Cathelicidin Contributes to the Restriction of Leishmania in Human Host Macrophages

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated-Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model.


INTRODUCTION
The disease Leishmaniasis is still affecting 12 million people worldwide, of which up to 30,000 cases die yearly (1,2). Up to date, no vaccine is available and treatment is not always evident due to the socioeconomic conditions in the affected countries (3,4). Our knowledge regarding the interaction of Leishmania with its human host cell, the macrophage, is still fragmentary, as little is known with respect to antimicrobial mechanisms restricting Leishmania growth in human primary macrophages. Moreover, few data is available demonstrating which macrophage phenotype is the most superior for Leishmania survival or killing. The human body comprises a broad spectrum of different macrophage phenotypes, related to distinct functional properties (5). Herein, the M1/M2 polarization has been the main framework for years in the field of immunology. In the murine system, "alternatively activated" type 2 macrophages are shown to support Leishmania parasite replication and persistence via an increased arginase I activity, which negatively correlates to the expression of nitric oxide synthase II (6)(7)(8)(9)(10). In contrast, "classically activated" M1 inflammatory macrophages enhance the production of free nitric oxide (NO) radicals, hereby eliminating intracellular parasites (11,12). In human macrophages however, NO-mediated killing of Leishmania is still under debate, underlying the controversy of extrapolating immunological aspects from mouse to man (13)(14)(15)(16). Nevertheless, antimicrobial peptides (AMPs), comprising defensins and cathelicidins, are key players in the human host's immune defense. In humans, only the cathelicidin antimicrobial protein hCAP18, encoded by the gene CAMP, has been identified. The CAMP gene product is cleaved to form the amphipathic, active LL37 peptide. LL37 can be found in various cell types, body fluids and tissues, such as the skin, where an increased production has been described to correlate with disease pathologies (17,18). As a key molecule in host defense, LL37 exerts antimicrobial properties toward bacteria (Staphylococcus spp., Pseudomonas spp., Mycobacteria spp.), viruses, fungi, as well as parasites (19)(20)(21)(22)(23)(24)(25)(26). Dos Santos et al. could demonstrate cathelicidin to exert anti-leishmanial activity in L. donovani infected macrophages, in line with data of Dos Santos et al. showing an IL-32/cathelicidinmediated control of L. braziliensis in THP-1 cells (27). This AMP, LL37, able to create pores, hereby disrupting membranes. Although the exact mode of action is unknown, two models have been widely accepted being the "carpet" and "toroidal" model (17,28). The toroidal model defines a pore architecture, formed by peptide channels, whereas the carpet model describes a more severe membrane perturbation, as seen for detergentinduced membrane destruction (29). In this study, we aimed to identify a role for the human cathelicidin during Leishmania infection. We could demonstrate CAMP to be upregulated in lesion material from Ethiopian individuals suffering from cutaneous Leishmaniasis. Using a human primary macrophage in vitro model, we identified CAMP to be upregulated specifically in pro-inflammatory macrophages and rLL37 was demonstrated to kill Leishmania in a dose dependent manner. By modulating the vitamin D pathway, we demonstrated CAMP expression to be upregulated, enhancing the macrophage's parasite killing capacity. In contrast, using a RNA interference (RNAi) approach in human primary macrophages targeting CAMP mRNA, the expression of hCAP18 was strongly reduced, enabling Leishmania parasites to survive better. In all, these data suggest an anti-parasitic activity of cathelicidin in a human primary in vitro cell model for cutaneous leishmaniasis and patient skin lesions.

An Increased Expression of Cathelicidin in Skin Biopsies of African Patients With Cutaneous Leishmaniasis
In search for antimicrobial mechanisms in self-healing cutaneous Leishmaniasis (CL), we investigated the expression of human cathelicidin hCAP18. In Addis Ababa, Ethiopia, clinical samples from patients with CL and controls were collected and tested using RT-PCR. Patients varied in age, ethnicity, disease duration, and wound location, as depicted (Figures 1A,B). All patients were tested positive for the presence of Leishmania aethiopica by PCR. Interestingly, a significantly higher transcript abundance of the human cathelicidin hCAP18 was detected in skin biopsies of patients with CL, compared to control samples ( Figure 1C).

