ABSTRACT
l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with Leishmania mexicana can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an in vivo model the capacity of two L. mexicana isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with L. mexicana isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that L. mexicana isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.
INTRODUCTION
The leishmaniases are a group of vector-borne neglected diseases caused by different species of a protozoan parasite that belongs to the genus Leishmania and represent an important public health problem worldwide. In humans, at least 20 different species of Leishmania can be etiological agents of the leishmaniases, originating different clinical manifestations that range from skin lesions (cutaneous leishmaniasis) and mucosal damages (mucocutaneous leishmaniasis) to life-threatening systemic infections that affect internal organs, principally liver, spleen, and bone marrow (visceral leishmaniasis) (1, 2). These different clinical forms result from complex interactions between the infecting Leishmania species and the host immune response to the infection. Leishmania parasites are transmitted between mammalian hosts, including humans, by the bite of female phlebotomine sand fly vectors. During a blood meal, sand flies inoculate the host skin with flagellated and motile Leishmania promastigotes, previously developed in the sand fly gut. Once in the skin of the host, Leishmania promastigotes invade several phagocytic cells, including neutrophils, dendritic cells, monocytes, and macrophages, the latter being the primary host cells for Leishmania (2, 3). Within macrophages, promastigotes transform into and multiply as amastigote forms, which possess a nonvisible external flagellum and are responsible for the disease onset and progression in the mammalian host.
Leishmania has developed multiple strategies to evade or benefit from the host immune response and ensure its survival within the host. Among these strategies, the regulation of the catabolism of l-arginine via the inducible nitric oxide synthase (NOS2) and arginase-1 (Arg-1) has arisen as a critical pathway involved in leishmaniasis establishment and progression. In this context, macrophages can either kill or host intracellular Leishmania parasites depending on their ability to metabolize l-arginine through NOS2 or Arg-1, whose expression is differentially induced and regulated in these phagocytes, according to their activation status (4–7). In classically activated macrophages (M1 or inflammatory macrophages), Th1 cytokines (e.g., gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) induce the expression and function of NOS2, which oxidizes l-arginine into l-citrulline and nitric oxide, the latter being a free radical that mediates one of the most potent mechanisms to eliminate intracellular Leishmania parasites (7–12). On the other hand, in alternatively activated macrophages (M2 or wound-healing macrophages), Th2 cytokines (e.g., interleukin 4 [IL-4], IL-10, and IL-13) induce the expression and function of Arg-1, which hydrolyzes l-arginine into urea and l-ornithine, the latter being a precursor for the synthesis of polyamines (small and polycationic molecules that participate in several cellular processes such as differentiation, DNA replication, and protein translation), which are essential for Leishmania intramacrophage survival and proliferation (5–7, 9, 11, 12).
Experimental models have demonstrated a critical role for l-arginine metabolism via Arg-1 in the pathogenesis of murine leishmaniasis. Indeed, in nonhealing cutaneous leishmaniasis models, high levels of Arg-1 and its activity have been detected locally within the lesions of mice, correlating this fact with an uncontrolled growth and elevated loads of Leishmania parasites (6, 13). In this line, in nonhealing lesions, an accumulation of high numbers of alternative activated macrophages has been revealed, which in turn promote Leishmania parasite growth via polyamine synthesis due to l-arginine metabolism through Arg-1 (6, 14). In addition to the direct promotion of Leishmania parasite multiplication via polyamine synthesis, high Arg-1 expression and activity in alternatively activated macrophages and other myeloid cells have also been shown to modulate T cell immune responses against Leishmania infection by reducing the bioavailability of l-arginine in the microenvironment (6, 14, 15). Due to the fact that T cells are extremely susceptible to l-arginine starvation, a low availability of this amino acid makes them hyporesponsive to antigen stimulation besides affecting their proliferation and effector functions (15). Moreover, Leishmania parasites harbor their own arginase, whose activity has been demonstrated to be essential for their replication and infectivity, both in vitro and in vivo (16–24).
Contrary to the identified role of Arg-1 in disease exacerbation, the expression and function of NOS2 has been demonstrated to be decisive for Leishmania parasite killing and infection resolution in experimental murine cutaneous leishmaniasis (25–27). Within this context, a marked expression and activity of NOS2, which correlates with few or no parasites, has been identified in skin lesions of mice resistant to Leishmania infection (26, 27). Additionally, macrophages infiltrating the lesions of these resistant mice have been shown to express NOS2 (27), a fact that suggests the presence and importance of classically activated macrophages in healing lesions. In the same way that Leishmania parasites harbor their own arginase, a Leishmania-derived nitric oxide synthase has also been reported and associated with the infectivity of the parasite and/or a mechanism to evade host immune responses (28, 29).
In Mexico, Leishmania mexicana is the main etiological agent responsible for cutaneous leishmaniasis and can cause two main different clinical manifestations in patients: localized cutaneous leishmaniasis (LCL), characterized by the development of an ulcer at the sites of parasite inoculation, and diffuse cutaneous leishmaniasis (DCL), where parasites spread throughout the skin, forming multiple disfiguring nodules (30, 31). The factors determining the development of LCL or DCL after infection with L. mexicana remain unclear. In patients, LCL has been associated with a polarized Th1 immune response against L. mexicana infection and low macrophage parasite loads, whereas DCL has been associated with a polarized Th2 response and high macrophage parasite loads (32–34).
In the present study, we tested in an in vivo murine model the hypothesis that an L. mexicana isolate obtained from a patient with LCL and an L. mexicana isolate obtained from a patient with DCL differentially regulate l-arginine metabolism through Arg-1 and NOS2, influencing the infection course and severity in the host. Furthermore, we examined the participation of macrophages and their activation status in this process. Finally, we analyzed the differential expression of Arg-1 and NOS2 in biopsy specimens obtained from Mexican patients with LCL and DCL.
