Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils

Norma Salaiza-Suazo; Roxana Porcel-Aranibar; Isabel Cristina Cañeda-Guzmán; Adriana Ruiz-Remigio; Jaime Zamora-Chimal; José Delgado-Domínguez; Rocely Cervantes-Sarabia; Georgina Carrada-Figueroa; Baldomero Sánchez-Barragán; Victor Javier Leal-Ascencio; Armando Pérez-Torres; Héctor A Rodríguez-Martínez; Ingeborg Becker

doi:10.1371/journal.pone.0296887

. 2024 Feb 15;19(2):e0296887. doi: 10.1371/journal.pone.0296887

Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils

Norma Salaiza-Suazo ¹, Roxana Porcel-Aranibar ¹, Isabel Cristina Cañeda-Guzmán ¹, Adriana Ruiz-Remigio ¹, Jaime Zamora-Chimal ¹, José Delgado-Domínguez ¹, Rocely Cervantes-Sarabia ¹, Georgina Carrada-Figueroa ², Baldomero Sánchez-Barragán ³, Victor Javier Leal-Ascencio ⁴, Armando Pérez-Torres ⁵, Héctor A Rodríguez-Martínez ¹, Ingeborg Becker ^1,^*

Editor: Vyacheslav Yurchenko⁶

PMCID: PMC10868813 PMID: 38359037

Abstract

Eosinophils are mainly associated with parasitic infections and allergic manifestations. They produce many biologically active substances that contribute to the destruction of pathogens through the degranulation of microbicidal components and inflammatory tissue effects. In leishmaniasis, eosinophils have been found within inflammatory infiltrate with protective immunity against the parasite. We analyzed the responses of eosinophils from patients with localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, as well as from healthy subjects, when exposed to Leishmania mexicana. All DCL patients exhibited blood eosinophilia, along with elevated eosinophil counts in non-ulcerated nodules. In contrast, only LCL patients with prolonged disease progression showed eosinophils in their blood and cutaneous ulcers. Eosinophils from DCL patients secreted significantly higher levels of IL-6, IL-8, and IL-13, compared to eosinophils from LCL patients. Additionally, DCL patients displayed higher serum levels of anti-Leishmania IgG antibodies. We also demonstrated that eosinophils from both LCL and DCL patients responded to L. mexicana promastigotes with a robust oxidative burst, which was equally intense in both patient groups and significantly higher than in healthy subjects. Coincubation of eosinophils (from donors with eosinophilia) with L. mexicana promastigotes in vitro revealed various mechanisms of parasite damage associated with different patterns of granule exocytosis: 1) localized degranulation on the parasite surface, 2) the release of cytoplasmic membrane-bound “degranulation sacs” containing granules, 3) release of eosinophil extracellular traps containing DNA and granules with major basic protein. In conclusion, eosinophils damage L. mexicana parasites through the release of granules via diverse mechanisms. However, despite DCL patients having abundant eosinophils in their blood and tissues, their apparent inability to provide protection may be linked to the release of cytokines and chemokines that promote a Th2 immune response and disease progression in these patients.

Introduction

In humans, Leishmania mexicana can cause localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). The most common clinical form of the disease occurring in over 90% of the cases is LCL. It is characterized by ulcers at the sites of parasite inoculation, accompanied by a robust cellular immune response and a scarce number of parasites within the lesions. Patients with LCL generally experience a relatively benign clinical course [1]. Less than 1% progress to DCL, which is characterized by multiple non-ulcerated nodules containing heavily parasitized macrophages that cover large areas of the body. These patients exhibit an ineffective cell-mediated immune response against Leishmania parasites, leading to uncontrolled and potentially fatal progression [2]. In Mexico, 500–1000 new cases of cutaneous leishmaniasis are diagnosed every year [3].

In the early stages of infection, the inflammatory infiltrate includes neutrophils, macrophages, and eosinophils. Under normal conditions, eosinophils constitute only 1–5% of circulating leukocytes, although they can also be found in tissues as resident cells. Eosinophils are well equipped to combat pathogens, such as helminths, and their numbers increase during infection [4]. Eosinophils play key roles in immune modulation, tissue remodeling, and repair, primarily through the secretion and degranulation of inflammatory mediators, including cytokines and chemokines. However, their effector mechanisms can also inadvertently damage bystander cells and tissues [4,5].

During the acute phase of leishmaniasis, eosinophils can be present in skin lesions, often alongside mast cells. Furthermore, a close association between eosinophils and parasitized macrophages has been reported in chronic lesions, suggesting that eosinophils may contribute to parasite destruction through cooperation with macrophages [6]. Tissue eosinophilia has been observed in DCL patients, with evidence of parasitized and lysed eosinophils and dispersion of their granules near parasitized macrophages [7]. Additionally, both in the mouse model of leishmaniasis and in humans with chronic phases of the disease, eosinophils are frequently found in granulomas [8–10]. One of the mechanisms through which eosinophils assist in controlling the parasite is the production of hydrogen peroxide, which has a detrimental effect on Leishmania amazonensis [11]. However, the precise physiological role of eosinophils in leishmaniasis remains incompletely understood.

In this study, we conducted a comparative analysis of the in vitro response of eosinophils isolated from LCL and DCL patients, following their co-incubation with L. mexicana. Our analysis included the measurement of IL-6, IL8 and IL-13 production, as well as assessment of their oxidative burst.

Additionally, we co-incubated eosinophils from non-Leishmania eosinophilic subjects with L. mexicana promastigotes and analyzed their defense mechanisms against the parasites. Our findings reveal that eosinophils employ various defense mechanisms when confronted with L. mexicana. These mechanisms include phagocytosis, contact degranulation, the release of granules directly onto the parasite, expulsion of “degranulation sacs” containing granules surrounded by a membrane, which are released into the immediate vicinity of the parasites, and the release of eosinophil extracellular traps (EETs) containing DNA and granules rich in major basic protein (MBP). All of these mechanisms result in irreversible damage to the parasite. Our data provide new insights into the innate defense mechanisms exerted by eosinophils against L. mexicana, as well as differences in the eosinophil response between LCL and DCL patients.

Materials and methods

Ethics statement

The study received approval from the Ethics and Research Committees of the Facultad de Medicina, UNAM (Universidad Nacional Autónoma de México) with reference FM/DI/088/2017. We adhered strictly to the guidelines established by the Ministry of Health in Mexico. All participants and healthy donors (controls) were informed and provided written consent to participate in the study.

