Ethics statement

All procedures involving animals were approved by the ethical review committees of the London School of Hygiene and Tropical Medicine (PPL: 70/8427), Imperial College (PPL: 70/6712) and the Liverpool School of Tropical Medicine (PPL: 40/2958) and performed in accordance with United Kingdom Government (Home Office) and EC regulations. Mice were anaesthetized by intraperitoneal injection of ketamine 150 mg/kg and xylazine 15 mg/kg, and all efforts were made to minimize any suffering.

Parasite preparation and inoculation into skin

Leishmania (Leishmania) mexicana (MNYC/BZ/62/M379) promastigotes were grown until exponential phase in M199 culture medium (HEPES modification with Earle’s salts, Sigma-Aldrich) supplemented with 10% v/v heat-inactivated foetal calf serum (hiFCS, Gibco, UK), 1% v/v penicillin-streptomycin (PS), 1 x BME vitamins (Sigma-Aldrich), pH 7.2 and then passaged at 5 x 10⁵ promastigotes/ml into Grace's insect culture medium (Invitrogen, supplemented as for M199 medium) pH 5.5 to reach stationary phase at 26°C. This method produces a high yield of L. mexicana metacyclic promastigotes (typically >85%). Metacyclic promastigotes were isolated from stationary phase cultures using a discontinuous density gradient [31]. One thousand metacyclic promastigotes in Dulbecco’s phosphate buffered saline (PBS) were injected intradermally in the ears of 6–8-week old female BALB/c mice, with or without 0.03 μg L. mexicana PSG. To minimise tissue damage for Affymetrix analysis, parasites and PSG were delivered by nanoinjection with bevelled fine tipped borosilicate glass microneedles (Drummond) attached to a Nanoject II Auto-Nanolitre injector [32] in a total volume of 160 nl, and for all other experiments in 10 μl using a 31 gauge, 0.3 ml insulin syringe and needle. After 4, 6, 24 or 48 hours post-inoculation, mice were humanely euthanized, and ears were recovered using 2 mm diameter punch biopsy, centred on the site of injection, for RNA extraction. In other experiments, the inoculation of ears with 0.5 μg PSG or PBS was followed 6 hours later by a full-thickness, 2 mm diameter punch biopsy to the site of injection. The diameter of the wounds were measured daily and expressed as proportion of the initial wound.

PSG preparation

Colony reared female Lutzomyia longipalpis (Jacobina strain) were infected with L. mexicana amastigotes freshly isolated from BALB/c rump lesions as previously described [9] and offered to sand flies through a membrane at 2 x 10⁶ amastigotes/ ml in fresh defibrinated rabbit blood (TCS Biosciences—without antibiotics). Flies were maintained at >70% relative humidity and offered 20% v/v sucrose solution to feed ad libitum. PSG was dissected from 7–8 day old fly infections, processed to remove parasites and contaminating proteins by passing through a 0.22 μm pore diameter syringe filter, then heat-treating for 1 hour at 60°C, followed by four freeze-thaws. To ensure minimal batch-to-batch variation, PSG from 200 flies were collected at a time in 400 μl PBS and adjusted to 0.2 μg/μl total protein using the BCA (bicinchoninic acid) protein assay (Pierce). PSG was stored at -20°C until use [12].

Saliva preparation

Sand fly saliva was obtained from 5 day-old female uninfected flies using the gland-piercing method into PBS [6] and stored at -20°C until use.

Sand fly bite transmission

Five-day-old Lutzomyia longipalpis (Jacobina strain) female sand flies were infected with L. mexicana amastigotes through by artificial membrane feeding. Blood-fed flies were separated and maintained under a 12-h light:dark cycle at 28–30°C, 80%–95% relative humidity, and supplied with 20% (w/v) sucrose. Anaesthetised, 6–8-week old female BALB/c mice were infected by allowing single infected sand flies to bite the ear.

RNA extraction

At different time points post-inoculation recovered mouse ears were fragmented using a MiniBeadBeater (1 min at 5000 rpm/min) in 1 ml of lysis buffer with Precellys ceramic 2.8 mm beads, as previously described [33]. RNA isolation was performed with the RNeasy Plus Mini kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. Evaluation of RNA quality was performed by optical density measurement using a Nanodrop (ThermoFisher Scientific) and/or their integrity was assessed using an Agilent-2100 Bioanalyzer (RNA integrity >9) [34]. RNA were either used for the GeneChip hybridization or were reverse transcribed in cDNA for real-time quantitative PCR (RTqPCR).

GeneChip hybridization and data analysis

RNAs were hybridized onto Affymetrix Mouse 430_2 GeneChips, using the Affymetrix Two-Cycle Eukaryotic Target Labelling procedure: http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf. MIAME-compliant data are available through ArrayExpress and GEO databases (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52101). Gene-level expression values were derived from the CEL file probe-level hybridization intensities using the model-based Robust Multichip Average algorithm (RMA) [35]. RMA performs normalization, background correction and data summarization. Pearson’s correlation coefficient were calculated and a heatmap representation was performed using the Expression Console Software. Local pooled error (LPE) tests [36] were performed to identify significant differences in gene expression between different conditions. The estimated false discovery rate of this analyse was calculated using the Benjamini and Hochberg approach [37] to correct for multiple comparisons. Ingenuity Pathway Analysis (IPA) software v 9.0 (http://www.ingenuity.com) was used identify canonical pathways regulated by PSG presence at 6 hours post-inoculation. Gene Ontology (GO) analysis was also performed to find gene pathways and networks enriched by PSG in the Affymetrix data set at 6 hours post-infection (http://www.geneontology.org). This service connects to the analysis tool from the PANTHER Classification System (http://pantherdb.org), which is maintained up to date with GO annotations.

