The sequence of the P17 peptide (LFKEILEKIKAKL-NH2) was characterized by de novo sequencing using mass spectrometry and Edman degradation (31). P17 peptide and its randomly designed C-terminal amidated scrambled counterpart (KIKEEKFLLKLI-NH2) were synthesized on a Liberty microwave assisted automated peptide synthesizer (CEM, Saclay, France) at a purity grade higher than 99% as previously described (31, 47). The authenticity and the molecular identity of the synthetic peptides were controlled by MALDI-TOF-MS.

Human peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by a density gradient centrifugation method on Lymphoprep (Abcys). Monocytes were isolated from mononuclear cells by adherence to plastic for 2 h in special macrophage serum-free medium (SFM; Life Technologies) with l-glutamine at 37°C in a humidified atmosphere containing 5% CO₂. Non-adherent cells were removed by washing with Hanks’ balanced salt solution (HBSS) (Gibco, Invitrogen), and the remaining adherent cells (>85% monocytes) were incubated in SFM medium. The h-MDMs were obtained after 24 h of culture in SFM medium. Adherent h-MDMs were pre-incubated 30 min or not before the addition of P17 (200 µg/ml) with GW9662 (1 nM; Santa Cruz Biotechnology), MAFP (20 µM; Calbiochem), MK-886 (10 µM; Calbiochem), N-acetyl-cysteine (10 mM; Sigma), or Z-Vad-FMK (ZVAD) (50 µM; Calbiochem) for 30 min.

The strain of C. albicans used throughout these experiments was isolated from a blood culture of a patient in the Toulouse-Rangueil University Hospital (98/26135). The isolate was identified as C. albicans based on common laboratory criteria and cultured on Sabouraud dextrose agar (SDA; Biorad, Hercules, CA, USA) plates containing gentamicin and chloramphenicol. C. albicans was maintained by transfers on SDA plates. Growth from an 18- to 24-h SDA culture of C. albicans was suspended in sterile saline buffer (HBSS; Life Technologies). In all experiments, the h-MDMs were challenged with blastospores.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and were allowed to interact for 40 min at 37°C with C. albicans blastospores (at a ratio of 0.3 yeast per macrophage) as previously described (36, 40). Unbound yeasts were removed by four washes with medium. h-MDMs were then incubated at 37°C for 4 h. After incubation, the medium was removed and cells were lysed. The CFU of C. albicans were quantified after plating on Sabouraud plates for 24–48 h at 37°C.

In some experiments, monocyte-derived macrophages were incubated with 1 µg per well of MR siRNA (Santa Cruz Biotechnology sc-45360) and/or Dectin-1 siRNA (Santa Cruz Biotechnology sc-63276) for 6 h into siRNA transfection medium (siRNA reagent system, Santa Cruz Biotechnology sc-45064) according to the manufacturer’s instructions before the addition of P17.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and were then challenged with GFP-labeled yeasts at a ratio of 6 blastospores per macrophage. In some experiments, the monocyte-derived macrophages were incubated at 4°C for 20 min with 500 µg/ml of mannans and/or laminarins before the addition of the yeasts. The binding was performed at 4°C and the phagocytosis was initiated at 37°C. The number of C. albicans bound or engulfed by macrophages was determined by fluorescence quantification using the Envision fluorimetry-based approach (Perkin Elmer).

