Deletion of Interleukin-4 Receptor Alpha-Responsive Keratinocytes in BALB/c Mice Does Not Alter Susceptibility to Cutaneous Leishmaniasis

The skin microenvironment at the site of infection plays a role in the early events that determine protective T helper 1/type 1 immune responses during cutaneous leishmaniasis (CL) infection. During CL in nonhealing BALB/c mice, early interleukin-4 (IL-4) can instruct dendritic cells for protective Th1 immunity.

to the disease (1)(2)(3). Th1 immunity during L. major infection is characterized by classical activation of macrophages via the cytokines interferon gamma (IFN-␥) and interleukin-12 (IL-12), while Th2 immunity is characterized by alternative activation of macrophages via the production of various cytokines, including IL-13, IL-5, and, primarily, IL-4, which signals via the IL-4 receptor alpha chain (IL-4R␣). Previous studies have demonstrated that a resistant phenotype was observed in C57BL/6 mice (healer strain) infected with L. major, while BALB/c mice (nonhealer strain) were susceptible to cutaneous leishmaniasis (CL) (2,(4)(5)(6)(7). While the general understanding is that IL-4 induces a Th2 response detrimental to CL, there have been studies demonstrating that the early production of IL-4 at the site of infection in BALB/c mice drives a beneficial Th1 response under the instruction of dendritic cells (DCs) (8,9). This phenomenon is further supported by the fact that both BALB/c and C57BL/6 mouse strains secrete IL-4 early after L. major infection, which is sustained in susceptible but transient in resistant mice (10). The skin, which serves as an immune organ (11), is the primary site of infection during cutaneous leishmaniasis (1). During a blood feed, the female phlebotomine sandfly deposits L. major promastigotes into the skin. The promastigote parasites must pass through this skin barrier and its components to establish an infection. The epidermal layer of the skin is composed primarily of keratinocytes, which produce factors such as cytokines, among others (12). Thus, keratinocytes could provide early signals at the site of L. major infection to initiate distinct immune effector responses. Indeed, infection with L. major IL-81 promastigotes has been shown to induce keratinocytes to rapidly secrete IL-12, IL-1␤, and IL-4 in C57BL/6 mice. This suggests that keratinocytes provide the source of early IL-4 that may instruct DCs to drive the host beneficial Th1/type 1 response (13). As keratinocytes express surface IL-4 receptor, these cells are capable of both autocrine and paracrine stimulation (14,15). We recently demonstrated that C57BL/6 mice deficient for IL-4R␣-responsive keratinocytes were able to develop a protective Th1/type 1 effector response to L. major LV39 infection (16). However, considering that the impact of IL-4-mediated DC instruction was most pronounced in the susceptible BALB/c background in response to more virulent and less virulent strains of parasites, the role of early IL-4 signaling on keratinocytes needs to be investigated on a nonhealer BALB/c genetic background during cutaneous leishmaniasis to fully elucidate effector immune responses in response to infection with more virulent and less virulent L. major strains. Here, we extended our recent study by generating keratinocyte-specific IL-4R␣-deficient mice on a BALB/c genetic background (KRT14 cre IL-4R␣ Ϫ/lox mice) to analyze disease progression and host immune responses following infection with the L. major strain IL-81 (a highly virulent strain) as well as LV39 (less virulent strain). We successfully showed that the IL-4R␣ signal on keratinocytes from KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice was absent, in contrast to the results for wild-type BALB/c mice. We found that during experimental cutaneous leishmaniasis, KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice were more susceptible to infection, similar to littermate control IL-4R␣ Ϫ/lox BALB/c mice, following subcutaneous (s.c.) infection in the footpad or intradermal (i.d.) infection in the ear. Furthermore, footpad swelling, parasite loads, IFN-␥/IL-4/IL-13 production, and type 1 and type 2 antibodies were similar between both groups. Despite a significant decrease in parasite burden seen at the site of infection after i.d. inoculation of L. major LV39, KRT14 cre IL-4R␣ Ϫ/lox mice on the BALB/c genetic background still developed a nonhealing response. Taking our results together, we revealed that deletion of IL-4R␣ signaling on keratinocytes does not influence susceptibility of genetically susceptible BALB/c mice to CL.

