In C57BL/6 Mice Leishmania Major Polarization Produced by a Strain Of

Nonhealing Infection despite Th1 Polarization Produced by a Strain of Leishmania major in C57BL/6 Mice

This information is current as Charles F. Anderson, Susana Mendez and David L. Sacks of September 29, 2021. J Immunol 2005; 174:2934-2941; ; doi: 10.4049/jimmunol.174.5.2934 http://www.jimmunol.org/content/174/5/2934 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Nonhealing Infection despite Th1 Polarization Produced by a Strain of Leishmania major in C57BL/6 Mice

Charles F. Anderson, Susana Mendez,1 and David L. Sacks2

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demon- strate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-␥ produced by Leishmania- specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively ␥ cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN- - Downloaded from activated macrophages in vitro. Infection of IL-10؊/؊ mice, blockade of the IL-10R, or depletion of CD25؉ cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms

of leishmaniasis in humans. The Journal of Immunology, 2005, 174: 2934–2941. http://www.jimmunol.org/

cquired resistance to the intracellular protozoan Leish- rine models has fostered the main conceptual framework for un- mania major is dependent on the development of a Th1- derstanding healing and nonhealing forms of clinical disease. type immune response, marked by the induction of However, it has not been possible to clearly associate a Th2 po- Aϩ ϩ CD4 and CD8 T cells mediating IFN-␥-dependent macrophage larity with nonhealing, systemic, or reactivation forms of leish- microbicidal activity. In the murine model for cutaneous leishman- maniasis in humans. IFN-␥-producing cells or mRNA remain iasis, the mechanisms of acquired resistance to L. major have been readily detectable in patients with kala-azar, postkala-azar dermal well documented using genetically resistant C57BL/6 mice, which leishmaniasis, or chronic cutaneous leishmaniasis, and the oppos- reproduce the self-cure disease outcome typically seen in humans ing cytokine most commonly found in these clinical settings is not by guest on September 29, 2021 (1, 2). Intradermal inoculation of these mice with a biologically IL-4, but IL-10 (10–21). Interestingly, IL-10 has also been found appropriate low dose of infectious stage parasites produces an ini- to contribute to BALB/c susceptibility to L. major (22), although tial prepatent phase of intracellular growth, absent of visible le- its influence is generally obscured by the Th2 bias that is intrinsic sions or pathology, followed by lesion development and the accu- to this mouse strain (23). In contrast, a role for IL-10 in regulating mulation of IFN-␥-producing CD4ϩ and CD8ϩ T cells in the immunity in resistant mice has been conclusively shown, in this lesion (3–5). Following resolution of the lesion, a low number of case mediating not susceptibility per se, but the persistence of low parasites persists indefinitely in the site, and the host is protected numbers of parasites in the skin following clinical cure (4). Thus, against a rechallenge infection at a distal site (3, 5). IL-10Ϫ/Ϫ mice achieve sterile immunity, as do healed C57BL/6 In contrast to the self-limiting infections with L. major observed mice treated during the chronic phase with anti-IL-10 receptor in C57BL/6 mice, BALB/c mice develop progressive, nonhealing Abs. More recently, IL-10-producing naturally occurring lesions that are associated with an early, sustained, and dominant CD4ϩCD25ϩ regulatory T cells have been shown to home to L. Th2 response, especially IL-4 (1, 2). Furthermore, ablation of the major lesions, and to be necessary for parasite persistence in cells or cytokines involved in normal Th1 development in resistant healed mice (24). These cells may fulfill a primarily homeostatic mice results in Th2 deviation and progressive infection (6–9). The function, preventing excessive pathology mediated by Leishma- Th1/Th2 balance that controls immunity to L. major in these mu- nia-specific Th1 cells in the inflammatory site. Although regulatory and effector T cell subsets appear to function in equilibrium to maintain latency in healed mice, these findings raise the possibility Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Dis- that an imbalance in regulatory cells or cytokines might control eases, National Institutes of Health, Bethesda, MD 20892 nonhealing or systemic disease outcomes. Received for publication September 8, 2004. Accepted for publication December In the present study, we have analyzed the role of these oppos- 16, 2004. ing host factors in the immune response of C57BL/6 mice to a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance strain of L. major that was isolated from a patient with nonhealing with 18 U.S.C. Section 1734 solely to indicate this fact. lesions (25). The lesions persisted for months following multiple 1 Current address: Department of Microbiology and Tropical Medicine, George courses of treatment, but the patient was found to have a positive Washington University, Washington, DC 20037. skin test and proliferative responses to Leishmania Ags. In the 2 Address correspondence and reprint requests to Dr. David L. Sacks, National In- current study, despite the induction of a strong and polarized Th1 stitute of Allergy and Infectious Diseases, National Institutes of Health, Laboratory of Parasitic Diseases, Building 4, Room 126, 4 Center Drive MSC 0425, Bethesda, MD response, the parasites are not effectively controlled, and the der- 20892-0425. E-mail address: [email protected] mal lesions fail to resolve. We examine the degree of suppression

