carlsbad, CA, USA) and electroblotted onto a nitrocellulose transfer membrane, according to the manufacturer’s instructions (Life Technologies). Membranes were soaked for 2 h in 0.05% TTBS (20 mM Tris-HCl, pH 7.5, with 0.5 M NaCl and 0.05% Tween 20) containing 3% fat-free dry milk and incubated overnight at 4°C with primary antibodies against human TLR2 or TLR4. Immunoreactivity was detected with a secondary anti-mouse or antirabbit antibody conjugated with HRP (GE Healthcare, Uppsala, Sweden). Immunoreactive bands were visualized with the ECLWestern blotting detection system (GE Healthcare).

Animals WT and Cnlp⫺/⫺ mice on a C57BL/6J background (obtained from Dr. Oliver Soehnlein Institute for Cardiovascular Prevention, Ludwig-MaximiliansUniversity, Munich , Germany, by courtesy of Dr. Richard L. Gallo, Division of Dermatology, UCSD, La Jolla, California, USA.) were kept in animal housing with constant temperature, humidity, 12-h light-dark cycles, and ad libitum food and water. All mice are male and age-matched (9 –12 weeks). Cnlp⫺/⫺ mice are deficient in CRAMP, the murine homologue of LL-37. The original phenotype of Cnlp⫺/⫺ mice is described in the previous

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Wan et al. LL-37 enhances bacterial clearance by macrophages publication [18]. All experiments were approved by the Regional Ethical Committee for Animal Experimentation.

LL-37 for 24 h, although the alterations of intracellular killing of E. coli did not reach statistical significance (Fig. 1D).

s.c. Air pouch

LL-37 enhances phagocytosis of IgG-opsonized bacteria by human macrophages in a dose- and time-dependent manner

As.c. air pouch was created as described previously [19]. In brief, at Days 0 and 3, sterile air (4 ml) was injected s.c. in the back of the mouse. Cell recruitment was induced at Day 6 by injection of murine TNF-␣ (50 ng) in 100 ␮l PBS into the pouch. After 24 h, the air pouch was lavaged with 3 ml PBS. A small portion of harvested cells was resuspended in PBS with 0.5% BSA and stained with anti-Gr-1 (BioLegend, San Diego, CA USA) and antiF4/80 (AbD Serotec, Düsseldorf, Germany) to discriminate among neutrophils, monocytes, and macrophages. Cell-surface molecule expression was assessed on a FACSort and analyzed with BD CellQuest software to determine the percentage of neutrophils (Gr-1⫹), monocytes (Gr-1⫹ and F4/ 80⫹), and macrophages (F4/80⫹) in the recruited cell population. The remaining cells were resuspended in RPMI-1640 medium, supplemented with 25 mM HEPES (Life Technologies). The same number of macrophages was seeded into 96-well culture plates, and unattached cells were washed away after incubation for 2 h at 37°C. Afterwards, the phagocytosis assay was carried out as described above.

Statistical analysis Results are presented as mean ⫾ sd. Differences between the means were evaluated using one-way ANOVA or Student’s t-test. A value of P ⬍ 0.05 was considered statistically significant.

RESULTS

LL-37 enhances bacterial clearance by dTHP-1 cells We analyzed the effects of LL-37 on phagocytosis of IgG-opsonized, complement-opsonized, and nonopsonized bacteria by dTHP-1 cells. Phagocytosis of IgG-opsonized E. coli and S. aureus was enhanced significantly by preincubation of dTHP-1 cells with LL-37 (Fig. 1A). However, LL-37 treatment did not alter the phagocytosis of complement-opsonized bacteria by dTHP-1 cells (Fig. 1B). Interestingly, LL-37 selectively enhanced phagocytosis of nonopsonized E. coli but not nonopsonized S. aureus (Fig. 1C). Furthermore, intracellular killing of E. coli and S. aureus by dTHP-1 cells was augmented after incubation with 1 or 10 ␮g/ml