RESULTS
Genotypic and functional characterization of KRT14 cre IL-4R␣ ؊/lox BALB/c mice. Genetically modified BALB/c mice expressing Cre-recombinase under the control of the keratinocyte cell-specific locus krt14 (Jackson Laboratory) were intercrossed with IL-4R␣ Ϫ/Ϫ BALB/c mice (17) and IL-4R␣ lox/lox BALB/c mice (18) to generate KRT14 cre IL-4R␣ Ϫ/lox mice (Fig. 1A). This breeding strategy avoids possible non-Mendelian Cre Infection and Immunity activities during early embryogenesis by reducing the substrate (lox), resulting in increased Cre efficiency (19,20). KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice were identified by PCR genotyping (Fig. 1B), as indicated by the presence of the 494-bp Cre band, 450-bp LoxP band, and both the deleted and wild-type IL-4R␣ allele. Analysis of IL-4R␣ cell surface expression on isolated ear keratinocytes (CD45 Ϫ CD49 ϩ K14 ϩ ) by flow cytometry demonstrated efficient IL-4R␣ depletion on KRT14 cre IL-4R␣ Ϫ/lox keratinocytes, which was apparent by the shift of IL-4R␣ expression and geometric mean fluorescence intensity (GMFI) compared to that of wild-type BALB/c keratinocytes (Fig. 1C). As expected, KRT14 cre IL-4R␣ Ϫ/lox mice were confirmed to have a regular cell surface expression of IL-4R␣ on CD4 ϩ T cells, CD19 ϩ B cells, MHCII hi CD11c hi dendritic cells, and MHCII hi CD11c Ϫ CD11b hi macrophages in the cervical lymph node after L. major infection (Fig. 1D). The expression of Desmocollin-1 (Dsc-1), a marker gene for keratinocytes, can correlate with normal epidermal differentiation and development of keratinocytes (21). Additionally, the presence of IL-4 has been shown to downregulate the expression of Dsc-1 (22,23). We therefore examined whether keratinocyte function would be altered due to loss of IL-4R␣ on keratinocytes by measuring Dsc-1 expression and treating with recombinant IL-4. IL-4 stimulation reduced mRNA expression of Dsc-1 in wild-type BALB/c but not keratinocytes isolated from KRT14 cre IL-4R␣ Ϫ/lox or IL-4R␣ Ϫ/Ϫ mice, confirming functionally unresponsive IL-4R␣ signaling in KRT14 cre IL-4R␣ Ϫ/lox keratinocytes (Fig. 1E). These data provide evidence of efficient deletion of IL-4R␣ on keratinocytes from KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice while showing intact IL-4R␣ surface expression on other lymph node cells and demonstrating no effect on keratinocyte functionality. KRT14 cre IL-4R␣ ؊/lox BALB/c mice remain susceptible to L. major infection, similar to littermate control IL-4R␣ ؊/lox BALB/c mice, during experimental cutaneous leishmaniasis in the footpad. To determine whether IL-4R␣ signaling on keratinocytes at the site of infection contributes to nonhealing disease during CL, KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice and appropriate controls (IL-4R␣ Ϫ/lox BALB/c, IL-4R␣ Ϫ/Ϫ BALB/c, and C57BL/6 mice) were infected s.c. in the left hind footpad with stationary-phase promastigotes, either with 2 ϫ 10 5 of the highly virulent L. major IL-81 strain ( Fig. 2A) or with a dose of 2 ϫ 10 6 of the less virulent L. major LV39 strain (Fig. 2B). Importantly, during both L. major IL-81 and L. major LV39 infections, KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice developed progressive footpad swelling, similar to littermate control IL-4R␣ Ϫ/lox BALB/c mice, and exhibited similarly high parasite loads in the infected footpads and draining popliteal lymph nodes and dissemination to the spleen (Fig. 2). Global IL-4R␣ Ϫ/Ϫ BALB/c mice, which are generally resistant to L. major infection, had significantly less footpad swelling than littermate control IL-4R␣ Ϫ/lox BALB/c mice during infection with either strain ( Fig. 2A). After infection with L. major IL-81, parasite loads of global