ϩ ϩ mediated by IL-10 and by CD4 CD25 regulatory T cells and erated from bone marrow in the presence of GM-CSF as previously de- argue that these are major contributing factors to the evolution of scribed (27). LN cells (3 ϫ 106) or dermal cells pooled from six ears were these nonhealing infections. resuspended in RPMI 1640 containing 10% FCS, 10 mM HEPES, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, and 1 ml of the cell suspen- sion was incubated with uninfected or amastigote-infected BMDCs at a

Materials and Methods ratio of 5:1 in 24-well plates at 37°C in 5% CO2 for 18 h, with brefeldin Mice A added during the last 5 h. The cells were then fixed and analyzed for surface markers and intracellular IFN-␥. In some experiments, unfraction- C57BL/6 mice were purchased from the Division of Cancer Treatment, ated LN or dermal cells were incubated for 48 h with BMDCs or amastig- National Cancer Institute (Frederick, MD). C57BL/10SgSnAi, C57BL/ ote-infected BMDCs, and IFN-␥, IL-10, and IL-4 from culture supernatants 10SgSnAi-[KO]IL-10, and C57BL/6SgSnAi-[KO]IL-4 mice were pur- were quantitated by ELISA (eBioscience) according to the manufacturer’s chased from Taconic Farms. All mice were maintained in the National protocol. The limits of detection for the cytokines were 4 pg/ml for IL-4, Institute of Allergy and Infectious Diseases animal care facility under spe- 15 pg/ml for IL-10, and 15 pg/ml for IFN-␥. cific pathogen-free conditions. Immunolabeling and flow cytometry Antibodies Before staining, LN or dermal cells were incubated with an anti-FcIIIR/IIR For in vivo Ab treatments, mice were given 1.0 mg i.p. injections at the (clone 2.4G2; BD Pharmingen) mAb in PBS containing 0.1% BSA and indicated times with anti-CD25 (PC61), anti-IL-10␣R (1B1.3a), or an iso- 0.01% NaN3. The staining of surface markers and intracellular cytokines type control (GL113). CD25 depletion was determined by staining cells was performed sequentially. The cells were stained first for their surface Ͼ with Ab clone 7D4, which showed depletion to be 80% compared with markers (TCR chain receptor, CD8, or CD4), followed by a permeabili- control-treated mice. The following fluorochrome-conjugated Abs for cell zation step and staining with anti-IFN-␥. The lymphocytes were identified surface and intracytoplasmic staining were purchased from BD Pharmin- by characteristic size (forward light scatter) and granularity (side light scat- Downloaded from gen: PE-anti-CD25 (PC61), FITC-anti-CD25 (clone 7D4), FITC-anti- ter), in combination with anti-TCR-␤ chain and anti-CD4 or anti-CD8, or ␤ ␣ TCR- (H57-597), PE-Cy5 anti-CD4 (L3T4), PE-Cy5 anti-CD8 anti-CD25 for surface staining. For each sample, 150,000 events were ac- ␥ (53–6.7), and PE anti-IFN- (XMG1.2). The isotype controls used (all quired for analysis. The data were collected and analyzed using CellQuest from BD Pharmingen) were rat IgG2b (A95-1), rat IgG2a (R35-95), rat Pro software and a FACSCalibur flow cytometer (BD Biosciences). IgG1 (R3-34), and hamster IgG group 2 (Ha4/8). For ELISA measurements of cytokines, the following Ab pairs were purchased from eBioscience. Real-time PCR IL-4: 11B11, BVD6-24G2; IL-10: JES5-16E3, JES5-2A5; IFN-␥:

XMG1.2, R4-6A2. For analysis of gene expression, ears were removed and immediately http://www.jimmunol.org/ placed in RNAlater stabilization buffer (Qiagen) to prevent degradation. Parasite preparation and intradermal inoculation Tissue was then disrupted by placing the ears in mortars containing liquid nitrogen and grinding with a pestle. The disrupted tissue was then homog- L. major clones V1 (MHOM/IL/80/Friedlin) and Sd (MHOM/SN/74/SD) enized using Qiashredder homogenizers and processed for RNA isolation were cultured in medium 199 with 20% heat-inactivated FCS (Gemini using the RNeasy minikit (Qiagen), following the manufacturer’s protocol, Bio-Products), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2 mM L- with all samples being treated with a DNase step to remove genomic DNA. glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml Reverse transcription was performed on 500 ng of total RNA from each ear hemin (in 50% triethanolamine), and 1 mg/ml 6-biotin (M199/S). The Sd sample using Superscript III First-Strand synthesis system for RT-PCR strain was isolated from a patient who was admitted to the National Insti- (Invitrogen Life Technologies). Real-time PCR was performed on an ABI tutes of Health for treatment of lesions that persisted following multiple Prism 7900HT sequence detection system (Applied Biosystems) using courses of therapy (25). Infective-stage metacyclic promastigotes of L. ma- by guest on September 29, 2021 10% of the cDNA reaction in a total volume of 20 ␮l of PCR mixture. jor were isolated from stationary cultures (4–5 days old) by density gra- FAM-MGB-labeled primer/probe sets for IFN-␥, IL-10, IL-13, inducible dient centrifugation as described previously (26). Metacyclic promastigotes NO synthase-2 (iNOS-2), and Foxp3 were designed from Applied Biosys- (1000) were inoculated into the ear dermis using a 30-gauge needle in a tems, and VIC-MGB-labeled 18 S rRNA was used as the endogenous volume of ϳ5 ␮l. The development of the lesion was monitored by mea- control. The relative quantitation of products was determined by the com- suring the diameter of the ear lesion with a direct-reading vernier caliper Ϫ⌬⌬ parative threshold cycle method using the equation 2 cT to determine the (Thomas Scientific). fold increase in product. Each gene of interest was normalized to the 18 S Processing of ear tissue and estimation of parasite load rRNA endogenous control, and the fold change in expression was displayed as relative to naive controls. Four ears were used for each time Parasite titrations were performed as previously described (3). The two point, and each sample was run in triplicate during the PCR. sheets of infected ears were separated, deposited in DMEM containing 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 0.2 mg/ml Liberase CI pu- In vitro killing assay rified enzyme blend (Roche Diagnostic Systems), and incubated for2hat 37°C. Sheets were then placed in a grinder and processed in a Medima- Peritoneal macrophages from C57BL/6 mice were plated in eight-chamber ϫ 4 chine tissue homogenizer (BD Biosciences) for 4 min. Tissue homogenates Lab-Tek Permanox tissue culture slides (Miles Laboratories) at 5 10 were then filtered through a 70-␮m cell strainer (Falcon Products), and cells/well in a volume of 0.4 ml of RPMI 1640 complete. V1 or Sd tissue- aliquots of these were serially diluted in 96-well flat-bottom microtiter derived amastigotes were added to the wells at a ratio of 5:1, and infection ␮ plates containing biphasic medium prepared using 50 ␮l of NNN medium was allowed to establish for 72 h at 35°C. At this time, 0.1 g/ml LPS and ␥ containing 30% of defibrinated rabbit blood overlaid with 50 ␮l of M199/S. titrating amounts of recombinant mouse IFN- were added. After an ad- The number of viable parasites in each sample was determined from the ditional 72 h, the slides were fixed for 1 min in anhydrous methanol and highest dilution at which promastigotes could be grown out after 7 days of stained with Diff-Quick solutions. The numbers of infected and non-in- incubation at 25°C. fected macrophages and the number of amastigotes were counted. The percentage of killing relative to untreated macrophages was calculated on Analysis of intradermal lymphocytes, APC preparation, and the basis of the comparison of total amastigotes per 100 macrophages. cytokine analysis Statistical analysis To characterize leukocytes in the inoculation sites, the ears were collected, and the ventral and dorsal dermal sheets were prepared as described above. To determine whether differences were statistically significant, Student’s t For cell surface phenotype, cells were fixed in PBS/2% paraformaldehyde test was performed, using a two-tailed distribution with unpaired samples. for 15 min at 25°C, washed, and resuspended in staining buffer (PBS with 0.1% BSA and 0.01% NaN3) for phenotypic analysis by flow cytometry. Results 3 For in vitro restimulation, unfractionated lymph node (LN) cells or dermal L. major Sd produces a nonhealing phenotype in C57BL/6 mice cells were incubated with uninfected or amastigote-infected bone marrow- derived dendritic cells (BMDCs) as a source of APCs. BMDCs were gen- To characterize the host response to the L. major Sd strain that produced nonhealing lesions in a patient, C57BL/6 mice were in- 3 Abbreviations used in this paper: LN, lymph node; BMDC, bone marrow-derived oculated with 1000 metacyclic promastigotes, and monitored for dendritic cell; iNOS, inducible NO synthase; DLN, draining LN. lesion development in reference to the L. major V1 strain. As 2936 A NONHEALING L. major INFECTION IN C57BL/6 MICE

FIGURE 2. Sd infection is not defective in the recruitment of lymphocytes to the lesion. At various times postinfection, dermal cells were im- munolabeled for TCR-␤ and CD4 or CD8 and analyzed by ﬂow cytometry. Lymphocytes were gated according to forward scatter and side scatter properties. The numbers shown in the upper right quadrant are the absolute numbers of cells per ear (ϫ104) and are the results of four to six ears pooled per time point. The numbers represent the mean and SD of three independent experiments. Downloaded from

an aberrant Th2 response in this normally resistant host. As early as 3 wk postinfection, Ag-speciﬁc IFN-␥ production was detectable in high levels in the DLN of the Sd-infected mice, at a time when Th1 priming was barely in evidence in the mice infected