Oligonucleotide primers and transcriptional analyses by RTqPCR

Primers were designed using OligoPerfect Designer (Invitrogen Corp. Carlsbad, CA). The primer sequences and melting temperature (Tm) are summarized in S2 Table. RNAs were reverse transcribed in cDNA using random hexamers (Roche Diagnostics) and Moloney Murine Leukaemia Virus Reverse Transcriptase (Invitrogen, Life Technologies). A SYBR Green-based real-time PCR assay (QuantiTech SYBR Green Kit, Qiagen) for relative quantification of mouse target genes [33,38] was performed on a white MicroAmp Optical 384-well 7900HT Fast Real-Time PCR System (Applied Biosystems). Cycle threshold values (Ct) were determined by the Applied Biosystems SDS2.4 Software. Ct values were input into Biogazelle qBasePLUS software, a flexible and open source program for qPCR data management and analysis [39]. For normalisation calculations, candidate control genes were tested [40] with geNorm [41] and Normfinder programs [42]. Nono and l19 were selected as the most stable reference genes for the BALB/c ears and ywhaz and nono for BALB/c bone-marrow macrophages.

Scratch wound model of wound healing

In order to study cell migration in response to PSG-induced inflammation, a simple and robust scratch wound model was used, based on published methods [43]. Mouse derived L929 fibroblast (ATCC) and Kera-308 keratinocyte cell lines (CLS) were cultured in 25 cm² flasks (Costar) in DMEM (Gibco) supplemented with 10% v/v hiFCS, 1% v/v PS, 1 x BME, 2 mM L-glutamine in a 5% CO₂ humidified incubator at 37°C, and passaged every 7 days. Fibroblasts and keratinocytes were seeded in 24 well plates at a density of 1.5 x 10⁵/ml and 5 x 10⁵/ml per well, respectively, and incubated at 37°C, in a humidified atmosphere of 5% CO for 24 hours to obtain confluent monolayers. Wells were washed gently three times in PBS warmed to room temperature and scraped down the centre using a sterilised 200 μl pipette tip. Following a scratch, wells were washed gently three times in DMEM, and incubated with 100 μl of complete 1% v/v/ hiFCS DMEM-media supplemented with or without 0.5 μg/ml PSG. Positive controls were treated with 10 μg/ml TGFβ2 and 10 μg/ml EGF (Peprotech) [43], and triplicate wells were tested. For orientation, marks were made on the underside of each well along the length of the scratch using permanent marker. A series of photographs were taken in exactly the same position at 0, 12 and 24 hours post-scratch. Photographs were taken using an OptixCam Summit OCS-10.0X 10MP camera on a Nikon inverted microscope, viewed in OCView software (v1.1) and the area of the scratch wound of each image measured in ImageJ using the polygon selection tool (http://rsb.info.nih.gov). This tool was used to outline the leading edge of the monolayer in order to obtain the area of the scratch. The rate of wound closure is expressed as a percentage of the area at time-point zero.

BrdU incorporation assay

Bromodeoxyuridine (BrdU) is a thymidine analogue that is incorporated into newly synthesized DNA strands of actively proliferating cells. BrdU incorporation was detected immunochemically using the Calbiochem BrdU Cell Proliferation Assay (HTS01), according to the manufacturer’s instructions. Briefly, 20 μl BrdU label was added to each well immediately following the scratch and addition of PSG. At the times indicated post-scratch, wells were incubated with 200 μl BrdU fixative/denaturing solution for 30 min, at room temperature, followed by incubation with 100 μl anti-BrdU antibody (1:100), at room temperature for 1 hour. Wells were washed three times with plate wash solution and 100 μl BrdU peroxidise-conjugated goat ant-mouse IgG (1:1000) added to each well. After 30 min of incubation at room temperature wells were washed three times in plate wash solution and once in dH₂O before adding 100 μl of fluorogenic substrate solution. Following 30 min incubation in the dark at room temperature, 100 μl stop solution was added and fluorescence intensity was read on a Spectramax M3 plate reader at 320 nm excitation, 460 nm emission. Wells for each treatment were assayed in quadruplicate.

HeLa/STAT3 luciferase reporter assay

HeLa/STAT3 cells stably express firefly luciferase reporter gene under the control of the STAT3 response element (Signosis). Cells were grown to confluence on square petri dishes in DMEM (high glucose+sodium pyruvate+L-glutamine) (Gibco) supplemented with 1% v/v PS, 10% v/v/ h.i.FBS, 100 μg/ml Hygromycin B in a humidified incubator at 37°C with 5% CO₂ for 5–6 days. The day before the assay the cells were trypsinised and plated in 96 well plates at 5 x 10⁴ cells per well and incubated as above. Promastigote secretory gel, recombinant human Oncostatin M (Sigma) and anti-IGF1R antibodies (Peprotech) were added to the wells as required and incubated for a further 24 hours. Following this, wells were aspirated and gently washed in PBS once before adding 50 μl of the lysis buffer. Cells were allowed to lyse for 5 mins at room temperature with gentle rocking. Lysing cells were passed up and down a pipette tip before being freeze-thawed once at -80°C and room temperature. Twenty microliters of the lysate was transferred to a new 96 well plate containing 100 μl of a Luciferase substrate (Promega) and mixed gently 2–3 times by pipette. Relative light intensity was measured in a Spectramax M3 plate reader.

Infection of dermal cells in dermal air pouches

3 ml of sterile air was injected into the backs of shaved BALB/c mice to inflate an air-pouch. Into each air-pouch 1 x 10⁶ L. mexicana metacyclic promastigotes with or without 1 μg PSG, 1:50 IGF1 Ab (Peprotech), or 1:50 IgG1 isotype control Ab were injected in a total of 150 μl endotoxin-free PBS using a 27-gauge needle. At 48 hours post-injection the cells from the air-pouch were recovered using a 5 ml ice-cold medium cavity lavage. Following this, the cells were concentrated to 0.5 ml by centrifugation (1800 rpm, 5 min) and live cells counted by diluting in trypan blue dye using a Neubauer improved haemocytometer. Following extensive washing to remove extracellular parasites macrophages were separated from neutrophils by adhesion to plastic for 2 hours at 37°C in DMEM medium supplemented with 20% hiFCS. Viable amastigote burdens were determined by transformation assay of amastigotes liberated from 2.5 x 10⁵ cells using the protocol below.