IL-4R␣ Ϫ/Ϫ BALB/c mice in the footpad, popliteal lymph node, and spleen appeared to be significantly lower than the loads in littermate control IL-4R␣ Ϫ/lox BALB/c mice ( Fig. 2A). Upon infection with L. major LV39, the parasite loads in the popliteal lymph nodes of global IL-4R␣ Ϫ/Ϫ BALB/c mice appeared to be significantly higher than those of the littermate control IL-4R␣ Ϫ/lox BALB/c mice, which has been observed previously (24), while parasite loads in the spleen were significantly lower, as anticipated (Fig. 2B). As expected, genetically resistant C57BL/6 mice controlled the development of lesions during acute infection with L. major IL-81 and LV39 ( Fig. 2A and B), correlating with low parasite loads in footpads, draining popliteal lymph nodes, and spleens. Together, these results suggest that deletion of the IL-4R␣ chain on keratin- Mice were infected subcutaneously in the left hind footpad (FP) with stationary-phase L. major promastigotes at either 2 ϫ 10 5 IL-81 (A) or 2 ϫ 10 6 LV39 (B) (n ϭ 5 to 7 mice per group). The change in footpad swelling (in mm) was measured at weekly intervals. Parasite burden was determined at week 6 for panel A and week 8 for panel B by limiting dilution of homogenized footpads, single-cell suspensions of the draining popliteal lymph nodes (pLN), and homogenized spleens (SP). A representative of two individual experiments is shown with mean values Ϯ SEM. Statistical analysis was performed with comparisons to the control IL-4R␣ Ϫ/lox littermate mouse group as significant (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001), using a two-way ANOVA with Bonferroni posttests for change in swelling data and one-way ANOVA with Dunnett's multiple-comparison test for parasite load data. infection to determine whether these mice elicited a polarized Th2 immune response. At 6 weeks postinfection, the frequencies of immune cell populations infiltrating the popliteal draining lymph nodes were similar between KRT14 cre IL-4R␣ Ϫ/lox BALB/c and the littermate control IL-4R␣ Ϫ/lox BALB/c mice, as determined by flow cytometry and cell surface staining ( Fig. 3A and B). C57BL/6 mice at week 6 presented lower percentages of the CD3 ϩ CD4 ϩ Th cells, possibly due to disease control at this stage, and higher CD19 ϩ B lymphocytes and macrophages than susceptible littermate control IL-4R␣ Ϫ/lox BALB/c mice ( Fig. 3A and B). Collectively, these results suggest that the immune cell repertoire in the popliteal lymph node developed independently of IL-4R␣-responsive keratinocytes during L. major infection in KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice.
previously reported (9), resistant C57BL/6 mice had higher production of IFN-␥ (although not significant) and significantly lower production of IL-13 by CD4 ϩ Th cells than the littermate control IL-4R␣ Ϫ/lox BALB/c mice (Fig. 3C) following IL-81 infection in the footpad. Total popliteal lymph node cells were also restimulated with soluble leishmanial antigen (SLA) or mitogenic anti-CD3 to detect overall cytokine production by the immune cell repertoire (Fig. 3D to F). Similar levels of production of IFN-␥ (Fig.  3D), IL-4 (Fig. 3E), and IL-13 (Fig. 3F) were observed in KRT14 cre IL-4R␣ Ϫ/lox BALB/c and littermate control IL-4R␣ Ϫ/lox BALB/c mice. In contrast, C57BL/6 mice showed significantly lower production of IFN-␥ and drastically lower quantities of Th2 cytokines IL-4/IL-13 than littermate control IL-4R␣ Ϫ/lox BALB/c mice when lymph node cells were stimulated with anti-CD3 (Fig. 3D to F), as previously observed (26). Together, these results suggest that IL-4R␣-responsive keratinocytes do not play a decisive role in driving a polarized Th2 response during L. major infection in BALB/c mice in the footpad.