Expression of Cathelicidin Is More Prominent in Pro-inflammatory Than in Anti-inflammatory Human Macrophages
Human macrophages are key players during Leishmania infection. In a next step, the suitability of both human primary monocyte derived macrophages type 1 (hMDM1) and type 2 (hMDM2) as host for Leishmania parasites was assessed. From human blood, monocytes were isolated and differentiated using rhGM-CSF (10 ng/ml) or rhM-CSF (30 ng/ml), to generate hMDM1 or hMDM2, respectively. The hMDM1 were characterized by their fried-egg shaped morphology and CD14 + MHCII + CD163 − phenotype (Figure 3A, upper lane). (C) Skin biopsies, from healthy (n = 10) or CL patients (n = 10), were collected from which CAMP gene expression was assessed by qRT-PCR. Relative gene expression was normalized against GAPDH and presented as mean ± SD. Statistical analysis (Mann-Whitney test) was used to compare groups using GraphPad statistical software (*p < 0.05).
shown). These data demonstrate that targeting the Vitamin D pathway strongly compromises Leishmania parasite survival, as cathelicidin is strongly upregulated.

Reduction of Endogenous Cathelicidin in Human Primary Macrophages Promotes Leishmania Infection
The human cathelicidin is expressed by monocytes, macrophages as well as neutrophils (17,33). We already demonstrated (i) cathelicidin to be higher expressed in skin biopsies of patients with the self-healing cutaneous leishmaniasis compared to healthy controls, (ii) hrLL37 to facilitate apoptosis among promastigotes and amastigotes and (iii) hMDM1 to express cathelicidin to a higher extent comparted to hMDM2. To demonstrate a role for the intracellular, endogenous cathelicidin of macrophages in the elimination of Leishmania parasites, knockdown (KD) experiments were performed. Using an RNAi approach, we could significantly reduce CAMP gene expression (0.15 ± 0.13), compared to the control (1.0 ± 0.0) and non-sense FIGURE 4 | Vitamin-D derivatives induce LL37 mediated Leishmania restriction in human primary macrophages. hMDM-1 were generated and treated with calcitriol (CCT) or calcipotriol (CPT) for 24 h, or left untreated. Subsequently, hMDM-1 were infected with transgenic dsRed expressing Lm or Lae (MOI10). After 3 h, extracellular parasites were removed by washing, following incubation at 37 • C, 5% CO 2 . After 24 h (early; black bars) or 6 d (late; white bars) CAMP gene (n = 1-5) (A) and cathelicidin protein expression (n = 5-7) (B) were by qRT-PCR and western blot analysis. CAMP expression was normalized against the house keeping gene GAPHD. Western blots were analyzed by densitometry (ImageJ analysis), normalizing cathelicidin against ß-actin protein expression. Gene and protein expression were normalized to the untreated control, which was set to 1 (dashed line). In addition, parasite survival (C,D) and infection rates of Lm dsRed (E), analyzed by the mean fluorescent intensity (MFI) of the dsRed protein, was assessed by flow cytometry. Western blots, FACS histograms and data, presented as mean ± SD, are representative for at least 3 independent experiments (Wilcoxon matched-pairs signed rank test; *p < 0.05; **p < 0.01).
siRNA control (1.04 ± 0.74) ( Figure 5A). We were not able to show a clear cathelicidin protein decrease in siRNA treated cells by western blot (data not shown), as cathelicidin is expressed at low levels under steady state conditions. Therefore, we assessed cathelicidin protein expression in knockdown cells by triggering the Vitamin-D pathway first with CPT and CCT, including βactin as loading control, demonstrating a strong reduction of cathelicidin protein amount, upon CCT and CPT treatment, in the KD cells compared to the control cells ( Figure 5B). Next, KD and control cells were infected with Leishmania promastigotes, after which intracellular survival was investigated using an end-point titration assay. We could demonstrate that the number of viable Leishmania in control (284 ± 228 Lm) and non-sense siRNA treated cells (421 ± 303 Lm) did not significantly differ. However, KD of CAMP resulted in a higher parasite survival (544 ± 388 Lm) (Figure 5C), although the level of significance was not reached. In all, these data show cathelicidin to play a role in the restriction of Leishmania promastigote survival.

