Lymphocytes influence Leishmania major pathogenesis in a strain-dependent manner.

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis and is caused by several species of Leishmania parasite. Clinical presentation of CL varies from a self-healing infection to a chronic form of the disease determined by the virulence of infecting Leishmania species and host immune responses to the parasite. Mouse models of CL show contradictory roles of lymphocytes in pathogenesis, while acquired immune responses are responsible for host protection from diseases. To reconcile the inconclusive roles of acquired immune responses in pathogenesis, we infected mice from various genetic backgrounds with two pathogenic strains of Leishmania major, Friedlin or 5ASKH, and assessed the outcome of the infections. Our findings showed that the genetic backgrounds of L. major determine the impact of lymphocytes for pathogenesis. In the absence of lymphocytes, L. major Friedlin induced the lowest inflammatory reaction and pathology at the site of infection, while 5ASKH infection induced a strong inflammatory reaction and severe pathology. Lymphocytes ameliorated 5ASKH mediated pathology, while it exacerbated pathology during Friedlin infection. Excess inflammatory reactions, like the recruitment of macrophages, neutrophils, eosinophils and production of pro-inflammatory cytokines, together with uncontrolled parasite growth in the absence of lymphocytes during 5ASKH infection may induce severe pathology development. Taken together our study provides insight into the impact of differences in the genetic background of Leishmania on CL pathogenesis.

Introduction

Leishmaniasis is a group of diseases caused by protozoan parasites belonging to Leishmania species. Leishmaniasis threatens more than 350 million people from 98 countries in tropical and subtropical regions of the world with an estimated 0.7–1 million new cases and 20,000 to 30,000 deaths annually [1]. The disease can be categorized into three major forms: cutaneous, mucocutaneous and visceral leishmaniasis [2]. Cutaneous leishmaniasis (CL) is the most common form of the disease. Clinical manifestations of CL vary from asymptomatic to a non-healing chronic form of the disease determined by the infecting Leishmania species and by the host immune response to the parasite [3, 4]. The biological processes that may help to explain why some cases are asymptomatic and others show a variety of clinical pathologies, across the spectrum of self-healing to severe forms of leishmaniasis, are ill-defined. The diverse clinical manifestations of CL demand a thorough investigation of the pathophysiology of the infection in varying host defenses and parasite virulence.

Mouse models of leishmaniasis have been extensively studied to understand and characterize the host-parasite interactions during infections [5]. In mouse models of CL, the mechanism of resistance to Leishmania major infection has been well established using genetically resistant C57BL/6 mice that reproduce the self-cure disease outcomes typically seen in humans [3]. Resistance to L. major infection is mainly mediated by the CD4+ and CD8+ T lymphocytes, represented by the induction of Th1 immune response and IFN-γ dependent killing of the parasite by macrophages [5, 6]. Conversely, L. major infection drives BALB/c mice towards a Th2 response that allows for parasite replication and persistence [5].

While the role of the acquired immune response in protection against leishmaniasis has been well documented, its role in pathogenesis is not fully understood. Experimental CL studies showed the discrepant role of T/B lymphocytes in disease pathology. Some studies showed that lesion development was independent of T/B cells in CL models using L. major [7] or Leishmania amazonensis [8]. Other studies showed that lesion development caused by L. major [9, 10] or L. amazonensis [11] infection was delayed and less severe in the absence of T/B cells. Soong et al. showed that CD4+ T lymphocytes are indispensable for pathogenesis during L. amazonensis infection [12]. In these studies, different species or isolates of Leishmania parasite were used. The discrepant roles of T/B cells in pathogenesis may be due to the differences in parasite species or strains. It is reasonable to hypothesize that the genetic background of the Leishmania strain is crucial to determine the variable influence of lymphocytes for pathology development.

To investigate our hypothesis, we infected mice of various background with two strains of L. major with different degrees of pathogenicity, namely strains Friedlin and 5ASKH, and measured the disease outcome. Both L. major Friedlin and 5ASKH infections were controlled in resistant C57BL/6 mice with transient lesion development and limiting parasite burden. Susceptible BALB/c mice showed progressive lesion development with uncontrolled parasite growth. In the absence of T/B lymphocytes, 5ASKH strain caused more progressive lesion development, while Friedlin strain caused limited lesion development whilst maintaining similar levels of parasite burden to 5ASKH strain. Our results indicated that lymphocytes suppressed pathogenesis during 5ASKH infection, while the presence of lymphocytes during Friedlin infection upregulated pathogenesis. We conclude that the genetic background of L. major determines the distinct influence of lymphocytes for the pathogenesis of this organism.

