Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice
Lucia E. Rosas1,
Tracy Keiser1,
Joseph Barbi1,
Anjali A. Satoskar2,
Alecia Septer1,
Jennifer Kaczmarek1,
Claudio M. Lezama-Davila1 and
Abhay R. Satoskar1,3
1 Department of Microbiology, 2 Department of Pathology and 3 Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA
Correspondence to: A. R. Satoskar; E-mail: satoskar.2{at}osu.edu
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Abstract
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The experimental model of high-dose Leishmania mexicana infection is used frequently to study molecular mechanisms regulating Th2 response since most inbred mice regardless of their genetic background display Th2 cytokine-dependent susceptibility to L. mexicana unlike Leishmania major. Here, we analyzed the course of L. mexicana infection in BALB/c, C57BL/6 and CBA/J mouse strains using low-dose ear infection model that mimics natural transmission. Although all three strains were equally susceptible to high-dose back rump L. mexicana infection, they displayed marked differences in their ability to control parasite growth after low-dose ear infection. Leishmania mexicana-infected BALB/c mice produced high levels of Th2-associated cytokines and developed non-healing lesions full of parasites, whereas CBA/J mice preferentially produced Th1-associated IFN-
but low levels of IL-4, and developed small self-resolving lesions. Both BALB/c and C57BL/6 mice produced comparable amounts of IFN-
following L. mexicana infection, but later produced less Th2-associated cytokines, and exhibited an intermediate susceptibility phenotype characterized by lesion sizes that were significantly smaller than BALB/c mice but larger than CBA/J mice. Interestingly, all three strains also showed marked differences in trafficking of macrophages, CD4+ T cells and CD8+ T cells into their lesions. Finally, we analyzed the course of low-dose L. mexicana infection in signal transducers and activators of transcription (STAT) 6/ and STAT6+/+ BALB/c mice. We found that STAT6/ mice mount a Th1 response, produce high levels of IL-12 and IFN-
and develop smaller lesions containing fewer parasites as compared with STAT6+/+ mice. Our findings demonstrate that genetic background plays a critical role in determining susceptibility of inbred mice to low-dose L. mexicana infection. Furthermore, together with our previous findings, they show that STAT6-mediated signaling is involved in mediating susceptibility to L. mexicana following both high-dose back rump and low-dose ear dermis infection.
Keywords: cutaneous leishmaniasis, Leishmania mexicana, Th1/Th2
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Introduction
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Leishmaniasis comprises a group of diseases caused by various species of the intracellular protozoan parasite Leishmania. It is characterized by a wide spectrum of clinical manifestations in the mammal host. American cutaneous leishmaniasis caused by Leishmania mexicana manifests as chronic infection of the ear pinna causing Chiclero's ulcer (1). While most inbred mice mount a Th1 response, produce IFN-
and spontaneously resolve their lesions following Leishmania major infection, almost all mice with the exception of female DBA/2 and NZB develop large lesions full of parasites following infection with high dose of L. mexicana (2).
Previous studies by us and others have shown that susceptibility of most inbred mice to L. mexicana is due to their inability to produce IL-12 required for the development of protective Th1 response and IFN-
production (1). Furthermore, we and others have also found that IL-4/, IL-13/, IL-4/13/ and signal transducers and activators of transcription (STAT) 6/ mice mount a Th1 response and efficiently control L. mexicana infection, indicating that IL-4, IL-13 and STAT6-mediated signaling mediate susceptibility to L. mexicana by preventing Th1 development (36). Nevertheless, one recent study using a footpad inoculation model has reported that IL-4 and IL-12 do not play a significant role in determining the outcome of L. mexicana infection (7). Interestingly, STAT4-mediated signaling pathway was essential for the control of L. mexicana lesion growth in this model (7).
The mouse model of L. mexicana infection used in almost all experimental studies involves subcutaneously (s.c.) infecting mice by inoculating high numbers of parasites (106107) into the footpad or the back rump. Studies using L. major and L. mexicana models have shown that factors such number of parasites and route of infection play a critical role in regulating host immune responses and determining the outcome of cutaneous leishmaniasis caused by these two species of Leishmania. For example, susceptible BALB/c mice infected with low number of L. major promastigotes mount a robust Th1 response and control the infection (8, 9). On the other hand, NZB mice develop non-healing lesions when inoculated with low number of parasites but are resistant to high-dose (107) L. mexicana infection (2). Furthermore, C57BL/6 x BALB/c F1 mice mount a Th1 response and resolve their lesions following L. major infection into footpad, but develop non-healing lesions associated with Th2 response when infected into back rump (10). Similarly, SWR mice also develop progressive lesions following s.c. L. major infection into back rump, but develop relatively less severe disease following footpad inoculation (11). Nonetheless, low-dose ear infection L. major model involving infecting mice with as low as 1000 promastigotes into dermis of ear closely mimics natural infection (12, 13). Under natural conditions, the sandfly delivers into the dermis of the mammal host a low dose of parasites (<1000 metacyclic promastigotes), >103 times less than the traditional experimental inoculum (14).