RESULTS
Molecular and morphological characterization of Leishmania mexicana isolates.Two isolates of L. mexicana obtained from Mexican patients, one with localized cutaneous leishmaniasis (LCL) and the other with diffuse cutaneous leishmaniasis (DCL), were used throughout the present study (Table 1). Both isolates were identified as L. mexicana after performing restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer 1 (ITS1; the DNA sequence separating the genes coding for the small subunit and for the 5.8S large subunit of rRNA), which was amplified by PCR and digested with HaeIII restriction enzyme (Fig. 1A and B). It has been shown that the ITS1 region is highly variable in size and nucleotide sequence among Leishmania species, and its digestion with restriction enzymes after PCR amplification allows identification of most Leishmania species of medical importance (35). Amplification by PCR of ITS1 from the two L. mexicana isolates obtained from patients, an L. mexicana reference strain (positive control), and a Leishmania major reference strain (nonrelated species control) resulted in a 300- to 350-bp-length product (Fig. 1A), the size reported for Leishmania ITS1 (35). Further digestion of ITS1 amplicons with the HaeIII restriction enzyme resulted in different Leishmania species RFLP patterns (Fig. 1B). A pattern with three fragments of 188, 88, and 60 bp each, which is characteristic for L. mexicana (35), was obtained for the two isolates obtained from patients and for the L. mexicana reference strain (Fig. 1B). In contrast, a pattern with two fragments of 220 and 130 bp each, which is characteristic for L. major (36), was obtained for the L. major reference strain (Fig. 1B).
Clinical data of patients from which Leishmania mexicana isolates were obtained
Characterization of Leishmania mexicana isolates. Two L. mexicana isolates were obtained from Mexican patients: one with LCL and the other with DCL. Isolates were adapted to in vitro culture and maintained as axenic amastigotes. Amastigotes of L. mexicana and promastigotes of L. major from reference strains were used as controls. DNA was purified from Leishmania parasites by using phenol chloroform and used for identification of isolates as L. mexicana species by Leishmania ITS1 PCR-RFLP analysis. (A) Leishmania ITS1 was amplified by PCR, and the obtained amplicons were resolved on 2% ethidium bromide-stained agarose gels. (B) Amplicons obtained from ITS1 PCR were further digested with HaeIII restriction enzyme, and obtained fragments giving RFLP patterns were separated on 3% ethidium bromide-stained agarose gels. Micrographs showing growth patterns of axenic culture-maintained amastigotes from isolates obtained from a patient with LCL (C) and from a patient with DCL (D). Bars, 20 μm.
For experiments, L. mexicana isolates obtained from patients were cultured and used as amastigotes, due to the fact that when Leishmania is maintained in culture, this stage of the parasite is more infective for experimental mouse inoculation than stationary-phase promastigotes (37). Interestingly, amastigotes of L. mexicana isolates displayed some different growth patterns when cultured in vitro. Among these, the most notable difference was in the contact between amastigotes (cell to cell) when growing (Fig. 1C and D). Amastigotes of the L. mexicana isolate obtained from the patient with LCL grew mainly as individual cells (Fig. 1C), while amastigotes of the isolate obtained from the patient with DCL grew mostly forming tight and large clusters (Fig. 1D). Importantly, these differences were maintained when the isolates were passaged periodically through mice to preserve virulence.
Susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit lesions with different sizes and parasite loads as well as a differential migration of parasites to draining lymph nodes.After characterization, we analyzed if the L. mexicana isolate obtained from the patient with LCL and the L. mexicana isolate obtained from the patient with DCL showed differences in behavior during infection in a murine leishmaniasis model. As a first approach to evaluate this issue, BALB/c mice, which have been demonstrated to be highly susceptible to L. mexicana infection (38–40), were inoculated in the footpad with both isolates, and sizes of developed lesions were measured every 7 days during a 4-week period, followed by a final evaluation made 8 weeks postinfection (Fig. 2A). Lesions in mice infected with the L. mexicana isolate obtained from the patient with DCL became apparent during the first week of infection, while lesion onset in mice infected with the L. mexicana isolate obtained from the patient with LCL occurred during the second week of infection (Fig. 2A). From the second up to the eighth week of infection, mice infected with the L. mexicana isolate obtained from the patient with DCL developed bigger lesions (almost twice the size) than mice infected with the L. mexicana isolate obtained from the patient with LCL (Fig. 2A). Furthermore, we determined the number of parasites present in the footpad lesions of mice at 4 and 8 weeks after infection, as another parameter to evaluate the differential behavior of the L. mexicana isolates during infection (Fig. 2B). At the fourth week of infection, footpad lesions of mice inoculated with the isolate obtained from the patient with DCL exhibited higher parasite loads (at least three times more) than the lesions of mice inoculated with the isolate from the patient with LCL (Fig. 2B). At the eighth week of infection, footpad lesions in both groups of mice showed higher parasite loads than those observed at the fourth week of infection; however, parasite numbers between the two groups were similar (Fig. 2B).
Susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit lesions with different sizes and parasite loads as well as differential migration of parasites to draining lymph nodes. BALB/c mice were infected subcutaneously into the right hind footpads with 5 × 106 L. mexicana axenic amastigotes from isolates obtained from LCL and DCL patients. (A) The course of lesion development (ten mice per group) was monitored every 7 days during a 4-week period, followed by a final evaluation made 8 weeks postinfection, by measuring the footpad thickness increase. Parasite loads were determined in footpad lesion (B) and popliteal lymph node (C) homogenates from mice (four mice per group) after 4 and 8 weeks of infection. Data are expressed as the means ± SEMs and are representative of at least two independent experiments. L.N., lymph node; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
In murine experimental models, cutaneous infection of BALB/c mice with L. mexicana generally results in the development of progressive, nonhealing, and chronic lesions (38). Moreover, it has been shown that in progressive forms of murine cutaneous leishmaniasis, parasites are continuously detected in lesions as well as in the lymph nodes draining them (41). Consequently, we also evaluated the presence and abundance of parasites in the popliteal lymph nodes (pLNs) of mice infected for 4 and 8 weeks with the L. mexicana isolate obtained from the patient with LCL and with the L. mexicana isolate obtained from the patient with DCL (Fig. 2C). At the fourth week of infection, we were able to detect the presence of parasites in the pLNs of mice infected with both L. mexicana isolates, and surprisingly, pLNs of mice inoculated with the isolate obtained from the patient with LCL exhibited higher parasite loads (almost ten times more) than the pLNs of mice inoculated with the isolate from the patient with DCL (Fig. 2C). However, at the eighth week of infection, pLNs of both groups of mice showed higher parasite loads than those observed at the fourth week of infection; nevertheless, parasite numbers between the two groups were similar (Fig. 2C).