Bone marrow macrophage culture and infection

Macrophages were differentiated from bone marrow precursor cells in the presence of L929 fibroblast cell culture supernatant as a source of macrophage colony stimulating factor (CSF-1-responsive bone marrow-derived macrophages) as previously described [12]. Briefly, bone marrow was obtained from femurs from 6–8 week old BALB/c mice and bone marrow cells were cultured in supplemented DMEM medium (Gibco) (10% v/v hiFCS, 5% v/v horse serum, 1% v/v PS, 2 mM L-glutamine, 5 x 10⁻⁵ M β-mercaptoethanol and 15% v/v L929 culture supernatant) at 37°C, 10% CO₂ in a humidified incubator. Following 8 days of differentiation in plastic petridishes the bone marrow derived macrophages (BMMΦ) were washed and seeded into 16 chambered slides at 5 x 10⁴ BMMΦ/200 μl (Lab-Tek, Nunc) for promastigote transformation assays, or in 24 well culture plates at 5 x 10⁵/ml for arginase assays and RTqPCR. In vitro generated macrophages were exposed to Leishmania at a parasite to macrophage ratio (multiplicity of infection) of 5 L. mexicana metacyclic promastigotes to 1 macrophage (MOI 5:1) for 4 hours to achieve an intermediary level infection to assess the impact of various activations or vector-derived components on infection. Macrophages were washed extensively in DMEM to remove external parasites, then activators (20 U/ml IL-4, Preprotech) or L. mexicana PSG (0.5 μg for 5 x 10⁵ MΦ and 0.25 μg for 5 x 10⁴ MΦ) were added and the cells were cultured for up to a further 48 hours. The average viable intracellular parasite load was determined by promastigote transformation growth assay, below. RNA were extracted from macrophages using the RNeasy Plus Mini Kit (Qiagen) and the samples were reverse transcribed as previously described [44].

Promastigote transformation growth assay

5 x 10⁵ BALB/c BMMΦ or 2.5 x 10⁵ dermal macrophages recruited to and infected for 48 hours in air-pouches were lysed to release their amastigotes and grown for a further 48 hours in promastigote medium. Macrophages were lysed with sterile 0.008% v/v SDS in simple DMEM medium for 5 minutes at room temperature. The lysis was stopped by the addition of 17% v/v hiFCS in simple DMEM medium and the cells were mechanically ruptured with a cell scraper. Released amastigotes were concentrated on the bottom of the wells by centrifuging the plate at 3100 rpm for 10 minutes, washed in PBS and resuspended in 200 μl M199 promastigote medium (M199 medium supplemented with 1% v/v PS, 20% v/v hiFBS, 1% v/v hemin, 1 x BME vitamins, 4.2 mM NaHCO3, pH 7.2). The density of viable amastigotes that transformed and grew as promastigotes after 96 hours of culture at 26°C in 96 well microtitre plates was counted by haemocytometer.

Arginase assay

Arginase activity was determined by measuring the amount of urea generated from the hydrolysis of L-arginine as described [23]. Briefly, 5 x 10⁵ BMMΦ were solubilised with 200 μl lysis solution (0.1% Triton X-100 with protease inhibitor cocktail tablets (Roche), 10 mM MnCl₂, 25 mM Tris-HCl) and the enzyme was activated by heating for 10 min at 56°C. To this 10 μl 0.5 M L-arginine (pH 9.7) was added to the preparations to allow hydrolysis of the substrate and incubated at 37°C for 15–120 min. The reaction was stopped with 400 μl of H₂SO₄, H₃PO₄, H₂O (1:3:7 v/v). The urea concentration was measured at 550 nm after the addition of 20 ml α-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 100°C for 45 min. One unit of enzyme activity is defined as the amount of enzyme that catalyses the formation of 1 μmol of urea per min.

Luminex

Skin biopsies were homogenized in 100 μl of lysis solution (Fisher Scientific) containing T-PER Tissue Protein Extraction Reagent, 1.0 mM dithiothreitol and 1:100 protease inhibitor cocktail III using a tissue homogenizer (Biomasher, Camlab) and diluted with Bio-Plex samples diluents. IL-13 and IL-17a levels were determined using a Bio-Plex Mouse cytokine assay (Biorad) according to the manufacturer’s protocols. Briefly, antibody-coupled beads (50 μl) and the tissue homogenate (50 μl) were added into each well of 96-well filter plate. After incubation at room temperature for 30 minutes with agitation on a plate shaker, the supernatants were washed three times by wash buffer. The detection antibodies (25 μl) were added, and incubated at room temperature for another 30 minutes on plate shaker. Finally, streptavidin—PE (50 μl) was added into each well and incubated at room temperature for 10 minutes before washing and then 125 μl of Assay Buffer was added into each well. The microspheres were quantified using a Luminex 100 System (Luminexcorp).

Statistical analyses

Mann Whitney t-test was used to test the statistical significance between groups using GraphPad PRISM (v 5.0). All comparisons were made to the saline control except those indicated on the graphs with a connecting line. Linear correlation coefficients were generated by Spearman-rank correlation to assess the association of sand fly transmitted dose with host cytokine expression at the bite site. Significance was considered as p<0.05.