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and ROS production was measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol; Sigma) using a thermostatically (37°C) controlled luminometer (Envision; Perkin Elmer). The generation of chemiluminescence was monitored continuously for 1 h and 30 min after challenge or not with C. albicans blastospores (yeast-to-macrophage ratio: 3:1). Statistical analysis was performed using the area under the curve expressed in counts × seconds.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and challenged with C. albicans blastospores at a ratio of 3 yeasts per macrophage for 8 h. The release of TNF-α, IL-1β, IL-12, and IL-10 in the cell supernatants was determined with a commercially available OptiEIA kit (BD Biosciences) according to the manufacturer’s instructions.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and challenged for 8 h with C. albicans blastospores at a ratio of 3 yeasts per macrophage. h-MDMs were lysed with RIPA buffer (Sigma) and protein extracts were separated in SDS-PAGE as previously described (37). After protein transfer, membranes were incubated overnight at 4°C with a rabbit anti-phosphorylated p47^phox (Assay biotechnology, A1171; 1/260), a rabbit anti-caspase-1 (Biovision, 3019-100; 1/100), or a goat anti-GAPDH antibody (Cell Signaling, #5174; 1/1,000) and then for 1 h at room temperature with a peroxidase conjugated secondary antibody. Membranes were washed, and proteins of interest were visualized with WesternBright™ ECL (Advansta) or the SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) for the GAPDH. Images have been cropped for presentation.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and then incubated with ice-cold PBS and gently scraped. Collected cells were centrifuged at 1,500 rpm for 10 min, and the cell pellet was suspended in PBS medium supplemented with 1% fetal calf serum (FCS). Surface expressed MR, Dectin-1, DC-SIGN, CD16, or CD36 was detected, respectively, using PerCP-Dectin-1 monoclonal antibody (mAb; R&D FAB1859C-100, 1/40), PE-DC-SIGN (mAb; BD Biosciences 551 265, 1/20), FITC-CD16 (mAb; PNIM 0814, 1/20), or APC-CD36 monoclonal antibodies (mAb; BD Biociences 550 956, 1/40). To evaluate the MR surface expression, we have used MR-specific ligand conjugated with FITC (Sigma A7790, 1 mg/ml, 1/100). All staining were performed on PBS^−/− 1% FCS medium. A population of 10,000 cells was analyzed for each data point. All analyses were carried out in a Becton Dickinson FACScalibur using the FACSDiva version 6.2 software.

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) and/or LTB4 (100 nM; Cayman Chemical Company) for 8 h.

The mRNA preparation was made using the EZ-10 Spin Column Total RNA Minipreps Super Kit (Bio Basic) using the manufacturer’s protocol. Synthesis of cDNA was performed according to the manufacturer’s recommendations (Thermo electron). RT-qPCR was performed on a LightCycler 480 system using LightCycler SYBR Green I Master (Roche Diagnostics). The primers (Eurogentec) were designed with the software Primer 3. GAPDH mRNA was used as the invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in Table 1.

Human monocyte-derived macrophages were pre-labeled with [³H]AA. Briefly, h-MDMs (5 × 10⁵ per well in 48-well plates) were cultured for 18 h at 37°C in the presence of P17 (200 µg/ml) and 1 μCi/ml [³H]AA. The culture medium was then removed and pre-labeled macrophages were washed three times with 0.5 ml SFM. The cells were challenged with C. albicans blastospores at a ratio of 3 yeasts per macrophage for 2 h. The [³H]AA metabolites released into the culture medium were quantified by measurement of the radioactivity by beta liquid scintillation counting using a 1217 Wallac Rackbeta LKB 1217, as previously described (37).

Intracellular calcium concentration was measured using the fluorescent probe Fluo 3-AM (Molecular Probe). Briefly, h-MDMs (1.5 × 10⁵) were incubated with 11.5 × 10⁻⁶ M Fluo 3-AM for 30 min at 37°C. The time course of the intracytosolic Ca²⁺ level was recorded every 0.5 s for a total period of 3 min after the addition of P17 (200 µg/ml). In desensitization experiments, a second injection of bacterial N-formylmethionine-leucyl-phenylalanine peptide (fMLP) or P17 was performed at the end of fluorescence record and the time course of the intracytosolic Ca²⁺ level was recorded for supplemental 3 min. In some experiments, h-MDMs were pre-incubated with U73122 (2 µM) or with calcium-free HBSS for 10 min before the addition of P17. Fluorescence was quantified using the Envision fluorimetry-based approach (Perkin Elmer).