It should be noted that the clear differences found in the parasite loads after 4 weeks of infection with the two L. mexicana isolates were probably masked at the eighth week of infection by the tendency of BALB/c mice to develop chronic lesions when infected with L. mexicana.
Arg-1 and NOS2 protein presence is differentially regulated in the lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients.Using in vivo murine experimental models, it has been demonstrated that Arg-1 is induced during disease development after infection with different Leishmania species (13, 42, 43); furthermore, the presence of this enzyme at the site of infection has been correlated with proliferation of the parasite and disease progression (6, 19, 21). On the other hand, NOS2 presence at the site of infection has been correlated with parasite clearance and disease resolution in the L. major murine model (13, 26, 27). Thus, we analyzed if infection with the L. mexicana isolate obtained from the patient with LCL and infection with the L. mexicana isolate obtained from the patient with DCL resulted in differential protein synthesis of Arg-1 and NOS2 in BALB/c mice. For this purpose, footpad lesions of infected mice were removed at 4 and 8 weeks of infection for tissue homogenization, and the presence of Arg-1 and NOS2 was detected by Western blotting (Fig. 3A). The assays demonstrated that infection with both L. mexicana isolates led to Arg-1 protein synthesis, with higher expression levels (almost two times more) of this enzyme detected in the lesions of mice infected with the isolate obtained from the patient with DCL than in the lesions of mice infected with the isolate obtained from the patient with LCL (Fig. 3A and B). Regarding NOS2 presence, interestingly, this protein was expressed at lower levels only in the lesions of mice infected for 4 weeks with the isolate obtained from the patient with DCL (Fig. 3A). In addition, lesions of C57BL/6 mice infected for 4 weeks with L. major were used as a positive control to analyze NOS2 protein presence by Western blotting, which showed an intense band for this protein (Fig. 3A).
Arg-1 and NOS2 protein presence and activity are differentially regulated in the lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients. BALB/c mice (four mice per group) were infected subcutaneously in the right hind footpads with 5 × 106 L. mexicana axenic amastigotes from isolates obtained from LCL and DCL patients. (A) Arg-1 and NOS2 protein levels were evaluated in footpad lesion homogenates by Western blotting after 4 and 8 weeks of infection. Footpad lesion homogenates from mice infected with L. major were used as control for NOS2 protein presence. Coomassie blue staining of total protein in the membrane was used for the loading control. For the Western blot image, lesion homogenates from mice belonging to each group were pooled, and the protein presence of Arg-1 and NOS2 was evaluated. (B) Densitometric values (expressed in arbitrary units [A.U.]) for Arg-1 protein presence were calculated as the ratio of Arg-1 and total protein band intensities detected in the lesion homogenates from all mice for each group. Arg-1 activity, expressed as units per milligram of protein (C) or units per milligram of tissue (D), was determined by enzymatic assay performed in footpad lesion homogenates from mice infected for 4 and 8 weeks. Data are expressed as the means ± SEMs and are representative of at least two independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Arg-1 enzymatic activity is differentially regulated in lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients.Once we determined that the L. mexicana isolates obtained from patients with LCL and DCL differentially upregulated Arg-1 protein synthesis at the site of infection, we evaluated if this upregulation at the protein synthesis level paralleled enzyme activity. For this aim, arginase activity was determined in the lesion homogenates of mice infected with these L. mexicana isolates for 4 and 8 weeks (Fig. 3C and D). The obtained data revealed, at both times postinfection, higher arginase activity values, expressed either as units per milligram of protein (Fig. 3C) or as units per milligram of tissue (Fig. 3D), in the lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL than in the lesions of mice infected with the L. mexicana isolate obtained from the patient with LCL. Interestingly, even though arginase activity in the lesions of mice infected with any of the isolates diminished at 8 weeks of infection compared to the levels found at 4 weeks, the activity registered in the lesions of mice infected with the isolate obtained from the patient with DCL was always higher than the one obtained after infection with the isolate obtained from the patient with LCL. In relation to NOS2 activity, although a minimal presence of NOS2 was detected in the lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL, we tried to determine the activity of this enzyme by quantifying nitrites present in the tissue using the Griess reaction; however, we were not able to detect any nitrite levels (data not shown).
Lesions and draining lymph nodes of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit similar production of IL-4 and IL-10 but different production of IFN-γ.The murine infection model with L. major has demonstrated a host resistance or susceptibility to the disease depending on the development of a polarized Th1 or Th2 immune response, respectively (44). In this regard, host ability to the control of infection has been associated with the expansion of CD4+ Th1 cells, which produce IFN-γ. In contrast, host impossibility to control the infection has been associated with the expansion of CD4+ Th2 cells, which produce IL-4, IL-10, and IL-13 (6, 44–46). Furthermore, it has been demonstrated that induction of Arg-1 in the host, driven by IL-4 and IL-10, is clearly associated with the susceptibility to the infection, where the presence and activity of this enzyme clearly parallel lesion development and parasite burden increase (6, 13). Consequently, we analyzed if the changes that we observed in the induction of Arg-1 protein and activity in the lesions and lymph nodes of susceptible BALB/c mice infected with the L. mexicana isolate obtained from the patient with LCL and the isolate obtained from the patient with DCL were related to changes in the production of Th1 and Th2 cytokines during the infection course. For this aim, we assessed by enzyme-linked immunosorbent assay (ELISA) the production of IL-4, IL-10, and IFN-γ in footpad lesions and pLN homogenates of infected mice. There were no significant differences in the production of IL-4 and IL-10 between footpad lesions of mice infected with each of the L. mexicana isolates, either at 4 or at 8 weeks postinfection; however, both cytokine levels increased according to disease progression (Fig. 4A and B). Importantly, when comparing the amounts of both cytokines, we noticed that IL-10 levels were around ten times higher than those observed for IL-4 (Fig. 4A and B). Regarding IL-4 and IL-10 production in the pLNs, also, no changes in the production of these cytokines were detected between the groups of mice after 8 weeks of infection (Fig. 4A and B). With respect to IFN-γ production, interestingly, the infection of mice with the isolate obtained from the patient with DCL elicited a higher production of IFN-γ than the infection with the isolate obtained from the patient with DCL, both in the footpad lesions and pLNs, after 8 weeks of infection (Fig. 4C). Contrarily, there were no differences in the production of IFN-γ in the footpad lesions of mice infected for 4 weeks (Fig. 4C).