Therefore, we used the low-dose intra-dermal challenge model and analyzed the course of L. mexicana infection in BALB/c (H-2d), C57BL/6 (H-2b) and CBA/J (H-2k) mice which are highly susceptible to high-dose L. mexicana infection and STAT6/ BALB/c mice which are resistant. We measured antibody levels in sera and cytokine production by the draining lymph node cells from these mice. In addition, we also analyzed cell populations in the ear lesions from L. mexicana-infected BALB/c, C57BL/6 and CBA/J mice at different time points during infection. Our results show that the development of protective immunity and the outcome of cutaneous L. mexicana infection following low-dose challenge are profoundly influenced by the genetic background in inbred mice. Moreover, they also show that STAT6-mediated signaling pathway is involved in the pathogenesis of low-dose L. mexicana infection.
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Methods
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Animals
Age- and sex-matched BALB/c, C57BL/6, CBA/J and STAT6/ BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The animals were used for infection at 810 weeks of age and were maintained in the specific pathogen free (SPF) facilities at the Ohio State University animal facilities in accordance with institutional guidelines. In each experiment, four to five mice per group were included.
Parasites and infection protocol
Leishmania mexicana (MNYC/BZ/62/M379) was maintained by serial passage of amastigotes inoculated s.c. into the shaven rumps of 129SvE mice. Amastigotes isolated from lesions of infected mice were grown in vitro. Mice were infected intra-dermally by inoculating 103 L. mexicana stationary phase metacyclic promastigotes in a volume of 10 µl into ear dermis using a 30-guage needle. For high-dose model, mice were infected by inoculating 5 x 106 parasites s.c. into shaven back rump. The lesion growth was monitored by measuring the thickness of the infected ear using a dial-gauge micrometer at weekly intervals and was expressed as the increase in thickness of the infected ear compared with the uninfected ear.
Quantification of parasite loads
On day 75 post-infection, all the mice infected with low-dose L. mexicana were euthanized and relative parasite burdens in the infected ears were determined by limiting dilution analysis. The two sheets of infected ears were separated, placed dermal side up and ground on a nylon cell strainer in M199 medium supplemented with 100 U ml1 penicillin, 100 µg ml1 streptomycin and 10% FCS. For high-dose L. mexicana infection studies, parasite loads were measured on day 45 post-infection. Limiting dilution analysis with parasite suspensions was carried out as described previously (15). The results were expressed as reciprocal log parasite titers.
Histopathology
Infected ears from L. major-infected BALB/c, C57BL/6, CBA/J and STAT6/ BALB/c mice were excised and fixed in formalin for 4 days. The tissues were processed and embedded in paraffin, and 4- to 8-µm sections were cut. The sections were hydrated and stained by routine hematoxylin eosin staining.
Isolation of cells from the ear lesions and phenotypic analysis of cell populations by flow cytometry
Cells from the ear lesions of L. mexicana-infected CBA/J, C57BL/6 and BALB/c mice were isolated on days 15, 30 and 45 post-infection as described previously (16). Briefly, two sheets of ear were separated and incubated with dermal side down on complete RPMI 1640 at 37°C for 16 h. The cells spontaneously migrating out from the dermis were collected and filtered through 70-µm pore size cell strainer for flow cytometric analysis. Flow cytometric analysis was carried out on cells pooled from the lesions of four to five mice to enumerate different T cell populations. A total of 12 x 105 cells were labeled using FITC- or PE-conjugated anti-CD4, anti-CD8a, anti-CD3 and anti-CD11b antibodies (all purchased from Biolegend, San Diego, CA, USA). Flow cytometry analysis was performed using a FACSCalibur flow cytometer and CellQuestPro software (Becton Dickinson, Mount View, CA, USA). Macrophages and lymphocytes were gated according to forward and side scatter and at least 10 000 events were acquired and analyzed. The cell numbers for each cell population were calculated from the total number of cells using percentages of specific cell population obtained by flow cytometry.