Materials and methods

Ethics statement

The study protocol was approved by the Committee for Ethics on Animal Experiments (approval number 1505181226, 1505181227) and Recombinant DNA experiments (1403041262) in Nagasaki University. All of the studies were conducted under the guidelines for animal experiments, Nagasaki University and according to Japanese law for Humane Treatment and Management of Animals (Law No. 105 dated 19 October 1973 modified on 2 June 2006).

Animals

Six to eight weeks old female mice were used in all experiments. In this study, we included wild-type (WT) and nude BALB/c, WT and recombination activating gene 2 (Rag2) knockout (KO) C57BL/6, and NOD-SCID mice. WT BALB/c (BALB/cCrSlc) and C57BL/6 (C57BL/6JJmsSlc) mice were purchased from SLC, Japan. Nude BALB/c (CAnN.Cg-Foxn1nu/CrlCrlj) and NOD-SCID (NOD.CB17-Prkdc scid/J) mice were purchased from Charles River Laboratories, Japan. Rag2 KO C57BL/6 (B6(Cg)-Rag2tm1.1Cgn/J) mice were purchased from the Jackson Laboratory. Experimental mice were maintained under specific pathogen-free conditions at the Animal Facilities of Nagasaki University. Experimental animals were fed with standard rodent pellets supplemented with grain and given water ad libitum. Throughout this study, the animals were housed in a climate-controlled (23±2°C; relative humidity, 60%) and photoperiod controlled (12-hours light-dark cycles) animal quarters. Only healthy mice were included in the study.

Leishmania parasite and infection

L. major clone V9 (MHOM/IL/80/Friedlin) and L. major (MHOM/SU/73/5-ASKH) strain abbreviated as L. major Friedlin and L. major 5ASKH respectively were used in this study. The parasite culture was maintained in medium 199 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (CCB, Japan), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) at 26°C in a biological incubator. Cultures were passaged to fresh medium at a 50-fold dilution in every 2–3 days. Stationary phase promastigotes were harvested and suspended in 1× PBS. Mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH by subcutaneous injections into their right hind footpad. Following infection, footpad thickness was measured weekly using an analog caliper. Footpad swelling was calculated by subtracting the thickness of the uninfected counter footpad from the thickness of the infected footpad. Parasite burdens in the footpads and popliteal lymph nodes (pLN) were quantified at 6 weeks post-infection. Parasite burdens in the footpads and draining lymph nodes were quantified by either limiting dilution assay (LDA) or homogenizing tissue in 10 ml of complete medium 199 and incubated in 25cm2 culture flasks at 26°C for the detection of parasites transformed in vitro from amastigotes to motile promastigotes. At 3 days of culture, promastigotes were counted microscopically. For LDA tissues were homogenized in 1 ml complete medium 199 following 2 fold serial dilution across 96-well culture plates. Culture plates were incubated at 26°C. At 1-week culture plates were observed under a microscope for the presence of motile promastigotes.

Isolation of cells from the footpad of mouse

Mice were euthanized and the footpads have collected aseptically. The footpads were cut tangentially to the bone and mechanically disrupted with surgical scissors in 5 ml cold RPMI medium supplemented with 1 mg/ml collagenase (WAKO, Japan) and 1 mg/ml DNase (Roche Diagnostics, Japan). Disrupted footpads were then incubated for 30 min in a 37°C water bath with continuous shaking. Following incubation, the reactions were stopped by adding cold RPMI medium with 2.5 mM EDTA and strained through a 70 μm cell strainer. The cell suspensions were centrifuged at 440g for 6 min at 4°C and re-suspended in complete RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Flow cytometric analysis

Footpad cells were stained separately with 2 sets of fluorescence-conjugated antibody cocktail. The first set of antibody cocktail consisted of PE-conjugated anti-CD86, APC-conjugated anti-MHC class-II, FITC-conjugated anti-CD11b, PE-Cy7-conjugated anti-F4/80, APC-Cy7-conjugated anti-CD45, and PerCp-Cy5.5-conjugated anti-CD11c. The second set of the antibody included PE-conjugated anti-SiglecF, APC-conjugated anti-Gr1, FITC-conjugated anti-CD11b, PE-Cy7-conjugated anti-F4/80, APC-Cy7-conjugated anti-CD45, and PerCp-Cy5.5-conjugated anti-NKp46 antibody. Cells were then analyzed on a FACSVerse™ (BD Bioscience, NJ, USA). Data were analyzed with FlowJo software v10.

Ex-vivo cytokine analysis

Cells isolated from mouse footpad were plated at the concentration of 5×105 cells per well in 96-well culture plates in 200 μl RPMI medium (supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin). After 72h of PMA (40 ng/ml)/Ionomycin (4 μg/ml) stimulation, culture supernatants were stored at -30°C. Concentrations of IFN-γ, TNF-α, IL-12 p40, and IL-10 were measured by sandwich ELISA as per manufacturer (R&D Systems) instructions.