Lesions and draining lymph nodes of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit similar production of IL-4 and IL-10 but different production of IFN-γ. Footpad lesions of mice infected with isolates obtained from LCL and DCL patients were removed after 4 or 8 weeks of infection, while popliteal lymph nodes were removed after 8 weeks of infection. Excised tissues were homogenized, and IL-4 (A), IL-10 (B), and IFN-γ (C) expression levels were quantified by ELISA. Data are the means ± SEMs from three independent experiments. *, P ≤ 0.05.
Lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients contain macrophages with an alternatively activated phenotype.Arg-1 has been found in different cell types, including macrophages, dendritic cells, myeloid-derived suppressor cells, and granulocytes, among others, and the expression of this enzyme in such myeloid cells has been proved to be induced by IL-4, IL-13, and IL-10 Th2 cytokines (12, 47, 48). Macrophages are the main host cells for Leishmania parasites and can be instructed to host or kill this parasite depending on their activation status, which influences l-arginine metabolism through Arg-1 and NOS2 (5, 6). Classically activated macrophages upregulate the NOS2 enzyme, which metabolizes l-arginine into nitric oxide, a powerful leishmanicidal agent that contributes to parasite killing. Contrarily, alternatively activated macrophages upregulate the Arg-1 enzyme, which metabolizes l-arginine into polyamines, which allow parasite persistence and proliferation (5, 6). Given these facts, after determining the presence and activity of Arg-1 and NOS2 in the homogenates of lesions from susceptible BALB/c mice infected with the L. mexicana isolates obtained from patients with LCL and DCL, we next decided to identify, by immunohistochemistry, the presence of both enzymes in the lesions and if macrophages were expressing them. Lesions of mice infected with both L. mexicana isolates showed abundant Arg-1-positive cells harboring Leishmania amastigotes and morphologically identifiable as macrophages (Fig. 5A). In addition, these cells were shown to be positive for the macrophage marker F4/80 (Fig. 5B, representative staining for F4/80 in a tissue section of a lesion from a mouse infected with the isolate obtained from the DCL patient is shown). Interestingly, in lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL, macrophages showed more intense and widespread Arg-1 staining and seemed more heavily parasitized than the macrophages present in lesions of mice infected with the LCL isolate (Fig. 5A). In murine macrophages, l-arginine metabolism through Arg-1 and NOS2 is one of the most-used criteria to identify alternatively activated macrophages and classically activated macrophages, respectively (12, 49). Thus, the presence of Arg-1 in the macrophages allowed us to suggest an alternative activation status for these cells. To further corroborate this idea, we evaluated the presence, in the lesions of infected mice, of another characteristic marker of alternative activated murine macrophages, the chitinase-like secretory protein YM-1 (50). In this line, we first detected by Western blotting the presence of YM-1 in lesion lysates from mice infected with both L. mexicana isolates (Fig. 5D), and thereafter we performed immunohistochemistry assays which allowed us to detect this protein in macrophages expressing Arg-1 and harboring Leishmania amastigotes (Fig. 5A). On the other hand, immunohistochemistry against NOS2 revealed no presence of this enzyme in the lesions of mice infected with either of the L. mexicana isolates (Fig. 5A). Detection by immunohistochemistry of NOS2 in lesions of C57BL/6 mice infected with L. major was used as a positive control for the experiments (Fig. 5C).
Lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients contain macrophages with an alternatively activated phenotype. BALB/c mice (three mice per group) were infected subcutaneously in the right hind footpads with 5 × 106 L. mexicana axenic amastigotes from isolates obtained from LCL and DCL patients or 5 × 106 L. major promastigotes. After 8 weeks of infection, footpad lesions were dissected and processed for immunohistochemistry as described in Materials and Methods. (A) Tissue sections were incubated with specific primary antibodies against arginase-1, NOS2, and YM-1, followed by incubation with an HRP-conjugated secondary antibody. Immunolabeling was developed with DAB and further counterstained with Gill’s hematoxylin. Sections incubated only with the secondary antibody were used as a control for unspecific staining. (B) F4/80 staining in a tissue section of a lesion from a mouse infected with DCL isolate is shown as representative for macrophage identification. (C) Staining for NOS2 in tissue sections of lesions from C57BL/6 mice infected with 5 × 106 L. major promastigotes was used as positive control for NOS2 detection. (D) YM-1 protein presence was also evaluated by Western blotting in footpad lesion homogenates from mice 4 and 8 weeks after infection. Coomassie blue staining of total protein in the membrane was used for the loading control. Bars, 10 μm. Micrographs are representative of the sections from two independent experiments.
Lesions of Mexican patients with LCL and DCL contain macrophages with different Arg-1 and NOS2 expression as well as parasite load.After immunohistochemical analysis of Arg-1 and NOS2 presence in lesions of mice infected with the L. mexicana isolates obtained from patients with LCL and DCL, we pursued the same analysis in lesion biopsy specimens obtained from Mexican patients diagnosed with LCL and DCL. The lesions of patients with both types of cutaneous leishmaniasis showed Arg-1-positive cells harboring Leishmania amastigotes and morphologically identifiable as macrophages (Fig. 6A). In addition, these cells were shown to be positive for the macrophage marker CD68 (Fig. 6B, representative staining for CD68 in a tissue section of a lesion from a patient with DCL is shown). Interestingly, lesions of DCL patients showed macrophages with greater Arg-1 expression and were more heavily parasitized than those found in the lesions of LCL patients (Fig. 6A). Regarding NOS2 detection, immunohistochemistry staining revealed that only macrophages in the lesions of patients with LCL were positive for this enzyme (Fig. 6A).
Lesions of Mexican patients with LCL and DCL contain macrophages with different Arg-1 and NOS2 expression as well as parasite load. Biopsy specimens from lesions of Mexican patients with LCL and DCL were taken and processed for immunohistochemistry as described in Materials and Methods. (A) Tissue sections of biopsy specimens were incubated with specific primary antibodies against arginase-1 and NOS2, followed by incubation with an HRP-conjugated secondary antibody. Immunolabeling was developed with DAB and further counterstained with Gill’s hematoxylin. Sections incubated only with the secondary antibody were used as control for unspecific staining. (B) CD68 staining in a tissue section of a lesion from the patient with DCL is shown as representative for macrophage identification. Bars, 10 μm. Micrographs are representative of the sections from two LCL patient biopsy specimens and from three DCL patient biopsy specimens.