The quantification of cytokine production after ex vivo stimulation may not provide a true indication of the type 1 and type 2 or Th1 and Th2 responses in vivo (25). As it is known that IL-4 promotes isotype switching to IgG1 and IgE and that IgG2a levels correlate with the activity of IFN-␥ in vivo (27), we measured antigen-specific type 1 (IgG2a) and type 2 (IgG1 and total IgE) antibody titers in the mouse sera by enzymelinked immunosorbent assay (ELISA) 6 weeks postinfection with L. major IL-81. KRT14 cre IL-4R␣ Ϫ/lox BALB/c and littermate control IL-4R␣ Ϫ/lox BALB/c mice had comparable levels of IgG2a, IgG1, and total IgE during L. major IL-81 infection (Fig. 3G to I). Collectively, the data suggest that systemic antibody responses in KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice were unaffected by the deletion of the IL-4R␣ signaling receptor on keratinocytes during subcutaneous infection with L. major IL-81 in the footpad.
Intradermal inoculation of L. major parasites in the ear does not radically alter the outcome of disease in KRT14 cre IL-4R␣ ؊/lox BALB/c mice. While s.c. injection of L. major in the footpad is widely used, recent studies have suggested that i.d. injection of a lower dose of parasites into the ear provides a more physiological mode of infection, as it mimics some of the events that occur during parasite inoculation by the sandfly (28,29). Hence, we investigated whether a change in the site of infection and parasite dose would alter the phenotype and immune response of the KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice. Mice were i.d. infected into the left ear with a low dose of 1 ϫ 10 4 stationary-phase L. major IL-81 or L. major LV39 promastigotes. Ear lesion diameter was measured to monitor disease progression during the infection, and immune responses were evaluated at 8 weeks postinfection. Ear lesion diameter and the parasite load in the ear, cervical lymph node, and spleen of KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice during L. major IL-81 infection were high, similar to those of littermate control IL-4R␣ Ϫ/lox BALB/c mice (Fig. 4A). Global IL-4R␣ Ϫ/Ϫ BALB/c mice had significantly smaller lesion diameters and lower parasite loads in the ear than littermate control IL-4R␣ Ϫ/lox BALB/c mice after infection with L. major IL-81 (Fig. 4A). As previously reported (30), C57BL/6 mice had significantly smaller lesion diameters and associated reduced parasite load in the ear, cervical lymph node, and spleen compared to littermate control IL-4R␣ Ϫ/lox BALB/c mice after L. major IL-81 infection (Fig. 4A). Infection with L. major LV39 resulted in similar ear lesion diameters and cervical lymph node parasite loads between KRT14 cre IL-4R␣ Ϫ/lox BALB/c and littermate control IL-4R␣ Ϫ/lox BALB/c mice, with the surprising exception of significantly reduced parasite loads in the ear (Fig. 4B). Both global IL-4R␣ Ϫ/Ϫ BALB/c and C57BL/6 mouse groups had significantly smaller lesion diameters in the ears and significantly lower parasite loads in the ear than littermate control IL-4R␣ Ϫ/lox BALB/c mice after L. major LV39 infection (Fig. 4B). In contrast to L. major IL-81 infection, L. major LV39 infection did not result in dissemination to the spleen, as demonstrated by the absence of parasites in the spleen in all mouse groups. This could be explained by the higher virulence of the former parasite (9) and the i.d. route of infection. Collectively, the data indicate that despite a change in the site/route of infection and the dose of parasites, KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice remain susceptible to L. major and remain unaffected by the abrogated IL-4R␣ signaling on keratinocytes.