DISCUSSION
In the present study, we were able to define a role for the human cathelicidin in human leishmaniasis, based on data from clinical samples. In addition, a human primary in vitro cell model was designed to better mimic the in vivo interaction between Leishmania parasites and their host cell, the human macrophage. The two phenotypes of macrophages were demonstrated to interact differently with Leishmania parasites, as in anti-inflammatory macrophages are more susceptible compared to pro-inflammatory human macrophages. Furthermore, the latter pro-inflammatory phenotype expressed the cathelicidin CAMP gene transcript and protein more strongly, which we demonstrated to contribute in controlling Leishmania infection.  (36). In line, CAMP was demonstrated to be crucial for the local control of cutaneous lesion development and parasite growth, using CAMP KO mice (25). Furthermore, progression of visceral Leishmaniasis was demonstrated to be associated with vitamin D deficiency in dogs (37). Few data however, evaluate the effect of cathelicidin and/or the vitamin D pathway in human patients and/or a human cell model. Das et al. could show cathelicidin to augment antileishmanial macrophage activating properties of Amphotericin B (38). In line, we identified a strong upregulation of the human CAMP mRNA transcript in clinical samples from African patients with cutaneous Leishmaniasis, suggesting cathelicidin to play a role in human CL in vivo.

Cathelicidin-Induced Apoptotic Death of Leishmania
We could demonstrate the human cathelicidin to induce an apoptosis-like phenotype in Leishmania parasites, in a dose dependent manner in both L. major and L. aethiopica promastigotes as well as in L. aethiopica amastigotes. Although the underlying mode of action remains elusive, rLL37 was demonstrated to induce phosphatidylserine exposure, a round shaped cell morphology and DNA fragmentation, all characteristics of apoptosis (39). Presumably, the amphipathic α-helical peptide LL37 interacts with the negatively charged phospholipids within the parasitic membrane by electrostatic forces, as described for the carpet and toroidal-pore model (17). Surprisingly, recombinant LL37 did not exert apoptosis-inducing effect on the L. major amastigote life stage, when looking at TUNEL positivity and DNA degradation. Of note, Kulkarni et al. could show antimicrobial peptides to differently induce parasitic cell death, by means of non-apoptotic (class I) or apoptotic (class II) mediated killing (26). One could speculate these mechanisms to be also applicable in our model, which will be the focus of future research.
Amastigotes also differ in their surface charge compared to promastigotes, as Pimenta et al. could show transformation of Leishmania mexicana amazonensis promastigotes to amastigotes to be associated with a shift in the electrophoretic mobility (40). Of general acceptance, is the fact that cationic antimicrobial peptides strongly bind negatively charged phospholipid moieties. Due to the different surface charge between Leishmania life stages, we speculate LL37 to only bind Lm promastigotes resulting in killing, whereas LL37 to be ineffective in binding Lm amastigotes. Overall, our data indicate rLL37 to induce cell death in Leishmania promastigotes.

Cathelicidin in Mammalian Innate Immune Defense
Cathelicidins have gained increasing attention, as being an important mediator during innate immunity. Although cathelicidins are primarily present in human neutrophils, also keratinocytes, monocytes and macrophages harbor this antimicrobial peptide (17,33,(41)(42)(43). These cells may indicate where the cathelicidin is originating from upon Leishmania infection. Whether macrophages and keratinocytes exert synergistic effects with regard to cathelicidin production and Leishmania elimination is yet to be defined. Focusing on human primary macrophages, we could demonstrate different macrophage phenotypes to express cathelicidin to a different extent. The human cathelicidin is more abundant in proinflammatory macrophages, which may not be surprising as it drives macrophages polarization to a pro-inflammatory phenotype (44). The anti-inflammatory macrophages were more susceptible for infection, a finding in agreement with previous data and studies (45,46). To define an active role for cathelicidin during Leishmania infection, we modulated its expression. In concordance with previous studies, we could show CAMP expression to be highly enhanced upon activating the vitamin D pathway, using calcitriol or calcipotriol (47)(48)(49)(50). Interestingly, the intracellular survival of Leishmania parasites was significantly impaired. The group of Agerberth could show LL37 induced expression to be associated with the control of M. tuberculosis in human macrophages (51). Furthermore, phenyl butyrate/vitamin D3 treatment, induced LL37-mediated elimination of M. tuberculosis by macrophages, strengthening the data of the Modlin's group, showing cathelicidin to be required for the 1,25D(3)-triggered antimicrobial activity against intracellular M. tuberculosis (52,53). In all, triggering the vitamin D pathway in human macrophages, hereby inducing cathelicidin expression, restricts Leishmania survival.