FIGURE 1. Sd strain of L. major produces a nonhealing phenotype in http://www.jimmunol.org/ C57BL/6 mice. A total of 103 V1 or Sd metacyclic promastigotes were with V1 (Table I). IFN-␥ production in the Sd-infected mice was injected in the ear dermis of mice. A, Development of ear lesions over the maintained at high levels at 6 and 11 wk, whereas IFN-␥ produc- course of 22 wk. The mean and SD of six ears per group are shown. B, tion peaked at around 6 wk in the V1-infected mice and declined Parasite loads in ear lesions, showing the mean and SD from six ears per signiﬁcantly by 11 wk, at a time when the majority of parasites had group. C, Photographs of the ears were taken 22 wk postinfection The been eliminated. Importantly, at no time point did the DLN cells results are representative of at least three independent experiments. from the Sd or V1 mice produce signiﬁcant amounts of IL-4. In contrast, IL-10 was detected by 3 wk in the Sd mice, with high previously described (3), mice infected intradermally with V1 par- levels being produced by 6 wk postinfection. Apart from the early

asites produced lesions that healed spontaneously in 8–12 wk fol- time point, IL-10 was secreted in even higher levels by the DLN by guest on September 29, 2021 lowing inoculation (Fig. 1). In contrast, when mice were infected cells from the V1-infected mice. with the L. major Sd strain, an initial prepatent phase was followed The number of CD4ϩ and CD8ϩ T cells in the ear dermis was by the development of lesions that failed to heal. Lesions grew analyzed to determine whether the Sd infection was inhibiting the slowly, but progressively larger in diameter, resulting in ulcer- recruitment of effector cells to the site of infection. Fig. 2 shows ations and eventual loss of dermal tissue (Fig. 1). The lesions did the frequency of TCR-␤ϩCD4ϩ and TCR-␤ϩCD8ϩ cells present not disseminate to other dermal sites nor was there evidence of in the lesions at 3, 6, and 10 wk postinfection, along with the total visceralization beyond the local draining LN (DLN) (data not numbers of these subsets. The recruitment of CD4ϩ T cells was shown). Lesions harboring the V1 strain showed a peak in parasite apparent in both groups by 3 wk, though it was slightly higher in number at ϳ6 wk, which by 10 wk had been 90% cleared from the the Sd lesions at 3 and 6 wk, and ϳ2-fold higher at 10 wk, when site, and by 22 wk fewer than 100 parasites remained in the healed the V1 dermal lesions were beginning to resolve. Recruitment of lesion. In contrast, lesions harboring the Sd strain had a slow pro- CD8ϩ T cells was generally comparable in both groups at the gressive increase in the number of parasites throughout the 22 wk earlier time points, with a slight decrease of CD8ϩ cells seen in the that lesions were monitored. Sd infections compared with V1 at 10 wk. The ability of the lesional CD4ϩ T cells to secrete cytokines in Priming and recruitment of Th1 cells is not deﬁcient in L. major response to infected DC was measured by intracellular staining Sd-infected mice and by ELISA. Ag-speciﬁc IFN-␥-producing cells were detected in Because the L. major Sd strain produced a nonhealing phenotype, both groups as early as 3 wk, increasing in absolute numbers at wk we investigated the possibility that this L. major substrain induces 6, and maintained at high frequency at 10 wk. Strikingly, the Sd

Table I. ELISA cytokine measurements of draining lymph node cellsa

V1 DLNs Sd DLNs

3 wpi 6 wpi 11 wpi 3 wpi 6 wpi 11 wpi

ϪϩϪϩϪϩϪϩϪϩϪϩ

IFN-␥b 88 Ϯ 992Ϯ 20 Ͻ15 11,321 Ϯ 58 Ͻ15 3,994 Ϯ 139 146 Ϯ 118 4,745 Ϯ 998 Ͻ15 11,112 Ϯ 625 Ͻ15 16,224 Ϯ 436 IL-10b 22 Ϯ 30 24 Ϯ 40 108 Ϯ 48 2,324 Ϯ 24 80 Ϯ 16 828 Ϯ 35 Ͻ15 160 Ϯ 28 132 Ϯ 58 1,675 Ϯ 198 41 Ϯ 23 257 Ϯ 11 IL-4b Ͻ4 Ͻ4 Ͻ481Ϯ 37Ϯ 132Ϯ 1 Ͻ4 Ͻ4 Ͻ429Ϯ 4 Ͻ4 Ͻ4

a A total of 3 ϫ 106 DLN cells/ml was restimulated with BMDCs (Ϫ) or amastigote-infected BMDCs (ϩ). Supernatants were collected 48 h later for measurement. The mean and SD of triplicates of one experiment are shown and are representative of at least three independent experiments. wpi, Weeks postinfection. b Values given as picograms per milliliter. The Journal of Immunology 2937

FIGURE 4. V1 and Sd strains are equally susceptible to killing by activated macrophages in vitro. Peritoneal macrophages were plated in 8-well chamber slides and infected with V1 or Sd amastigotes. Macrophages were activated by the addition of LPS and titrated amounts of rIFN-␥, 72 h after infection. After an additional 72 h, macrophages were ﬁxed and stained for parasite quantitation. The percentage of killing was determined by the number of amastigotes per 100 macrophages and was based on the com- FIGURE 3. Sd-infected ears contain high numbers of IFN-␥ secreting parison with unactivated macrophages. effector cells. Dermal cells from six V1- or Sd-infected ears were pooled and restimulated for 18 h in the presence of BMDCs or amastigote-infected

BMDCs (BMDCs/Lm). Brefeldin A was added during the last 5 h, and Downloaded from cells were then fixed and permeabilized for intracellular cytokine staining. defined the LD for each of the strains, the Sd strain did not The numbers in the upper right quadrant are the percentages of CD4ϩ cells 50 ␥ display a greater resistance to killing at any of the concentrations secreting IFN- . The data are representative of at least three independent ␥ experiments. of rIFN- used. Thus the resistance of the Sd strain to killing in vivo is apt to reflect differences in the activation state of the infected cells in the inflammatory site. infection contained more than twice the number of Th1 effectors at ϩ ϩ http://www.jimmunol.org/ 10 wk, suggesting the higher parasite load at this time was driving The role of IL-10 and CD4 CD25 Treg cells in the the continued expansion and recruitment of effector cells (Fig. 3). development of nonhealing lesions due to L. major Sd Cytokines were also measured by ELISA (Table II). An earlier, Because the presence of IL-10 has been shown to permit the per- more elevated and sustained Ag-specific IFN-␥ response by Sd sistence of L. major in the face of a robust Th1 response in healed lesional cells was also noted by ELISA. IL-4-producing cells were mice (4), we considered the possibility that the nonhealing phe- not detectable in either group, either by intracellular staining (data notype might be due to a modest shift in the balance between not shown), or by ELISA, except for the slightly elevated levels IFN-␥ and IL-10 cytokines, not revealed in the ELISA assays of produced by the V1 lesional cells at wk 6 (Table II). In contrast, Ag re-stimulated dermal cells. As IL-10 has been shown to be substantial amounts of IL-10 were produced by ear lesional cells produced by keratinocytes in dermal lesions (13), and by Leish- by guest on September 29, 2021 from both groups by 3 wk, and high levels continued to be pro- mania-infected macrophages in vitro (22, 28), real-time RT-PCR duced by the V1 and Sd dermal cells throughout the healing and on RNA extracted directly from the ears was used to better quan- chronic phase, respectively. Interestingly, the majority of the IL-10 titate total IL-10 produced by the different possible sources of produced by the dermal cells did not require Ag re-stimulation for IL-10 in the inflammatory site. Compared with V1, the Sd strain release. Taken together, these results indicate that there is not a activated elevated levels of IFN-␥ mRNA, in agreement with the defect in Th1 priming, nor a defect in recruitment of Th1 cells to flow cytometry and ELISA data, but also contained significantly the site of the infection. The Sd parasites persist and produce non- increased message for IL-10 at 11 and 14 wk postinfection (Fig. 5). healing lesions despite the development of a polarized Th1 The difference seen at these time points, especially at wk 14, is apt response. to be secondary to the inflammatory conditions associated with To determine whether the Sd strain might persist in the lesion active vs healed lesions. No difference was observed at the 6-wk due to an intrinsic resistance to NO-dependent killing by activated time point, around the time when the clinical outcomes were be- macrophages, we performed an in vitro killing assay, in which ginning to diverge. Additionally, IL-13 was measured by real-time infected macrophages were activated with LPS and titrated PCR to exclude the possible role of this Th2-associated cytokine. amounts of rIFN-␥, providing both the priming and triggering At both 6 and 12 wk postinfection, neither group expressed sig- stimuli needed to generate optimal levels of NO (Fig. 4). Both nificant IL-13 messages above that detectable in naive ears. The strains were efficiently killed when the infected macrophages were data reinforce the absence of a Th2 immune deviation in the L. activated using 10 ng/ml rIFN-␥, and although a broad dose range major Sd-infected ears.

Table II. ELISA cytokine measurements of cells from ear lesionsa

V1 Ear Lesions Sd Ear Lesions

3 wpi 6 wpi 11 wpi 3 wpi 6 wpi 11 wpi

ϪϩϪϩϪϩϪϩϪϩϪ ϩ

IFN-␥b 197 Ϯ 45 170 Ϯ 10 962 Ϯ 287 6,533 Ϯ 50 3,369 Ϯ 303 8,110 Ϯ 795 56 Ϯ 18 5,625 Ϯ 715 2,916 Ϯ 324 13,200 Ϯ 50 14,704 Ϯ 1,000 21,852 Ϯ 164 IL-10b 147 Ϯ 20 506 Ϯ 102 1,435 Ϯ 24 2,213 Ϯ 115 705 Ϯ 4 987 Ϯ 13 207 Ϯ 3 699 Ϯ 26 957 Ϯ 39 1,263 Ϯ 26 684 Ϯ 6 787 Ϯ 53 IL-4b Ͻ48Ϯ 235Ϯ 1 138 Ϯ 223Ϯ 534Ϯ 1 Ͻ436Ϯ 4 Ͻ421Ϯ 214Ϯ 718Ϯ 6

a Six ears from each group were pooled and restimulated with BMDCs (Ϫ) or amastigote-infected BMDCs (ϩ). Supernatants were collected 48 h later for measurement. The mean and SD of triplicates from one experiment are shown and are representative of at least three independent experiments. wpi, Weeks postinfection. b Values given as picograms per milliliter. 2938 A NONHEALING L. major INFECTION IN C57BL/6 MICE

FIGURE 5. Sd lesions contain elevated levels of mRNA for IFN-␥, IL- 10, and Foxp3, and low amounts of IL-13. Total RNA was isolated directly from the ears at the indicated times during the infection, reverse transcribed, and analyzed by real-time PCR. The target genes were normalized to the endogenous control, and the values shown are the fold increase Downloaded from relative to expression in naive ears. Each bar is the geometric mean and SD .(p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) .of four individual ears

A potential source of IL-10 in the nonhealing lesions is CD4ϩCD25ϩ regulatory T cells. The presence of these cells has http://www.jimmunol.org/ been shown to control the persistence of L. major in healed mice (24), and adoptive transfer of these cells, or their Ag-induced expansion, has been recently shown to reactivate parasite growth and FIGURE 7. IL-10 deficiency or anti-CD25 treatment promotes parasite dermal pathology in healed lesions (29). Thus it was important to killing in the Sd-infected mice. Parasite loads in ear lesions in C57BL/10 Ϫ/Ϫ Ϫ/Ϫ 3 determine whether there was an increased frequency of or IL-10 mice or C57BL/6 or IL-4 mice infected with 10 metacy- ϩ ϩ clics and sacrificed at wk 10 for parasite enumeration (A), C57BL/6 mice CD24 CD25 Treg in the Sd mice. The forkhead/winged helix treated with anti-IL-10␣R Ab every 4 days during the last 2 wk of infection transcription factor Foxp3 has been shown to be specifically ex- ϩ Ϫ and sacrificed at wk 10 for parasite enumeration (B), and C57BL/6 mice pressed by CD25 Treg, as well as by CD25 T cells with reg- treated with anti-CD25 Ab every 4 days during the last 2 wk of infection ulatory activity (30, 31). Compared with V1, the Sd lesions con- and sacrificed at wk 12 for parasite enumeration (C). The data are repre- by guest on September 29, 2021 p Ͻ 0.05 compared with ,ء) tained a significantly increased level of Foxp3 mRNA at 11 and 14 sentative of two independent experiments wk postinfection and a small, though nonsignificant, increase at 6 control mice). wk (Fig. 5). And whereas the number of Foxp3 transcripts remained steady throughout the acute, healing, and latent stages of V1 infection, there was a steady increase during progressive lesion in the lesions revealed no major differences between the two in- development in the Sd non-cure mice. Flow cytometry analysis to ϩ ϩ ϩ Ϫ fections during the first 11 wk (Fig. 6). There was a modest in- measure the frequency of CD4 CD25 and CD4 CD25 T cells crease in the total number of CD25ϩ Treg in the Sd lesions at 6 wk, and especially at 11 wk when there was an ϳ3-fold increase compared with V1, consistent with the Foxp3 real-time PCR data. To determine what role IL-10 may play in prevention of healing, we first took a genetic approach by infecting IL-10Ϫ/Ϫ mice with either V1 or Sd (Fig. 7). At 10 wk postinfection, there was a 2-log reduction in L. major Sd parasite burden in the IL-10Ϫ/Ϫ mice compared with the wild-type B/10 mice (Fig. 7B). However, sterile cure of the Sd strain was not achieved as was apparent with V1, consistent with previous observations (4). Despite the reduced parasite load, the Sd lesions in the IL-10Ϫ/Ϫ mice were larger than in the wild-type mice throughout the course of infection, consistent with a critical role of IL-10 in modulating immune-mediated pathology (Fig. 7A). In contrast to the IL-10Ϫ/Ϫ mice, the IL-4Ϫ/Ϫ mice were found to be equally susceptible to L. major Sd as was wild-type B/6 mice, both in terms of the severity of their nonhealing lesions (not shown) and the failure to effectively reduce the parasite burden during the chronic stage (Fig. 7B). To determine FIGURE 6. V1- and Sd-infected ears contain high numbers of ϩ ϩ the effect of a transient block in IL-10 signaling, mice were treated CD4 CD25 cells. Dermal cells were removed from ear lesions at the ␣ indicated times and stained for CD4 and CD25. The numbers in the upper with anti-IL-10 R Ab during the chronic phase of the infection. right quadrant represent the percentage of total CD4ϩ cells that express Mice were sacrificed 1 wk after the course of treatment, and the CD25 and the absolute numbers of CD4ϩCD25ϩ cells (ϫ104) per ear. The parasite burden was measured (Fig. 7C). The treatment had a dra- data are from one representative experiment of at least three independent matic curative effect in the Sd-infected mice, with 99% of parasites experiments. being cleared from the site. The treatment also reduced the parasite The Journal of Immunology 2939

following the last treatment. The anti-CD25-treated mice had a signiﬁcant, 10-fold reduction in parasite number compared with the isotype controls (Fig. 9), and slight increases in both IFN-␥ and iNOS-2 expression in the lesion, though again the levels of both transcripts were already high in the untreated mice.

Mice with nonhealing L. major Sd infection develop concomitant immunity to reinfection Mice that developed nonhealing L. major Sd lesions at 14 wk were rechallenged in the contralateral ear with the Sd strain to determine whether protective immunity against reinfection had been gener- ated during the primary infection. Despite the inability to heal the primary lesion, the rechallenge ears did not develop measurable pathology, and 8 wk after rechallenge they had a 10-fold reduction in parasite burden compared with naive-infected controls (Fig. 9). FIGURE 8. ␣ Anti-IL-10 R- and anti-CD25-treated mice have increased Coinfection with the Sd and V1 strains using 1000 metacyclics in expression of IFN-␥ and iNOS-2 mRNA. At 12 wk postinfection, mice each ear did not compromise the ability of the mice to control and were given two i.p. injections of Ab 3 days apart. Three days after the second treatment, total RNA was isolated from ears, reverse transcribed, resolve the V1 lesions, again despite development of nonhealing and analyzed by real-time PCR. The target genes were normalized to the Sd lesions in the contralateral ear (data not shown). Downloaded from endogenous control, and the values shown are the fold increase relative to expression in naive mice. Each bar is the geometric mean and SD of four Discussion p Ͻ 0.05 compared with control mice). Using low dose intradermal inoculation in conventionally resistant ,ء) individual ears C57BL/6 mice, we have characterized the host immune response to a strain of L. major that produces a nonhealing phenotype. This