Quantitative RT-PCR

Mice footpads were homogenized with 1 ml TRIzol (Thermo Fisher Scientific) and φ1.0 stainless steel beads in the 2 ml tube using Micro Smash MS100R (TOMY, Tokyo, Japan) at 4°C using tissue homogenizer (Tomy, Micro Smash MS-100). RNA was isolated using Trizol Reagent according to manufacturer instructions. RNA yield was measured by a spectrophotometer (Beckman Coulter DU-730). We found 30–53 μg RNA from each sample, and 2 μg of total RNA was used as the template for the synthesis of 20 μl cDNA. Synthesized cDNA was analyzed for mouse TNF-α, IL-1β, IL-6, IL-12 p40, inducible nitric oxide synthase (iNOS), and arginase 1 by reverse transcriptase real-time PCR. PCR method and primer sequences were described before [13]. Briefly real-time polymerase chain reaction (PCR) assay was carried out using 1 μl of cDNA as the template, 10 μl of SYBR Select Master Mix (Thermo Fisher Scientific) and primers on the ABI Prism 7000 Sequence Detection System (Thermo Fisher Scientific). Data were analyzed by 2−ΔΔ Ct methods and normalized by GAPDH. The thermal cycling conditions for the PCR were 94°C for 10 min, followed by 40 cycles of 94°C for 15 s and 60°C for 1 min.

Statistical analysis

All statistical analysis was performed using GraphPad Prism Software Version-5. To check data distribution we had performed the Shapiro-Wilk normality test. Statistical analysis of the lesion kinetics was performed with two-way ANOVA to test differences between groups. In all cases, a P-value of less than 0.05 was considered statistically significant.

Results

Parasite strain-dependent influence of T lymphocytes on pathogenesis in susceptible BALB/c mice

To characterize the role of T lymphocytes and L. major strains on pathogenesis, we infected susceptible BALB/c WT and nude mice with 5×106 stationary phase promastigotes of either L. major strain, Friedlin or 5ASKH. Following infections, the lesions were measured weekly, until 6 weeks post-infection. The parasite burden in footpads and pLN were also measured at 6 weeks post-infection, at the end of the experiment. Both L. major Friedlin and 5ASKH infection showed progressive lesion development in WT mice (), where three of eight mice infected with 5ASKH developed ulcerative lesions compared with one of nine for Friedlin-infected mice (). In nude mice, L. major 5ASKH infection caused more severe ulcerative lesions in all 10 mice compared to 3/8 in WT mice (). Conversely, L. major Friedlin infection caused minimal lesion development with no ulcer formation in all 11 nude mice and 1/9 in WT mice (). It is worth noting that both Friedlin and 5ASKH showed a similarly high level of parasite burden in the footpads and pLN not only in nude mice but also in WT mice (Figs ). These results showed that T lymphocytes reduced immunopathology during 5ASKH infection, while T lymphocytes exacerbated lesion development during Friedlin infection.

Parasite strain-dependent influence of T lymphocytes on pathogenesis in susceptible BALB/c mice.

BALB/c WT and nude mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. (A) Footpad swelling after L. major infection. Each group contains 8–11 mice. All the data are from normal distribution except data of BALB/c mice at 1 and 3 weeks and BALB/c-nude mice at 2 weeks post-infection of L. major Friedlin have deviated from normal distribution. Data deviated from normal distribution were excluded from statistical comparison (B) Representative photographs of L. major-infected footpads at 6 weeks post-infection. (C) Parasite burden in footpads at 6 weeks post-infection. Each mouse footpad was homogenized in 10 ml culture media and cultured for 3 days then viable promastigotes were counted microscopically. Each group contains four–six mice. Symbols: BALB/c mice infected with L. major Friedlin blue circle or 5ASKH green triangle, BALB/c-nude mice infected with L. major Friedlin black circle or 5ASKH red triangle; Data are the mean ± SEM. Statistical analyses for lesion size by ANOVA. *†¶# means P<0.05 between BALB/c-Friedlin×BALB/c-5ASKH: *; Nude-Friedlin×Nude-5ASKH: †; BALB/c-Friedlin×Nude-Friedlin: ¶ and BALB/c-5ASKH×Nude-5ASKH: #.

Parasite strain-dependent influence of T/B lymphocytes on pathogenesis in resistant C57BL/6 mice

Next, we examined the L. major strain-dependent influence of lymphocytes for lesion development in resistant C57BL/6 mice that allows transient L. major infection. WT and Rag2 KO C57BL/6 mice were infected with either L. major Friedlin or 5ASKH. The WT mice showed transient non-ulcerative lesion development during infection with both L. major strains () and controlled parasite growth (). In Rag2 KO mice, which lack both conventional T/B lymphocytes, 5ASKH infection caused delayed but progressive and non-ulcerative lesion development, whereas Friedlin infection caused delayed and lesser lesion development () despite a similar level of parasite burden in Friedlin- or 5ASKH-infected footpads and pLN (Figs ).