In the following experiments, we focused on LL-37-enhanced phagocytosis of IgG-opsonized bacteria. Our results showed that incubation of dTHP-1 cells with LL-37 up to 20 ␮g/ml for 8 h enhanced the phagocytic capacity of IgG-opsonized S. aureus and E. coli with an optimal concentration of LL-37 at 1 and 10 ␮g/ml, respectively (Fig. 2A). At the optimal dose, LL-37 was equipotent to 10 nM LTB4 on enhancing phagocytosis of IgG-opsonized S. aureus. At a higher concentration of LL-37 (50 ␮g/ml), no effects on phagocytosis were observed (Fig. 2A). Although LL-37 has been claimed to exert cytotoxic effects on eukaryotic cells at high concentrations, we could not detect any detrimental effect of LL-37 up to 50 ␮g/ml on dTHP-1 viability (Supplemental Fig. 1), which excludes the possibility that lower phagocytosis-inducing activity of LL-37 at high doses is caused by cytotoxic effects of LL-37 on the cells. The phagocytic response of dTHP-1 cells at 1 ␮g/ml LL-37 peaked after 8 h, and no further increase was observed at later time-points up to 24 h (Fig. 2B). Under the same conditions, HMDMs also displayed enhanced phagocytosis of IgGopsonized E. coli and S. aureus, with the most significant effects on phagocytosis of IgG-opsonized E. coli (Fig. 2C). In addition, sLL-37 cannot induce bacterial phagocytosis by dTHP-1 cells compared with LL-37 (Fig. 2D). It has been reported that ␣-defensins (HNP-1–3) and HBP, two PMN-derived granule proteins, enhance bacterial phagocytosis by human macrophages [17]. LL-37 was found to be essentially equipotent as these two polypeptides with respect to their ability to increase bacterial phagocytosis by dTHP-1 cells (Fig. 2E). Combinations of these three peptides were more effective than each of them separately (Fig. 2E). Thus, the combinations, LL-37/HBP, LL-37/HNP-1, and LL-37/HNP-1/ HBP, were found to be most effective.

Figure 1. LL-37 enhances phagocytosis of bacteria by human macrophages. THP-1 cells (2⫻105) were differentiated by PMA in 96-well plates. Afterwards, dTHP-1 cells were incubated with 1 ␮g/ml LL-37 for 8 h, followed by washing with PBS three times. Fluorescent E. coli and S. aureus, opsonized with IgG (A), complement (B), or without (w/o) opsonization (C), were added to the wells at a ratio of 20 bacteria/cell for 1 h at 37°C. Ctrl, Control. Phagocytosis was measured and calculated as described in Materials and Methods. The results are from four separate experiments. (D) dTHP-1 cells (1⫻106) were treated with 1 ␮g/ml LL-37 for 24 h. Cells were washed three times with PBS, and 1 ⫻ 106 of live bacteria (E. coli strain D21 or S. aureus strain B5381), without opsonization, was added. After 2 h of incubation, macrophages were lysed using ice-cold water, and intracellular killing of E. coli (n⫽4) and S. aureus (n⫽7) was assessed, as described in Materials and Methods. ***P ⬍ 0.001, **P ⬍ 0.01, *P ⬍ 0.05.

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Figure 2. LL-37 elevates phagocytosis of IgG-opsonized bacteria by human macrophages. dTHP-1 cells (2⫻105) or HMDMs were cultured in 96-well plates. (A) dTHP-1 cells were treated with LL-37 at different concentrations (0 –50 ␮g/ml) for 8 h. Separately, dTHP-1 cells were treated with 10 nM LTB4 for 30 min as a positive control. After cells were washed three times with PBS, phagocytosis of IgG-opsonized E. coli (〫) or IgG-opsonized S. aureus () was analyzed (n⫽3). (B) dTHP-1 cells were incubated with 1 ␮g/ml LL-37 for different times (0 –24 h), and phagocytosis of added IgG-opsonized S. aureus was analyzed (n⫽4). C) Human primary monocyte-derived macrophages were treated with 1 ␮g/ml LL-37 or PBS (Ctrl) for 8 h. After washing of cells with PBS three times, phagocytosis of IgG-opsonized bacteria was measured (n⫽3). (D) dTHP-1 cells were incubated with 1 or 10 ␮g/ml LL-37 or sLL-37 for 8 h, and phagocytosis of IgG-opsonized S. aureus was analyzed (n⫽4). (E) dTHP-1 cells were treated separately with LL-37 (1 ␮g/ ml; ⬃0.2 ␮M), HNP-1 (0.5 ␮g/ml; ⬃0.2 ␮M), HBP (1 ␮g/ml; ⬃0.03 ␮M), or combinations of two or three polypeptides for 8 h, and phagocytosis of IgG-opsonized S. aureus by polypeptide-treated THP-1 cells was analyzed (n⫽3). HNP-1 (also called ␣-defensin-1); HBP (also called cationic antimicrobial protein of 37 kd or azurocidin). ***P ⬍ 0.001, **P ⬍ 0.01, *P ⬍ 0.05, NS: non significance.