Early IL-4 production in the ear appears unaffected by loss of signaling of IL-4R␣ on keratinocytes. The early source of IL-4 can contribute to susceptibility to L. major infection (13). Thus, we measured IL-4 expression directly in the skin of L. major-infected mice. Here, mice were infected with L. major LV39 in the ear dermis, and at 2 weeks postinfection the ears were retrieved and processed for the detection of IFN-␥, IL-4, and IL-13 at mRNA and protein levels ( Fig. 5A to C). IFN-␥, IL-4, and IL-13 were found to be undetectable at the mRNA level, as measured by quantitative PCR (qPCR) (data not shown). Detection of IFN-␥, IL-4, and IL-13 by ELISA showed no Statistical analysis was performed with comparisons to the control IL-4R␣ Ϫ/lox littermate mouse group as significant (*, P Ͻ 0.05; ***, P Ͻ 0.001), using a two-way ANOVA with Bonferroni posttests for change in ear lesion data and one-way ANOVA with Dunnett's multiple-comparison test for parasite load data. significant differences between control IL-4R␣ Ϫ/lox BALB/c littermates and KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice in the L. major LV39-infected ears (Fig. 5A to C). However, IFN-␥ and IL-4 levels in the ears were significantly decreased in C57BL/6 mice compared to those in littermate control IL-4R␣ Ϫ/lox BALB/c mice ( Fig. 5A and B). Additionally, at week 1 postinfection, IL-4 was not detectable at the mRNA or protein level in the ears of KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice infected with L. major LV39 (data not shown). While IL-4 was not detectable at the time analyzed, this finding suggests that it is produced locally or at very low doses that are not detectable. Additionally, IL-4 was reported to At 8 weeks postinfection, mice were sacrificed and cervical draining lymph nodes (cLN) retrieved. Single-cell suspensions obtained from the lymph nodes were extracellularly stained for CD4 (CD3 ϩ CD4 ϩ ) T cells, CD8 (CD3 ϩ CD8 ϩ ) T cells, and B cells (CD19 ϩ B220 ϩ ) (D), DC (dendritic cells) (CD11c hi MHCII hi ) and MF (macrophages) (CD11b hi CD11c Ϫ MHCII hi ) (E), and stimulated with PMA-ionomycin-monensin, followed by staining for intracellular cytokine production (F). Cells were restimulated with anti-CD3 and SLA for 72 h, after which cytokine production was measured by ELISA for IFN-␥ (G), IL-4 (H), and IL-13 (I).
(J) L. major-specific type 1 (IgG2a) and (K and L) type 2 antibodies (IgG1 and total IgE) were measured by ELISA of sera from mice infected for 6 weeks. Data are representative of two experiments (n ϭ 3 to 5 mice per group). Statistical analysis was performed with comparisons to the control IL-4R␣ Ϫ/lox littermate mouse group as significant (*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.001), using 2-way ANOVA with Dunnett's multiple-comparison test (A to I), two-way ANOVA with Bonferroni posttests (J and K), and Mann-Whitney test (nonparametric, unpaired t test) (L). be produced at an earlier time and produced at low quantities at this earlier time point (13).
Together, these findings highlight that during intradermal infection in the ear, IL-4R␣ signaling on keratinocytes in BALB/c mice does not affect protective immune responses, as determined by cellular responses, cytokine production, and type 1 and type 2 antibody titers.

DISCUSSION
Previous studies have supported a role for IL-4 in driving a polarized Th1 response and conferring resistance to BALB/c mice during L. major infection (8,25). Conversely, during experimental cutaneous leishmaniasis in C57BL/6 mice with a global deletion of IL-4R␣, it was seen that the deletion had no impact on resistance in these mice (16). Dendritic cells are the primary source of IL-12 and initiate antigen-specific immunity to Leishmania (31). Biedermann et al. incubated bone marrow-derived DCs with a Th1priming adjuvant in the presence of recombinant IL-4 and found increased IL-12 production upon IL-4 stimulation. Ova-specific CD4 ϩ T cells primed with activated dendritic cells also had significantly upregulated IFN-␥ under these conditions, indicating that IL-4 could instruct dendritic cells to secrete IL-12, thereby inducing Th1 cell differentiation (8). Biedermann et al. also demonstrated this IL-4 