PSG induces rapid expression of many genes in L. mexicana infected BALB/c mouse skin associated with different innate immune pathways and wound healing processes

Using a low dose model of infection with physiologically relevant concentrations of PSG, we compared the transcriptional profiles of BALB/c ears 6, 24 and 48 hours post-inoculation (p.i.) of 1 x 10³ Leishmania (L.) mexicana metacyclic promastigotes with and without 0.03 μg of PSG. Prior to this, footpad infections confirmed that PSG was able to exacerbate L. mexicana over a wide range of doses, including 0.03 μg, found to be egested by infected sand flies [12] (Fig 1A–1C). Sand flies are pool-feeding vectors, which lacerate the upper dermal capillary bed with their barbed mouthparts to obtain blood. As a result, the damage made to the skin is disproportionately larger than other vessel-feeding vectors with hypodermic-like mouthparts (e.g. mosquitoes), yet the damage is still very focal. This is hard to replicate using needles since even the smallest (34 gauge) are some 9.5 times bigger than a sand fly’s piercing-proboscis [45], which will lead to extensive tissue damage. Furthermore, studies that use the intradermal route of needle-infection to the ear typically inject 10–20 μl, which separates the two dermal sheets over a large area of the ear, causing even more damage. Therefore, for our Affymetrix gene transcription study we used nanoinjection to minimise the wound created and volume injected (160 nl), to focus on those interactions driven by the PSG; combined with a dose of PSG (0.03 μg) equivalent to the upper-limit egested by L. mexicana-infected Lu. longipalpis [12]. The expression profiles of individual mice after PSG injection were comparable, both in quantity and quality, as illustrated by a heat map of the top 50 responsive genes (Fig 1A) and from the high degree of correlation in expression levels between mice after PSG administration (Fig 1B).

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Fig 1. PSG exacerbates Leishmania in skin over a wide physiological range and induces different gene modulation at different time points post-infection in skin.

(A-C) BALB/c mice were infected with 1 x 10³ L. mexicana metacyclic promastigotes with or without 1.0 μg (A), 0.5 μg (B) or 0.03 (C) μg L. mexicana PSG, s.c in to the left footpad and the evolution of the lesions, compared to the contralateral foot, recorded over 80 days. Final average parasite burdens ± S.D. for these lesions: PBS: 5.167 x 10⁷ ± 9.79 x 10⁶; 1.0 μg PSG: 4.74 x 10⁸ ± 3.21 x 10⁸; 0.5 μg PSG: 3.27 x 10⁸ ± 3.59 x 10⁸; 0.03 μg PSG: 1.96 x 10⁸ ± 3.05 x 10⁸ amastigotes per footpad. BALB/c mice received co-inoculation of either PBS or 0.03 μg PSG with 1 x 10³ L. mexicana metacyclic promastigotes between the dermal sheets of the ear pinna using a nanoinjector. D) Heat map representation of normalised expression values. Genes shown are the top 50 differentially expressed at 6 hours (FC >1.5 at 5% FDR). E) Heat map representation of the Pearson correlation coefficients between replicates for all modulated genes. The colour gradient extends from red, representing perfect correlation (correlation distance, r, of 1), to cyan for low correlation. Chip coding: Experiment/Time/Replicate number, e.g. MT6-2 represents the 2^nd chip from a control ear injected with PBS and parasites, sampled at 6 hours post-infection; chips coded PSG are from PSG-inoculated ears. (F and G) Venn diagrams of the number of overlapping genes differentially expressed (FC > 1.5 at 5% FDR) at 6, 24 and 48 hours between ears injected with PBS and metacyclic promastigotes versus PSG and metacyclic promastigotes. F) Total number of modulated genes. G) Genes involved in different phases of wound healing. H) Validation of microarray gene expression following i.d. injection of 0.5 μg PSG per ear. Real-time quantitative PCR of 34 randomly selected up- and down-regulated genes, among the differentially expressed gene list of 6 hours p.i. (FC>1.5, FDR 5%).

https://doi.org/10.1371/journal.ppat.1006794.g001

Out of a total of 28,853 mouse genes assayed 7,907 with known function showed ≥1.5-fold change (FC) at a 5% false discovery rate (FDR) that were differentially expressed in response to PSG. The vast majority of these genes (99.8%) were expressed during the first 6 hours of infection (Fig 1C). Focussing on this early time point, we subjected the transcripts to a biological interaction network analysis using the Ingenuity Pathway Analysis (IPA) software v 9.0. Using a cut-off of ≥FC5 at 5% FDR, 5,312 genes with known function were used to analyse the most significant canonical pathways.

The top 168 most enriched pathways are listed in Supplementary Table 1 (S1 Table), where a greater—ln (p-value) reflects increasing enrichment of PSG targets within a pathway. Of these 43 (26%) (7.94E-16 < p value < 5.00E-2; FC5, 5% FDR) are involved in innate immunity and one or more phases of wound healing (Table 1). Among these, 2 are involved in haemostasis, 24 in inflammation, 36 in epithelial cell proliferation and 10 in fibrosis and wound remodelling (Fig 1D). Gene Ontology (GO) analysis of the Affymetrix data revealed many genes involved in growth factor signalling and polyamine metabolism were enriched in skin by the presence of PSG (S2 Table). Thus, the majority of genes modulated by PSG following infection with L. mexicana are involved in the inflammation and proliferation phases of wound healing. [46]. The proliferation phase can be further sub-divided into: (i) angiogenesis, (ii) fibroplasia and granulation tissue formation, (iii) epithelialisation and (iv) contraction. Among the 39 pathways involved in the proliferation phase, 14, 27, 29 and 5 differentially expressed genes are involved in these four sub-phases, respectively. Thus, at 6 hours p.i. PSG induces different modulated canonical pathways predominantly involved in: (i) inflammation, (ii) fibroplasias and granulation tissue formation and (iii) epithelialisation.