Parasite strain-dependent influence of T/B lymphocytes on pathogenesis in resistant C57BL/6 mice.

C57BL/6 WT and Rag2 KO mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. (A) Footpad swelling after L. major infection. All the data are from normal distribution except data of C57BL/6 mice at 1 week post-infection of L. major Friedlin have deviated from normal distribution. Data deviated from normal distribution were excluded from statistical comparison. Symbols: WT mice infected with L. major Friedlin blue circle or 5ASKH green triangle, Rag2 KO mice infected with L. major Friedlin black circle or 5ASKH red triangle; 3–10 mice per group. (B) Representative photographs of a L. major-infected footpad at 6 weeks post-infection. (C) Parasite burden at 6 weeks post-infection. Each mouse footpad was homogenized in 10 ml culture media and cultured for 3 days then viable promastigotes were counted microscopically. Three–five mice per group. Data are the mean ± SEM. Results are representative of two independent experiments with similar outcomes. *†¶# means P<0.05 between WT-Friedlin×WT-5ASKH: *; Rag2KO-Friedlin×Rag2KO-5ASKH: †; WT-Friedlin×Rag2KO-Friedlin: ¶ and WT-5ASKH×Rag2KO-5ASKH: #.

L. major Friedlin and 5ASKH caused minimum or severe pathology respectively in NOD-SCID mice

Lack of pathogenesis by Friedlin strain in severe immunodeficient condition was also confirmed in NOD-SCID mice. NOD-SCID mice lack T/B cells and also defects in functions of NK, NKT, macrophage, and dendritic cells. In NOD-SCID mice, there was no difference in parasite burden between Friedlin and 5ASKH infected footpad and pLN (). However, L. major 5ASKH showed non-ulcerative and progressive lesion development, but Friedlin showed almost no lesion development (Table 1 and ).

Table 1

Ulcer formation at 6 weeks post-infection with L. major Friedlin or 5ASKH.

Mice	Ulcer formation % (number)
	Friedlin	5ASKH
BALB/c	11.1 (1/9)	37.5 (3/8)
BALB/c-nude	0 (0/11)	100 (10/10)
C57BL/6	0 (0/8)	0 (0/8)
C57BL/6-Rag2 KO	0 (0/5)	0 (0/3)
NOD-SCID	0 (0/8)	0 (0/8)

Defective infiltration of immune cells to the site of L. major Friedlin infection

Our study showed that both parasite and host factors affected the clinical presentation of CL. Inflammatory responses are mediated by immune cells that control not only parasite growth but also contribute to the pathogenesis of CL [3, 14]. To characterize the inflammation induced by either L. major Friedlin or 5ASKH infections, we isolated immune cells from Rag2 KO-infected footpads at 4 weeks post-infection and analyzed these cells by flow cytometry. It is worth noting that, L. major Friedlin infection failed to recruit immune cells to the site of infection, while a 25 times higher number of cells were recruited to the footpads infected with L. major 5ASKH. Among the cells recruited to the site of L. major 5ASKH infection, macrophages (CD11b+F4/80+), neutrophils (CD11b+Gr1+), and eosinophils (CD11b+SiglecF+) were the major cell populations. In addition, L. major 5ASKH infected footpad also contained a higher proportion and number of activated macrophages (CD11b+F4/80+CD86+) compared to that with L. major Friedlin (Figs ).

L. major Friedlin infection failed to augment the recruitment of macrophages in Rag2 KO mice.

Rag2 KO C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection, cells from the mouse footpads were isolated and analyzed by flow cytometry. The total number (A) and percentages (B) of the cell populations are shown. Four–five mice per group. Data are the mean ± SEM.

Defective infiltration of neutrophils, eosinophils, and NK cells during L. major Friedlin infection in Rag2 KO mice.

Rag2 KO C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection, cells from the mouse footpads were isolated and analyzed by flow cytometry. The total number (A) and percentages (B) of the cell populations are shown. Four–five mice per group. Data are the mean ± SEM.

Lower activation of host immune responses by L. major Friedlin infection

Immune cells were isolated from the footpads of Rag2 KO mice 4 weeks after infection with L. major Friedlin or 5ASKH and were cultured for 72 h with or without PMA-ionomycin stimulation. Culture supernatants were analyzed for IFN-γ, TNF-α, IL-12, and IL-10 cytokines by ELISA. L major infection augmented the production of cytokines and L. major 5ASKH induced significantly higher cytokine production than Friedlin (). After stimulation with PMA-ionomycin, a similar trend was observed ().

Lower activation of host immune responses by L. major Friedlin infection.