instruction theory in mouse studies, showing that exogenous IL-4 administered during the period of dendritic cell activation was required to drive dendritic cells to elicit the Th1 response in usually susceptible BALB/c mice (8). The study by Biedermann et al. highlighted that a high dose of IL-4 administered exogenously was needed to observe a shift to protective Th1 immunity; however, the study did not provide insight on physiological doses of endogenous IL-4 during infection. Hurdayal et al. (9) addressed this using BALB/c mice with abrogated IL-4R␣ expression on CD11c ϩ dendritic cells. During L. major infection these mice were hypersusceptible to infection, showing increased footpad swelling and parasite loads and increased Th2 immune responses. This indicated that IL-4R␣-responsive dendritic cells play an important role in the elicited immune response. Ehrchen et al. further explored the IL-4 instruction theory by focusing on the microenvironment of the infected tissue (13). Importantly, various cytokines, including IL-4, were induced in L. major-infected C57BL/6 mice and L. major-infected BALB/c mice (13). Additionally, cytokine induction, including IL-4 in the skin and by keratinocytes, was higher in C57BL/6 mice than in BALB/c mice, and genes involved in keratinocyte differentiation were found to be overrepresented, suggesting an influence on their expression by the presence of L. major parasites. While Biedermann et al. showed that exogenous recombinant IL-4 could drive dendritic cells to instruct a Th1 response (8), Ehrchen et al. illustrated that blocking endogenous IL-4 with exogenous anti-IL-4 antibody in L. majorinfected C57BL/6 mice caused these mice to switch from a genetically resistant Th1 to a susceptible Th2 phenotype (8). Following on from Biedermann et al. (8) and Ehrchen et al. (13), we aimed to investigate whether IL-4R␣-responsive keratinocytes influence the outcome of disease during CL. Our initial study showed that in genetically resistant C57BL/6 mice, IL-4R␣-responsive keratinocytes did not affect the resistant phenotype (16), as seen by Ehrchen et al. in these resistant mice (13). The next step was to investigate whether deletion of IL-4R␣-responsive keratinocytes would contribute to nonhealing disease during experimental CL in BALB/c mice. If IL-4R␣ is indeed demonstrated to be important for disease outcome, IL-4/IL-13 released by these keratinocytes could signal and influence keratinocytes in an autocrine manner or influence dendritic cells in a paracrine manner. A keratinocyte-specific IL-4R␣-deficient mouse model was generated using the Cre/ FIG 6 Low-dose infection in the ear dermis does not significantly alter immune response between control littermates and KRT14 cre IL-4R␣ Ϫ/lox mice on a BALB/c genetic background during L. major IL-81 infection. At 8 weeks postinfection, mice were sacrificed and cervical draining lymph nodes (cLN) retrieved. Single-cell suspensions obtained from the lymph nodes were extracellularly stained for CD4 (CD3 ϩ CD4 ϩ ) T cells, CD8 (CD3 ϩ CD8 ϩ ) T cells, and B cells (CD19 ϩ B220 ϩ ) (A), DC (dendritic cells) (CD11c hi MHCII hi ) and MF (macrophages) (CD11b hi CD11c Ϫ MHCII hi ) (B), and also stimulated with PMA-ionomysin-monensin (C), followed by staining for intracellular cytokine production. Cells were restimulated with anti-CD3 and SLA for 72 h, after which cytokine production was measured with ELISA for IFN-␥ (D), IL-4 (E), and IL-13 (F). (G to I) L. major-specific type 1 (IgG2a) (G) and type 2 antibodies (IgG1 and total IgE) (H and I) were measured by ELISA of sera from mice infected for 6 weeks. Data are representative of two experiments (n ϭ 3 to 5 mice per group). Statistical analysis was performed with comparisons to the control IL-4R␣ Ϫ/lox littermate mouse group as significant (*, P Ͻ 0.05; ***, P Ͻ 0.001), using 2-way ANOVA with Dunnett's multiplecomparison test (A to F), two-way ANOVA with Bonferroni posttests (G and H), and Mann-Whitney test (nonparametric, unpaired t test) (I).
loxP system under the control of the krt14 locus. These mice were infected with L. major either s.c. in the footpad or i.d. in the ear, after which disease progression and cellular and humoral immunity were evaluated.