Patients

The number of participants varied across different study sections and these numbers are specified in each respective section. Patients diagnosed with LCL and DCL were residents of the State of Tabasco, an endemic area for L. mexicana in southeastern Mexico. All patients received anti-Leishmania treatment with pentavalent antimonials, as mandated by National Health Authorities. LCL patients presented active lesions with relatively few parasites. In contrast, DCL patients exhibited multiple nodular lesions characterized by heavily parasitized macrophages. The severity of DCL cases ranged from patients with nodules confined to the upper body to those with nodules affecting the entire body, limbs and oral and nasal mucosae. Leishmania parasites were detected in smears created from biopsy punches taken from the lesions, stained using Giemsa and subjected to histopathological analysis with hematoxylin and eosin (H&E) staining. For in vitro experiments, eosinophils from individuals with eosinophilia (n = 8) of unknown origin who sought care at the allergy ward in the Hospital General de México, were utilized. Only individuals with eosinophilia exceeding 6% were included in this study.

Peripheral blood eosinophils were quantified via flow cytometry, using FCS and SSC parameters, in patients with LCL (n = 30) and DCL (n = 4). Punch skin biopsies (Stiefel Laboratories, Inc., Coral Gables, FL, USA) were obtained from the lesions of LCL (n = 35) and DCL patients (n = 5), using 2% xylocaine as a local anesthesia. The biopsy specimens were fixed in 10% formalin and embedded in paraffin (Sigma-Aldrich, St. Louis, Mo, USA), cut into 4 μm thick sections, and stained with H&E, to evaluate eosinophil infiltration. Eosinophils were counted in eight images of each tissue, with a final area corresponding to 1 mm², using an Axio imager M1 microscope equipped with an MRc5 digital camera and Axiovision 4.8 software (Carl Zeiss, Industrielle Messtechnik GmbH, Oberkochen Germany).

Detection of IgE and IgG in human serum against Leishmania mexicana antigens

We determined IgE and IgG levels against L. mexicana antigens through ELISA in LCL (n = 8) and DCL patients (n = 8), as well as in healthy controls (n = 8). Each sample was analyzed in triplicate. Briefly, 96-well EIA/RIA plates (Corning, NY, USA) were coated with 0.6 μg/100 μl of lysed promastigotes in each well and incubated for 1 hour. The solution was removed and the plates were blocked with 200 μl of 0.5% casein for 1 hour. After blocking, 100 μl of diluted (1:5) sera from leishmaniasis patients or healthy donors were added and incubated overnight at 4°C. The plates were washed with PBS-Tween. For IgE detection, 100 μl of rabbit anti-human IgE (Dako, Agilent Technologies, Sta Clara, Ca, USA) (1:500) was added and incubated for 1 hour, followed by washing and the addition of 100 μl of biotin goat anti-rabbit IgG (1:8000) (Thermo Fisher Scientific, Waltham, Ma, USA) for 30 minutes. After washing, 100 μl of peroxidase-conjugated streptavidin (Zymed) (1:2500) was added and incubated for 30 min. For IgG detection, 100 μl of peroxidase-conjugated goat anti-human IgG (Zymed) antibody (1:4000) was added to the plates and incubated for 1 hour. Following washing,100 μl of TMB (Tetramethylbenzidine) peroxidase substrate (Becton Dickinson, NJ, USA) was added for 20 minutes. The reaction was halted with 100 μl of 1 M phosphoric acid. Absorbance was measured at 450 nm using an ELISA microplate reader (BIO-TEK).

Eosinophil isolation

Human eosinophils were obtained from peripheral blood samples of patients with LCL (n = 30) and DCL (n = 4). Cells were isolated through gradient centrifugation using Ficoll Hypaque (Sigma-Aldrich, St. Louis, Mo, USA). The blood was diluted 1:2 with sterile PBS pH 7.4 and layered over 20 mL Ficoll-hypaque (density ρ = 1.077 g/mL) in a 50 mL conical tube. Centrifugation was performed at 600×g for 30 min at 20°C using a swinging bucket rotor. The subsequent steps involve removing and discarding the plasma. Mononuclear cells were located at the plasma:Ficoll interface, granulocytes and erythrocytes were in the pellet. The pellet was resuspended in 1X Red Blood Cell Lysis Solution, and the complete 50 mL conical tube was filled with lysis solution. Incubation occurred for 10 min on ice, followed by centrifugation at 300×g for 10 min at 20°C. The supernatant was carefully removed entirely. Cells were washed adding 50 mL of buffer and the cell number was determined. The cell suspension was centrifuged at 300×g for 10 min. The supernatant was aspirated completely, and the cells were resuspended in 50 μl of buffer per 5x10⁷ cells. To this suspension, 50 μl of anti-CD16 conjugated microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) were added per 5x10⁷ cells and incubation occurred for 30 min at 4–8°C. Cells were washed by adding 1–2 mL of buffer per 10⁷ cells, followed by centrifugation at 300×g for 10 min. The supernatant was discarded, and the cells were resuspended to a final volume of up to 10⁸ cells in 500 μL of buffer. The suspension was then placed in a purification column, which had been previously washed with a buffer and positioned in the magnetic field of a MACS separator. Unlabeled cells, including eosinophils, were obtained by depletion of magnetically labeled cells. The total effluent containing the unlabeled cell fraction harbored the enriched eosinophil fraction. The purity and viability of eosinophils were >99%, as determined by Giemsa staining and by Trypan blue exclusion, respectively.

Leishmania mexicana promastigotes culture

L. mexicana promastigotes were cultured in blood agar (NNN medium) overlaid with Schneider’s Drosophila medium (Life Technologies), supplemented with 10% heat inactivated FBS (Biowest, Riverside, MO, USA) and 1% antibiotics (penicillin/streptomycin (Gibco, Life Technologies) at 28°C. Parasites were subcultured every 3–4 days and grown to a density of 1 x 10⁷/ml.

Cytokine measurement

The impact of L. mexicana on the production of IL-6, IL-8, and IL-13 by eosinophils from LCL (n = 7) and DCL (n = 7) patients, as well as healthy controls (n = 7) was analyzed as follows: 1 x 10⁶ cells were incubated for 18 hours at 37°C and 5% CO₂ with 10x10⁶ promastigotes in 1 ml of RPMI-1640 medium, supplemented with 10% heat-inactivated FBS. The cell-free supernatants from the cultures were collected and the concentrations of the cytokines were determined using human IL-6 and IL-13 Quantikine ELISA Kits (R&D Systems Minneapolis, MN, USA). IL-8 was analyzed by a standard sandwich ELISA. Briefly, 96-well flat-bottom microtiter plates (Costar, Corning, NY, USA) were coated with 1 μg/ml unconjugated anti-cytokine capture antibodies (Purified mouse anti-human IL-8 monoclonal antibody Clone G265-5, PharMingen) diluted in 0.1 M Na2HPO4 (pH 9) and incubated overnight at 4°C. The plates were blocked with PBS (pH 7.4) supplemented with 0.5% casein dissolved in 0.1 N NaOH. Cell-free supernatants and recombinant hIL-8 standard (PharMingen) were incubated in RPMI-1640 medium supplemented with 10% FBS overnight at 4°C. Bound human IL-8 was detected using 0.5 μg/ml biotin-labeled detection antibodies (Biotinylated mouse anti-human IL-8 monoclonal antibody Clone G265-8, PharMingen) diluted in 1% BSA with 0.05% Tween 20 and incubated for 1 hour at room temperature. The plate was developed using AP-Streptavidin Conjugate (Life TechnologiesTM) and phosphatase substrate (0.005 mg/mL, Sigma-Aldrich). Absorbance was read at 405 nm and the IL-8 concentration of each sample was calculated by regression analysis based on a standard curve. The detection limit of this assay ranged from 15.6 to 1000 pg/ml. The cut-off point is determined from the lower detection limit, values below this limit considered negative. Unstimulated eosinophils were included in all cytokine production determinations; however, they were not included in the plot as they were below the cut-off point.