LL-37 enhances the expression of CD32 and CD64 on human macrophages We next analyzed the expression of three major Fc␥Rs (CD16, CD32, and CD64) on the surface of macrophages, before and 974 Journal of Leukocyte Biology

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after LL-37 treatments. Our results showed that dTHP-1 cells expressed high levels of CD32 and CD64, whereas CD16 expression was in a very low level (data not shown). After dTHP-1 cells were incubated with 1 ␮g/ml LL-37 for 8 h,

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Wan et al. LL-37 enhances bacterial clearance by macrophages

CD64 expression on the cell surface was increased significantly compared with control cells (Fig. 3A and B). However, after 24 h, CD64 expression had declined; instead, the expression of CD32 was elevated (Fig. 3A and B). When HMDMs were treated with 1 ␮g/ml LL-37 for 24 h, the expression of CD32 and CD64 was elevated as well (Supplemental Fig. 2). In concordance, it was detected that a CD64 antibody blocked LL-37induced bacterial phagocytosis (Fig. 3C), which further confirmed the involvement of Fc␥R in LL-37-induced phagocytosis of IgG-opsonized bacteria. It is noteworthy that only a part of LL-37-treated dTHP-1 cells exhibited elevated CD64 and CD32 expression, as detected by confocal microscopy (Fig. 3B). We believe that it this is a result of heterogeneous dTHP-1, an assumption supported by the fact that we could observe enhanced expression of CD32 and CD64 in most of LL-37treated HMDMs (data not shown). Moreover, we also analyzed the expression of some molecules on LL-37-treated dTHP-1 cells related to activation and phagocytosis of macrophages. As the results showed (Fig. 3C), among these molecules, only CD68 expression was elevated in LL-37-treated dTHP-1. Notably, the complement receptor CD11b is constitutively expressed by dTHP-1 cells (Fig. 3D). However, no alterations of the expression of CD11b, CD11c, CD16, or the mannose receptor CD206 were detected upon treatment of dTHP-1 cells with LL-37 (Fig. 3D).

LL-37 modulates the expression of TLR4 and CD14 in dTHP-1 cells We evaluated the expression of TLR2 and TLR4 in dTHP-1 cells treated with LL-37. We found elevated protein expression of TLR4 rather than TLR2 in LL-37-treated dTHP-1 cells in a dose-dependent manner, both at 8 and 24 h (Fig. 4A). CD14 is involved in recognizing LPS, the component of the outer membrane of Gram- bacteria [20]. Accordingly, we found that protein expression of CD14 on the surface of dTHP-1 cells is also augmented by LL-37 treatment (Fig. 4B and C). Similar results of TLR4 and CD14 expression were also detected in LL-37-treated HMDMs (Fig. 4D and E). Interestingly, the phagocytic capacity against IgG-opsonized and nonopsonized E. coli was suppressed significantly by blocking TLR4 on dTHP-1 cells with a specific antibody, whereas no effect on phagocytosis of IgG-opsonized S. aureus was detected (Fig. 4F), indicating the involvement of TLR4 in phagocytosis of E. coli by macrophages. Furthermore, blocking TLR4 also displayed significant influence on LL-37-induced phagocytosis of E. coli by dTHP-1 cells (Fig. 4F).