Amastigotes in lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit different protein presence and enzymatic activity of Leishmania-harbored arginase.Due to the fact that Leishmania parasites also express an arginase and a nitric oxide synthase, which have been proposed as virulence factors (16–24, 28, 29), we analyzed the presence and functionality of these enzymes in amastigotes directly purified from lesions of mice infected with L. mexicana isolates obtained from patients with LCL and DCL. In Western blot experiments, a Leishmania-harbored arginase was only detected in the extracts of amastigotes purified from lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL (Fig. 7A). Arginase activity assays detected a much higher arginase activity (almost 20 times more) in the extracts of amastigotes purified from lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL than in those purified from lesions of mice infected with the L. mexicana isolate obtained from the patient with LCL (Fig. 7B), in which Leishmania arginase protein was not detected by Western blotting (Fig. 7A). Regarding Leishmania-harbored nitric oxide synthase, neither the protien (Fig. 7A) nor activity of the enzyme (data not shown) was detected in the extracts of amastigotes purified from lesions of mice infected with any of the L. mexicana isolates.
Amastigotes in lesions of susceptible mice infected with Leishmania mexicana isolates obtained from LCL and DCL patients exhibit different protein presence and enzymatic activity of Leishmania-harbored arginase. BALB/c mice (three mice per group) were infected subcutaneously in the right hind footpads with 5 × 106 L. mexicana axenic amastigotes from isolates obtained from LCL and DCL patients. After 8 weeks of infection, Leishmania amastigotes were purified from footpad lesions and lysed. (A) Amastigote lysates were tested for Leishmania arginase and NOS protein presence by Western blotting. Indian ink staining of total proteins in the membrane was used for the loading control. (B) Arginase activity, expressed as milliunits (mU) per milligram of protein, was determined in amastigote lysates by enzymatic assay. For the Western blot image, the protein presence of Arg-1 and NOS2 in the homogenates from only two mice is shown. Data are expressed as the means ± SEMs and are representative of at least two independent experiments.
DISCUSSION
In the present study, we examined in an in vivo model the ability of two Leishmania mexicana isolates, one obtained from a patient with LCL and another obtained from a patient with DCL, to differentially regulate l-arginine metabolism through Arg-1 and NOS2. In our model, we inoculated BALB/c mice with these isolates, due to the high susceptibility of this mouse strain to L. mexicana infection (39). First, we assessed the pathogenic behavior of the isolates during experimental infection by analyzing the size and parasite loads of the developed lesions as well as the dissemination of the parasites to the lymph nodes. We observed that, despite belonging to the same Leishmania species, the L. mexicana isolate obtained from the patient with DCL exhibited greater virulence than the L. mexicana isolate obtained from the patient with LCL. This was reflected by the development of lesions that were bigger and harbored higher parasite numbers in mice infected with the isolate obtained from the patient with DCL than those developed in mice infected with the isolate obtained from the patient with LCL. In agreement with our observations, other studies have also highlighted differences in the evolution time of lesions and their parasitic loads in mice infected with different strains and isolates of L. major and Leishmania amazonensis (51–53). Regarding the dissemination of parasites from lesions to the lymph nodes, contrary to our findings in the footpad lesions, we detected greater parasite loads in the lymph nodes of mice infected with the isolate obtained from the patient with LCL than in the lymph nodes of mice infected with the isolate obtained from the patient with DCL. This fact could be related to reported findings that propose an impairment in the ability of phagocytes heavily infected with Leishmania, which we have routinely observed in the lesions of mice infected with the isolate obtained from the patient with DCL, to exit from the infection site and migrate to the lymph nodes (41, 54, 55).
A clear association between Arg-1 presence and activity, uncontrolled replication of parasites, and increase in size has been found in the lesions of susceptible mice infected with Leishmania parasites (6, 13, 56). Here, we demonstrate higher Arg-1 expression and activity levels, accompanied by an increase in size and parasite load, in the lesions of susceptible mice infected with the L. mexicana isolate obtained from the DCL patient than in the lesions of mice infected with the isolate obtained from the patient with LCL. Due to the fact that we found the isolate obtained from the patient with DCL more pathogenic than the isolate obtained from the patient with LCL, our findings are in line with others where it has been shown that lesions of susceptible mice infected with a more pathogenic Leishmania species, e.g., L. major, exhibit higher arginase activity and bigger size than the lesions of mice infected with a less pathogenic species, e.g., Leishmania tropica (56).
On the other hand, NOS2 expression and subsequent nitric oxide production in the tissues of mice clinically resistant to L. major infection have been closely associated with disease amelioration and lesion healing in experimental cutaneous leishmaniasis (26, 27, 57–59). In our experiments, we found very little (compared to the lesions of C57BL/6 resistant mice infected with L. major, used here as positive controls for NOS2 expression) or no presence of NOS2 in the lesions of mice infected with the L. mexicana isolates obtained from patients with LCL and DCL, with no impact in the progression of the infection. Given the fact that a reciprocal regulation of NOS2 and Arg-1 exists (9, 48), we find possible that the increased upregulation of Arg-1 induced during the infection of susceptible mice infected with the L. mexicana isolates could have impaired NOS2 expression by decreasing the availability of l-arginine, which has been demonstrated to be needed for NOS2 translation (60), or by the production of polyamines such as spermine and spermidine, which have been found capable to suppress the induction of NOS2 (61–63).
In experimental murine cutaneous leishmaniasis, progressive infection in mice is associated with IL-4, IL-10, and IL-13 production (44–46), which in turn induce Arg-1 expression and function, leading to an excessive parasite proliferation and uncontrolled disease pathology (4–6, 13). In this line, we detected similar amounts of IL-4 or IL-10 in the lesions and in the lymph nodes of mice infected with either of the two L. mexicana isolates obtained from patients. This observation suggests that the differential expression and function of Arg-1 observed in the lesions of mice was not dependent on the capability of the isolates to induce the production of higher or lower levels of each of these Th2 cytokines. Given the pivotal role of IFN-γ in leishmaniasis, we also evaluated the presence of this cytokine. Regardless, we detected higher levels of IFN-γ in the lesions and lymph nodes of mice infected with the isolate obtained from the patient with DCL than in those of mice infected with the isolate obtained from the patient with LCL; these differences were not reflected either in NOS2 expression and function or disease amelioration. Our findings agree with different clinical studies that have demonstrated the presence of IFN-γ in the lesions of patients with cutaneous leishmaniasis; moreover, a positive correlation of lesion size with IFN-γ expression levels has been well documented (64–67).