Rag2 KO C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection, cells from mouse footpads were isolated and cultured for 3 days. Culture supernatants were analyzed for (A) IFN-γ, (B) TNF-α, (C) IL-12p40, and (D) IL-10 cytokines by ELISA. Three–four mice per group. Data are the mean ± SEM. *, P<0.05.

Discussion

The distinct impact of lymphocytes for the pathogenesis of CL was confirmed to be attributed to the genetic background of L. major. Our results clarified the contradictory findings of previous independent studies, in which lesion development by CL was either similar [7, 8], delayed or less pronounced [9, 10, 12] in the absence of T/B lymphocyte-mediated immune responses compared to wild-type mice. The L. major 5ASKH infection model was consistent with reports that lesion development by either L. major (MHOM/UZ/91/PM2) or L. amazonensis (MPRO/BR/72/M1845) was similar or less in the presence of lymphocytes [7, 8]. The L. major Friedlin infection model also explains the previous report that delayed or no lesion development in the absence of T/B lymphocytes by L. major (WHOM/IR/-/173) [9, 10] or L. amazonensis (MHOM/BR/77/LTB0016) [12] infection. Thus, the impact of lymphocytes for pathogenesis can be determined by the genetic background of L. major. In this study, the genetic background of L. major strains was not analyzed, which is one of the limitations of the study. We compared only two strains of L. major we had no comparison with other strains of L. major or different species of Leishmania. Further immunological analyses are needed to inquire into the differential lymphocyte response towards different Leishmania strains and species.

In Rag2–deficient C57BL/6 mice, 5ASKH infection caused progressive lesion development. In the early phase of its infection, the degree of footpad swelling was delayed in the absence of T/B lymphocytes indicating that even during 5ASKH infection, lymphocytes could be attributed to lesion formation to some extent. In fact, 5ASKH-infected footpads of WT mice showed a trend of higher expression of pro-inflammatory molecules, such as IL-6, TNF-α, IL-1β, IL-12p40 and iNOS compared with Friedlin-infected footpads (). The preceding studies also independently showed delayed lesion development after L. major (WHO strain WHOM/IR/-/173) infection in C.B-17 SCID mice compared with BALB/c mice [9, 10].

Pathogenesis can be caused by either the direct tissue-damaging effect of the parasites, parasite-induced immune-mediated skin inflammation or their combined effects. Belkaid et al. showed that lesion development starts after the parasite burden reaches its peak and that it coincides with leukocyte trafficking in resistant C57BL/6 mice [15], which emphasized the importance of parasite-induced inflammatory reactions for pathogenesis. We showed that L. major 5ASKH infection induced higher inflammatory reactions both in the presence and absence of T/B lymphocytes. Massive recruitment of immune cells, mainly macrophages, neutrophils and eosinophils, was observed at the site of 5ASKH infection even in the absence of lymphocytes. It is consistent with the prior study which showed that lesion development was associated with acute infiltration of leukocytes, primarily macrophages, neutrophils and eosinophils [15]. Immune cells recruited to the site of 5ASKH infection were also highly augmented with a larger number of CD86+-activated macrophages and a higher level of cytokine production was observed compared with Friedlin infection. The inflammatory cytokines are able to activate the macrophages for intracellular killing of the parasite. In wild-type mice, T-lymphocytes are known to be the major source of IFN-γ which is central for the protective immunity. In the absence of T/B lymphocytes, mice failed to control parasites. It indicates that T/B lymphocytes are critical for the control of the parasites, mainly through the production of sufficient amount of critical cytokines, like IFN-γ and TNF-α. In the absence of T/B lymphocytes, macrophages, dendritic cells, NK cells, and group 1 innate lymphoid cells may be one of the sources of IFN-γ, which is insufficient for the protection (). It may explain why the immunodeficient mice infected with both L. major strains showed no difference in parasite burden. However, augmented inflammatory responses after 5ASKH infection may have caused the severe pathology observed. Strong inflammatory reactions at the site of L. major 5ASKH infection may explain the severe pathology observed even in the absence of T/B lymphocytes. L. major Friedlin infection failed to induce inflammatory reactions, like recruitment of immune cells and pro-inflammatory cytokines production at the site of infection in the absence of T/B lymphocytes, which may explain the minimum pathology observed. The outcome of Friedlin infection in the immunodeficient mice was consistent with the reported no pathology associated with less infiltration of monocytes at the site of L. amazonensis infection in mice lacking T cells [12]. Although we compared inflammatory responses only at 4 weeks after L. major Friedlin/5ASKH infection, it is also important to compare early inflammatory responses to address the mechanisms responsible for the different disease outcomes. Different Leishmania parasites may have diverse molecules or strategies determining the impact of lymphocytes for immuno-pathogenesis. Comparative analysis showed a significant number of virulence factors or molecules differentially expressed by Leishmania isolates [16-18]. We also analyzed only a few cytokines or host molecules after L. major infection. Further studies are required to identify the molecules and pathways involved.