Subcutaneous infection of L. major IL-81 or LV39 in the footpad of KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice revealed no role for IL-4R␣-responsive keratinocytes in modulating nonhealing disease to L. major. KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice were susceptible to infection, showing progressive footpad swelling and parasite loads, characteristic of genetically susceptible BALB/c mice (32). L. major IL-81-infected KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice showed dissemination of parasites to the spleen, similar to that of littermate control IL-4R␣ Ϫ/lox BALB/c mice and as expected in susceptible wild-type BALB/c mice (33,34). In these mice, the mounting of a Th2 response, along with the alternative activation of macrophages, is the reason why parasites disseminate and cause severe disease (35)(36)(37)(38). The parasites can use the polyamines generated from alternative activation of macrophages for their growth and survival (9). Disease progression in BALB/c mice infected with L. major corresponds to an upregulation of Th2 cytokines, CD4 ϩ Th2 cells, and type 2 antibody responses (9,26,(39)(40)(41)(42). Increased infiltration of macrophages to the draining lymph node in C57BL/6 mice could be linked to the roles they play in protection, being essential not just for uptake of parasites and establishment of an immune response but also for their killing function by classically activated macrophages (1,6,(43)(44)(45). Together, the data suggested that BALB/c mice with abrogated IL-4R␣ were susceptible to L. major infection and that the absence of IL-4R␣ expression on keratinocytes did not allow for protection against L. major.
An intradermal inoculation of low-dose stationary-phase promastigote L. major parasites in the ear better mimics natural infection by the sandfly than s.c. inoculation (28)(29)(30)(46)(47)(48)(49). KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice infected i.d. with 10 4 L. major parasites showed swelling progression and parasite burden similar to that of littermate control IL-4R␣ Ϫ/lox BALB/c mice. L. major LV39-infected KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice showed significantly fewer parasites at the site of infection (the ear) than the littermate control IL-4R␣ Ϫ/lox BALB/c mice. This suggests that KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice were able to control parasite replication in the ear and that IL-4/IL-13 signaling through IL-4R␣ on keratinocytes hampers immune regulatory functions when present. Upon investigation of the early production of IL-4 in the ear after 2 weeks of infection with L. major LV39, we found no significant change in KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice compared to results for littermate control IL-4R␣ Ϫ/lox BALB/c mice, while resistant C57BL/6 mice appeared to have significantly lower production of IL-4. Overall, our data suggested that while IL-4R␣-responsive keratinocytes were not contributing to the development of the lesions or overall T helper immune responses of KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice in the ear infection model, parasite replication at the site of the L. major LV39 infection was more controlled in these mice than in littermate control IL-4R␣ Ϫ/lox BALB/c mice.
Hence, our findings indicate that while IL-4/IL-13 signaling via IL-4R␣ on keratinocytes in BALB/c mice does not contribute to their nonhealing phenotype, it could contribute to parasite control or replication at the site of infection in the ear model during L. major LV39 infection but not during L. major IL-81 infection. This finding needs to be further explored. This study therefore provides some understanding of the early immune responses occurring in the dermis and during cutaneous leishmaniasis in nonhealer BALB/c mice, and it also complements our recent findings in healer C57BL/6 mice (16). Additionally, our data highlight the importance of the strain of the L. major parasite, as well as the use of a more physiological route of infection, when studying cutaneous leishmaniasis in experimental mouse models. Generation and genotyping of KRT14 cre IL-4R␣ ؊/lox mice. Keratinocyte cell-specific IL-4R␣deficient (KRT14 cre IL-4R␣ Ϫ/lox ) BALB/c mice were generated using the Cre/loxP system and characterized by our laboratory. Briefly, KRT14 cre mice (Jackson Laboratory) were crossed with IL-4R␣ Ϫ/Ϫ BALB/c mice (17) and transgenic IL-4R␣ lox/lox mice (18) to generate hemizygous KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice after nine generations of breeding. All mice were kept under specific-pathogen-free conditions in individually ventilated cages. Experimental mice were age and sex matched and used between 8 and 10 weeks of age. Genotyping of KRT14 cre IL-4R␣ Ϫ/lox BALB/c mice was carried out using the following specific primers: KRT14 P1, forward primer, 5=-TTC CTC AGG AGT GTC TTC GC; KRT14 P2, reverse primer, 5=-GTC CAT GTC CTT CCT GAA GC; KRT14 P3, forward primer, 5=-CAA ATG TTG CTT GTC TGG TG; KRT14 P4, reverse primer, 5=-GTC AGT CGA GTG CAC AGT TT.