Oxidative burst

The oxidative burst was measured by chemiluminescence immunoassay with eosinophils obtained from the same group of patients and controls as in the cytokine analysis. Briefly, 1x10⁶ eosinophils were incubated with 10x10⁶ promastigotes in 80 μl of RPMI 1640 medium, 210 μl of luminol (Sigma-Aldrich, St. Louis, Mo, USA) at a concentration of 1 mg/ml, and 60 μl AB+ serum-opsonized zymosan (12.5 mg/ml). Chemiluminescence was analyzed for 30 min (Luminoskan Labsystem, Finland) at 37°C with a 550 nm filter [12]. The values obtained were expressed in mVolt.

Interaction of Leishmania promastigotes and blood eosinophils from healthy controls

The interaction between L. mexicana and eosinophils was analyzed using 5-carboxyfluorescein diacetate (CFDA) staining. Leishmania promastigotes (10x10⁶) were washed twice with cold RPMI and stained with 5 μM CFDA (Sigma-Aldrich, St. Louis, Mo, USA) for 10 min in RPMI at 37°C. The parasites were washed three times with PBS at 26°C and incubated with 1x10⁶ eosinophils for 2 hours at 37°C, protected from light. They were then centrifuged and resuspended in 10–20 μl of PBS and analyzed with an epifluorescent microscope [13]. Some of the smears were stained with Giemsa.

Transmission electron microscopy

Following careful washing with PBS, co-cultures of eosinophils (1x10⁶) and promastigotes (10x10⁶) were fixed in 2.5% glutaraldehyde diluted in 0.2 M cacodylate buffer, pH 7.4, for 2 hours, at 4°C. After three washes of 10 minutes each in 0.15 M cacodylate buffer, pH 7.4 at 4°C, the pellets of the cocultures were postfixed with 1% osmium tetroxide in 0.2 M cacodylate buffer, pH 7.4, for 30 min at 4°C. Dehydration was carried out in gradually increasing concentrations of ethanol, followed by transfer to absolute toluene, using two changes of 10 min each. Infiltration was done with araldite 6005-toluene solution (1:1) for 24 hours at room temperature and then in pure araldite 6005 (two changes) at 60°C for 2 hours. Specimens were embedded in pure araldite 6005 at 60°C, for 36 hours. Ultrathin sections were obtained with a diamond knife, contrasted with uranyl acetate and lead citrate, and examined using an Electron Microscope (EM) 109 (Carl Zeiss, Germany). All reagents used were from Electron Microscopy Science, Hatfield, PA, USA.

Immunocytochemistry for detection of eosinophil major basic protein (MBP)

For the immunocytochemical analysis, 1x 10⁶ purified eosinophils were incubated in a chamber slide (Nunc Lab-Tek, Sigma-Aldrich, St. Louis, Mo, USA) for 2 hours at 37°C and 5% CO₂ with 10x10⁶ L. mexicana promastigotes in 1 ml of RPMI-1640 medium supplemented with 10% heat-inactivated FBS. After incubation, they were washed with PBS and fixed with 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Mo, USA). Nonspecific antigen sites were blocked with 2% bovine serum albumin, dissolved in Tris-HCl, pH 7.6. Thereafter, they were stained with mouse anti-human MBP at a dilution of 1:100 (Santa Cruz Biotechnology, Ca, USA) for 30 min. After washing, eosinophils were incubated with the secondary antibody, biotin goat anti-mouse at a dilution of 1:100 (Invitrogen, Waltham Ma. USA) for 30 min, washed and incubated with streptavidin AP (Alkaline Phosphatase) at a dilution of 1:100 (Thermo Fisher Scientific, Waltham Ma. USA) for 30 min. After washing, they were counterstained with hematoxylin and mounted with resin. Additionally, the incubation of eosinophils with promastigotes was also stained with DAPI in separate samples (Sigma-Aldrich, St. Louis M. USA) to verify the formation of DNA nets.

Statistical analysis

Comparisons between experimental groups and controls were performed using Mann–Whitney U-test. A value of p < 0.05 was considered statistically significant, using Prism 8 for Windows.

Results

Eosinophils in peripheral blood and lesions of LCL and DCL patients

Blood samples were collected from 30 LCL patients (19 males and 11 females), with a mean age of 28 years (range: 16–78 years) and disease durations ranging from 1.5 to 18 months (mean: 16 ± 3.98). Four DCL patients (three males and one female) with a mean age of 37 years (range: 20–45 years) and disease durations ranging from 40–120 months (mean: 67 ± 18.14 months) were also included. Eosinophils were quantified in peripheral blood using flow cytometry. The mean percentage of eosinophils in LCL patients was 11.1 ± 1.51 with 60% of LCL patients exhibiting eosinophilia ranging from 5% to 32% (Fig 1A). There were no significant correlations between the number of eosinophils in peripheral blood and disease duration, age or gender (data not shown). Notably, all female patients had a shorter disease duration compared to male patients (Fig 1B), with females showing an average disease evolution of 6.5 ± 1.45 months, while males had an average of 21.4 ± 5.95 months. All DCL patients exhibited blood eosinophilia, with a mean percentage of eosinophils in the blood of 16 ± 5.8%, ranging from 7% to 34% (Fig 1A). It is noteworthy that all DCL and few LCL patients with chronic infections lasting over two years had blood eosinophilia (Fig 1B).

Fig 1 — A. Percentage of blood eosinophils from patients with LCL (black circles, n = 30) and DCL (blue squares, n = 4). B. Blood eosinophils and disease evolution time according to disease form and gender are shown: LCL male patients are indicated with green filled circles; DCL male patients are indicated by filled red squares. LCL female patients are indicated by empty green circles; DCL female patients are indicated with empty red squares). The horizontal line represents the mean, and each symbol represents a patient (female or male). Bars represent the mean ± SEM. ns: non-significant.