It is well known that macrophages are the main host cells for Leishmania parasites, and elegant studies using in vitro approaches have demonstrated that, depending on their activation status, they can either kill (in classically activated macrophages) or host (in alternatively activated macrophages) intracellular Leishmania parasites via the metabolism of l-arginine through NOS2 or Arg-1, respectively (4, 5, 17). Here, we identified, in vivo, infected macrophages with an alternatively activation phenotype (F4/80+ Arg-1+ YM-1+) in the lesions of mice inoculated with either of the two L. mexicana isolates obtained from patients. However, alternatively activated macrophages present in the lesions of mice infected with the isolate obtained from the patient with DCL seemed to exhibit higher Arg-1 expression and to harbor larger numbers of amastigotes than those present in lesions of mice infected with the L. mexicana isolate obtained from the patient with LCL. It is well known that metabolism of l-arginine through Arg-1 in macrophages leads to the production of polyamines, which in turn favors Leishmania intracellular growth (4–6). In this regard, we propose that the isolate obtained from the patient with DCL could be more efficient in inducing Arg-1 expression in macrophages, leading to both higher polyamine synthesis and proliferation of the parasite in these host cells, than the isolate obtained from the patient with LCL. On the other hand, the presence of NOS2 was not observed in the lesion macrophages from mice infected with either of the two isolates.
In human leishmaniasis, Arg-1 protein and activity have been detected in the lesions of patients with cutaneous leishmaniasis infected with different Leishmania species such as L. aethiopica, L. major, L. tropica, and L. amazonensis (68–70); however, this issue has not yet been studied in the lesions of patients infected with L. mexicana. In this work, we detected infected macrophages (CD68+) expressing Arg-1 in lesion biopsy specimens from Mexican patients suffering LCL and DCL due to L. mexicana infection. Interestingly and in a similar manner to our observations in the murine model, detected macrophages in the lesions of patients with DCL exhibited higher Arg-1 expression and greater numbers of amastigotes than the macrophages found in the lesions of patients with LCL. These findings are in line with what has been shown in L. amazonensis-infected patients suffering LCL and DCL, where higher expression of Arg-1 has been detected in the lesions of patients with DCL than in those of patients with LCL (69). On the other hand, and differently from what we found in the lesions of infected mice, we were able to detect macrophages expressing NOS2 in the biopsy specimens of patients with LCL, an observation that coincides with the work performed by Qadoumi and colleagues, which reports the presence of macrophages expressing NOS2 in skin biopsy specimens from Mexican patients with LCL (71).
A large body of evidence supports the existence of a functional parasite-harbored arginase in different Leishmania species, including L. mexicana, and this enzyme has been found to be essential for parasite replication and infectivity as well as for modulating host immune response and disease pathogenesis (16–24). In this study, we were able to detect a Leishmania-harbored arginase only in the extracts of amastigotes purified from lesions of mice infected with the L. mexicana isolate obtained from the patient with DCL, which also exhibited far higher activity than the one detected in the extracts of amastigotes purified from lesions of mice infected with the isolate obtained from the patient with LCL. Because very little enzymatic activity was detected in the latter, we believe that arginase was expressed in these extracts of amastigotes, but the concentration of this protein was not enough to be detected by our Western blot procedure. Consistent with previous observations proposing that Leishmania-harbored arginase enhances disease pathogenesis by augmenting host cellular arginase activities (19), we found a possible correlation between the arginase expression and activity levels present in the L. mexicana amastigotes from the isolates obtained from the patients with DCL and LCL and the Arg-1 expression and activity levels present in the lesions of mice infected with these isolates.
In conclusion, our results emphasize the preponderant role of l-arginine metabolism through Arg-1 during infection with L. mexicana. Specifically, the evidences shown here tempt us to suggest a differential regulation of this novel pathway during the immunopathogenesis of LCL and DCL, where pathogenic factors intrinsic to the parasites isolated from patients with each of these two clinical manifestations may be involved. Undoubtedly, further thorough studies involving experimentation with other isolates obtained from patients, gene expression profile identification during experimental and human infection, and evaluation of new parameters will be needed to strengthen our findings.
MATERIALS AND METHODS
Mice.BALB/c and C57BL/6 mice were purchased from Charles Rivers Laboratories (Wilmington, MA, USA) and bred at the animal facility of the Unidad de Investigación en Medicina Experimental, Facultad de Medicina, UNAM. Mice were maintained in accordance with the national guidelines for animal care (NOM 062 ZOO 1999) and used for experiments at 8 to 10 weeks of age, according to protocols that previously were approved by the Ethical Committee of the Facultad de Medicina, UNAM (approval number 004/2014).
Parasites.Two Leishmania mexicana isolates were used in this study: MHOM/MX/2012/CGR, obtained from a Mexican patient diagnosed with localized cutaneous leishmaniasis (LCL), and MHOM/MX/2012/MRB, obtained from a Mexican patient diagnosed with diffuse cutaneous leishmaniasis (DCL). The LCL patient was a resident of the Mexican state of Campeche, while the DCL patient was a resident of the Mexican state of Tabasco, two states with high leishmaniasis endemicity in the southeast region of Mexico. After obtaining informed written consent from the patients, asepsis of the lesions, and local anesthesia administration, a 3-mm-punch biopsy sample was taken from the active edge of the skin ulcer of the LCL patient, while needle aspirations were taken from nodular lesions of the DCL patient. The biopsy specimen obtained from the LCL patient was homogenized in phosphate-buffered saline (PBS) by grinding the tissue with a pestle in a 1.5-ml conical tube in order to release amastigotes from infected cells. Both biopsy specimen homogenate and aspirates containing amastigotes were inoculated in Sloppy Evans semisolid medium, maintained at 26°C, and observed every 48 h until the transformation of amastigotes into promastigotes was detected. Upon transformation, promastigotes were cultured at 26°C in medium 199, pH 7.2, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, 100 μg/ml streptomycin, 2 mM l-glutamine, and 1% basal medium Eagle (BME) vitamins (all from Gibco of Thermo Fisher Scientific, Waltham, MA, USA). Once reaching the stationary phase of growth, promastigotes were inoculated subcutaneously into the right hind footpads of BALB/c mice. After lesion development, amastigotes were isolated from infected footpads and cultured as axenic amastigotes at 33°C in Grace’s insect medium, pH 5.4, supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 μg/ml streptomycin (all from Gibco of Thermo Fisher Scientific), as previously described (37, 72). As controls, Leishmania mexicana reference strain MNYC/BZ/62/M379 and Leishmania major reference strain MHOM/IL/81/Friedlin (both kindly donated by Paul A. Bates, Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, United Kingdom) were used for some experiments. The infectivity of Leishmania strains and isolates was maintained by regular passage through BALB/c mice.