CL has a wide spectrum of clinical manifestations depending on the host immunity and the infecting Leishmania species. CL is caused by several species of Leishmania parasite. In the new world, L. mexicana, L. amazonensis, L. panamensis, L. brazilienesis, L. guyanensis, L peruviana and L. venezuelensis are the etiological agents of CL, whereas in the old world L. tropica, L. killicki, L. aethiopica, L. arabica, L. infantum and L. major are responsible for CL [19, 20]. Many studies have been reported on CL, but few studies focused on the diversity of the clinical presentations. We extensively studied infection outcomes caused by two different strains of L. major in animal models with different genetic backgrounds and varying immune competency. L. major Friedlin and 5ASKH were originally isolated from patients with CL in the old world, Israel and Turkmenistan respectively, then adapted to the conditions in the Laboratory and established as the strains [21]. In immune-competent mice, we observed different infection outcomes in different host genetic backgrounds. Friedlin infection caused almost no ulceration in any genetic background studied, whereas 5ASKH infection caused ulceration in BALB/c mice and non-ulcerative lesions in C57BL/6 mice. 5ASKH infection caused more ulcerative lesions in T lymphocyte-deficient BALB/c mice compared with WT mice. Terabe et al. showed that T/B lymphocytes are a prerequisite for ulcer formation during L. amazonensis infection in BALB/c mice [8]. Therefore, particular Leishmania species may have distinct immunopathologies of ulcer formation.

Conclusion

Different species or strains of Leishmania were associated with a wide spectrum of diseases in human [19, 20, 22, 23] and mouse models. Previous studies showing that L. major LV39 caused a non-healing infection [24-26] whereas other L. major strain (MHOM/IL/81/FEBNI) caused healing infections in IL-4 deficient BALB/c mice [25-26]. Furthermore, it now appears that strains of L. major can cause variant pathogenesis even in the absence of lymphocytes. Lymphocytes differentially regulate pathogenesis during L. major Friedlin/5ASKH infection. Leishmania has mechanisms to evade host immune protection, thereby aiding survival inside the host [27]. L. major Friedlin may have a superior immune evasion mechanism compared with strain 5ASKH that allows it to survive within the host by eliciting minimum immune-mediated pathogenesis. Understanding the basis for the diversity in pathogenesis is important in determining how to apply our knowledge for the development of new approaches for the treatment of leishmaniasis.

BALB/c WT and nude mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. Parasite burden in popliteal lymph nodes (pLN) at 6 weeks post-infection. Each mouse footpad was homogenized in 10 ml culture media and cultured for 3 days then viable promastigotes were counted microscopically. Each group contains 8–11 mice. Symbols: BALB/c mice infected with L. major Friedlin ○ or 5ASKH △, BALB/c-nude mice infected with L. major Friedlin ● or 5ASKH ▲; Data are mean ± SEM.

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C57BL/6 WT and Rag2 KO mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. Parasite burden in popliteal lymph nodes (pLN) at 6 weeks post-infection. Each mouse footpad was homogenized in 10 ml culture media and cultured for 3 days then viable promastigotes were counted microscopically. 3–9 mice per group. Symbols: wild-type mice infected with L. major Friedlin ○ or 5ASKH △, Rag2 KO mice infected with L. major Friedlin ● or 5ASKH ▲. Data are mean ± SEM. Results are representative of 2 independent experiments with a similar outcome.

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L. major Friedlin or 5ASKH infection in NOD-SCID mice.

NOD-SCID mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. (A) Footpad swelling after L. major infection. Symbols: NOD-SCID mice infected with L. major Friedlin ● or 5ASKH ▲; 8 mice per group. (B) Representative photographs of L. major infected footpad at 6 weeks post-infection. (C) Footpad parasite burden at 6 weeks post-infection. Each mouse footpad was homogenized in 10 ml culture media and cultured for 3 days then viable promastigotes were counted microscopically. 4–8 mice per group. Data are mean ± SEM. Results are representative of two independent experiments with a similar outcome.

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Flow cytometric analysis of mouse footpad cells.

Rag2 KO C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection, cells were isolated from mouse footpads and analyzed by flow cytometry. Gating strategies for (A) macrophages; (B) neutrophils, eosinophils, and NK cells.

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Less activation of host immune responses by L. major Friedlin infection.

Rag2 KO C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection cells were isolated from mouse footpads and stimulated with PMA-ionomycin for 3 days. Culture supernatants were analyzed for cytokines by ELISA. 3–4 mice per group. Data are mean ± SEM. *, P<0.05.

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L. major Friedlin infection induced a lower level of the immune response.