Eosinophils in lesions of LCL and DCL patients

Skin biopsies were obtained from 35 LCL patients and 5 DCL patients. When analyzing skin lesions, significant differences were observed between eosinophil numbers of both clinical forms (Fig 2A). Eosinophils were observed in skin lesions of all 5 DCL patients with a mean of 14 ± 1.15 eosinophils/mm² (ranging from 11 to 18). In contrast, only 20% of LCL patients had eosinophils in their tissues, with a mean of 3 ± 1 eosinophils/mm² (ranging from 0 and 18). When comparing eosinophils in tissues between LCL and DCL patients, a significant difference was found between the groups, with significantly higher numbers in DCL patients (Fig 2A). However, when specifically comparing LCL patients with tissue eosinophils to all DCL patients, no significant differences were found between the two groups of patients. LCL patients with tissue eosinophils showed an average of 13 ± 1.6 eosinophils/mm² (ranging between 5 and 18), while DCL patients showed an average of 14 ± 1.1 eosinophils/mm² (ranging between 11 and 17). When comparing eosinophils in tissues between female and male of LCL and DCL patients, a significant difference was found only between males (Fig 2B). Histopathological analysis of infected tissues revealed that DCL patients had loss of tissue integrity and a higher parasite load, compared to those of LCL patients (Fig 2. bottom panel).

Fig 2 — A. Eosinophils in the tissue were quantitated and expressed as eosinophils/mm²_. LCL patients (black circles, n = 35) and DCL patients (blue squares, n = 5). B. Eosinophils/mm² in tissue lesions according to disease form and gender. Male LCL patients (n = 25, filled green circles) and female LCL patients (n = 10, empty green circles); Male DCL patients (n = 4, filled red triangles) and female (n = 1, empty red triangle). Bottom panel: Representative H&E staining of biopsies taken from LCL and DCL patients. Black arrows indicate *Leishmania* amastigotes and blue arrows indicate eosinophils. Scale bar = 20 μm. Bars represent the mean ± SEM, and asterisks represent statistically significant differences (P < 0.05).

Anti-Leishmania IgE and IgG in sera from LCL and DCL patients

Serum levels of IgE and IgG were measured in LCL (n = 8) and DCL (n = 8) patients to determine the serological response to L. mexicana. Both antibodies were identified in all patients, with no significant differences in IgE levels between DCL (1.43 ± 0.18) and LCL patients (0.95 ± 0.19). However, both patient groups produced significantly more IgE than controls (0.167 ± 0.068) (Fig 3A). When comparing IgG production, DCL patients (1.73 ± 0.05) produced significantly more than LCL patients (1.31 ± 0.11), and both patient groups produced more IgG than controls (0.293 ± 0.074) (Fig 3B).

Fig 3 — A. IgE levels. B. IgG levels. Each symbol represents a patient, and the horizontal line represents the mean of each group. LCL (n = 8) and DCL (n = 8) patients, healthy controls (n = 8). *All symbols represent statistically significant differences (P < 0.05).

Cytokine (IL-6, IL-13, and IL-8) production and oxidative burst by EOSINOPHILS of LCL and DCL patients incubated with L. mexicana promastigotes

Our data indicate that IL-6 and IL-13 production by eosinophils from patients with DCL (n = 7) was significantly higher, compared to those from LCL patients (n = 7) or healthy control subjects (n = 7), when incubated with L. mexicana promastigotes. IL-6 production by DCL eosinophils (45 ± 13.2 pg/mL) was significantly higher compared to LCL patients (7.4 ± 1 pg/mL). Control subjects did not exhibit IL-6 production (Fig 4A).

Fig 4 — Eosinophils from LCL and DCL patients and from healthy controls were incubated with L. *mexicana* promastigotes. A. IL-6 production. B. IL-8 production. C. IL-13 production. D. ROS production measured by luminol reaction (in mV). Bars represent the mean ± SEM (n = 7 for each group of patients and healthy control subjects). All symbols represent statistically significant differences (P < 0.05).

IL-8 production by eosinophils of DCL patients (45 ± 2.6 pg/mL) was also significantly higher compared to LCL patients (29 ± 2 pg/mL) and control subjects (40 ± 1.7 pg/mL). Interestingly, eosinophils of control subjects produced nearly the same amount of IL-8 as those of DCL patients) (Fig 4B).

In the case of IL-13 production, eosinophils of DCL patients showed a significantly higher production (1000 ± 11.5 pg/mL) compared to LCL patients and controls (50 ± 2.8 pg/mL) (Fig 4C). These findings demonstrate that eosinophils from DCL patients produce significantly more IL-6, IL-8, and IL-13 when exposed to L. mexicana promastigotes compared to those of LCL patients. Unstimulated eosinophils did not exhibit IL-6, IL-8, and IL-13 cytokine production and were below the ELISA cut-off point.

The analysis of the oxidative burst in eosinophils of LCL (n = 7), DCL (n = 7) and healthy subjects (n = 7) co-incubated with L. mexicana promastigotes showed that the parasite enhances the magnitude of the oxidative burst in eosinophils of both groups of patients to an equal extent, which was significantly higher compared to eosinophils from healthy controls (Fig 4D). However, no significant differences between both clinical forms.

Phagocytosis and lysis of Leishmania mexicana promastigotes by eosinophils

Microscopic analysis revealed that purified eosinophils do not release granules in the absence of parasites (Fig 5A). When co-incubated with L. mexicana, eosinophils exhibited the ability to damage extracellular promastigotes by releasing cytoplasmic granules. This damage resulted in the alteration of parasite morphology and the acquisition of a deformed phenotype (Fig 5B). Giemsa staining further demonstrated that damage occurred when Leishmania was in close contact with the eosinophils, as well as when parasites were surrounded by clouds of massively released granules (Fig 5B). Staining with CFDA revealed that L. mexicana promastigotes began to form clusters in the culture medium (Fig 5C). After incubation with eosinophils, the promastigotes are phagocytosed and transformed into intracellular parasites, ultimately leading to their degradation (Fig 5D). The percentage of eosinophil degranulation was significantly higher in eosinophils co-incubated with Leishmania (Fig 5E).

Eosinophils release cytoplasmic “degranulation sacs” with granules in response to Leishmania

Co-incubation of eosinophils with L. mexicana revealed that, in addition to diffusely releasing cytoplasmic granules near parasites, eosinophils also release cytoplasmic “degranulation sacs’’ containing granules with electron-dense characteristics, surrounded by a membrane (Fig 6). Subsequently, as the cell membrane was lost, individual granules came into close contact with the parasites, leading to damage (Fig 6A and 6B). Electron microscopy analysis further illustrated that eosinophils could shed substantial portions of their cytoplasm, including granules, in the form of “degranulation sacs’’ onto the parasite. This phenomenon was evidenced by the presence of submembrane microtubules and caused significant damage to the parasites. Signs of parasite damage included diffuse osmophilic areas, indicating damage to intracellular membranes (Fig 6C and 6D).