FPR2/ALX mediates LL-37-up-regulated bacterial phagocytosis by dTHP-1 cells It has been reported that various immunomodulatory functions of LL-37 are mediated by several receptors, such as FPR2/ALX [11, 21], P2X7 [13], and EGFR [22]. Here, we found that pretreatment of dTHP-1 cells with the FPR2/ALX antagonist peptide WRW4 totally abolishes LL-37-enhanced bacterial phagocytosis of dTHP-1 cells (Fig. 5A), and the GPCR inhibitor pertussis toxin showed a similar effect (Supplemental Fig. 3). In contrast, inhibitors of P2X7 or EGFR had www.jleukbio.org

no effect on LL-37-promoted bacterial phagocytosis (Fig. 5B and C). Enhanced bacterial phagocytosis was also detected after dTHP-1 cells were incubated with the specific FPR2/ALX agonist WKYMVm peptide (Fig. 5D). Finally, WRW4 also blocked LL-37-induced CD32 expression on dTHP-1 cells (Fig. 5E).

Macrophages from Cnlpⴚ/ⴚ mice exhibit suppressed bacterial phagocytosis To obtain evidence for the involvement of LL-37 in regulating bacterial phagocytosis in vivo, Cnlp⫺/⫺ mice (deficient in CRAMP, murine homologue to LL-37) were used. Considering the potential effects of LL-37 (or mCRAMP in mice) on chemotaxis of leukocytes in vivo [23, 24], we induced acute inflammation by TNF-␣ injection into the dorsal s.c. air pouch of WT and Cnlp⫺/⫺ mice, and we collected leukocytes after 24 h and counted the subtypes of leukocytes (⬃15% neutrophils, ⬃10% monocytes, and ⬃60% macrophages). The relative amounts of different subtypes of leukocytes were similar in WT and Cnlp⫺/⫺ mice (data not shown). In our assay, the collected leukocytes were allowed to adhere to culture plates for 2 h, and then, the unbound cells were washed away, and only the bacterial phagocytosis of adherent cells was analyzed. We detected the composition of the adherent cells, in which monocytes and neutrophils accounted for only ⬃5% and ⬃3%, respectively. Therefore, in our assay, it was mainly macrophages to phagocytize bacteria. Bacterial phagocytosis of macrophages from WT and Cnlp⫺/⫺ mice was evaluated, and our result revealed that phagocytosis of IgG-opsonized S. aureus was significantly suppressed in macrophages from Cnlp⫺/⫺ mice compared with WT mice (Fig. 6A). Moreover, the expression of CD14 and Fc␥Rs on leukocytes from Cnlp⫺/⫺ mice was significantly lower than that on corresponding cells from WT mice (Fig. 6B and C).

DISCUSSION In this study, we found that preincubation of human macrophages with LL-37 significantly induced the following bacterial clearance by macrophages. Furthermore, we corroborated our in vitro data using WT and Cnlp⫺/⫺ mice. In WT mice, mCRAMP was detected in air-pouch lavage at 24 h after TNF-␣ injection (Supplemental Fig. 4), indicating that after migration into the cavity of the air pouch, macrophages in WT mice were exposed to mCRAMP. Our results showed that phagocytosis of IgG-opsonized S. aureus by macrophages from Cnlp⫺/⫺ mice was significantly lower than that from WT (Fig. 6), which supports the conclusion that cathelicidin is involved in regulating bacterial phagocytosis by human macrophages. The prophagocytic response triggered by LL-37 was observed at low levels of the peptide, whereas LL-37 failed to elevate bacterial phagocytosis at the highest dose (50 ␮g/ml). Our results showed that LL-37, at concentrations up to 50 ␮g/ ml, had no cytotoxic effects on dTHP-1 cells (Supplemental Fig. 1), indicating that lower phagocytosis-inducing activity of LL-37 at higher concentrations was not a result of cytotoxic activity of LL-37 on macrophages. It has been reported that Volume 95, June 2014