C57BL/6 mice were infected with 5×106 stationary phase promastigotes of L. major Friedlin or 5ASKH subcutaneously into the hind footpad. At 4 weeks post-infection total RNA was isolated from mouse footpads and analyzed for mRNA of targeted molecules by real-time RT-PCR. (A) Footpad parasite burden at 4 weeks post-infection measured by limiting dilution assay. (B) mRNA expression in the infected footpad at 4 weeks post-infection. 3–4 mice per group. Data are mean ± SEM.

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5 Aug 2019

Dear Prof. Hamano:

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

We hope to receive your revised manuscript by Oct 04 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

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Sincerely,

Syamal Roy

Guest Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The studies by Abu Musa et al are of interest. Their findings help reconcile discrepant observations in the literature.

Reviewer #2: The objectives of the study are clearly defined.

The study design is appropriate, but there are problems in the interpretation of data.

The same previous answer

The number of experiments and mice used in the experiments is fine.

The authors used ANOVA, but no test for normality of the data was done

No concerns about ethical or regulatory requirements

Reviewer #3: Although the methods are suitable, can the authors please clarify/rectify the following points?

1) In regards to Fig 1A, Fig 2A and S3A Fig, why was one-way ANOVA used to determine statistical significance? Since their phenotype depends time and experimental condition, two-way ANOVA seems more appropriate.

2) In Figs 1A and 2A, errors bars are too thick, which makes it confusing for readers to properly visualize and assess the data. Since there are four different time series in the same graphs, the authors should use different colours to better differentiate the lines.

3) Numerical data in Figs 3-5 and S5 Fig should also be depicted in the form of scatter plots, as done in Fig 1C, S1 Fig and elsewhere.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Overall data and conclusions are clearly presented and discussed.

Reviewer #2: Yes, it does.

The Figures presented low quality, it is not clear the statistical differences shown in the Figures

Reviewer #3: 1) Overall, results are laid out well, but the description of the data is often incorrect (see ‘Summary and General Comments’). For example, Figs 1-2 do not really show a ‘T lymphocyte-dependent pathogenesis by L. major Friedlin and independent in 5ASKH’, but rather whether lymphocytes worsen/help control disease by those strains.

2) In Fig 1B, the photos pertaining to the 5ASKH strain do not recapitulate the data shown in 1A, which show that 5ASKH-induced lesions worsened in BALB/c nude mice. Since the photos in 1B show the opposite, perhaps the authors accidentally flipped them?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The paragraph beginning on line 361 discusses observations with the different Leishmania species; however, the authors may consider including a bit more information about the pedigree of both of the strains that were tested, in other to give some context of the diversity of disease presentations that may be seen in L. major endemic regions.

Reviewer #2: The conclusions are supported by the data, but the discussion is a summary of the results.

The limitations of analysis are not described.

Yes, they do, but some of the statements are not in agreement with the literature,

Yes, it is

Reviewer #3: The conclusions are partially supported by the authors’ findings. Section 5 (Conclusions) should focus on the positive and negative influence that lymphocytes have on the pathologies caused by the strains that the authors employed. As such, that section should be written concisely. The text pertaining to parasite virulence factors and whatnot should be integrated within the main body of the Discussion (section 4).

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No concerns noted

Reviewer #2: (No Response)

Reviewer #3: 1) Lines 379-392 ramble too much about the link that their mouse-based work has with Leishmania/HIV co-infected patients. In fact, lines 387-392 contain a very speculative and incoherent argument.

2) Authors should employ current and proper genetic terminology to describe their mouse strains throughout the manuscript.

3) In the title, the word ‘the’ should be omitted. As per my major concerns (outlined in the ‘Summary and General Comments’ section), the word ‘requirement’ should be replaced by ‘influence’.

I suggest the following title: ‘Lymphocytes influence Leishmania major pathogenesis in a strain-dependent manner’.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The results of the study are significant as they bring to the fore observations that cutaneous disease presentation in endemic areas is variable. The variability could apparently be be due to strain differences within parasite species that impact immune responses.

Reviewer #2: The manuscript showed differences in the development of lesions by two different strains of L. major (Friedlin and 5ASKH) using different mouse strains such as BALB/c and nude BALB/c, as well as C57Bl/6, gene 2 (Rag2) knockout (KO) C57BL/6, and NOD-SCID mice. The authors observed that in mice lacking T/B responses, 5ASKH developed a larger lesion, with ulceration whereas Friedlin strain induced in these mice smaller lesions, with less inflammation.They also performed experiments employing flow cytometry, and there was a large recruitment of cells to the lesion caused by 5ASKH compared to Friedlin. Concerning cytokines, the results were similar, with an increase in IFN-g, IL-10, TNF and IL-12p40 in the cells from lesions induced by 5ASKH. One intriguing fact is that there was no statistical difference in the parasite load from the lesions caused by the two different strains. Although, the results are interesting, this observation of development of pathology caused by Leishmania has been discussed for a long time, as the author stated, and the results of this manuscript reinforce this idea.