Fig 6 — A. Eosinophil (green arrow) and release of cytoplasmic “degranulation sacs’’. B. Degranulation sacs (black arrows) and parasites (pink arrow). C and D. Ultramicroscopic images showing released specific or secondary granules (g) or within cytoplasmic pockets limited by an intact membrane (black arrows). These “degranulation sacs” were in close position to distorted L. *mexicana* (Lm). Parasites, identified by their submembranous microtubule cytoskeleton (arrowheads) show signs of damage as evidenced by osmiophilic areas (white asterisk), nuclear fragmentation (n), and a clear or aqueous cytoplasm denoting an extensive cytoplasmic vacuolization. (D) Some eosinophil specific granules still retain the characteristic electron-dense central crystalline core, surrounded by a clearer peripheral matrix (red arrows). Light microscopy scale bar = 20 μm. TEM scale bar = 500 nm.

Extracellular traps are induced by L. mexicana

Another leishmanicidal mechanism observed in vitro was the release of extracellular traps (EETs) by eosinophils, which entrapped the parasites and brought them into contact with released granules such as MPB (7A). Under resting conditions, only eosinophil nuclear DNA was stained blue (7B). However, after coincubation with Leishmania, eosinophils released nets containing DNA, as indicated by blue DAPI staining, which binds DNA (Fig 7C). These DNA nets effectively trapped the parasites (Fig 7D).

Fig 7 — A) Extracellular traps (black asterisk) containing MBP-positive granules (black arrow) and parasites within the nets (red arrow). B) Purified eosinophils show their nuclei stained blue with DAPI (green arrow). No nets are formed in the absence of *Leishmania*. C) Eosinophils co-incubated with L. *mexicana* promastigotes show the formation of extracellular DNA traps (white arrow). D) The same image (C) was analyzed with light and fluorescence microscopy, showing the parasites (red arrow) in contact with nets (white arrow). Scale bar = 20 μm.

Discussion

Eosinophils, constituting 1–5% of human blood leukocytes, play a multifunctional role in defending against various infections, including parasites, viruses, bacteria, and fungi [4,5,14]. However, the understanding of eosinophils’ role in patients infected with L. mexicana, presenting varying disease severity, such as localized (LCL) or diffuse (DCL) cutaneous leishmaniasis, remains limited.

In our study, we observed that all DCL patients, and a subset of LCL patients with prolonged disease evolution exhibited both blood and tissue eosinophilia. Blood eosinophilia has previously been reported in L. mexicana-infected DCL patients and recently cured LCL patients [15], as well as in patients infected with high numbers of L. donovani parasites [16]. Mouse models of leishmaniasis have also demonstrated that the eosinophil responses depend on mouse strains, Leishmania species, and the phase of infection [10,17,18]. Elevated eosinophil numbers, persisting throughout the infection, have been described in susceptible mouse strains infected with L. mexicana and L. amazonensis, which correlated with enhanced tissue damage and high numbers of intracellular amastigotes [6,10].

Eosinophils employ various mechanisms to control pathogens, including oxidative bursts, phagocytosis, and the release of eosinophil extracellular traps (EETs). EETs contain histones, eosinophil cationic proteins, and cytotoxic granules such as MBP [19,20] within “degranulation sacs’’, which are released through a single fusion pore [21]. Furthermore, the shedding of clusters of free extracellular eosinophil granules, as well as of plasma membrane-enveloped structures containing cytoplasmic granules, has been reported. After membrane lysis, the granules are released into the extracellular space, where they function as free granules [22].

Our findings demonstrate that eosinophils phagocytize L. mexicana promastigotes in vitro (Supplementary 1), which is accompanied by various forms of granule exocytosis that inflict damage to the parasite, as confirmed by electron microscopy and Giemsa staining. The contact between the parasites and eosinophils induced the release of free granules and the extrusion of membrane bound “degranulation sacs’’ containing numerous granules, which are released near the parasites, leading to damage (Supplementary 2). This phenomenon resembles a piecemeal degranulation process [21,22]. Furthermore, eosinophils released EETs containing MBP in response to L. mexicana parasites. Our data align with previous reports of eosinophils attaching to and phagocytizing Leishmania mexicana amazonensis parasites, resulting in oxidative bursts and the release of extracellular traps containing membrane-bound clusters of granules, as well as free granules, both of which potentially contribute to parasite elimination [23–25]. Although our observations were conducted in vitro, this phenomenon may be expected to occur in infected tissues, thus contributing to tissue damage [26,27].

Eosinophil cytokines modulate the immune response, but their involvement in defining the virulence of cutaneous leishmaniasis is not well understood. Leishmaniasis, a disease with divergent outcomes based on the balance of pro- and anti-inflammatory cytokines, is regulated by cytokine-producing cells. Th1 cytokines support parasite control, while a Th2 response is associated with susceptibility [5,14]. Given that the immune response in DCL patients cannot effectively control the extensive spread of parasites, we were interested in examining whether eosinophil responses to L. mexicana differ between DCL and LCL patients, potentially favoring disease progression.

Our data now show that eosinophils from DCL patients secrete significantly higher levels of IL-6, IL-8 and IL-13 in response to L. mexicana promastigotes, compared to eosinophils from LCL patients. This differential response between DCL and LCL eosinophils, may be related to the genetic background of patients, as previous studies have reported differential expression of inflammatory genes in NK cells from DCL and LCL patients following TLR stimulation with LPG, with LCL NK cells expressing more genes associated to inflammatory responses compared to DCL NK cells [28].

Previous studies have reported the presence of IL-6 during the active phases of infection by several Leishmania species [29–31]. IL-6 along with IL-1 and IL-23 regulates Th17 cell differentiation [31]. The Th17 response promotes the chemotaxis of neutrophils through the induction of IL-8 on fibroblasts, epithelial, and endothelial cells [32]. In experimental cutaneous leishmaniasis, the Th17 response has been associated with an increase in pathological conditions, primarily due to the neutrophil-mediated promotion of Th17 response [33].

Early recruitment of neutrophils to the infection site mediated by IL-8 also leads to their activation and enhanced phagocytosis of the parasite. Neutrophils not only harbor the parasite, shielding it from the deleterious effects of complement, but they have also been described to aid in parasite spread through “silent” phagocytosis that occurs after apoptotic cells containing the parasites are phagocytosed by non-activated macrophages (“Trojan horse” strategy) [34–36]. Our results of enhanced production of this chemokine are in line with the literature, where infections by L. infantum, and L. major have all been shown to enhance the production of IL-8 [37–39]. Additionally, aside from disease modulation resulting from enhanced IL-8 production, polymorphisms in IL-8 (-251A/T) or IL-6 (174G/C) genes should also be considered in future studies, as they have been shown to influence the outcome of mucosal leishmaniasis [40–42]. This aspect remains to be analyzed in cutaneous leishmaniasis caused by L. mexicana.