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Figure 3. The expression of CD32 and CD64 on the surface of dTHP-1 is regulated by LL-37. dTHP-1 cells were incubated with 1 ␮g/ml LL-37 for 8 or 24 h and washed three times with PBS. (A and B) Cells were incubated with FITC-labeled CD16, CD32, or CD64 antibodies in PBS/1% BSA for 30 min at RT. After washing, fluorescence was detected by POLARstar fluorometry (n⫽4; A) or confocal microscopy (representative of at least three independent experiments; B). Green, CD32 and CD64; blue, DAPI. Bars indicate 10 ␮m. ***P ⬍ 0.001. MFI, Mean fluorescence intensity. (C) dTHP-1 cells (2⫻105) were incubated with 1 ␮g/ml LL-37 or 1 ␮g/ml LL-37 plus an antibody against human CD64 (anti-CD64, 2 mg protein/ml) or isotype control antibody (control antibody, anti-mouse CD4, 2 mg protein/ml) for 8 h, followed by washing three times with PBS. Fluorescent S. aureus opsonized with IgG was added to the wells at a ratio of 20 bacteria/cell for 1 h at 37°C. Phagocytosis was measured (n⫽3). (D) dTHP-1 cells were incubated with 1 ␮g/ml LL-37 for 24 h and washed three times with PBS. Afterwards, cells were incubated with fluorescence-conjugated CD44, CD54, CD68, or CD163 antibodies in PBS/1% BSA for 30 min at RT. After washing, fluorescence was detected by POLARstar fluorometry (n⫽3). (E) Cells were collected and incubated with fluorescently labeled CD11b, CD11c, or CD206 antibodies in PBS/1% BSA for 30 min at RT. After washing with PBS, fluorescence was detected by FACS. Each figure is representative of three independent experiments. isotype ctrl, Cells were incubated with corresponding fluorescence-conjugated isotype control antibody.

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Figure 4. LL-37 augments the expression of TLR4 and CD14 on human macrophages. (A) dTHP-1 cells (1⫻106) were treated with 0, 0.5, 1, 5, or 10 ␮g/ml LL-37 for 8 or 24 h. The cells were lysed in RIPA buffer, and the expression of TLR2 and TLR4 was detected by Western blot analyses. The image is representative of four independent experiments. (B and C) dTHP-1 cells were incubated with 1 or 10 ␮g/ml LL-37 for 8 or 24 h. After washing with PBS three times, cells were incubated with FITC-labeled CD14 in PBS/1% BSA for 30 min at RT. Afterwards, the fluorescence was detected by (B) fluorometry (n⫽3) or (C) confocal microscopy (green, CD14; blue, DAPI; the image is representative of at least three independent experiments; bars indicate 10 ␮m). (D and E) HMDMs were treated with 1 or 10 ␮g/ml LL-37 for 24 h. After washing with PBS three times, (D) the cells were lysed in RIPA buffer, and the expression of TLR2 and TLR4 was detected by Western blot analyses, or (E) cells were incubated with FITC-labeled CD14 in PBS/1% BSA for 30 min at RT. Afterwards, the fluorescence was detected by fluorometry (n⫽3). (F) dTHP-1 cells were treated with 1 ␮g/ml LL-37 or PBS for 24 h. Cells were then incubated with specific anti-TLR4 antibody (2 ␮g/ml protein) or control buffer (0.1% gelatin in PBS) for 30 min at 37°C before adding IgG-opsonized S. aureus, IgG-opsonized E. coli, or nonopsonized E. coli. Phagocytosis was measured as described in Materials and Methods. ***P ⬍ 0.001, **P ⬍ 0.01, *P ⬍ 0.05.

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Figure 5. FPR2/ALX is engaged in LL-37-enhanced bacterial phagocytosis by dTHP-1 cells. (A–C) dTHP-1 cells (2⫻105) in 96-well plates were preincubated with the FPR2/ALX antagonist WRW4 (1 ␮M, n⫽4; A), the P2X7 inhibitor KN-62 (1 ␮M, n⫽4; B), or the EGFR inhibitor AG1478 (1 ␮M, n⫽4; C) for 1 h at 37°C, followed by incubation with 1 ␮g/ml LL-37 for 8 h. (D) dTHP-1 cells were incubated with the specific FPR2/ALX agonist WKYMVm peptide in various concentrations for 8 h at 37°C. After washing with PBS, IgG-opsonized S. aureus was added, and phagocytosis was measured, as described in Materials and Methods. (E) dTHP-1 cells were incubated with LL-37 (1 ␮g/ml), WRW4 (1 ␮M), LL-37 (1 ␮g/ml) plus WRW4 (1 ␮M), or culture medium for 24 h. Afterwards, cells were washed three time with PBS and then incubated with FITC-labeled CD32 antibody in PBS/1% BSA for 30 min at RT. After washing, fluorescence was detected by POLARstar fluorometry. ***P ⬍ 0.001, **P ⬍ 0.01.