Antibody ELISA
Peripheral blood was obtained from infected mice by tail snapping at various time points after infection. Plasma samples were obtained following centrifugation at 200 x g, and were used to analyze the levels of L. mexicana-specific Th2-associated IgG1 and IgE, and Th1-associated IgG2a antibodies by ELISA (17).
T cell proliferation assay and cytokine ELISA
On days 15, 30, 45 and 75 post-infection, the retromaxilar draining lymph nodes were removed in sterile conditions. Single-cell suspensions were prepared by gentle teasing in RPMI 1640 supplemented with 2 mM L-glutamine, 10% fetal bovine serum (heat inactivated), 100 U ml1 of penicillin, 100 µg ml1 of streptomycin and 0.5 mM ß-mercaptoethanol (GIBCO BRL, Grand Island, NY, USA). Viable cells were counted by trypan blue exclusion and adjusted to 3 x 106 cells ml1 for lymph nodes in the same medium. Aliquots of 100 µl were placed into 96-well flat bottom culture plates (Costar, Cambridge, MA, USA) and stimulated with 20 µg ml1 of Leishmania mexicana antigen (LmAg) prepared by seven cycles of repeated freezing in liquid nitrogen (70°C) and thawing. Proliferation of lymphocytes was determined after 60 h of incubation at 37°C, using the non-radioactive AlamarBlue Assay (BioSource International, Inc., Camarillo, CA, USA) as described previously (18). Supernatants were collected from parallel cultures after 60 h of incubation for ELISA quantification of cytokine production. Levels of cytokines (IL-12, IFN-
, IL-10 and IL-4) in supernatants were measured using sandwich ELISA method according to the manufacturer's instructions (BD PharMingen, San Diego, CA, USA). Antibody clones used were as follows: IL-12, clone C15.6 (capture) and clone C17.8 (detection); IFN-
, clone R4-6A2 (capture) and clone XMG1.2 (detection); IL-4, clone BVD4-1D11 (capture) and clone BVD6-24G2 (detection), and IL-10, clone JES5-2A5 (capture) and clone SXC-1 (detection). Reagents for IL-13 ELISA were purchased from R&D Systems, Minneapolis, MN, USA.
Statistical analysis
All the data showed in this study were obtained from two or three different experiments unless specified otherwise. Comparisons between strains considered in this work were made using Student's unpaired t-test. P < 0.05 was considered significant. The statistical significance of the sera titers was determined by using MannWhitney U prime test.
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Results
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Course of low-dose L. mexicana ear infection in BALB/c, C57BL/6 and CBA/J mice
Following inoculation with 1000 metacyclic L. mexicana promastigotes into ear dermis, BALB/c mice developed large ulcerating lesions full of parasites, whereas similarly infected CBA/J mice were highly resistant to infection and developed self-resolving small lesions containing few parasites (Fig. 1A and C). On the other hand, C57BL/6 mice displayed an intermediate susceptibility to L. mexicana and developed lesions that were significantly smaller as compared with those in BALB/c mice but larger than CBA/J mice. All three mouse strains were equally susceptible to high-dose back rump L. mexicana infection and developed rapidly progressing large lesions full of parasites (Fig. 1B and D).

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Fig. 1. Course of low- and high-dose Leishmania mexicana infection in BALB/c, C57BL/6 and CBA/J mice. (A) Low-dose ear lesion growth was monitored by measuring the thickness of the infected ear using a dial-gauge micrometer at weekly intervals and was expressed as the increase in thickness of the infected ear compared with the uninfected ear. Data are expressed as mean lesion size (millimeters) ± SE. The data are representative of three independent experiments (n = 15 mice per group). (B) High-dose back rump lesion growth was monitored by measuring lesion diameter at weekly intervals and was expressed as mean lesion diameter (millimeters) ± SE. The experiment was terminated on day 45 post-infection as most mice begin to ulcerate their lesions at this time. Five to six mice were used in each group. (C) Parasite burdens in ear lesions were determined on day 75 by limiting dilution analysis and data are expressed as mean log dilution ± SE. (D) Parasite burdens in the back rumps were determined on day 45 by limiting dilution analysis and data are expresses as mean log dilution ± SE. (n = 10).