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Table 1. In skin, PSG induces gene pathways involved in wound healing.

https://doi.org/10.1371/journal.ppat.1006794.t001

Validation of gene expression profiling

To validate the microarray data, we selected 35 genes associated with wound healing with different abundance levels (S3 Table) and quantified them by Real-Time reverse transcription quantitative PCR (RTqPCR) from samples collected at 6 hours p.i. All primers had amplification efficiencies close to 2. All threshold cycle (CT) values were normalized with two reference genes, nono and l19, that tested as the most stable. RTqPCR confirmed a high correlation (p<0.0001) with the corresponding microarray expression. (Fig 1H).

PSG accelerates wound healing in skin

As PSG up-regulates transcripts involved in wounding, when inoculated together with L. mexicana metacyclics, we hypothesized that PSG could promote wound healing. In a series of in vitro and in vivo experiments, we tested the ability of PSG to influence some of the key stages in skin injury and repair. We adapted an in vitro model of wounding using fibroblast and keratinocyte cell lines monolayers to test the effect of PSG on epithelial cell proliferation and migration [42]. Using a scratch wound model, we measured cell proliferation (BrdU incorporation) and cell migration over a 24 hour period (Fig 2A to 2C). The addition of PSG resulted in accelerated scratch closure (Fig 2A & 2B) following enhanced cell proliferation (Fig 2C) and migration of fibroblasts. In contrast, PSG did not exert any effect on keratinocyte monolayers (S1 Fig). To translate these in vitro results to wound healing in skin, we conditioned mouse ears with the intradermal injection of 0.5 μg PSG or PBS 4 hours prior to making 2 mm diameter full-thickness wounds and measured wound closure over 10 days (Fig 2D). PSG accelerated wound healing from day 2 onwards, leading day on day to significantly smaller wounds when compared to the PBS-injected controls (p<0.05).

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Fig 2. PSG improves wound healing in vitro and in skin.

Monolayers of L929 fibroblasts were scratched in the presence of culture media supplemented with or without 0.5 μg/ml L. mexicana PSG. Positive controls were treated with 10 μg/ml TGFβ2 and 10 μg/ml EGF. A) At 0, 12 and 24 hours post-wound, photomicrographs were taken and scratch closure was determined from using ImageJ. B) Representative images for the closure of a PSG-treated scratch. C) In replicate experiments, cell proliferation of fibroblasts was determined by measuring the incorporation of the fluorescent thymidine analogue, BrdU. Statistical analyses were performed between Media vs. PSG at each time point. Each in condition was performed in quadruplicate, data is pooled from 3 experiments. D) Daily wound closure following intradermal injection of PBS (open symbols) or 0.5 μg L. mexicana PSG (closed symbols) in the ears of BALB/c mice. Ears were wounded with a 2 mm diameter full-thickness ear punch 6 hours after the intra-dermal injection of PBS or PSG. Inset photographs are ears representative of each group at day 10 post-wound. Average wound closure ±SEM is shown from 12 mice per group (*: p<0.05; **: p<0.005; ***: p<0.0005 by Mann Whitney t-test); data is pooled from duplicate experiments.

https://doi.org/10.1371/journal.ppat.1006794.g002

PSG augments the early inflammatory response of the dermis to injury

Next, we investigated the role of PSG to modulate chemokines, cytokines and growth factors in skin over the course of a host’s response to a wound. We found that PSG strongly upregulated the early expression and secretion of a wide range of chemokines and cytokines in BALB/c ear dermis following a full-thickness wound (Fig 3A & 3B). Previously, we demonstrated that L. mexicana PSG rapidly (within 4 hours) recruits both neutrophils and macrophages to BALB/c mouse dermal air-pouches; and this was synergistic with Lu. longipalpis sand fly saliva [12]. Here we extend this finding by demonstrating that within 4 hours PSG upregulated the expression of the macrophage-recruiting chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and the neutrophil-recruiting chemokine CXCL2 (KC) in skin following a wound (Fig 3A). Similarly, PSG also increased the expression of a large number of pro-inflammatory cytokines, including IL-1β, IL-6and TNFα (Fig 3B). Collectively, these results show that PSG favours an early pro-inflammatory response in skin.

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Fig 3. The presence of PSG in a dermal wound differentially modulates the expression of genes involved in various stages of healing.

Ears of BALB/c mice were intra-dermally inoculated with 0.5 μg L. mexicana PSG (grey bars) or PBS (white bars) 4 hours prior to a full-thickness 2 mm diameter punch biopsy. A-C) 4 hours and 6 days post-wound ears were measured for transcripts involved in the inflammation and cell proliferation phases of wound healing by real-time quantitative PCR. A) Chemokines: CCL2, CCL3, CCL4 and CXCL2, B) pro-inflammatory-modulating cytokines: IL-1α, IL-1β, IL-6, IL-10 and TNFα, and C) epidermal growth factors and receptors: EGF, IGF1, EGFR, IGF1R and FGFR2. Relative expression was normalised to the housekeeping genes nono and l19 and is presented as the mean ±SD with 9–12 mice per group (*: p<0.05, **: p<0.005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g003

Macrophages provide a large proportion of pro-inflammatory cytokines [reviewed by 46] and their presence is not only important for the control of inflammatory cell migration but also for the stimulation of fibroblast and keratinocyte proliferation [47]. All the pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, TNFα) fell in the PSG-treated ears 6 days into wound healing. This may result from the PSG’s ability to promote macrophage alternative activation [12]. Alternatively activated (M2) macrophages (AAMΦ) predominate during the resolution phase of wound healing and secrete IL-10, an inhibitor of neutrophil and macrophage infiltration toward the site of injury [48]. During leishmaniasis, IL-10 plays an important role in suppressing the immune response and maintaining parasite persistence [49]. In the ears of BALB/c mice we found an early 7-fold increase in the expression of IL-10 (p = 0.052) in response to PSG following a wound, and by 6 days post-wound IL-10 was the only cytokine that remained upregulated (Fig 3B). IL-10 produced by resident skin cells [48] can inhibit the production of CCL2 and CCL3 chemokines and the pro-inflammatory cytokines IL-1β, IL-6 and TNFα [47,50]. Therefore, IL-10 may act as a negative regulator of the inflammatory response to promote the proliferative phase of wound healing.