Cathelicidin Contributes to a Reduced Parasite Survival
Vitamin D derivatives induce expression of diverse immune modulators, such as cathelicidin, IL-1ß, etc. (54). To target the CAMP gene more specifically, a RNA interference (RNAi) approach was chosen. Our data showed Leishmania parasite survival to be enhanced. Of note, no significant difference was observed between non-target and anti-CAMP siRNA treatment. One should keep in mind, that all human macrophages were derived from human blood donors, which may differ in gender, immune status, etc., having an impact on host pathogen interactions (55). Furthermore, also transfection as a treatment, may result in RNAi associated immune stimulation through activation of IFN signaling cascades (31). Both aspects, might "bias" our results, with regard to the comparison to the untreated control, as type I interferons have been demonstrated to increase superoxide dismutase (SOD) expression in macrophages, favoring parasite survival (56). Besides the restrictions of the employed methodology, a stronger tendency toward parasite survival, upon CAMP RNAi was observed, suggesting cathelicidin to contribute in restricting Leishmania parasite survival. Indeed, McGwire's group could show the corresponding murine cathelicidin (CRAMP) to control Leishmania parasite infection in a mouse infection model (25). Knockout mice for CRAMP were reported to develop exacerbated lesions combined with a higher parasites distribution upon L. major infection as compared to wild type mice (25). Of note, Gombart et al. showed a vitamin D response element (VDRE) to be conserved in the CAMP promotor of primates. The absence of the VDRE region in the genomes of mouse, rat and canine makes the expression of CRAMP not tunable by the vitamin D pathway (57). In humans, the great potential of cathelicidin is also highlighted in other disease pathologies. A deficiency of cathelicidin may impede the outcome of inflammation in the lungs of patients with severe sarcoidosis (58). Furthermore, Searing et al. propose an increased production of LL37 to prevent patients with atopic dermatitis from herpes infection (59).

CONCLUSION
In the current study, we revealed the CAMP transcript to be strongly upregulated in skin lesion material from cutaneous leishmaniasis patients. Using an in vitro model, we demonstrated pro-inflammatory human macrophages to be able to control Leishmania infection more efficiently compared to antiinflammatory macrophages, to which cathelicidin expression is contributing. In addition to the NO-based anti-leishmanial mouse effector mechanism, we propose that vitamin D-inducible cathelicidin expression in combination with GM-CSF polarized macrophages to be a unique mechanism, which contributes to the restriction of Leishmania in human macrophages.
Upon transformation into the amastigote life stage, a 1 logscale higher dsRed fluorescence is present, due to the highlevel expression in the amastigote life stage (31). Logarithmicphase or stationary-phase promastigotes were obtained after 2 (log-phase) or 7 (stat-phase) days of culture, respectively. Lm and Lae axenic amastigotes were generated by incubating logphase promastigotes in pH 5.5 at 33 • C and isolated using a discontinuous Histopaque R 1119 (Sigma Aldrich, Germany) density gradient as described (61).

Assessing Apoptosis
To assess apoptosis, Lm and Lae promastigotes, which resided in a logarithmic growth phase, were treated with 30 and 60 ng/ml hrLL37 (PeptaNova GmbH, Sandhausen, Germany) for 72 h. Leishmania axenic amastigotes were treated with 10 and 100 ng/ml hrLL37 or 25 µM staurosporine for 24 h. Subsequently, DNA fragmentation was assessed by flow cytometry or immunofluorescence imaging using an in situ cell-death detection kit, based on terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), as described (30). Exposure of phosphatidylserine (PS) was assessed by AnnexinA5 binding using flow cytometry.

Sample Collection
Clinically suspected CL patients, who visited the Ankesha and Kela health centers consented to participate in the study, were clinically examined for CL. Ankesha and Kela health centers are found in the leishmaniasis endemic regions in East Gojam Zones of Amhara region and in Gurage zone of Southern regional state of Ethiopia, respectively. Patients, diagnosed for active CL, were recruited to this study prior to treatment. Diagnosis was confirmed by microscopy or culture from skin lesion scraping. After the skin lesion was cleaned, the boarder of lesion was collected for microscopy analysis. Healthy controls were recruited from patients admitted for minor surgery ALERT hospital. All study participants were seronegative for HIV. Skin biopsies from CL patients were taken from the border site of the lesion, using a disposable punch (3 mm in diameter). Local anesthesia with 2% lidocaine was applied. Control skin biopsies were obtained from the leftover samples taken for skin graft of selected individuals (without infection or immunological disorder) visiting the ALERT hospital surgery department.
In addition, from skin samples Leishmania promastigotes were cultivated. DNA was extracted from culture and biopsy samples using QIAamp R DNA Mini Kit according to manufacturer's procedure. PCR amplification was performed with 100 ng template and the HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) using the primers Lae speciesspecific primers V5F 5 ′ -GGTGATGTGCCCGAGTGCA-3 ′ and V10R 5 ′ -CGTGCACATCAGCACATGGG-3 ′ .