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Analysis of the histopathology of inoculation sites in L. mexicana-infected BALB/c, C57BL/6 and CBA/J mice
On day 75 post-infection, skin lesions from CBA/J mice showed preserved skin with some foci of inflammation comprised primarily of lymphocytes and macrophages with few parasites (Fig. 2A and D). In contrast, ear lesions from BALB/c and C57BL/6 mice demonstrated skin ulceration with dermal inflammatory infiltrate comprised of neutrophils, eosinophils and heavily parasitized macrophages (Fig. 2B, C, E and F). Interestingly, C57BL/6 mice displayed significantly higher number of lymphocytes in their lesions as compared with BALB/c mice (Fig. 2B and E). Ears from uninfected CBA/J, C57BL/6 and BALB/c mice showed only few scattered macrophages and occasional lymphocyte (data not shown).

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Fig. 2. Histopathological analysis of infected ears from (A and D) CBA/J, (B and E) C57BL/6 and (C and F) BALB/c mice on day 75 after infection. Hematoxylin- and eosin-stained ears from BALB/c and C57BL/6 mice showed ulceration and extensive tissue destruction with inflammatory infiltrate comprised of parasitized macrophages (arrows) and neutrophils. In contrast, similarly stained sections from CBA/J mice displayed more preserved skin with lymphocytes and macrophages containing few parasites.
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Analysis of cell populations in the lesions from L. mexicana-infected BALB/c, C57BL/6 and CBA/J mice
Cell populations in the lesions of L. mexicana-infected CBA/J, C57BL/6 and BALB/c mice on days 15, 30 and 45 post-infection were analyzed and enumerated using flow cytometry. Lesions from BALB/c mice contained relatively more CD4+ T cells on day 15 post-infection (Fig. 3A), but all three strains contained comparable numbers of CD8+ T cells and CD11b+ macrophages at this time (Fig. 3B and C). On day 30, all three strains showed only a marginal increase in the number of CD4+ and CD8+ T cells in their lesions (Fig. 3A and B), but C57BL/6 showed nearly 3-fold increase in the number of intra-lesional CD11b+ macrophages. By day 45 post-infection, BALB/c mice recruited large number of CD4+ CD3+ cells in their lesions as compared with CBA/J and C57BL/6 mice (Fig. 3A), but the lesions from CBA/J mice contained nearly 2-fold more CD8+ cells than the other two strains (Fig. 3B). Both BALB/c and C57BL/6 mice also displayed an increase in the number of CD11b+ macrophages in their lesions as compared with CBA/J mice (Fig. 3C). However, lesions of C57BL/6 mice contained nearly 5- to 7-fold more macrophages than the other two strains (Fig. 3C). Infected lesions from all three mouse strains contained detectable number of 
T cells, but the differences within the groups were not significant statistically (data not shown).

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Fig. 3. Enumeration of (A) CD4+ CD3+, (B) CD8+ CD3+ and (C) CD11b+ cells in the lesions of Leishmania mexicana-infected CBA/J, C57BL/6 and BALB/c mice. On days 15, 30 and 45 after infection, cells isolated from the ear lesions of L. mexicana-infected CBA/J, C57BL/6 and BALB/c mice were enumerated and analyzed by flow cytometry. The cell numbers for each cell population were calculated from the total number of cells using percentages of specific cell population obtained by flow cytometry. Data expressed as the number of cells per ear lesion. Four to five mice were used per group at each time point.
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Antibody responses to L. mexicana in BALB/c, C57BL/6 and CBA/J mice
LmAg-specific serum IgG1, IgG2a and total IgE levels were determined in L. mexicana-infected BALB/c, C57BL/6 and CBA/J mice at 30, 55 and 70 days post-infection (Fig. 4). On day 28 after infection, neither IgG1 nor IgG2a was detectable in sera from all three groups. However, on day 55 post-infection and thereafter, L. mexicana-infected BALB/c mice displayed significantly higher levels of LmAg-specific IgG1 as compared with similarly infected C57BL/6 and CBA/J mice. On day 70 post-infection, BALB/c also displayed higher titers of IgG2a when compared with other groups but the difference was not statistically significant. Furthermore, at this time point, all three strains displayed comparable levels of total IgE.

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Fig. 4. Antibody responses in Leishmania mexicana-infected CBA/J, C57BL/6 and BALB/c mice. Mice were bled by tail snipping at different time points following L. mexicana infection and serum levels of Th2-associated IgG1 and total IgE as well as Th1-associated IgG2a were measured by ELISA. Data expressed as reciprocal endpoint titer ± SE for IgG1 and IgG2a. ND, no antibody detected at the 1 : 100 dilution. **P < 0.05, ***P < 0.01.