Identification of Leishmania isolates by ITS1 PCR-RFLP.Leishmania isolates obtained from patients were confirmed as Leishmania mexicana species by restriction fragment length polymorphism (RFLP) analyses of PCR-obtained amplicons of the internal transcribed spacer 1 (ITS1), the sequence of DNA that separates the genes coding for the rRNA forming the ribosome small subunit (SSU) and for the 5.8S rRNA forming the ribosome large subunit (LSU), as previously described (35), with minor modifications. After being washed three times in PBS, 1 × 108 L. mexicana axenic amastigotes or L. major promastigotes were resuspended in lysis buffer (50 mM NaCl, 50 mM EDTA, 1% SDS, 50 mM Tris-HCl [pH 8.0], and 100 μg/ml proteinase K) and incubated at 60°C overnight. DNA was purified from the lysates by phenol-chloroform extraction and ethanol precipitation according to standard operating procedures. Isolated DNA was used as the template for PCR amplification of Leishmania ITS1 employing the primers LITSR, 5′-CTG GAT CAT TTT CCG ATG-3′, and L5.8S, 5′-TGA TAC CAC TTA TCG CAC TT-3′. PCRs were performed in a final volume of 50 μl containing 1× Phusion HF buffer, 0.5 μM each primer, 200 μM deoxynucleoside triphosphate (dNTP) mixture, 1 U Phusion HF DNA polymerase (all from New England BioLabs, Ipswich, MA, USA), and 1 μg of Leishmania DNA. The thermocycling profile consisted of an initial denaturation step at 94°C for 5 min, 35 cycles of 94°C for 40 s (denaturation), 53°C for 30 s (annealing), and 72°C for 60 s (extension), and a final extension step at 72°C for 7 min. PCR products were resolved on 2% ethidium bromide-stained agarose gels. After PCR amplification, RFLP analysis of ITS1 amplicons was performed by digesting 10 μl of PCR products with BsuRI (HaeIII) restriction enzyme (Thermo Fisher Scientific) according to the manufacturer’s instructions. The obtained restriction fragments were analyzed on 3% ethidium bromide-stained agarose gels.
Infection of mice, disease course evaluation, and parasite load assessment.Female BALB/c and C57BL/6 mice were inoculated subcutaneously in the right hind footpads with 5 × 106 L. mexicana amastigotes and L. major promastigotes, respectively, both from stationary phase of growth. The course of disease was monitored weekly by measuring the increase in footpad thickness with a Vernier caliper (model DTG-001; iGaging, San Clemente, CA, USA). Footpad thickness increase was calculated by subtracting the thickness value of the uninfected contralateral footpad from that of the infected footpad. Parasite burdens in the footpad lesions and popliteal lymph nodes (pLNs) of infected mice were determined by direct counting of amastigotes in a Neubauer chamber, after tissue homogenization (37, 73). For this purpose, footpads and pLNs were excised and forced, in the presence of PBS, with a 10-ml syringe plunger through a 100-μm nylon cell strainer (Falcon; Corning, Corning, NY, USA) coupled to a 50-ml conical tube in order to disrupt the tissue and achieve mechanical lysis of infected cells. The resultant homogenate, containing free amastigotes and some undisrupted host cells, was passed five times through a 25-gauge needle to ensure release of amastigotes from the infected cells that were not lysed during the previous step and to break amastigotes clumps to proceed with parasite counting.
Western blotting.Footpads of infected mice were excised and homogenized in 0.1% Triton X-100, 25 mM Tris-HCl (pH 7.4) buffer, containing 1× EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Resultant homogenates were clarified by centrifugation at 12,000 × g for 15 min at 4°C and assayed for total protein concentration using the bicinchoninic acid (BCA) assay protein kit (Novagen-Merck KGaA, Darmstadt, Germany). Equal amounts of protein (20 μg) were boiled with reducing Laemmli buffer and resolved on 7.5% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred (25 V for 60 min) onto Immobilon-P membranes (Millipore-Merck KGaA, Darmstadt, Germany) using a wet Mini Trans-Blot Cell apparatus (Bio-Rad Laboratories, Hercules, CA, USA). After transference, membranes were blocked with 10% nonfat dried milk (Bio-Rad Laboratories) diluted in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) for 30 min at room temperature. Membranes were then incubated overnight at 4°C with 1:50,000 monoclonal mouse anti-arginase-1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 1:1,000 polyclonal rabbit anti-iNOS (GeneTex, Inc., Irvine, CA, USA), or 1:10,000 polyclonal rabbit anti-YM-1 (STEMCELL Technologies, Vancouver, Canada) primary antibodies diluted in TBST containing 10% nonfat dried milk. Then, membranes were washed with TBST (three times for 5 min each) and incubated for 60 min at room temperature with either 1:50,000 horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG or 1:10,000 HRP-conjugated goat anti-rabbit IgG secondary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) diluted in TBS containing 10% nonfat dried milk. After incubation with secondary antibodies, membranes were washed with TBST (three times for 10 min each), developed with a chemiluminescent substrate for HRP (Millipore-Merck KGaA), and exposed to X-ray films (Santa Cruz Biotechnology, Inc.). Coomassie blue staining of membranes was used as a loading control when comparing protein expression levels. Densitometric analyses of bands were performed using the ImageJ software (Image Analysis Processing in Java rings; NIH, Bethesda, MD, USA).