In visceral leishmaniasis (VL) caused by L. infantum, higher IL-6 and IL-17 levels were found in male patients, as compared to females, which correlated with enhanced disease severity in males [43,44]. We now show that IL-6 production by eosinophils was higher in DCL patients as compared to LCL patients, suggesting that IL-6 plays a role in disease severity in cutaneous leishmaniasis. Furthermore, our finding of elevated production of IL-13 by eosinophils of DCL patients is another factor that might contribute to enhanced disease severity. IL-13 activates eosinophils and promotes the development of alternatively activated macrophages, which have been shown to promote disease progression in cutaneous leishmaniasis [14,31,37]. This cytokine is functionally redundant with IL-4 and inhibits the production of inflammatory cytokines, thereby interfering with resistance to Leishmania [45]. Furthermore, IL-13 can promote the isotype switching of B cells to produce IgE and IgG4 in humans [46]. Our observations reveal that both DCL and LCL patients have elevated anti-Leishmania IgE and IgG antibodies. Although no differences were observed in IgE production between both clinical forms, it is noteworthy that both patient groups tested positive for IgE, contrasting with other reports of IgE in L. amazonensis infections, where only 40% of patients were positive for this antibody isotype [47]. However, we found that DCL patients had significantly higher levels of IgG, compared to LCL patients. This is noteworthy, as higher amounts of IgG in leishmaniasis have been associated with susceptibility, as IgG induces the production of regulatory IL-10 in monocytes, contributing to the inhibition of cellular responses in chronic DCL patients [48].

In summary, these three cytokines have been associated with disease susceptibility, and our data align with the literature, showing their enhanced production by eosinophils from DCL patients under in vitro conditions. It is possible the higher eosinophil numbers in the infiltrated inflammatory areas of DCL patients contribute to the production of these cytokines, further exacerbating disease severity. However, this hypothesis remains to be investigated in the context of cutaneous leishmaniasis caused by L. mexicana.

We found that all female patients had a shorter disease duration compared to male patients. This could be due to differences in the immune system between females and males, that have already been well documented [49]. These differences possibly affect disease susceptibility to leishmaniasis. In male mice infected with L. major, parasite load was shown to be related to eosinophils. A significant correlation between elevated eosinophil infiltration and increased parasite loads was only found in male mice infected with L. major, suggesting that eosinophils seem to be related to disease severity in males [50]. Thus, circulating sex hormones, and possibly additional parasite factors, affect the immune response and play a role in the disease outcome [51,52]. The influence of sex hormones on Leishmania parasites was shown in experiments where Leishmania mexicana was incubated with dihydrotestosterone, which led to their enhanced growth and infectivity and may be one of the possible factors associated with male susceptibility to Leishmania [53]. It remains to be analyzed whether men with a longer disease course, are also more likely to develop eosinophilia.

The results obtained shed new light on the apparent dual role of eosinophils in leishmaniasis: they can inflict crucial damage to the parasite showing a healing effect, whereas they can also cause worsening of the disease by producing cytokines such as IL-6, IL-8 and IL-13 upon contact with the parasite, associating eosinophils with disease severity and pathological conditions.

Conclusions

Eosinophils employ diverse effector mechanisms when co-incubated with Leishmania mexicana, including the exocytosis of granules onto the parasite, the release of membrane bound “degranulation sacs”, and the formation of EETs, all of which contribute to parasite damage. However, despite presenting eosinophilia, DCL patients exhibit a high parasite load and uncontrolled disease progression. Given that eosinophils from DCL patients also release significantly higher amounts of cytokines IL-6, IL-13, and the chemokine IL-8, compared to LCL patients, these mediators may contribute to the disease progression observed in DCL patients. Our data provide additional insights into the innate defense mechanisms exerted by eosinophils against Leishmania mexicana. To further clarify the biological significance of eosinophils in leishmaniasis it would be interesting to study other effector mechanisms, including signaling pathways, signal transductions, and polymorphisms in the eosinophils of these patients, in order to enhance our understanding of eosinophils in this disease.

Supporting information

S1 Fig. Phagocytosed parasites by eosinophils.

A) Parasites stained with CFDA (green color) phagocytosed by eosinophils are observed inside these cells. Black arrows show phagocytized parasites. B) Remains of degraded parasites (black arrows) are observed already inside the eosinophils (green color granules). The co-incubation ratio was 1:10 for 2 hours. Scale bar = 20 μm.

(TIF)

Click here for additional data file.^{(5.2MB, tif)}

S2 Fig. Leishmania mexicana damaged by eosinophil contact.

A) Normal shape and size of a viable Leishmania promastigote. B) Morphological changes in parasite size, damage to membranes, formation of small vacuoles within the parasite, and loss of flagellum were observed in parasites co-incubated with eosinophils in a 1:10 ratio for 1 hour. Black arrows show damaged parasites. Scale bar = 20 μm.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by UNAM-PAPIIT IG201221 and CONACyT-Fronteras 6682. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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PONE-D-23-05814Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophilsPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The article “Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils “ by Norma Salaiza-Suazo et al., describe in several experiments the presence and response of eosinophils in LCL and DCL. Eosinophils have been neglected in the leishmaniasis contexts, probably because their significance is overshadowed by the importance of macrophages and T cells. This study address a relevant gap in the knowledge. However results here shown are rather descriptive than conclusive.

Major comments:

Methods section is in many points poorly described:

-Need more specification on the eosinophil isolation: How long was centrifuged? How was done the RBC lysis? How many washes/time/g-force?

-Leishmania was cultured in the absence of antibiotics?

-Also in-house method for IL-8 measurement lacks important information: which capture/detection antibody was used? Please describe the protocol.

-How was the cytometry done? What was the antibodies used and gating strategy applied?

-How was the cut-off in ELISA calculated?

-Why is the luminescence in oxidative burst measured as Mmvolt?

Line 309 – 310 “These findings demonstrate that eosinophils from patients with

DCL produce significantly more IL-6, IL-8, and IL-13 when exposed to L. mexicana

promastigotes, compared to those of LCL patients.” Cytokine measurement should have been done with unstimulated eosinophils. This is important to be sure that the cytokine production is not due to a pre-activated status. How could the authors be sure the cytokines produced were induced by parasites rather than being already activated?

Line 318-330 – To assume that eosinophils are being harmful against leishmania, the authors should show quantitatively increased death of promastigotes while in contact with Leishmania. They could use cellular death markers (Propidium iodide for example) to quantify the number of dead promastigotes in contact with eosinophil.

Lines 407-409 – It is not correct to associate IL-6 with TH2 responses. IL-6 is mainly produced by myeloid cells (mostly macrophages) and this cytokine is increased during VL together with a TH2 response, independently. IL-6 is required to a TH17 response and whether their production by eosinophils is important/significant during cutaneous leishmaniasis is yet to be shown.

Line 435: The authors says that “it is noteworthy that 70% LCL and 90% DCL were positive for IgE”, although figure 3 clearly shows (as well as is described in the text) that all patients were IgG and IgE positive. Please revise this excerpt and figure.