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Kinetics of in vitro cytokine production by LmAg-stimulated draining lymph node cells from BALB/c, C57BL/6 and CBA/J mice following low-dose L. mexicana infection
LmAg-stimulated lymph node cells from L. mexicana-infected CBA/J, C57BL/6 and BALB/c mice displayed significant proliferative responses at all the time points examined. There was no significant difference in the magnitude of LmAg-specific proliferation of lymph node cells between groups, indicating that differences in cytokine levels in vitro measured by ELISA were not reflective of a higher proliferative response of one group over the others (data not shown).
Culture supernatants from the above assay were analyzed by ELISA for the presence of the Th1-associated cytokines IL-12 and IFN-
and the Th2-associated cytokines IL-4, IL-10 and IL-13. LmAg-stimulated lymph node cells from all three strains produced comparable levels of IFN-
on day 15, but those from CBA/J mice produced significantly more on day 30 (Fig. 5A). On day 45, IFN-
production by lymph node cells from C57BL/6 mice increased significantly and was 2- to 3-fold higher as compared with CBA/J and BALB/c mice (Fig. 5A). At this time, culture supernatants from CBA/J and BALB/c mice contained comparable amounts of IFN-
(Fig. 5A). On day 75 post-infection, all strains produced less IFN-
as compared with day 45, but levels of IFN-
were significantly higher in culture supernatants from CBA/J mice (Fig. 5A). LmAg-stimulated lymph node cells from BALB/c mice produced significantly more IL-4 as compared with C57BL/6 and CBA/J mice on day 30 post-infection and thereafter (Fig. 5B). Furthermore, the lymph node cell culture supernatants from BALB/c mice also contained significantly more IL-10 on days 30 and 45, and IL-13 on days 45 and 75 (Fig. 5C and D). There were no significant differences in the levels of IL-12p70 produced by the lymph node cells of all the groups at all the time point examined (data not shown).

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Fig. 5. Kinetics of (A) IFN- , (B) IL-4, (C) IL-10 and (D) IL-13 production by LmAg-stimulated lymph node cells from CBA/J, C57BL/6 and BALB/c mice infected with low-dose Leishmania mexicana. The data shown for days 30, 45, and 75 are means of two independent experiments at each time point (n = 810). The data shown for day 15 are form one experiment (n = 5). Asterisks indicate statistically significant (P < 0.05) differences between the groups.
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In vitro cytokine production by LmAg-stimulated draining lymph node cells from BALB/c, C57BL/6 and CBA/J mice following high-dose back rump L. mexicana infection
On day 45 after infection, LmAg-stimulated lymph node cells from BALB/c mice produced significantly less IFN-
as compared with those from CBA/J and C57BL/6 mice which produced comparable amounts of IFN-
(Fig. 6A). At this time, both BALB/c and CBA/J mice produced significantly more IL-4 than the C57BL/6 mice (Fig. 6B), but CBA/J mice produced significantly more IL-10 as compared with the other two strains (Fig. 6C). The culture supernatants from BALB/c mice also contained more IL-13 as compared with CBA/J and C57BL/6 mice, but these differences were statistically not significant (Fig. 6D). All three strains produced low levels of IL-12p70 (data not shown).

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Fig. 6. (A) IFN- , (B) IL-4, (C) IL-10 and (D) IL-13 production by LmAg-stimulated lymph node cells from CBA/J, C57BL/6 and BALB/c mice following high-dose Leishmania mexicana infection on day 45 post-infection. Data expressed as means ± SE (n = 45; * denotes P < 0.05).
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Course of low-dose L. mexicana infection in STAT6/ BALB/c mice
Following intra-dermal inoculation with 1000 L. mexicana promastigotes into ear pinna, as expected, STAT6+/+ mice developed large non-healing lesions. In contrast, STAT6/ BALB/c developed either no lesions or small lesions that contained significantly fewer parasites (Fig. 7A and B). Histopathological analysis of infected ears from STAT6/ mice showed a preserved skin with some foci of inflammation comprised primarily of lymphocytes and macrophages (data not shown). Similar results were obtained in three independent experiments.

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Fig. 7. Course of low-dose Leishmania mexicana ear infection in STAT6/ and STAT6+/+ BALB/c mice. (A) Mice were infected by inoculating 1000 L. mexicana stationary phase promastigotes into ear dermis and disease progression was monitored by weekly measurements of the lesion size. (B) On day 75, parasite burdens in infected ears were determined by limiting dilution analysis. Similar results were observed in three independent experiments.