Generation of Human Monocyte-Derived Macrophages
Human peripheral mononuclear cells (PBMCs) were isolated from buffy coats (DRK-Blutspendedienst Hessen GmbH) by passage over a Leukocyte Separation Medium gradient as described previously (30). Monocytes, obtained by plastic adherence or CD14 selection were incubated either with 10 ng/ml rhGM-CSF (Leukine R Sanofi-Aventis, Bridgewater, US) or 30 ng/ml rhM-CSF (R&D Systems, Abingdon, UK) for a period of 5 to 7 d at 37 • C, 5% CO 2 to generate hMDM1 or hMDM2, respectively. Cells were generated in 6 w plates or in 25 cm 2 culture flasks and were detached by cooling cells down on ice, following detachment with a cell scraper. Experimental data, conducted with monocytes obtained from human donors, are depicted as dot plots, in which each dot presents data from a single donor.

Infection of hMDM With Leishmania Parasites
HMDM were harvested and transferred into 1.5 ml microcentrifuge tubes, to which cells do not attach. The cells were co-incubated with stationary phase Lm or Lae promastigotes or axenic amastigotes at a MOI ratio of 1:10 in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 50 µM β-mercaptoethanol (all from Sigma Aldrich), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, 10 mM HEPES (all from Biochrom) for 3 h at 37 • C in a humidified atmosphere in a CO 2 incubator. Extracellular parasites were removed by centrifugation and washing the cells. During infection experiments, cathelicidin expression was induced by incubation of hMDM with 100 nM calcitriol or calcipotriol for 24 h. For transgenic LmdsRed flow cytometry was used to analyze infection rates. These transgenic parasites can also be used as a model to follow the parasite propagation, which is based on the development and replication of amastigotes. The LmdsRed promastigotes increase their fluorescence intensity when transforming into amastigotes, which enables the quantification of the parasite propagation by measuring the dsRed mean fluorescence intensity using FACS (62,63).

Western Blot
A total number of 0.5 × 10 6 hMDM were lysed in Lämmli-Buffer (A. bidest supplemented with 0.7 M Glycerol, 1.7% SDS, 0.1 M DTT and 30 µM Bromphenol blue), denaturated at 95 • C for 10 min and loaded onto a 15% SDS-polyacrylamide gel. The separated proteins were blotted onto a nitrocellulose transfer membrane at 145 mA constant voltage for 1 h. The membrane was blocked with WB-Block-Solution (GE Healthcare, Buckinghamshire, United Kingdom) washed with WB-Wash-Buffer (A. bidest supplemented with 0.5% Tween, 0.14 M NaCl, 10 mM Tris, 1 mM NaN3; pH 8) and subsequently incubated with an anti-LL37 primary antibody (kindly provided by Prof. B. Agerberth) overnight at 4 • C. After extensive washing, the membrane was incubated with a HRP-conjugated secondary antibody (1:1000, from Cell Signaling, Danvers, USA) for 1 h at room temperature. The membrane was washed once more and the protein bands were detected using an ECL substrate (GE Healthcare, Buckinghamshire, United Kingdom). Using ImageJ, densitometry analysis was performed to quantify the intensities of the protein bands.

End-Point Titration Assay
The amount of viable intracellular parasites inside hMDM1 (control, non-sense siRNA or anti-CAMP treated) was determined. After 3 h of infection, MOI10, cells were washed to remove extracellular parasites. After 48 h of infection, the end-point titration assay was carried out. Therefore, cell scraper detached hMDM1 were counted and 2000 hMDM1 were seeded, in quadruplicates, in a 96 w plate containing biphasic Novy-Nicolle-McNeal blood agar medium. Wells were serial diluted (factor 1.5) for 24 times. Plates were incubated for 7-10 days at 27 • C. By microscopical analysis, plates were analyzed to assess at which dilution growth was seen. Based on the dilution factor and the amount of hMDM1 that were seeded in the first well, the amount of parasites per 1,000 hMDM1 was calculated (63). The formula applied to calculate the amount of parasites per 1,000 macrophages is 1.5 x /2 were x is the dilution step in which still parasite growth was observed.

Statistical Analysis
Numerical data are presented as the mean ± standard deviation (SD). For statistical analysis, data were tested for their normal distribution, using the D'Agostino and Pearson omnibus normality test. If passed, statistical analysis was determined by a paired Student t-test. If data were not normally distributed or in case to few biological replicates were present to test normal distribution, a non-parametric test (Mann-Whitney test or Wilcoxon matched-pairs signed rank test) was used.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/supplementary material.