LL-37 is produced constitutively and is found at mucosal surfaces and in most body fluids at concentrations of 2–5 ␮g/ml [25]. Furthermore, Byfield et al. [26] reported that the range of LL-37 concentrations in saliva is 0.7–27 ␮g/ml, 0.9 –2.3 ␮g/ml in plasma of healthy individuals, 0.1–3.2 ␮g/ml in cerebrospinal fluid, and 0 –20 ␮g/ml in ascites from patients diagnosed with liver cirrhosis or abdominal cancer. Although LL-37 has a minimal inhibitory concentration ⬍10 ␮g/ml against a variety of common bacteria in media containing low or high ionic strength [27], LL-37, at concentrations as high as 100 ␮g/ml, has little or no antimicrobial activity in tissue culture media [28]. Therefore, the capacity of LL-37 to kill 978 Journal of Leukocyte Biology

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bacteria directly may be limited in vivo; instead, LL-37 exhibits immunomodulatory effects under the same conditions [28]. As we demonstrate in this study, LL-37 promotes bacterial phagocytosis and killing by human macrophages under conditions that may be relevant to normal physiological states and to certain infections. On the other hand, LL-37 has been found at much higher concentrations at sites of chronic inflammation; for example, 30 ␮g/ml LL-37 was detected in the CF lung [29]. However, the accumulated levels of LL-37 may be too high to significantly promote bacterial phagocytosis by macrophages, which may partly explain why CF patients always suffer from chronic bacterial lung infections [30].

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Wan et al. LL-37 enhances bacterial clearance by macrophages

Figure 6. Macrophages from Cnlpⴚ/ⴚ mice exhibit lower phagocytosis of IgG-opsonized S. aureus. TNF-␣ (50 ng) in 100 ␮l PBS was injected into the dorsal air pouch of WT and Cnlp⫺/⫺ mice. Twenty-four hours later, the air pouch was lavaged with 3 ml PBS. The cells in the lavage fluid were separated by centrifugation at 300 g for 10 min. The numbers and composition of the harvested cells were analyzed by FACS. Afterwards, (A) the cells containing 2 ⫻ 105 monocytes/macrophages from WT (n⫽7) and Cnlp⫺/⫺ mice (n⫽6) were seeded into black 96-well plates, and nonadherent cells were washed off after 2 h. Phagocytosis of IgG-opsonized S. aureus was measured as mean fluorescence intensity (MFI), or (B and C) whole leukocytes from WT (n⫽5) and Cnlp⫺/⫺ mice (n⫽5) were incubated with fluorescence-labeled antibody against murine CD14 (B) or murine Fc␥Rs plus fluorescence-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; C). After washing, the MFI of the cells was analyzed by FACS.