Male mice aged 12 weeks on C57BL/6 background were used for in vivo experiments. Mice were bred and handled by the protocols approved by the Conseil Scientifique du Centre de Formation et de Recherche Experimental Medico Chirurgical and the ethics board of the Midi-Pyrénées ethic committee for animal experimentation (Approval no B3155503). All cages were changed twice weekly, and all manipulations of the animals were carried out in a laminal blow hood under aseptic conditions. The photoperiod was adjusted to 12 h light and 12 h dark. C57BL/6 mice were purchased from Janvier (France).

The gastrointestinal infection (GI) with the C. albicans was established by oral infection with 50 × 10⁶ C. albicans per mouse. To establish esophageal and GI candidiasis in mice, we performed intra-esophageal infection with 5 × 10⁷ viable cells of C. albicans in sterile saline solution. No antibiotic or immunosuppressive treatment was used to facilitate mucosal infection by C. albicans of the oral cavity and GI tract.

Mice were treated intraperitoneally with P17 (10 µg per mouse) 1 day before the day of the infection with C. albicans and then every 2 days (four injections). Control groups received saline solution. Therapeutic studies were performed on separate groups of six mice infected with C. albicans treated or not with P17.

The body weight of each mouse was recorded daily, and the condition of each mouse was assessed twice daily. Feces were collected on days 4 and 5 post-infections. At day 6 post-infection, all mice were euthanized using CO₂ asphyxia and the cecum and colon were aseptically removed to evaluate C. albicans colonization. To quantify the number of viable C. albicans in cecum, colon, and feces, each tissue sample removed was mechanically homogenized in 1 ml of saline with 100 U of penicillin/ml and 100 µg of streptomycin/ml. Serial dilutions of homogenate were plated onto SDA for quantitative determination of the number of C. albicans in the tissue samples. Plates were incubated at 37°C for 1–2 days and the number of colonies was counted.

After euthanasia, resident peritoneal cells were harvested by washing the peritoneal cavity with 5 ml of sterile 199 medium with Hanks’ salts as previously described (6). Collected cells were centrifuged at 400 × g for 8 min, and the cell pellet was suspended in SFM optimized for macrophage culture (Invitrogen Life Technologies). Cells were allowed to adhere for 2 h at 37 C and 5% CO₂. Non-adherent cells were then removed by washing with HBSS. The macrophage monolayers were used to investigate their capacity to bind, to engulf and to kill C. albicans, and to produce ROS and IL-1β in response to yeast challenge.

For each experiment, the data were subjected to one-way analysis of variance followed by the means multiple comparison method of Bonferroni–Dunnett. p < 0.05 was considered as the level of statistical significance.

In order to assess the impact of P17 in h-MDMs differentiation, we evaluated the expression of specific markers of classical and alternative activation in P17-treated h-MDMs. Overall, P17-treated h-MDMs displayed a downregulation of membrane receptors characteristics of classical M1 polarization, such as CD11b (Itgam), CD16 (Fcgr3), and CD32 (Fcgr2), which was mirrored by an upregulation of MR (Mrc1), Dectin-1 (Clec7a), DC-SIGN (Cd209), and CD36 (Cd36) alternative activation markers (Figure 1A). This finding was further supported by the reciprocal increase of anti-inflammatory IL-10 (Il10), IL-1 receptor antagonist (Il1ra), TGFβ (Tgfb1), CCL-17 (Ccl17), and CCR2 (Ccr2) and reduction of IL-12 (Il12) and CCL-2 (Ccl2) mRNA expression. Moreover, consistent with increased mRNA encoding alternative M2 activation markers, protein levels of MR, Dectin-1, and IL-10 were significantly increased (Figures 1B,C). The acquisition of this alternative phenotype upon P17 activation is reinforced by the strong decrease of IL-12 protein level (Figure 1C). We observed unaffected protein amounts for DC-SIGN, CD16, and CD36 (Figure 1B).