Lines 442 – 444 – authors must be careful to assume “an enhanced production of cytokines” in DCL patients, since they only measured eosinophil derived cytokines and this does not mean that these cytokines are in fact increased in this patients or even related to susceptibility (for example, IFN-g is associated with resistance and it is also increased during VL). The production of these cytokines by eosinophils compared to other cells (macrophages and monocytes) might be insignificant in natural conditions.

Figure 5 - Authors claim to demonstrate phagocitozed leishmania by eosinophil but the image has bad quality. Do authors have clear evidence of phagocytosis by eosinophils (stained slides would be enough).

Authors should discuss why an innate immune cell would respond differently in DCL and LCL. Which possible mechanisms would allow eos from DCL to be more prone to respond to promastigotes than LCL?

Line 449: “their release of the cytokines IL-6, IL-8 and IL-13 promotes disease severity”: the data here shown do not support this statement.

Reviewer #2: The authors report on an important and up-to-now largely neglected aspect of a cellular repsonse to Leishmania infection. This study certainly will be of great interest.

Major concerns:

- The statistical method employed should be detailled in the respective Fig. legends. The authors might want to use multivariate testing.

- The authors should demonstrate a by FACS that their MACS-based enrichment of eosinophils worked.

- The Fig. 5 - 7 should be analyzed using a readout that allows statistical analysis. Otherwise these findings seem very anectodal.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2024 Feb 15;19(2):e0296887. doi: 10.1371/journal.pone.0296887.r002

Author response to Decision Letter 0

28 Sep 2023

Major comments:

1) Methods section is in many points poorly described:

a) Need more specification on the eosinophil isolation: How long was centrifuged? How was done the RBC lysis? How many washes/time/g-force?

R= New information was added on lines 168-190

b) -Leishmania was cultured in the absence of antibiotics?

R=The information was added on lines 196-197.

c) -Also, in-house method for IL-8 measurement lacks important information: which capture/detection antibody was used? Please describe the protocol.

R=The information was added on lines 207-226.

d) -How was the cut-off in ELISA calculated?

R= The cut-off point information was added on lines 223-224.

e) - How was the cytometry done? What was the antibodies used and gating strategy applied?

R= Eosinophils in peripheral blood were determined by FCS and SSC by flow cytometry, this information was added on lines 133-134. The figure below represents the gating strategy.

f) -Why is the luminescence in oxidative bursts measured as Mmvolt?

R= The Luminoskan's light detector has a photomultiplier tube (photomultiplier tube), a signal acquisition and processor system, which are in a closed compartment, where all the chemiluminescence is captured by the photomultiplier tube which sends the light signal and according to the configuration of our device (Luminoskan) the maximum light signal generated in the chemiluminescence reaction is reported in mVolts.

Line 309 – 310 “These findings demonstrate that eosinophils from patients with

DCL produce significantly more IL-6, IL-8, and IL-13 when exposed to L. mexicana

R= Unstimulated eosinophils were included in all cytokine production determinations. However, they were not included in the plot because they were below the cut-off point. The information was added on lines 352-354.

R= No staining with supravital dyes was performed, however, to demonstrate cell damage we are now including a new supplementary figure 2 showing Giemsa-stained viable intact parasites compared to damaged parasites incubated with eosinophils, where morphological changes and cell damage of the parasites are visible. This observation was also evidenced in the electron microscopy photographs of Figure 6, showing damage of the external membrane of the parasite.

R= We agree and have removed the statement regarding the association of IL-6 with a Th2 response. Instead, we added the relevance of IL-6 in the Th17 response in cutaneous leishmaniasis on lines 459-464.

R= We thank the reviewer and made the correction on line 488.

R= We thank the reviewer and have changed the text on lines 496-501.

R= Figure 5 was modified with a better photograph in panel 5D that shows parasites phagocyted by eosinophils. Additionally, we added a new supplementary figure 1 with more photographs showing the phagocytosis and possible intracellular degradation of the parasite.

Authors should discuss why an innate immune cell would respond differently in DCL and LCL. Which possible mechanisms would allow eos from DCL to be more prone to respond to promastigotes than LCL?

R= We now added a possible explanation for the differential response between LCL and DCL patients on lines 452-457, including a new reference (30) where differences in genetic background between both patients have been reported.

Line 449: “their release of the cytokines IL-6, IL-8 and IL-13 promotes disease severity”: the data here shown do not support this statement.

R= We agree with the reviewer and changed the statement on lines 503-505.

Reviewer #2: The authors report on an important and up-to-now largely neglected aspect of a cellular repsonse to Leishmania infection. This study certainly will be of great interest.

Major concerns:

- The statistical method employed should be detailled in the respective Fig. legends. The authors might want to use multivariate testing.

R= The objective of the work was to compare the response between eosinophils of LCL and DCL patients and for this approach we decided use to the Mann–Whitney U-test.

New information was added in Figure legends 1, 2 and 5, detailing the statistical significance of the figures.

- The Fig. 5 - 7 should be analyzed using a readout that allows statistical analysis. Otherwise these findings seem very anectodal.

R= We agree and modified Figure 5, adding the panel 5E, that quantitatively describes the % of degranulation induced by parasite incubation. This result was also included in line 375-376.

- The authors should demonstrate a by FACS that their MACS-based enrichment of eosinophils worked.

R= We don´t have a FACS analysis of the enrichment of eosinophils, but include for the reviewer the figure below that shows a Giemsa stain of peripheral blood cells from patients before (A, B) and after the MACS-based enrichment (C, D). The purity of eosinophils was >99%. The information was described on lines 190-191. This purity was analyzed by a microscopical analysis of the characteristic acidophilic granules and the polymorphic nuclei as markers of eosinophils.

Figure 1. Giemsa stain of eosinophil MACS-based enrichment. A and B show peripherical blood cells from eosinophilic patients. C and D show eosinophil enrichment. Scale bar = 20 µm.

I am sorry, but I could include the image to this text to show the enriched eosinophils.

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PLoS One. doi: 10.1371/journal.pone.0296887.r003

Decision Letter 1

Vyacheslav Yurchenko

10 Nov 2023

PONE-D-23-05814R1Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophilsPLOS ONE

Dear Dr. Becker,

I invite authors to revise their work but I do not anticipate another round of review if all the comments are properly addressed in the manuscript and rebuttal letter.

Please submit your revised manuscript by Dec 25 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Vyacheslav Yurchenko, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The entire manuscript has been improved, and the questions were mostly solved. However a final adjustment must be done. The cytometry plot is very strange and unusual. Is there a better figure that could be alternatively used? I recommend that the authors report more details about the method used: how flow cytometry as performed? How RBC were lysed? How do they processed the blood? Important to mention which equipment was used for flow cytometry.

Reviewer #3: The study by Salaiza-Suazo et al. addresses the important yet mostly unknown aspect of eosinophils in the immune response against CL in the new world. Furthermore, this study uses patient samples to compare various parameters of eosinophil responses among those experiencing the localized (LCL) versus the severe diffuse (DCL) form of disease. Overall, the study is a useful step towards elucidating the role of eosinophils in L. mexicana infection as well as in sorting out differences contributing to LCL versus DCL.