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Antibody and cytokine responses in L. mexicana-infected STAT6/ BALB/c mice
Levels of LmAg-specific Th1-associated IgG2a and Th2-asociated IgG1 were measured in L. mexicana-infected STAT6+/+ and STAT6/ mice at 6- and 10-weeks post-infection. At both these time points, STAT6+/+ mice consistently displayed higher titers of LmAg-specific IgG1 as compared with STAT6/ mice, although the difference between the groups was statistically significant only at later time point. Both STAT6+/+ and STAT6/ mice displayed comparable titers of IgG2a at both the time points (data not shown).
On day 75 after infection, LmAg-stimulated lymph node cells from STAT6/ mice produced significantly more Th1-associated IFN-
as compared with STAT6+/+ mice which secreted significantly more IL-4 (Fig. 8A and B). Similarly, STAT6/ lymph node cells also produced significantly more IL-12p70 as compared with those from STAT6+/+ mice (294 ± 117 pg ml1 and 39 ± 19 pg ml1 in STAT6/ and STAT6+/+ mice, respectively; P < 0.01). Although STAT6/ mice also produced relatively more IL-13 than the STAT6+/+ mice, the difference was statistically not significant (Fig. 8C).

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Fig. 8. (A) IFN- , (B) IL-4 and (C) IL-13 production by LmAg-stimulated lymph node cells from STAT6/ and STAT6+/+ BALB/c mice on day 75 post-infection. Data are expressed as means ± SE (n = 5). Similar results were observed in three independent experiments (* denotes P < 0.05).
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Discussion
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The interesting finding in this study is that BALB/c, C57BL/6 and CBA/J mice which develop large non-healing lesions following s.c. high-dose L. mexicana infection mount diverse immune responses and show marked differences in their ability to control L. mexicana growth when infected with low parasite inoculum into ear dermis. This indicates that genetic background plays an important role in regulating in vivo immune responses and influencing the outcome of low-dose L. mexicana infection.
The clinical outcome of Leishmania infection is undoubtedly dependent in part on the parasite species initiating infection. However, genetically regulated immunoregulatory factors operating within the host also play a role in determining the disease outcome by controlling growth of different species of Leishmania even when these species may infect the same tissue sites. For example, natural resistance-associated macrophage protein 1 (Nramp1) gene which controls early growth of Leishmania donovani in liver plays no role in controlling growth of L. major in that organ (19, 20). Furthermore, cutaneous growth of L. major has been shown to be under genetic and immunoregulatory controls that are different from those associated with cutaneous growth of L. mexicana (19, 21). Thus, while majority of inbred mice except those on BALB genetic background develops self-healing lesions when infected s.c. with L. major, virtually all mice develop rapidly growing large non-healing lesions full of parasites following s.c. L. mexicana infection. Nevertheless, in humans, L. mexicana infection commonly manifests as the chronic and progressive infection of ear dermis (Chiclero's ulcer) which is difficult to treat than those involving other cutaneous sites, in part due to the lower vascularity (22). Hence, we used experimental model of intra-dermal low-dose Leishmania infection in ear which mimics these conditions of natural challenge and was originally reported for L. major (12). As reported previously, we found that BALB/c, C57BL/6 and CBA/J mice were highly susceptible to high-dose L. mexicana challenge (23), but displayed distinct patterns of lesion growth and susceptibility to infection following inoculation of 1000 metacyclic promastigotes into ear dermis. Our observations are partly similar to those reported in a recent study which found that CBA/Ca mice are relatively resistant to low-dose footpad L. mexicana infection as compared with BALB/c mice (24). Nonetheless, taken together, these findings indicate that genetic background plays a critical role in regulating host immunity and controlling parasite replication following low-dose dermal L. mexicana infection.
There may be several factors why our study and that by Roger et al. (24) were able to reveal genetic differences in susceptibility to L. mexicana which were not evident in the previous studies. First, unlike conventional model which involves using 15 x 106 parasites for infection, we and Rogers et al. (24) used low parasite dose (1000 promastigotes) similar to the parasite inoculum that a host is likely to receive through the bite of a sandfly. Second, we used intra-dermal rather than s.c. route of infection to mimic the natural transmission. Both these factors, the site of infection and the number of inoculated parasites, have been shown to influence the immune mechanisms that determine susceptibility or resistance to infection (25, 26). Additionally, cell populations in the epidermal compartment such as keratinocytes and Langerhans cells also secrete a distinct set of cytokines when activated following infection.