PSG promotes the conditions for proliferation of fibroblasts and epithelial cells in skin

During the epithelialisation phase of wound healing, keratinocytes migrate to the wound edge and proliferate to form a new epidermis [51–55]. Re-epithelialisation is under the tight control from a variety of growth factors. Affymetrix data and IPA analysis revealed that after 6 hours of injection of PSG into the dermis, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) signalling pathways were enhanced (Table 1). This was supported by GO analysis, which revealed a wide range of growth factor signalling pathways to be enriched by PSG, including: VEGF, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), Wingless-related integration site (Wnt), insulin growth factor-1 (IGF1), EGF and Nerve growth factor (NGF) (S2 Table). By 4 hours post-wound, TNFα, EGF and EGFR expression increased 4-, 2- and 1.5-fold, respectively, compared to the PBS controls (Fig 3B and 3C). EGF acts in a paracrine fashion on keratinocytes by increasing their proliferation and their migration in wounds [51,52]. The influence of PSG in skin was to show a general increase in the expression of EGF and its receptor EGFR (Fig 3C). VEGF is expressed in keratinocytes [53] and is induced by HGF [54], resulting in increased migration and epithelialisation [55]. Inside a wound, AAMΦ initiate angiogenesis by releasing TNFα and VEGF [53].

In the proliferation phase of wound healing fibroblasts migrate, proliferate and synthesize extracellular matrix components including type I collagen [56]. This phase is induced by different cytokine and growth factors such as transforming growth factor beta (TGFβ), PGDF, FGF and IGF1 [43,56]. These signalling pathways were significantly upregulated in the presence of PSG (Table 1). In addition, some cytokine pathways involved in fibroplasia and granulation tissue formation were upregulated by the presence of PSG (Table 1). In particular, IL-4 and IL-6 signalling favour the synthesis of extracellular matrix molecules, including type I collagen and FGF and IGF1 promote the recruitment and differentiation of fibroblasts, and the production of extracellular matrix components [44,57,58]. Transcripts of FGFR2, IGF1 and IGF1R were significantly upregulated in presence of PSG (Fig 3C). Interestingly, we observed that in wounds PSG enhanced the early expression of the IGF1 receptor, followed six days later by enhanced the expression of IGF1 (Fig 3C); indicating that IGF1-signalling was promoted throughout the whole healing process.

PSG enhances the arginase activity of macrophages, production of arginase-inducing mediators and Leishmania survival

Alternatively activated macrophages and L-arginine metabolism are instrumental for efficient wound healing and Leishmania growth in the vertebrate host [44,57,58]. To assess whether PSG promoted the viability of parasites in the early intradermal injection site we measured small subunit ribosomal RNA (ssrRNA) parasite transcripts by RTqPCR in the same samples in which mouse transcripts were quantified. At 6 hours post-infection, we could detect an 8-fold increase of parasite transcripts in those ears co-injected with PSG, translating to a 4.3-fold increase in parasite burden (Fig 4A). From our Affymetrix and RTqPCR results of PSG inoculated into skin we found that PSG significantly increased the early expression of IL-10 and IGF1 (9 and 4-fold change respectively), both known inducers of arginase activity in macrophages [59,60]. This was confirmed in vitro when unstimulated bone-marrow macrophages (BMMΦ) were co-incubated with increasing amounts of PSG, IL-10 or IGF1 for 48 hours (Fig 4B). Arginase 1 in murine macrophages can be induced by Th2 cytokines or pathogen-induced TLRs via signal transducer and activator of transcription (STAT) factors STAT6 and STAT3, respectively [61,62]. Expression of classically activated/M1- and alternatively activated/M2-specific STAT transcription factors confirmed that BMMΦ exposed to the highest concentration of PSG suppressed interferon gamma (INF-γ)-responsive STAT1 and enhanced STAT6 and STAT3 (Fig 4C). This was associated with increases in the expression of STAT-signalling transcription factors: nuclear transcription factor kappa-B (NF-κB), inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β) and the TLR adaptor protein myeloid differentiation primary response gene 88 (MyD88)—used by almost all TLRs to activate NF-κB (Fig 4D). Exposure of STAT3 luciferase reporter HeLa cells to 1 μg PSG for 24 hours resulted in 3.1-fold enhanced luciferase activity, showing that the JAK-STAT receptor signalling pathway was activated by PSG and this could be partially blocked by inclusion of antibodies against the IGF1 receptor (S2 Fig). These data indicate that PSG promotes the alternative activation of macrophages in skin. Supporting this, we could detect an increased expression of many markers of alternatively activated myeloid cells in BALB/c ears 24 hours post-infection with or without PSG, namely: arginase 1 (Arg1) (but not mitochondrial arginase 2, Arg2), ornithine decarboxylase 1 (Odc1), chitinase-like protein 3 (Ym1), fibronectin 1 (Fn1); Arg1-inducers: IL-10, IL-6 and IGF1; L-arginine transporter Slc7a2 and L-arginine metabolism: inducible nitric oxide synthase (iNOS); but not expression of the prototypical alternative activator IL-4, production of IL-13 protein or expression of the IL-4/13 receptor IL-4Rα (Fig 4E). To test the role of IL-4 or IL-13 for the action of PSG during cutaneous leishmaniasis we co-infected WT and IL-4Rα^-/- BALB/c mice with a low dose of L. mexicana metacyclics in the presence or absence of PSG (Fig 5). This showed that PSG could exacerbate cutaneous lesion pathology of L. mexicana (Fig 5A & 5B) and promote parasite replication (Fig 5C) in the absence of IL-4 or IL-13 signalling, similar to WT mice. Taken together, these in vivo and in vitro results reinforce the PSG’s ability to manipulate macrophage alternative activation in favour of early Leishmania infection and survival potentially through STAT6 and STAT3 pathways, independently of IL-4Rα-signalling.