Surprisingly, the increase in alternative markers in P17-treated h-MDMs was accompanied by an inflammatory signature, as demonstrated by an augmentation in the mRNA level of the pro-inflammatory cytokines IL-1β (Il1b), TNF-α (Tnfα), and IL-6 (Il6). Consistently, the production of IL-1β and TNF-α was also induced in P17-treated h-MDMs (Figure 1C). In line with increased IL-1β production in P17-treated h-MDMs, the processing of pro-caspase-1 into its p20 subunit, which is a hallmark of caspase-1 activation, was significantly increased in P17-treated h-MDMs, demonstrating that the caspase-1-induced IL-1β release is activated by P17 (Figure 1D).

To further explore the pro-inflammatory impact of P17 on h-MDMs, we next assessed ROS production by P17-treated h-MDMs. We demonstrated that ROS release was strongly increased in P17-treated h-MDMs (Figure 1E). In line, the mRNA level and the amount of phosphorylated p47^phox, a cytosolic subunit of the NADPH oxidase complex whose activation is essential to ROS release, were significantly increased in h-MDMs activated by P17 (Figures 1F,G).

Taken together, these results indicated that P17 induces an alternative phenotype characterized simultaneously by a CLRs signature composed of MR and Dectin-1 and a pro-inflammatory profile.

The nuclear receptor PPARγ is a key component of the signaling pathway leading to alternative activation of macrophages and directly controls the expression of CLRs (34–36). To identify how P17 may have an impact on MR and Dectin-1 overexpression, we evaluated whether P17 can regulate PPARγ activation. The mRNA levels of PPARγ (Pparg) and of its target gene SRB1 (Scarb1) were significantly increased in P17-treated h-MDMs, suggesting that P17 improved PPARγ activation.

To further explore whether P17-induced PPARγ activation was involved in MR and Dectin-1 overexpression, we evaluated the gene expression of these two CLRs in the presence of GW9662, a selective PPARγ antagonist. The treatment of P17-activated h-MDMs with GW9662 inhibited Mrc1 and Clec7a gene overexpressions, demonstrating that PPARγ is critically required for P17-induced Dectin-1 and MR expression on human h-MDMs (Figure 2A).

Peroxisome proliferator-activated receptor gamma is activated by endogenous ligands derived from AA (34, 35, 43, 48). The COX-1/2 cyclooxygenase, 5- and 12/15-LOX are considered to be critical for the conversion of AA into endogenous PPARγ ligands. To assess whether P17 can coordinate PPARγ ligand availability, we evaluated the gene expression of enzymes involved in both COX and LOX signaling pathways. The mRNA levels of cPLA2 (Pla2g4a), enzyme needed for AA release from membrane phospholipids, 5-LOX (Alox5), FLAP (5-LOX activating enzyme) (Alox5ap), and LTA4 hydrolase (Lta4h), critical for LTB4 synthesis, were strongly increased in P17-treated h-MDM (Figure 2B). Moreover, the mRNA levels of COX-2 (Ptgs2), PGES (Ptges), and 15-LOX (Alox15) were not differentially expressed in untreated and P17-treated h-MDMs, supporting that P17 had no incidence on COX-2 and 12/15-LOX signaling pathways.

Then, we determined whether these effects on gene expression also translate into changes in ligand availability. Consistent with cPLA2 gene overexpression, the mobilization of AA was induced in P17-treated h-MDMs (Figure 2C). Furthermore, in line with LTA4 hydrolase expression in P17-treated h-MDMs, we observed strong increase of LTB4 release (Figure 2D). These data suggest that the generation of LTB4 metabolites by h-MDMs upon P17 treatment is dependent both on AA mobilization and metabolism.

To confirm that P17 positively regulates MR and Dectin-1 expressions through the AA metabolism, we determined Mrc1 and Clec7a gene expression in the presence of MAFP, a specific cPLA2 inhibitor, and MK-886, a FLAP inhibitor, which prevents 5-LOX activation. The increase of MR and Dectin-1 mRNA levels in P17-treated h-MDMs was completely lost in the presence of MAFP and MK-886 (Figure 2E), further supporting that P17 controls MR and Dectin-1 expressions through AA metabolism.