Previous studies have shown that C57BL/6 mice infected by intra-dermal inoculation of low number of L. major promastigotes into ear develop dermal leishmaniasis which is characterized by an initial silent phase favoring parasite replication with no lesion formation followed by the development of skin lesion associated with the development of host immunity and killing of parasites at the site of infection (12). However, we found that the development of dermal lesions in C57BL/6 mice following low-dose L. mexicana infection was not associated with control of parasite replication in skin. In fact, as the infection progressed, L. mexicana-infected C57BL/6 mice developed ear lesions full of parasites destroying the pinna (our unpublished results). Nonetheless, BALB/c mice were significantly more susceptible to L. mexicana and developed significantly larger dermal lesions containing more parasites as compared with C57BL/6 mice. These results are consistent with findings in a previous study which demonstrated that C57BL/6 mice are relatively resistant to L. mexicana and control lymph node parasite replication and visceral dissemination more efficiently than BALB/c mice (27). Moreover, it is also likely that difference in susceptibility of these two inbred strains to L. mexicana may be due to genetically influenced functional differences in skin macrophages which have been shown to affect parasite replication and survival (17). Indeed, in the present study, we found that the ear lesions from C57BL/6 mice contained nearly 5-fold more CD11b+ macrophages than the BALB/c mice, suggesting that these macrophages may be responsible for controlling parasite replication in C57BL/6 mice.
Several recent studies using low-dose L. major ear infection model have shown that macrophages, CD4+ T cells and CD8+ T cells recruited to the infected skin play a critical role in determining the outcome of infection (25). We found that susceptible BALB/c and C57BL/6 mice recruited 5- to 8-fold more macrophages as compared with CBA/J mice as the infection progressed. Furthermore, C57BL/6 mice contained 3-fold more macrophages in their lesions as compared with BALB/c mice. Hence, it is possible that the difference in the L. mexicana lesion growth between BALB/c and C57BL/6 mice may be due to relatively more killing of parasites by macrophages in lesions of C57BL/6 mice. Interestingly, BALB/c mice, which developed largest lesions, contained relatively high number of CD4+ T cells in their lesions throughout the course of infection, suggesting that CD4+ T cells recruited to the site of infection in these mice are likely to be non-Th1 cells. On the other hand, resistant CBA/J mice showed a significant influx of CD8+ T cells into their lesions on day 45 and contained nearly 2-fold more CD8+ T cells than the other two strains, indicating that CD8+ T cells are to likely play an important role in restricting L. mexicana growth in skin. Thus, it is likely that impaired trafficking of CD8+ T cells into the lesions is also at least in part responsible for the failure of C57BL/6 and BALB/c mice to control parasite growth. This is perhaps not surprising as several studies have reported that CD8+ T cells not only play a critical role in host defense against primary cutaneous L. major infection (16) but also are required for vaccine-induced protection against Leishmania amazonensis (28). Furthermore, we recently showed that lack of CXCR3 impaired trafficking of CD8 T cells into the lesion during L. major infection and rendered resistant C57BL/6 mice susceptible to infection, suggesting that CXCR3 and other T cell-associated chemokine receptors may also contribute to trafficking of CD8 T cells during L. mexicana infection (29). Ongoing studies in our laboratory are examining these possibilities and are characterizing the cell populations in lesions during L. mexicana infection.
Using conventional high-dose s.c. infection model, several studies have shown that susceptibility of most inbred mice to L. mexicana is due to their inability to produce IL-12 rather than lack of IL-12 responsiveness (30, 31), although NZB and female DBA/2 mice do mount a Th1 response and successfully control high-dose L. mexicana infection (2, 32). Furthermore, we and others have also shown that Th2-type cytokines IL-4 and IL-13 play a critical role in the pathogenesis of cutaneous L. mexicana infection by inhibiting Th1 development by signaling via STAT6-mediated pathway (36). In the present study, we found that LmAg-stimulated lymph node cells from CBA/J infected with low dose of L. mexicana produced significantly higher amounts of Th1-associated IFN-
on days 30 and 75 after infection as compared with similarly infected C57BL/6 and BALB/c mice, but those from susceptible BALB/c mice produced significantly more Th2-associated IL-4, IL-10 and IL-13 at most time points. Interestingly, C57BL/6 mice produced significantly more IFN-
on day 45, but were unable to maintain it. In this context, it is important to note that BALB/c and C57BL/6 lymph node cells did secrete significant quantities of IFN-
on day 30 post-infection and thereafter. All three strains produced low but comparable levels of IL-12 at all the time points. This finding supports our previous observations as well as the results reported by Bennett et al. who found that L. mexicana-infected bone marrow-derived dendritic cells fail to activate and secrete IL-12 (30, 33). Taken together, these findings suggest that enhanced production of Th2-associated cytokines IL-4, IL-10 and IL-13 in BALB/c mice may be responsible for their relatively increased susceptibility to low-dose L. mexicana infection as compared with CBA/J and C57BL/6 mice.