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Fig 4. The presence of PSG during an early Leishmania infection promotes the expression of genes and signalling pathways involved in macrophage alternative activation.

Ears of BALB/c mice were intra-dermally infected with 1 x 10³ L. mexicana metacyclic promastigotes with 0.5 μg L. mexicana PSG (grey bars) or PBS (white bars), or 5 x 10⁵ BMMΦ from BALB/c mice were exposed to L. mexicana PSG or PBS with or without L. mexicana metacyclic promastigotes (MOI 5:1). A) 48h post-infection expression of Leishmania ssrRNA in ears and the extrapolated parasite burdens, n = 6/group. B) Arginase activity of uninfected BMMΦ exposed to increasing concentrations of PSG, IL-10 or IGF1 for 48 hours. C) 48 h expression of signal transducer and activator of transcription (STAT) factors 1, 3 and 6 in 5 x 10⁵ infected BMMΦ in response to PBS, 0.5 μg/ml PSG or uninfected BMMΦ exposed to 20 U/ml recombinant murine IL-4. D) 48 h expression of STAT-signalling transcription factors: NF-κB, Ikk-β and MyD88 in infected BALB/c macrophages co-incubated with PBS or 0.5μg PSG.E) 48 h expression of markers of macrophage alternative activation or arginine metabolism measured in infected BALB/c ears in response to intradermal injection of PBS or 0.5 μg/ml PSG by real-time quantitative PCR. IL-13 production was determined from whole cell lysates using Luminex. Data is representative of triplicate experiments. Relative expression was normalised to the housekeeping genes ywhaz and nono and is presented as the mean ±SD (*: p<0.05, **: p<0.005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g004

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Fig 5. PSG does not require IL-4Rα signalling to exacerbate cutaneous leishmaniasis in mice.

A and B) Footpad infections of WT (A) and IL-4Rα^-/- (B) BALB/c mice with 1 x 10³ L. mexicana metacyclic promastigotes intradermally injected with PBS or 0.5 μg L. mexicana PSG, 6 mice per group. C) Final parasite burdens of footpads from B) at the end of the experiment (WT: 80 d post-infection, IL-4Rα^-/-: 151 d post-infection). Data pooled from two replicate experiments. A&B) Averages ±SEM are shown, C) Each point represent individual mice, bars indicate the mean (*: p<0.05; **: p<0.005; ***: p<0.0005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g005

PSG enhances both wound healing and Leishmania infection by sand fly bite through insulin-like growth factor 1 signalling in vivo

Knowing that PSG promoted dermal wound repair and enhanced L. mexicana infections in vivo, we next wanted to test the role of IGF1 on both these outcomes. IGF1 is known to promote the arginase activities of both parasite and host macrophage alike to enhance Leishmania infection in vitro and in vivo [63–65]. From our microarray results, IGF1-signalling was one of the highest-ranking pathways influenced by PSG (Table 1). By combining PSG with anti-IGF1R Ab we could significantly reduce the gel’s ability to promote the healing of 2 mm diameter full-thickness wounds to BALB/c mouse ears (Fig 6A). Furthermore, conditioning skin with anti-IGF1R Ab 4 hours prior to L. mexicana infection by a single infected Lu. longipalpis bite (average ± SD, 4.1 x 10⁴ ± 1.18 x 10⁴ promastigotes; 65% ± 17% metacyclics per gut) resulted in a significant delay in cutaneous lesion appearance and consistently weaker lesion growth (Fig 6B). At the end of the experiment, approximately 60 days post-infection, the average lesion size in the antibody-treated mice were some 53% smaller than the isotype antibody controls. Analysis of the parasite burdens of these footpad lesions confirmed that sand fly delivery of Leishmania to skin pre-treated with anti-IGF1R Ab significantly reduced their ability to proliferate (Fig 6C, 20-fold decrease, p = 0.021). Collectively, these results show that IGF1-signalling significantly contributes to the PSG’s ability to enhance wound healing and Leishmania growth in skin.

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Fig 6. Insulin-like growth factor 1-signalling controls the wound healing and Leishmania-exacerbating properties of PSG in mice.

A) Rate of 2 mm diameter wound closure in BALB/c ears preconditioned with PBS, 0.5 μg L. mexicana PSG or 0.5 μg L. mexicana PSG + 1:50 anti-IGF1R antibody, 12 mice per group. B) Single L. mexicana-infected sand fly bite infections in footpads of BALB/c mice treated with or without 1:50 anti-IGF1R antibody, 6 mice per group. C) Final parasite burdens of footpads from B) at the end of the experiment. Data pooled from two replicate experiments. A&B) Averages ±SEM are shown, C) Each point represent individual mice, bars indicate the mean (*: p<0.05; **: p<0.005; ***: p<0.0005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g006

PSG regurgitated during infected sand fly bite promotes IGF1 and IL-10 expression and enhances transmissible macrophage infections via arginase