Peroxisome proliferator-activated receptor gamma activity, as determinate by the induction of MR-, Dectin-1- and SRB1-specific PPARγ target genes, was increased similarly with P17 and LTB4 (Figure 2F). Furthermore, the addition of both P17 and LTB4 did not showed any additive effect, suggesting that P17 regulates MR and Dectin-1 surface expression by controlling PPARγ activation through the LTB4 production. Interestingly, the inhibition of P17-induced PPARγ target genes expression by MAFP was restored by the addition of LTB4, clearly establishing that P17-mediated PPARγ activation through LTB4 synthesis. Overall these data showed that P17 induces alternative activation of h-MDMs characterized by Dectin-1 and MR expressions through LTB4-mediated PPARγ activation.

Previous works from our laboratory established the importance of alternative activation in the fungicidal functions of macrophages (34, 35, 43). On the basis of the current findings demonstrating an effect for P17 on alternative polarization, we next investigated whether P17-treated h-MDMs could have an impact on the C. albicans clearance. P17 did not exhibit an effective anti-fungal activity against C. albicans relative to the conventional amphotericin B anti-fungal agent. Furthermore, the association of P17 with amphotericin B did not improve the AMB anti-fungal activity, supporting that this peptide could not be used as a direct anti-fungal agent (Figure S1A in Supplementary Material). After P17 treatment, h-MDMs showed a robust increase in their ability to kill C. albicans, demonstrating that P17 promotes macrophage-intrinsic anti-fungal activity and supporting the use of P17 as a promising immunomodulatory compound to restrain fungal infections (Figure 3A). Consistent with our observation, P17-treated h-MDMs were more efficient to bind and to engulf C. albicans (Figures 3B,C). Interestingly, the induction of ROS production by P17 is essential in in vitro fungicidal activity of P17-activated h-MDMs, since the use of antioxidant N-acetyl cysteine (NAC) totally abolished the fungicidal effect of P17-treated h-MDMs against C. albicans (Figure 3D).

Then, we examined whether ROS production, which act as a common cellular signal upstream of the inflammasome activation (49), was responsible for IL-1β induction by P17 in response to Candida. While the antioxidant NAC suppressed Candida-induced IL-1β secretion by P17-activated h-MDMs, the inhibition of caspase activation by the addition of ZVAD did not change ROS production by P17-activated h-MDMs after Candida challenge (Figures 3E,F). Therefore, these results showed that in response to Candida, ROS production occurred upstream of the caspase-1-induced IL-1β production.

To determine whether P17-mediated PPARγ activation dependently on LTB4 synthesis was involved in fungicidal activity of P17-treated h-MDMs, we evaluated their ability to kill C. albicans in the presence of a selective PPARγ antagonist GW9662, a specific inhibitor of AA mobilization MAFP, or an inhibitor of 5-LOX activation MK-886. The increased capacity of P17-treated h-MDMs to kill C. albicans was totally inhibited in presence of GW9662, MAFP, and MK-886 (Figure 3G). Consistent with these findings, the addition of these inhibitors abolished P17-mediated ROS, IL-1β release, and caspase-1 activation of h-MDMs in response to C. albicans (Figures 3H–J).

To explore whether the increased expression of Dectin-1 and MR on P17-treated h-MDMs has any functional consequence in C. albicans elimination, we assessed the ability of P17-treated h-MDMs deficient for Dectin-1 and/or MR to kill the yeast. The gene silencing for Dectin-1 and/or MR in P17-treated h-MDMs resulted in the abolition of the ability of these cells to kill more efficiently C. albicans (Figure 3K). In terms of recognition of C. albicans, the pre-treatment of P17-activated h-MDMs with soluble MR and Dectin-1-blocking agents (mannan and/or laminarin, respectively) abrogated the increased capacity for P17-treated h-MDMs to interact with the yeast (Figure 3L). Moreover, this pre-treatment totally abolished the induction of ROS production after C. albicans challenge (Figure 3M).

Taken together, these data provided evidence that MR and dectin-1 are critical in fungicidal properties of h-MDMs mediated by P17 and support the importance of AA/LTB4/PPARγ/Dectin-1-MR axis in the strengthening of anti-fungal functions of h-MDMs by this HDP.

Several studies demonstrated that HDPs induce intracellular calcium mobilization in immune cells (50–53). On this basis, we evaluated cytosolic calcium concentration in P17-activated h-MDMs. P17 treatment induced a significant augmentation of intracellular calcium concentration in h-MDMs, indicating that this HDP triggers intracellular calcium signal (Figure 4A). In order to determine the source of this calcium release, we assessed calcium mobilization in calcium-deprived medium to inhibit extracellular calcium influx (HBSS) and in the presence of U73122, an inhibitor of the intracellular pools calcium release. Interestingly, calcium mobilization was abolished both in presence of HBSS and U73122 (Figure 4A), suggesting that the P17-increase intracellular calcium concentration in h-MDMs was dependent on extracellular calcium influx and intracellular calcium stores mobilization.

Most of the HDPs immunomodulatory functions are mediated through GPCRs (27–29, 51). In order to evaluate the involvement of these receptors in the activation of h-MDMs mediated by P17, we used pertussis toxin (PTX), known to inhibit some GPCRs. PTX treatment totally inhibited the mobilization of calcium in P17-activated h-MDMs (Figure 4B), suggesting that P17 activity on h-MDMs was mediated by PTX-sensitive GPCR interaction. Interestingly, although PTX-sensitive fMLP receptors 1 and 2 (FPR1 and FPR2) are the main GPCRs involved in the modulation of immune response by HDPs, we demonstrated here that the desensitization of these receptors by the addition of fMLP did not affect P17-induced calcium mobilization (Figure 4C).

To investigate the role of P17-induced calcium mobilization in the fungicidal properties of P17-treated h-MDMs, we evaluated their capacity to kill the yeasts in the presence of an intracellular calcium chelator BAPTA-AM. The increase of ability to kill C. albicans of P17-activated h-MDMs was completely lost in the presence of BAPTA-AM (Figure 4D). These findings were consistent with the lack of induction of ROS and IL-1β production in BAPTA-AM pretreated h-MDMs activated with P17 (Figures 4E,F), further supporting that the intracellular mobilization of calcium is essential in fungicidal properties of P17-treated h-MDMs. Altogether, these data highlight that P17 modulates anti-fungal immune response of h-MDMs through PTX-sensitive GPCR-triggered intracellular calcium mobilization.

On the basis of the current findings demonstrating the microbicidal activity of P17-treated h-MDM against C. albicans, we next explored whether the P17 treatment could have an impact on the in vivo candidiasis outcome. In this context, we evaluated the fungal burden in the intestinal tract and the macrophage microbicidal functions in a murine experimental model of gastrointestinal candidiasis. P17-treated mice (P17) infected with C. albicans developed less severe gastrointestinal infection than untreated mice (NaCl), as demonstrated by lesser weight loss and by reduced fungal burden as reflected by a decreased number of CFU in the colon, cecum, and feces of mice treated with P17 (Figures 5A,B).

To investigate whether the effect of P17 on decreased Candida gastrointestinal colonization can be correlated to its impact on fungicidal functions of macrophages, we evaluated the capacity of macrophages from P17-treated mice to kill yeasts in vitro. Compared to macrophages from untreated mice, macrophages from P17-treated mice showed an increase in their ability to kill C. albicans (Figure 5C). Consistent with our observation, macrophages from P17-treated mice were more efficient in engulfing C. albicans and producing ROS and IL-1β (Figures 5D–G). Moreover, IL-12 release was similar in macrophages from untreated and P17-treated mice, demonstrating that P17 in vivo administration did not impact on IL-12 macrophage production (Figure 5G).

Taken together, these data provide in vivo evidence that P17 improves macrophage-intrinsic anti-fungal activity and support that P17 may constitute promising compound to restrain gastrointestinal fungal infection.