The pattern of cytokine production during high-dose L. mexicana infection differed from those observed after low-dose infection. Although CBA/J mice produced significant amounts of IFN-
, they produced as much IL-4 as BALB/c mice and significantly more IL-10 than C57BL/6 as well as BALB/c mice, suggesting that these cytokines may be responsible for the susceptibility of CBA/J mice to high-dose L. mexicana infection. On the other hand, the pattern of cytokine secretion in BALB/c mice was similar to that observed in low-dose model as they failed to produce significant amounts IFN-
but displayed high levels of IL-4 and IL-13. These observations suggest that the mechanisms mediating susceptibility of BALB/c mice to high- and low-dose L. mexicana infection are likely to be similar. Interestingly, C57BL/6 mice produced as much IFN-
as CBA/J mice but displayed significantly lower levels of IL-4, suggesting that other immune mechanisms may be involved in the pathogenesis of high-dose L. mexicana infection in C57BL/6 mice.
The B cells and antibodies play a limited role in mediating protection against Leishmania. In fact, a previous study has shown that antibodies play a role in the pathogenesis of cutaneous leishmaniasis caused by the parasites of L. mexicana complex by facilitating internalization of parasites by FcR (34). However, levels of Th1-associated IgG2a and Th2-associated IgG1 and IgE are also measured as an indicator of in vivo Th1 and Th2 development. In the present study, serum levels of LmAg-specific IgG2a and IgG1 as well as total IgE in mice infected with low dose of L. mexicana were lower as compared with those typically observed in high-dose infection. Furthermore, the levels in these mice did not correlate clearly with the cytokine responses probably due to poor systemic B cell response due to low dose of L. mexicana.
Although previous studies by us and others have shown that IL-4 and IL-13 play critical roles in mediating susceptibility to L. mexicana following high dose s.c. infection into back rump (5, 6), recent studies using high-dose footpad infection model reported that L. mexicana-infected IL-4/ as well as STAT6/ mice fail to heal their lesions (7, 35). However, in the present study, we found that STAT6/BALB/c mice mount a Th1-like response, produce high levels of IL-12p70 and IFN-
and develop either no lesions or small self-resolving lesions containing few parasites following low-dose L. mexicana dermal ear infection. Increased IL-12p70 production in STAT6/ BALB/c mice suggests that STAT6 signaling inhibits Th1 development during L. mexicana infection by inhibiting IL-12p70 production. These findings are similar to our observations in previous experiments with conventional high-dose model of infection (6) and indicate that STAT6-mediated pathway also plays a role in the pathogenesis of dermal leishmaniasis caused by L. mexicana.
In conclusion, CBA/J mice predominantly produce Th1-associated IFN-
, recruit relatively more CD8+ T cells into the site of infection and effectively control the parasite growth following inoculation with low-dose L. mexicana into ear dermis, while BALB/c mice mount a Th2-like response, recruit macrophages and CD4+ T cells into their lesions and develop large lesions full of parasites. On the other hand, C57BL/6 mice are more susceptible to low-dose L. mexicana infection as compared with CBA/J mice but fail to display a clear Th1- or Th2-biased response, recruit large numbers of macrophages into the site of infection and develop relatively smaller lesions containing fewer parasites as compared with highly susceptible BALB/c mice. Moreover, intra-dermal infection of STAT6/ BALB/c mice with low number of L. mexicana induces a robust Th1-like response and renders them resistant to infection.
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Acknowledgements
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This work was supported by National Institutes of Health research grant AI51823-01 to A.R.S.
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Abbreviations
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LmAg | Leishmania mexicana antigen |
s.c. | subcutaneously |
SPF | specific pathogen free |
STAT | signal transducers and activators of transcription |
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Notes
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Transmitting editor: C. Terhorst
Received 6 June 2005,
accepted 29 July 2005.
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