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We compared the phagocytic effects of LL-37 on IgG-opsonized, complement-opsonized, and nonopsonized bacteria in dTHP-1 cells. In contrast to IgG-opsonized bacteria, LL-37 treatment did not affect phagocytosis of complement-opsonized bacteria (Fig. 1B), which was consistent with the effects of LL-37 on expression of Fc␥Rs and complement receptors (CD11b and CD11c). However, phagocytosis of nonopsonized E. coli, but not nonopsonized S. aureus, was affected by LL-37 treatment (Fig. 1C). Actually, we also analyzed the expression of some molecules on LL-37-treated dTHP-1 cells related to activation and phagocytosis of macrophages (CD44, CD54, CD68, CD163, and CD206). Among these molecules, only expression of CD68 was elevated in LL-37-treated dTHP-1 cells. CD68 is a human macrophage marker related to lysosomal glycoproteins, and it may be to bind to tissue- and organ-specific lectins or selectins, allowing homing of macrophage subsets to particular sites [31]. Additionally, it was reported that CD68 could be involved in binding and uptake of oxidized LDL in macrophages [32]. Further studies are required to determine whether CD68 or other molecules mediate the direct phagocytosis of nonopsonized E.coli by dTHP-1 cells. There are several reports demonstrating that TLRs are involved in bacterial phagocytosis and intracellular bacterial killing [33–35]. Our results showed that expression of TLR4 and CD14 was up-regulated in LL-37-treated dTHP-1 cells and HMDMs (Fig. 4A–E). Interestingly, protein expression of TLR4 rather than TLR2 was increased in LL-37-treated human macrophages in a dose-dependent manner, which may explain why LL-37 only elevates phagocytosis of nonopsonized E. coli, whereas phagocytosis of nonopsonized S. aureus was unaffected. In our hands, phagocytosis of IgG-opsonized or nonopsonized E. coli by dTHP-1 cells was significantly suppressed by blocking TLR4 with a specific TLR4 antibody, irrespective of LL-37 treatment, while blocking TLR4 displayed no effects on phagocytosis of IgG-opsonized S. aureus (Fig. 4F), in line with the notion that TLR4 signaling is important for phagocytosis of Gram- bacteria. However, whether TLR4 is involved in LL-37-induced phagocytosis of E.coli is uncertain. In a previous report, it was shown that among various TLRs, LL-37 selectively increases the levels of TLR4 mRNA and protein in human mast cells [36]. In addition, the human lactoferrin-derived 1–11 AMP enhances TLR4 and CD14 expression on macrophages [37]. On the other hand, LL-37 has the capacity to block TLR4 activation on DCs by altering receptor motility [38]. LL-37 also inhibited macrophage activation by LPS [39 – 41] via blocking the interaction between LPS and LPS-binding protein [42] and mechanisms other than direct binding to LPS [40 – 42]. Detailed analysis showed that the effects of LL-37 on cells of the myeloid lineage were dependent on the time when LL-37 was added, the concentration of LL37, the stimuli and maturation state of the monocytes or macrophages [42]. Apparently, LL-37 may promote protective and destructive facets of host defense, and the net effect seems to be dependent on several factors, including cellular context, dose, and timing. In addition, all of our evidence indicates that FPR2/ALX mediates LL-37-induced bacterial phagocytosis. FPR2/ALX beVolume 95, June 2014

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longs to the GPCR family and binds the proresolving LXA4, as well as fMLP and related peptides. This receptor plays a role in chemotaxis and activation of phagocytes [43], with expression in neutrophils, monocytes, NK cells [44], and macrophages [45]. Maderna et al. reported that FPR2/ALX mediates LXA4-stimulated phagocytosis of opsonized zymosans by dTHP-1 cells or apoptotic PMNs by mouse bone marrow-derived macrophages [46], lending further support to our conclusion that FPR2/ALX is functionally involved in LL-37-enhanced bacterial phagocytosis by human macrophages. In conclusion, we demonstrate that LL-37 activates human macrophages to elevate the expression of Fc␥Rs and TLR4 in macrophages, resulting in enhanced capacity to phagocytize bacteria. Hence, this property of LL-37 will facilitate bacterial clearance and strengthen host defense.

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AUTHORSHIP M.W. designed and performed experiments, analyzed data, and wrote the manuscript. A.M.v.D. performed animal experiments and wrote the manuscript; X.T. performed experiments; L.L., B.A., and J.Z.H. supervised the project and revised the manuscript.

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GRANTS This study was supported by the Swedish Research Council (10350, 11217, 20854, 04342, Linneus Grant CERIC), CIDaT, EC FP7 funding (201668), Torsten och Ragnar Söderberg Foundation, Swedish Foundation for Strategic Research, and Cancer Foundation. J.Z.H. is supported by a Distinguished Professor Award from Karolinska Institutet. We thank Dr. Antonio Di Gennaro for his contributions to method development and the Center for Live Imaging of Cells (CLICK) at Karolinska Institutet for the technical support with confocal microscopy.

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DISCLOSURES

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The authors declare no competing financial interests.

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KEY WORDS: cathelicidin 䡠 Fc␥R 䡠 TLR 䡠 innate immunity 䡠 infection

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