Having confirmed a role for IGF1-signalling for natural Leishmania infection by sand fly bite, we next dissected the contribution made by the various components of transmission: parasites, PSG and sand fly saliva. During transmission, infected sand flies generate a unique wound to a very specific compartment of skin that is difficult to replicate using needles. Using uninfected and L. mexicana-infected sand flies (with mature, transmissible infections: average ± SD, 3 x 10⁴ ± 1.63 x 10⁴ promastigotes; 58% ± 9% metacyclics per gut), we compared dermal IGF1 expression in response to individual sand fly bites. In this way, each bite received the same wound and deposition of saliva yet the infected bites would additionally receive parasites and PSG. RTqPCR of bitten skin revealed that IGF1, IGF1R and IL-10 expression were significantly higher following infected sand fly bites compared to uninfected bites (IGF1 and IGF1R: p <0.001; IL-10: p <0.05) (Fig 7A). Further analysis of the infected bites by RTqPCR to determine the number of L. mexicana parasites transmitted (<10 to >10³) revealed an association between the dose of parasites delivered per bite with IGF1, IGF1R and IL-10 expression (Fig 7B). Using the L. mexicana-Lu. longipalpis model of natural infection we previously demonstrated a positive relationship between the infective dose and amount of egested PSG [12], which is consistent with one of the main functions of the PSG plug—to block the anterior sand fly midgut for parasite regurgitation. Collectively, this points toward PSG as the main component of natural Leishmania infection able to manipulate IGF1 expression and signalling at the site of transmission. To test this, we intradermally injected PSG, saliva and a mixture of PSG and saliva without Leishmania into BALB/c ears and found that PSG was the only component of transmission could increase local IGF1, IGF1R and IL-10 expression 6 hours later (Fig 7C: 28-, 2- and 13-fold increase respectively, p <0.005). In contrast, sand fly saliva was only able to moderately enhance IL-10 expression. Furthermore, the combination of PSG with sand fly saliva was still capable of promoting IGF1, IGF1R and IL-10 expression. These data show that regurgitated PSG manipulates IGF1-signalling at the site of transmission.

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Fig 7. PSG enhances IGF1 expression in skin following an infected fly bite and enhances macrophage alternative activation.

A) IGF1, IGF1R and IL-10 expression in BALB/c mouse ears 6 hours post-bite from single uninfected or L. mexicana-infected Lu. longipalpis sand flies (Day 8 post-infection), one bite per ear (12 uninfected bites, 35 infected bites). B) IGF1, IGF1R and IL-10 expression in bitten ears in relation to the dose of transmitted parasites, determined from Leishmania ssrRNA expression in ears. C) IGF1, IGF1R and IL-10 expression in BALB/c ears 6 hours following intradermal injection of with PBS, 0.5 μg L. mexicana PSG, 0.5 μg Lu. longipalpis saliva or PSG and saliva combined. Data is pooled from duplicate experiments with 12 mice per group. Relative expression was normalised to the housekeeping genes nono and l19 and is presented as the mean ±SD (*: p<0.05, **: p<0.005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g007

PSG can manipulate host macrophage arginase activity to promote the intracellular growth of L. mexicana [12]. To test the role of PSG-IGF1-signalling for macrophage alternative activation we repeated our experiments using in vitro BMMΦ and assessed the role of PSG and IGF1 on parasite viability and host cell arginase activity (Fig 8A & 8B). To determine parasite viability amastigotes were liberated from infected BMMΦ and allowed to transform into flagellated promastigotes in vitro–mimicking their onward transmission to sand flies. Similar to our previous findings [12], we found that PSG could promote the survival and growth of amastigotes by enhancing the arginase activity of BMMΦ, which was sensitive to the host-specific arginase inhibitor nor-NOHA. Similarly, the presence of a monoclonal antibody to the IGF1R reduced macrophage infection and arginase activity in PSG-treated cells but not in the saline-treated or isotype antibody controls. Moreover, this could be partially restored by adding L-ornithine—the product of arginase activity on its substrate L-arginine; thereby bypassing arginase for the biosynthesis of polyamines. This indicates that IGF1-blockade affected the macrophage or parasite’s polyamine biosynthetic pathways, reducing the benefit of PSG to amastigotes growth by an average of 72% (p = 0.026). These data show that PSG potentiates host cell activation and polyamine metabolism through IGF1-signalling.

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Fig 8. PSG manipulates macrophage arginases to promote intracellular Leishmania growth in vitro and in vivo via IGF1 signalling.

A) Viable amastigote burdens of macrophages infected in vitro was determined by transformation assay of amastigotes into promastigotes liberated from 5 x 10⁶ BALB/c BMMΦ. Macrophages were infected with L. mexicana metacyclic promastigotes (MOI 1:1) in the presence of ±1 μg L. mexicana PSG; ±1:100 anti-IGF1R Ab or isotype control; ±100 μM nor-NOHA; ±100 μM L-ornithine for 48 hours. B) Arginase activity of duplicate infected macrophages from (A). C) Parasite burden of BALB/c dermal air-pouches infected with 1x10⁶ L. mexicana metacyclic promastigotes ±1 μg PSG, ±1:50 anti-IGF1R Ab; ±100 μg L-ornithine for 48 hours. Macrophages were recovered from infected air-pouches and separated by adherence to plastic. Data is representative of triplicate experiments (A&B) or pooled from duplicate experiments (C, 8 mice per group). Unless they are linked with a bar all test groups are compared to their relevant saline control; (ns, not significant p>0.05; *: p<0.05; **: p<0.005; ***: p<0.0005 by Mann Whitney t-test).

https://doi.org/10.1371/journal.ppat.1006794.g008

Lastly, to demonstrate that higher IGF1 expression by macrophages exposed to PSG and IGF1-signalling exacerbated Leishmania infections in vivo we infected dermal air-pouches with 10⁶ L. mexicana metacyclic promastigotes on BALB/c mice treated with or without anti-IGF1R Ab (Fig 8C). Transformation and growth of amastigotes (as promastigotes) obtained from the infected macrophages recruited to and recovered from the air-pouches revealed that parasite burdens were 8.6-fold higher in those infections receiving PSG without antibody (p = 0.0003), confirming the exacerbatory role of PSG for macrophage infection in vivo in skin. In contrast, blockade of IGF1-signalling reduced the exacerbating effect of PSG by 36%, which could be restored with L-ornithine supplementation. Collectively, these data demonstrate the importance of IGF1 for the Leishmania-enhancing properties of PSG in skin shortly after transmission—in order to benefit Leishmania infection and onward transmission.