MATERIALS AND METHODS
Functional characterization of KRT14 cre IL-4R␣ ؊/lox mice. IL-4R␣ deletion was confirmed with flow cytometry, with staining for IL-4R␣ on ear-isolated keratinocytes. Briefly, ears were digested in complete Dulbecco's modified Eagle's medium (cDMEM) containing 0.2 mg/ml Liberase (TL research grade; Roche) and filtered through a 40-m filter. Keratinocytes were then isolated, counted, and labeled. Labeling was performed by adding the antibodies for 20 min on ice, followed by the addition of the secondary antibody. Keratinocytes were gated as CD45 Ϫ and were double positive for CD49 and K14. Antibodies included CD45-peridinin chlorophyll protein (PerCP), CD49f-fluorescein isothiocyanate (FITC), mouse anti-keratin 14, and goat anti-mouse A555 (ThermoFisher Scientific). CD45 Ϫ CD49 ϩ K14 ϩ keratinocytes were then stained for the presence of IL-4R␣ with IL-4R␣-phycoerythrin (PE) (BD, Pharmingen), and samples were acquired on a Fortessa machine (BD, San Jose, CA, USA). Flow data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Cell surface IL-4R␣ expression was further analyzed in total lymphocytes, Th cells (CD3 ϩ CD4 ϩ ), B cells (CD19 ϩ B220 ϩ ), dendritic cells (CD11c ϩ MHCII ϩ ), and macrophages (CD11b ϩ MHCII ϩ ) in the draining lymph node of L. major-infected IL-4R␣ Ϫ/Ϫ , IL-4R␣ Ϫ/lox , and KRT14 cre IL-4R␣ Ϫ/lox mice by flow cytometry. For Dsc-1 mRNA expression in keratinocytes, primary keratinocytes were isolated from the tail skin of adult mice. Briefly, the skin from the tail was collected and incubated at 4°C for 16 h in 5 U/ml dispase (STEMCELL) supplemented with 1% penicillinstreptomycin-neomycin (PSN) (Gibco) and 0.5% gentamicin (Gibco). The epidermis was next separated from the dermis and treated with 0.2% trypsin (Gibco) for 5 min at 37°C. The reaction was stopped with fetal calf serum (FCS), and cells were then collected by crushing the epidermis through a 100-m cell strainer. A total of 0.75 ϫ 10 6 cells/ml were plated in a 6-well plate coated with type 1 collagen (STEMCELL). Cells were grown for 8 days at 37°C in CnT-57.S medium (CELLnTEC) supplemented with 1% PSN and 0.5% gentamicin. The medium was discarded and cells were detached with 200 l 0.2% trypsin for 5 min at 37°C. Cells were counted and plated at 0.3 ϫ 10 6 cells/ml in a 96-well plate coated with type 1 collagen. Isolated keratinocytes were then stimulated with 20 ng/ml of mouse IL-4 recombinant protein (Affymetrix, eBioscience) or left untreated for 24 h at 37°C as previously described (22). mRNA was isolated thereafter using an RNA Minikit (Qiagen) according to the manufacturer's instructions, cDNA synthesized, and quantitative reverse transcription-PCR (RT-PCR) performed using a LightCycler (Roche). Values were normalized to hprt level and are presented as fold induction compared to the level for unstimulated keratinocytes. The primer sequences for Dsc-1 were the following: forward (F), 5=-GGGAG CACCTTCTCTAAGCA-3=; reverse (R), 5=-TTTTGACAGGCATCACAAAATAA-3= (22).
Detection of viable parasite burden. Infected footpad, ear, draining lymph node, and spleen cell suspensions were cultured in Schneider's culture medium (Sigma). Parasite burden was determined with the limiting dilution assay (LDA) that has been previously described (17).