Protein presence of Leishmania-harbored arginase and nitric oxide synthase was also evaluated in lesion-derived amastigotes. For this purpose, amastigotes were purified from footpad lesions as previously described (74), whole-cell lysates were prepared, and 20 μg of protein was used for Western blot assays, according to the method described above. To detect arginase protein in amastigote lysates, 1:5,000 polyclonal rabbit anti-arginase-1 (GeneTex, Inc.) primary antibody was used, while 1:100 monoclonal mouse anti-NOS2 (Santa Cruz Biotechnology, Inc.), 1:100 monoclonal mouse anti-NOS1 (Santa Cruz Biotechnology, Inc.), and 1:100 polyclonal rabbit anti-NOS2 (GeneTex, Inc.) primary antibodies were used to detect nitric oxide synthase protein. Indian ink staining of membranes was used for the loading control.
Determination of arginase enzymatic activity.Arginase enzymatic activity was determined in the lesions of infected mice by measuring the amount of urea generated from the hydrolysis of l-arginine, as previously described (6, 75) with some modifications. Briefly, footpads of infected mice were excised, weighed, and homogenized in 0.1% Triton X-100, 25 mM Tris-HCl (pH 7.4) buffer, containing 1× EDTA-free protease inhibitor cocktail. Resultant homogenates were clarified by centrifugation at 12,000 × g for 15 min at 4°C and assayed for total protein concentration as described above. A volume of 1 to 5 μl of footpad lesion homogenates was diluted to a 50-μl final volume, per sample, with 0.1% Triton X-100, 10 mM MnCl2, 25 mM Tris-HCl (pH 7.4) buffer, followed by heating for 10 min at 56°C to achieve enzyme activation. A volume of 50 μl of l-arginine (0.5 M, pH 9.7) was added to each sample, and arginine hydrolysis was conducted at 37°C for 15 min. The reaction was stopped by adding 400 μl of a H2SO4-H3PO4-H2O (1:3:7 [vol/vol]) mixture. The concentration of produced urea was measured at 550 nm, according to the Archibald colorimetric method, after the addition of 20 μl of 7% α-isonitrosopropiophenone (dissolved in absolute ethanol) and heating at 100°C for 30 min. Arginase activity was reported as units per milligram of tissue or units per milligram of protein, where one unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of urea per min.
Assessment of arginase enzymatic activity in lysates of purified lesion-derived amastigotes was performed according to the method described above, with the exception that l-arginine hydrolysis was conducted for 120 min instead of 15 min.
Quantification of cytokines by ELISA.Cytokine levels were measured in homogenates of footpad lesions and popliteal lymph nodes of L. mexicana-infected mice by standard sandwich ELISA. Tissue homogenates were prepared as described above and assayed to quantify IL-4, IL-10, and IFN-γ levels by using specific ELISA kits for IL-4 and IL-10 (BioLegend, San Diego, CA, USA) and IFN-γ (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturers’ instructions.
Immunohistochemistry.Footpads of infected mice or 4- to 6-mm-diameter lesion biopsy specimens from Mexican patients with LCL and DCL were fixed for 24 h in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA), followed by standard processing for paraffin embedding. For immunostaining, 4-μm-thick sections were mounted on charged microscope glass slides (Thermo Fisher Scientific), deparaffinized, and rehydrated. To unmask epitopes, antigen retrieval was performed either by heating the tissue slides in sodium citrate buffer (10 mM, pH 6) at 95°C for 20 min or by incubation with proteinase K (20 μg/ml in Tris-EDTA [TE] buffer, pH 8) for 10 min at room temperature. Endogenous peroxidase activity in the tissue was quenched by incubation in 3% H2O2 in TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl) for 20 min at room temperature. Nonspecific antibody binding was blocked with 10% normal serum from the species in which the secondary antibody was generated and 1% bovine serum albumin, both diluted in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20). Sections were then incubated overnight at 4°C with 1:500 polyclonal rabbit anti-arginase 1, 1:100 polyclonal rabbit anti-iNOS, 1:50 polyclonal rat anti-F4/80 (all from GeneTex, Inc.), 1:200 polyclonal rabbit anti-YM-1 (STEMCELL Technologies, Vancouver, Canada), and monoclonal mouse anti-CD68 (Abcam, Cambridge, UK) primary antibodies diluted in TBST supplemented with 1% bovine serum albumin (BSA). After washing with TBST (three times for 5 min each), sections were incubated for 60 min at room temperature with 1:500 polyclonal HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich), 1:100 HRP-conjugated goat anti-rat IgG, or 1:500 HRP-conjugated goat anti-human IgG (both from Jackson ImmunoResearch Inc., West Grove, PA, USA) secondary antibodies diluted in TBST supplemented with 1% BSA. Afterwards, sections were washed with TBST, and 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) was added as the substrate for HRP to visualize immunodetection. Finally, sections were counterstained with Gill’s hematoxylin, dehydrated, cleared, and mounted. Tissue sections were observed by light microscopy (Microphot-FXA microscope; Nikon Instruments Inc., Melville, NY, USA) and digital images were acquired using a Nikon DXM1200F digital camera and the ACT-1 software (Nikon Instruments Inc.).
Data analysis.Data are reported as the means ± standard errors of the means (SEMs) and were analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical differences between groups were evaluated using Student’s t tests (when comparing two groups) and one-way analyses of variance (ANOVAs) followed by Bonferroni post hoc tests (when comparing multiple groups). Differences between groups were considered significant when the P value was <0.05.
ACKNOWLEDGMENTS
This work was supported by grants IN225116 and IN218119 from PAPIIT, DGAPA, UNAM, to L.G.-K. Arturo A. Wilkins Rodríguez is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and received fellowship 269394 from CONACYT.
We thank Ingeborg Becker Fauser (Unidad de Investigación en Medicina Experimental, Facultad de Medicina, UNAM) and Norma Galindo Sevilla (Instituto Nacional de Perinatología) for providing the biopsy samples from patients with leishmaniasis and the technician in pathology, Victor Hugo Torres (Hospital General de México), for sectioning paraffin-embedded tissues for immunohistochemistry.
FOOTNOTES
- Received 5 January 2020.
- Returned for modification 27 January 2020.
- Accepted 31 March 2020.
- Accepted manuscript posted online 20 April 2020.
- Copyright © 2020 American Society for Microbiology.