I consider that in the R1 version of the manuscript, the authors have appropriately addressed the initial set of concerns and suggestions raised by the reviewers. There are no major concerns.

Minor concerns: A total of 25 minor concerns have been added as comments in the pdf version of PONE-D-23-05814R1.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

**********

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PLoS One. 2024 Feb 15;19(2):e0296887. doi: 10.1371/journal.pone.0296887.r004

Author response to Decision Letter 1

16 Dec 2023

Dear Editor,

we thank the reviewers for the valuable comments that have improved the manuscript. We have made the suggested corrections and addressed the points raised by the reviewer and hope the manuscript is now suitable for publication.

Corrections are listed according to the lines in the original manuscript, which are shown in the parentheses.

1.- (Line 34) extra spaces were removed in line 34.

2.- (Line 98) “mexicana” was corrected to lower case in line 97.

3.- (Line 188-190) Font size was corrected lines 189-190.

4.- (Line 244) the text was corrected to “intracellular parasites” in line 375.

5.- (Line 289) The age range of LCL patients was included in line 289.

6.- (Line 296) the correction was made changing 1B to “1A” in line 295.

7.- (Lines 300, 304) New information and 4 new references (49-52) suggested by the reviewer were included. Furthermore, an additional reference (53) on male susceptibility was also included. The new text included in the manuscript is found in lines 508-522.

8.- (Line 306) We have modified figure 2, including a new graph of eosinophil counts in tissues (2B) of males and females with LCL and DCL. Due to the modifications of Figure 2, changes were made in line 308, where the word “Fig. 2A” was inserted and in line 314. Furthermore, the Figure legend for Figure 2 was modified in lines 816-820. Unfortunately, we do not have all the ages of the patients available at the moment.

9.- (Lines 318-320) A text describing results in Figure 2B was included in lines 815-819.

10.- (Line 353) the correction was made in line 354.

11.- (Line 374) The word “amastigote” was substituted for “parasites” in line 375.

12.- (Line 376) The correction was made and now reads Fig 5“E” in line 377.

13.- (Line 402) The correction was made stating “1-5 %” in line 403, ref. #4 is cited.

14.- (Lines 404 and 405) the corrections were made in lines 405 and 406.

15.- (Line 406) The comment was addressed in lines 508-522.

16.- (Line 451)

#1. The observation was addressed in lines 508-522 of the discussion and 4 new references (49-52) suggested by the reviewer were included. Furthermore, an additional reference (53) on male susceptibility was also included. The new text added to the manuscript is found in lines 508-522.

#2. The observation was addressed in the discussion lines 479-483. The corrected Figure 2B now includes information on eosinophilia and male patients with localized or diffuse cutaneous leishmaniasis and 2 new references (43, 44) were added.

17.- (Line 479) The “L” was corrected and now reads IL-13 in line 485

18.- (Line 503) The sentence was corrected in lines 523-527.

19.- (Line 758) Figure Legend 1 was corrected (lines 806-809). With regard to the suggestion of illustrating males vs females in panel 1A, we believe that the information is already presented in panel 1B, and prefer not to overload panel A.

20) (line 760) The word “dot” was substituted by “symbol” in line 810.

21)- (line 769) We have included more information in new Figure 2B.

22) (Line 800) The additional space was eliminated in line 859.

23) (Line 807) The information of the scale bar was included in line 861.

24) (Line 824) The information for supplementary Figure 1 was added in line 878.

25) (Line 830) The information for supplementary Figure 2 was added in line 885-886.

26) The symbols were modified in Fig.1A and the Figure legend was adjusted (lines 806-810).

Thank you very much.

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PLoS One. doi: 10.1371/journal.pone.0296887.r005

Decision Letter 2

Vyacheslav Yurchenko

20 Dec 2023

Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils

PONE-D-23-05814R2

Dear Dr. Becker,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0296887.r006

Acceptance letter

Vyacheslav Yurchenko

7 Feb 2024

PONE-D-23-05814R2

PLOS ONE

Dear Dr. Becker,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Phagocytosed parasites by eosinophils.

(TIF)

Click here for additional data file.^{(5.2MB, tif)}

S2 Fig. Leishmania mexicana damaged by eosinophil contact.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

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Submitted filename: Response to reviewers.docx

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Attachment

Submitted filename: PONE-D-23-05814_R1.pdf

Click here for additional data file.^{(1.4MB, pdf)}

Attachment

Submitted filename: Response to the reviewer.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils

Norma Salaiza-Suazo

Roxana Porcel-Aranibar

Isabel Cristina Cañeda-Guzmán

Adriana Ruiz-Remigio

Jaime Zamora-Chimal

José Delgado-Domínguez

Rocely Cervantes-Sarabia

Georgina Carrada-Figueroa

Baldomero Sánchez-Barragán

Victor Javier Leal-Ascencio

Armando Pérez-Torres

Héctor A Rodríguez-Martínez

Ingeborg Becker

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Patients

Detection of IgE and IgG in human serum against Leishmania mexicana antigens

Eosinophil isolation

Leishmania mexicana promastigotes culture

Cytokine measurement

Oxidative burst

Interaction of Leishmania promastigotes and blood eosinophils from healthy controls

Transmission electron microscopy

Immunocytochemistry for detection of eosinophil major basic protein (MBP)

Statistical analysis

Results

Eosinophils in peripheral blood and lesions of LCL and DCL patients

Fig 1. Eosinophils in peripheral blood of patients with cutaneous leishmaniasis.

Eosinophils in lesions of LCL and DCL patients

Fig 2. Eosinophils in lesions of patients with cutaneous leishmaniasis.

Anti-Leishmania IgE and IgG in sera from LCL and DCL patients

Fig 3. Anti-Leishmania IgE and IgG in sera from patients with cutaneous leishmaniasis.

Cytokine (IL-6, IL-13, and IL-8) production and oxidative burst by EOSINOPHILS of LCL and DCL patients incubated with L. mexicana promastigotes

Fig 4. Cytokine production and oxidative burst were analyzed in eosinophils from patients with LCL and DCL.

Phagocytosis and lysis of Leishmania mexicana promastigotes by eosinophils

Fig 5. Phagocytosis and lysis of Leishmania mexicana by eosinophils.

Eosinophils release cytoplasmic “degranulation sacs” with granules in response to Leishmania

Fig 6. Release of cytoplasmic “degranulation sacs” by eosinophils co-incubated with parasites.

Extracellular traps are induced by L. mexicana

Fig 7. Extracellular eosinophil traps induced by co-incubation with L. mexicana.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 0

Decision Letter 1

Vyacheslav Yurchenko

Roles

Author response to Decision Letter 1

Decision Letter 2

Vyacheslav Yurchenko

Roles

Acceptance letter

Vyacheslav Yurchenko

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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