However, there are some aspects that deserve discussion:

1-There was no explanation about the assay to calculate the parasite load.

2- the amount of cells collected from the footpad of infected mouse. Is this material also used to evaluate the parasite load?

3- Which type of response was observed in the draining lymph nodes of the mice infected by the two strains of L. major?

4- The authors did not analyze the inflammatory responses before 6 weeks or 4 weeks of infection in some experiments. It would be interesting to perform these experiments, comparing with the wild type mouse.

5-One important aspect to discuss, is related to the control of L. major Friedlin in the lesion, since the recruitment of cells is really low and also, the inflammatory cytokines able to activate the macrophages to eliminate the parasite.

6- Important data that are not present in the manuscript is related with the source of cytokines in the mice without T/B. Which cell is producing IFN-g?

7- The Discussion needs improvement. It is a summary of results without any discussion. The conclusions are really repetitive.

Reviewer #3: The study by Abu Musa et al. sought to elucidate how parasite strains influence the role of lymphocytes during Leishmania major infection. To this end, the authors used two strains of differing virulence (Friedlin and 5ASKH) to infect mice that contained or lacked lymphocytes.

Although interesting, I have significant concerns regarding how the authors interpreted their data.

1) Throughout the manuscript, the authors repeatedly claim that while the pathogenesis caused by the Friedlin strain is dependent on lymphocytes, that of the 5ASKH strain is largely not. The authors gone on the argue that the **requirement** for lymphocytes in pathogenesis is strain-dependent. Nonetheless, these statements are not really backed by the data. Instead of portraying a requirement for lymphocytes, the data in Figs 1A and 2A clearly show that lymphocytes influence (either exacerbate or diminish) infection outcome depending on parasite strain. This is further nuanced by the time-dependent development of the lesions, which should be more explicitly described in the results section.

2) The finding that lymphocyte presence helps control 5ASKH infection does not mean that pathogenicity is independent of lymphocytes (or the opposite in the case of the Friedlin strain). In order to prove that there really is a requirement for those cells, authors must perform adoptive cell transfer experiments (eg. introduce WT lymphocytes into nude/RAG2 mice or vice-versa).

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

6 Sep 2019

Submitted filename: Response to reviewers.docx

Click here for additional data file.

3 Oct 2019

Dear Prof. Hamano:

Thank you very much for submitting your manuscript "Lymphocytes influence Leishmania major pathogenesis in a strain-dependent manner" (PNTD-D-19-01017R1) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) Appropriate Figure Files

Please remove all name and figure # text from your figure files upon submitting your revision. Please also take this time to check that your figures are of high resolution, which will improve both the editorial review process and help expedite your manuscript's publication should it be accepted. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plosntds/s/figures

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We hope to receive your revised manuscript by Dec 02 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit your revised files, please log in to https://www.editorialmanager.com/pntd/

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Syamal Roy

Guest Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Please respond to the minor comments by reviewer 3. Thank you!

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: Yes

Reviewer #3: The authors have properly addressed my comments in regards to methodology.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: Yes

Reviewer #3: Overall, the layout and description of results have been improved.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: Yes

Reviewer #3: Although the discussion and conclusion sections have been enhanced, the authors can still trim off a lot of experimental detail that is already presented in the results and figure legends.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: The authors answered all the questions raised up by the reviewer including new experiments that improve the study and the manuscript.

Reviewer #3: Prior to formal acceptance, the authors should thoroughly proofread their manuscript to correct errors such as the ones found on line 45 (may induced -> may induce), line 349 (were not analyzed -> was not analyzed), line 385 (may caused -> may have caused), etc. […]

The graphs appear to be blurry in the PDF version. Authors should ensure that their GraphPad-generated images have been exported in TIFF format at 600dpi, and that their multi-panel montages are saved in a non lossy compression format.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: The authors answered all the questions raised up by the reviewer including new experiments that improve the study and the manuscript.

Reviewer #3: In this revised submission, Abu Musa and colleagues have addressed most of my concerns. Prospective immunological studies are needed to decipher the differential lymphocyte response towards different Leishmania strains and species.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: Yes: Guillermo Arango Duque

22 Oct 2019

Dear Prof. Hamano,

We are pleased to inform you that your manuscript, "Lymphocytes influence Leishmania major pathogenesis in a strain-dependent manner", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Syamal Roy

Guest Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

8 Nov 2019

Dear Prof. Hamano,

We are delighted to inform you that your manuscript, "Lymphocytes influence Leishmania major pathogenesis in a strain-dependent manner," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases