C57BL/6 mice were purchased from Charles Rivers. 4get, and KN2 mice (22, 23) were a gift from Markus Mohrs, Trudeau Institute, USA. Keratinocyte cell-specific 4get/KN2 mice were obtained by the breeding of 4get and KN2 mice. IL-4Rα-deficient (KRT14^creIL-4Rα^−/lox) mice on the C57BL/6 genetic background were generated using the Cre/loxP system. Briefly, KRT14^cre mice on the BALB/c genetic background (Jackson Laboratory) were first backcrossed on the C57BL/6 genetic background for nine generations. The progeny were crossed with IL-4Rα^−/− C57BL/6 mice and transgenic IL-4Rα^lox/lox mice (24) to generate hemizygous KRT14^creIL-4Rα^−/lox C57BL/6 mice. Genotyping of KRT14^creIL-4Rα^−/lox C57BL/6 mice was performed using specific primers: KRT14 P1, 5’- TTC CTC AGG AGT GTC TTC GC; KRT14 P2, 5’- GTC CAT GTC CTT CCT GAA GC; KRT14 P3, 5’- CAA ATG TTG CTT GTC TGG TG; KRT14 P4, 5’- GTC AGT CGA GTG CAC AGT TT. Characterization was also performed by flow cytometry using the anti-IL-4Rα mAb [anti-CD124, anti-IL-4Rα-PE from (BD, Pharmingen)]. Mice were bred under pathogen-free conditions at the Epalinges Center. 5- to 8-week-old females were used.

All animal experimental protocols were approved by the veterinary office regulations of the State of Vaud, Switzerland, authorization 1266.6-7 to FTC and performed in compliance with Swiss ethics laws for animal protection. All mouse experiments performed at the University of Cape Town were performed in strict accordance with the South African national guidelines, as well as the Animal Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town (license no. 015/034). All efforts were made to minimize and reduce suffering of animals.

Leishmania major (LV39, MRHO/Sv/59/P strain) or in selected experiments GFP-expressing L. major IL-81 (MHOM/IL/81/FEBNI) parasites were cultured at 26°C in complete M199 medium (10% fetal bovine serum, 4% HEPES, 1% penicillin, streptomycin, and neomycin). 10⁴ or 2 × 10⁵ L. major metacyclic promastigotes were prepared from confluent stationary phase promastigotes by Ficoll gradient density centrifugation as previously described (25) and injected in the ear dermis or footpad. Lesion development was monitored using Vernier caliper, ear lesion diameter was measured for intradermal infection. For IL-4 depletion, 2 × 10⁵ metacyclic parasites (±1 µg 11B11 antibody) were needle injected into the ear dermis in iDMEM in a final volume of 10 µL, or in the hind footpad in a volume of 50 µL. Four hours later, 1 µg of 11B11 mAb was injected intradermally (i.d.) in a final volume of 10 µL. For the induction of a Th2 response in C57BL/6 4get/KN2 mice, 1 mg of αIFNγ (XMG1.2) and αIL-12 were injected intraperitoneally 24, 2 h prior and 48 h post intradermal infection of 2 × 10⁵ L. major metacyclic promastigotes.

Leishmania major-specific IgG1 and IgG2c levels were determined by ELISA in the sera of mice harvested at the termination of the experiment, as previously described (26), Biotinylated goat anti-mouse IgG2c (Southern Biotech) and biotinylated rat anti-mouse IgG1 (BD Pharmingen) were used. Plates were read at the optical density of 490 nm. Titration curve were performed for all samples.

Infected ears, spleen, and dLNs were isolated and processed to single cell suspensions. Briefly, ears were recovered, homogenized in iDMEM containing 0.2 mg/mL Liberase TL (Roche) for 2 h at 37°C and then homogenized and filtered using 40-µm filters (Falcon). dLN and spleen cells were isolated, homogenized, and stained for flow cytometry analysis. For intracellular cytokine staining, 1 × 10⁶ dLN cells were stimulated with 50 µg/mL PMA, 500 µg/mL Ionomycin, and 1 µg/mL Brefeldin A for 4 h prior staining. Live parasites were determined by limiting dilution assay (LDA) as described previously (27).

10⁶ dLN cells were cultured in presence or absence of UV-irradiated stationary phase parasites (parasites were exposed to UV.C for 3 min, at a distance of 10 cm from the UV lamp) L. major promastigotes (MOI of 5:1) for 72 h at 37°C. Supernatants were recovered and IL-4 production measured by ELISA following manufacturer’s instructions (BD Biosciences; R&D). IFNγ secretion was detected using a homemade ELISA kit as previously described (28).

Primary keratinocytes were isolated from the skin of adult mice tail. Briefly, the skin from the tail was collected and incubated at 4°C for 16 h in a solution of 5 U/mL Dispase (STEMCELL) supplemented with 1% PNS (Gibco) and 0.5% Gentamicin (Gibco). The epidermis was separated from the dermis and treated with 0.2% trypsin (Gibco) for 5 min at 37°C. The reaction was stopped with FCS, and cells were collected by crushing the epidermis with a 100-µm cell strainer (Falcon). 7.5 × 10⁵ cells/mL were plated in a 6-well plate coated with Type I collagen (STEMCELL). Cells were grown for 8 days at 37°C in CnT-57.S medium (CELLnTEC) supplemented with 1% PNS and 0.5% Gentamicin. Medium were removed and cells detached with 200 µL 0.2% trypsin for 5 min at 37°C. Cells were counted and plated at 0.3 × 10⁶ cells/mL in a 96-well plate coated with Type I collagen.

Stained cells were analyzed using a BD LSR II-SORP or Fortessa system (Becton Dickinson) and analyzed with FlowJo software (Tree Star). The following anti-mouse mAbs were used: CD45- PerCPCy5, CD45-APC, CD8-APC, anti-IFNγ-PECy7, and anti-IL-4-FITC (BD Biosciences), CD4-AF700, CD11b APC-eFluor780, and c-kit PECy7 all from (eBiosciences). Anti human CD2 (BioLegend) was used to detect IL-4 production in KN2 mice. To detect IL-4Rα on primary keratinocytes, the following anti-mouse mAbs were used: CD124, anti IL-4Rα-PE (BD, Pharmingen), and CD49f-Brillant Violet 421 (BioLegend). Imaging flow cytometry was performed using an ImageStream cytometer (Amnis, Millipore) at low speed. Data were analyzed with the IDEAS software.

All p values were determined with Prism software (GraphPad Software, Inc.) using the Student’s t-test for unpaired data or paired data, following prior testing for normal distribution. The degree of significance was indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

To investigate the impact of IL-4Rα signaling on keratinocytes in a protective Th1 cell differentiation against cutaneous leishmaniasis, we generated mice on the C57BL/6 genetic background that are genetically deficient in IL-4Rα expression on keratinocytes (KRT14^CreIL-4Rα^−/lox) using the Cre/LoxP recombination system under control of the KRT14 locus. In these mice, keratinocytes cannot respond to IL-4 and IL-13, while other cells can. The absence of skin IL-4Rα expression was verified by PCR of naïve ears. KRT14^CreIL-4Rα^−/lox expressed both the deleted and WT IL-4Rα allele in addition to the LoxP and Cre bands. The genotype was also validated for IL-4Rα^−/lox, IL-4Rα^+/+ (C57BL/6 wild type) control mice as well as for IL-4^−/− on the C57BL/6 genetic background (Figure 1A). The selective absence of IL-4Rα on KRT14^CreIL-4Rα^−/lox keratinocytes was further analyzed by flow cytometry on primary keratinocytes derived from the skin of these mice. IL-4Rα expression was not detected on CD45⁻CD49f⁺ keratinocytes of KRT14^CreIL-4Rα^−/lox mice. As controls, expression of IL-4Rα was detected on keratinocytes of wild-type C57BL/6 mice and was absent on keratinocytes of IL-4Rα^−/− mice (Figure 1B). As expected, expression of IL-4Rα was, however, detected in splenic CD4⁺ T cells, CD19⁺ B cells, and F480⁺ macrophages of KRT14^CreIL-4Rα^−/lox mice (Figure 1C). The slightly lower IL-4Rα surface expression levels observed in KRT14^CreIL-4Rα^−/lox CD4⁺ T cells, CD19⁺ B cells and F480⁺ macrophages compared to those observed in C57BL/6 wild-type cells are in line with the IL-4Rα^−/lox genotype of the control mice. No IL-4Rα expression was observed in IL-4Rα^−/− CD4⁺ T cells and CD19⁺ B cells. These data show that KRT14^CreIL-4Rα^−/lox mice do not express IL-4Rα selectively on keratinocytes.

To investigate if IL-4Rα signaling in keratinocytes could have an impact on the local microenvironment driving Th1 differentiation during L. major infection, KRT14^CreIL-4Rα^−/lox on the C57BL/6 genetic background were infected subcutaneously in the footpad with 2 × 10⁵ L. major. Lesion development and parasite load in the dLNs and spleen were analyzed and compared to lesion developing in control IL-4Rα^−/lox, IL-4Rα^+/+, and IL-4Rα^−/− mice all on the C57BL/6 genetic background. L. major susceptible BALB/c mice were similarly infected and used for comparison. No difference in lesion development was observed between all groups of mice on the C57BL/6 genetic background, and BALB/c mice developed a significantly larger lesion (Figure 2A). Parasite load was also similar in the dLNs of these mice and no parasite dissemination to the spleen was observed, while significantly higher dLN parasite load and the presence of parasite dissemination to the spleen were observed in BALB/c mice (Figures 2B,C). Phlebotomine sand flies deposit significantly lower dose of parasites in the mammalian dermis, and it was previously reported that the cellular recruitment differ between subcutaneous infection in the footpad or intradermal infection in mouse ear dermis (29). To assess a potential role for IL-4Rα signaling in keratinocytes on disease outcome in more physiological conditions, we needle inoculated mice with 1 × 10⁴ L. major LV39 metacyclic parasites, in the ear dermis of KRT14^CreIL-4Rα^−/lox mice. Lesion development and parasite load were compared to those observed in similarly infected IL-4Rα^−/lox, IL-4Rα^−/− mice, IL-4Rα^+/+ mice all on the C57BL/6 genetic background. No difference in lesion size was observed in all groups of mice. In contrast, similarly infected BALB/c mice developed non-healing lesions (Figure 2D). All groups of mice on the C57BL/6 genetic background also controlled parasite load as assessed in the dLN and no parasite dissemination to the spleen was observed while similarly infected BALB/c mice showed higher parasite load in dLN and parasite disseminated to the spleen (Figures 2E,F). These data demonstrate that IL-4Rα signaling in keratinocytes is not required for control of lesion development and parasite burden. In addition, total lack of IL-4Rα signaling did not have an impact on the resolution of the infection. The control of parasite load in KRT14^CreIL-4Rα^−/lox and IL-4Rα^−/− mice suggested that an efficient protective Th1 immune response could develop in these mice in absence of IL-4Rα signaling.

To further investigate in details if IL-4Rα signaling in keratinocytes impacted Th1 cell differentiation during cutaneous leishmaniasis, KRT14^CreIL-4Rα^−/lox mice and control IL-4Rα^−/lox were infected with 10⁴ L. major (LV39). Four weeks after infection, the frequency of IFNγ and IL-4 producing cells was analyzed by flow cytometry at the site of infection. A high frequency of CD4⁺IFNγ⁺ was detected in the ear dermis of KRT14^CreIL-4Rα^−/lox mice, with no difference compared to that measured in similarly infected IL-4Rα^−/lox control mice (Figure 3A). Only very low levels of CD45⁺CD4⁺IL-4⁺ cells were detected in both groups (Figure 3B). The frequency of CD4⁺IFNγ⁺ cells observed in dLN cells of both groups of mice was also similar (Figure 3C) and more elevated than the frequency of CD45⁺CD4⁺IL-4⁺ cells observed in dLN cells (Figure 3D) revealing that the CD4⁺ Th1 cells are able to differentiate in absence of IL-4Rα signaling in keratinocytes.

In line with the elevated levels of IFNγ detected at the site of infection and in dLN cells, high levels of serum IgG2c were observed in KRT14^CreIL-4Rα^−/lox and control IL-4Rα^−/lox mice (Figure 3E). BALB/c mice showed elevated levels of IgG1, correlating with the high production of IL-4 observed in their CD4⁺ T cells, while sera from KRT14^CreIL-4Rα^−/lox and control mice did not show significant IgG1 levels (Figure 3F), in line with the absence of Th2 cells detected in dLN and at the site of infection. Similar data were obtained following s.c. infection in the footpads with a higher dose of parasites (2 × 10⁵ L. major, data not shown). Taken together, these results show that the absence of IL-4Rα signaling in keratinocytes does not impair the development of a protective Th1 type of immune response during early L. major infection.

The lack of impact of IL-4Rα signaling on lesion development, parasite control, and the differentiation of CD4⁺ Th1 cells at the site of infection and in the dLN firmly demonstrated that IL-4 signaling on keratinocytes was not involved in Th1 cell differentiation following L. major infection. However, it did not rule out an early role for keratinocyte-derived IL-4 in Th1 cell differentiation. To visualize the presence of il-4 mRNA expressing cells in the mouse ear dermis cells of L. major-infected mice, we used the bicistronic IL-4 reporter (4get) mice on the C57BL/6 genetic background (22). We first determined if il-4 mRNA expression was detectable in naive ear skin of C57BL/6 mice. A clear e-GFP-positive population was observed selectively in the CD45⁺ hematopoietic population. These cells did not express CD11b at their cell surface and stained positive for c-kit, a marker specific for mast cells (Figure 4A). Further analysis by imaging flow cytometry confirmed that the e-GFP il-4-expressing cells corresponded to mast cells characterized by the expression of CD45, the lack of expression of CD11b, their large size (average of 14.5 µm of diameter) and the presence of numerous granules (Figure 4B). We then analyzed the modulation of il-4 mRNA expressing cells 4 and 16 h following i.d. infection with L. major, at a time when expression of il-4 mRNA by keratinocytes was reported to be the highest following subcutaneous infection in the footpad (21). No e-GFP⁺ cells were detectable in the CD45-negative cell population, suggesting that keratinocytes did not express detectable levels of il-4 mRNA at the time analyzed. Mast cells were the only cell population where il-4 mRNA, as assessed by e-GFP expression, was detectable 4 and 16 h p.i. (Figure 4A lower panels). The frequency of e-GFP⁺ cell (approximately 1.5% of total ear cells) between infected and the contralateral uninfected ear was analyzed for each mouse. It did not vary between infected and uninfected ears at the analyzed time points (Figure 4C). These data show that most of the il-4 mRNA expressing cells in the ear during the first 16 h post infection are mast cells and that L. major does not modulate il-4 mRNA expression in these cells early during infection.

Mast cells appeared to be the major dermal cells expressing IL-4 early after infection, suggesting that this source of IL-4 could play a role locally. To visualize IL-4 cytokine production, C57BL/6 KN2 (knockin huCD2) IL-4 reporter mice that specifically report the production of IL-4 protein were used. Cells producing IL-4 express a fragment of the human CD2 molecule at the cell surface making it easily detectable by flow cytometry (23). We crossed these KN2 mice with 4get mice to simultaneously detect IL-4 expression and production. First, to ensure that digestion of the ear would not impact IL-4 detection, KN2/4get mice, all on the C57BL/6 genetic background, were treated with anti-IFNγ and anti-IL-12 mAbs at the onset of infection to induce Th2 cell differentiation. Four weeks post infection, ears from mAb-depleted or control mice were isolated, processed, and analyzed by flow cytometry. Treatment with anti-IFNγ and anti-IL-12 promoted the differentiation of Th2 cells. il-4 mRNA expression (e-GFP⁺ cells) and IL-4 protein production (detected by hCD2 expression) were easily detectable (7.9%) in the ear dermis of CD4⁺ T cells (Figures 5A,C). Minimal IL-4 expression or production (0.4%) was detectable in ear cells of similarly infected KN2/4get mice treated with a control mAb (Figure 5B) in line with the differentiation of Th1 cells in these mice. 4get/KN2 naïve ears (0 h p.i.) showed detectable il-4 mRNA expression in CD45⁺ but not in CD45⁻ cells and no IL-4 proteins as no huCD2⁺ cells were detected in either population, suggesting a lack of IL-4 production (Figure 5D). Infection did not change the frequency of cells transcribing il-4 and no IL-4 production was observed 12 and 24 h after L. major infection (Figures 5E–G). These data suggest that ear skin mast cells transcribe the il-4 gene but do not produce detectable IL-4 proteins at steady-state conditions, and L. major does not induce IL-4 production in the ear dermis early during infection.

Taken together, these results demonstrate that during the first hours of infection, il-4 gene expression can be detected at the infection site selectively in mast cell, but il-4 mRNA expression was not modulated during L. major infection (analyzed at 0, 4,12,16, and 24 h p.i.). Furthermore, no IL-4 protein production was detectable in the ear dermis during the first 24 h after L. major infection.

To further investigate if skin-derived IL-4 played a role in Th1 cell differentiation, C57BL/6 wild-type mice were infected with 2 × 10⁵ L. major in the ear dermis following the protocol described in Figure 6A, which is similar to that described previously (21). Two weeks post infection, the frequency of CD4⁺IFNγ⁺ and CD4⁺IL-4⁺ dLN T cells represented in average 0.6 and 0.13% of total CD4⁺ T cells, respectively. These values were similar to those obtained in mice similarly infected but treated with a control mAb (Figure 6B). The levels of IFNγ and IL-4 secretion in dLN cells stimulated ex vivo with UV-treated L. major 2 weeks after infection also showed no significant differences between the anti-IL-4 and control groups (Figure 6C). In line with these data, no difference in parasite load was observed between groups depleted or not of IL-4 at the onset of infection (Figure 6D). These results show that neutralization of IL-4 locally in the ear dermis during the first hours of infection at the site of infection does not have an impact on the development of the Type-1 immune response during cutaneous leishmaniasis.