burden in the healing V1 lesions, as previously reported. In neither occurs despite the development of a strong and polarized Th1 re- http://www.jimmunol.org/ infection did the treatment result in sterile cure, though it is pos- sponse, as measured by IFN-␥ production in the DLN as well as in sible that more prolonged treatment, or treatment during more la- the site of the infection, and the correspondingly low levels of IL-4 tent stage of infection with V1, might have produced the complete and IL-13 produced in these sites. In addition, the nonhealing phe- clearance of V1 achieved in the prior studies. The more effective notype developed despite normal recruitment of CD4ϩ and CD8ϩ clearance of parasites following anti-IL-10R treatment was asso- lymphocytes, and despite high level induction of iNOS in the le- ␥ ciated in both groups with increased IFN- and iNOS expression sion, conditions that are collectively associated with effective con- in the lesion, as measured by real-time PCR. However, it should be trol of L. major infection in the skin (2). The possibility that the Sd noted that the increased iNOS expression in the L. major Sd le- strain is intrinsically more resistant to IFN-␥-mediated killing was sions was slight, and perhaps more importantly, the levels in the addressed by determining the concentrations of IFN-␥ required to by guest on September 29, 2021 untreated Sd-infected mice were already high (Fig. 8). Sd-infected ϩ activate peritoneal macrophages for leishmanicidal capacity in mice were also depleted of CD25 cells during the chronic stage vitro, and no significant difference compared with the V1 strain of the infection by treating with anti-CD25 Ab over a course of 2 was observed. The non-cure phenotype seen in this model is wk, and analyzing the parasite load and immune response 1 wk clearly divergent from the BALB/c model of susceptibility, in which the infection is marked by a strong Th2 bias. The disease- promoting cytokine that could be identified in this model is not IL-4, but IL-10, as indicated by the enhanced host resistance observed in Sd-infected IL-10Ϫ/Ϫ B10 mice, or in B6 mice treated with anti-IL-10R Ab. CD4ϩCD25ϩ regulatory T cells were also shown to play a role, as the in vivo depletion of CD25ϩ cells promoted greater clearance of parasites from the skin. It is important to consider that the L. major BALB/c model has in general failed to adequately explain severe forms of clinical disease, which seem not to be associated with a Th1 response defect per se, but with concomitant expression of IL-10 (10, 11, 13–21). Although IL-10 and CD4ϩCD25ϩ T cells are induced by L. major Sd and are required for the evolution of the nonhealing lesions, these responses also accompany the dermal infections with V1 that produce a healing phenotype in B6 mice. In this setting, IL-10 produced by naturally occurring regulatory T cells has been shown to prevent sterile cure, and the regulatory cells seem to function in equilibrium with Leishmania-specific effectors to establish and maintain latent infection in the skin (24). Might the inability to heal the primary Sd lesion be the result of an imbalance FIGURE 9. Sd-infected mice develop protective immunity to a rechal- in suppressor cells or cytokines in the inflammatory site? While lenge. Fourteen weeks following primary infection, mice were rechallenged in the opposite ear with 103 metacyclics, along with naive animals IL-10 production and both IL-10 and Foxp3 mRNA were main- as a comparison. A, Ear lesion development in rechallenged and naive tained at elevated levels in the nonhealing compared with the heal- mice. The mean and SD of five mice per group are shown. B, Parasite ing L. major lesions, at the critical earlier stage of infection, around burdens in individual ears in the primary site and secondary rechallenge wk 6, when the V1 parasites began to be effectively controlled, the site 8 wk after rechallenge. inability of an equally potent Th1 response to clear the Sd parasites 2940 A NONHEALING L. major INFECTION IN C57BL/6 MICE was not associated with increased IL-10 production or an increased animals were rechallenged in the contralateral ear, they were sub- frequency of CD4ϩCD25ϩ T cells in the site. In fact, the most stantially protected. Thus the IL-10, Treg, and possibly other sup- striking difference between the immune response induced by these pressive mechanisms that function within the primary inflamma- two strains is the high level IFN-␥ production by cells from the tory site to prevent effective clearance by Th1 cells do not inhibit DLN or ear lesions in the Sd-infected mice as early as 3 wk post- the expression of Th1 effector activity in a naive re-challenge site. challenge, at a time when the Th1 response had yet to develop in We have argued that the activation of Treg during infection pro- the mice infected with V1. How this paradoxical finding relates to vides a benefit to the host by not only controlling the severity of the evolution of the non-cure phenotype is not known. It is of inflammation, but by promoting parasite persistence the Treg will course possible that additional suppressor factors other than IL-10 indirectly maintain a memory pool necessary for resistance to re- might accompany the early, elevated IFN-␥ response in the Sd infection (24). Since Treg may be preferentially recruited to or mice to shift the balance in favor of parasite survival. One indi- expanded by signals that accumulate within inflamed tissue, they cation that the L. major Sd strain induces factors in addition to may not be able to home to naive rechallenge sites in sufficient IL-10 that promote infection is the observation that a low level time or numbers to suppress the expression of concomitant infection was maintained in the IL-10Ϫ/Ϫ mice, whereas complete immunity. cure was achieved in the IL-10Ϫ/Ϫ mice infected with V1, con- The availability of a non-cure phenotype of L. major infection firming our prior studies (4). associated not with aberrant Th2 development but with ongoing Although IL-10 production may not be a sufficient condition for Th1 responses rendered ineffective by an imbalance in homeostatic the evolution the non-cure phenotype, it nonetheless contributes in suppressive cells or cytokines may better reflect the mechanisms an essential way to this outcome, as indicated by the more effective controlling non-curing forms of cutaneous visceral leishmaniasis Downloaded from clearance of the Sd strain in the IL-10-deficient and anti-IL-10R- in humans. treated mice. The source of the IL-10 induced by Sd infection is not clear from these studies. Depletion of the CD25ϩ cells did not Disclosures appreciably reduce the amount of IL-10 produced in the site (data The authors have no financial conflict of interest. not shown). Furthermore, the IL-10R blockade consistently pro- ϩ moted greater parasite clearance than did the CD25 cell deple- References http://www.jimmunol.org/ tion, suggesting alternative sources of IL-10. It has been shown 1. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leish- that amastigote-infected macrophages produce IL-10 (28, 32), as mania major. Annu. Rev. Immunol. 13:151. ␥ 2. Sacks, D., and N. Noben-Trauth. 2002. The immunology of susceptibility and do macrophages following Fc R ligation (22), which in this con- resistance to Leishmania major in mice. Nat. Rev. Immunol. 2:845. text might involve anti-Leishmania Abs and released Ags. Kera- 3. Belkaid, Y., S. Mendez, R. Lira, N. Kadambi, G. Milon, and D. Sacks. 2000. A tinocytes are also known to produce IL-10 (13, 33), and their abil- natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and ity to do so in visceral leishmaniasis patients is associated with immunity. J. Immunol. 165:969. reactivation disease (postkala azar dermal leishmaniasis) (13). It is 4. Belkaid, Y., K. F. Hoffmann, S. Mendez, S. Kamhawi, M. C. Udey, T. A. Wynn, interesting that in contrast to the IFN-␥ response, the IL-10 pro- and D. L. Sacks. 2001. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti- by guest on September 29, 2021 duced by the lesional cells was consistently at high levels in the IL-10 receptor antibody for sterile cure. J. Exp. Med. 194:1497. absence of Ag restimulation, suggesting that non-Ag-specific 5. Belkaid, Y., E. Von Stebut, S. Mendez, R. Lira, E. Caler, S. Bertholet, M. C. Udey, and D. Sacks. 2002. CD8ϩ T cells are required for primary immu- sources of IL-10 are present in the inflammatory site. It is possible nity in C57BL/6 mice following low-dose, intradermal challenge with Leishma- that differences in the frequency or activation state of these IL-10- nia major. J. Immunol. 168:3992. producing cells, obscured by the quantitation of total IL-10, rep- 6. Belosevic, M., D. S. Finbloom, P. H. Van Der Meide, M. V. Slayter, and C. A. Nacy. 1989. Administration of monoclonal anti-IFN-␥ antibodies in vivo resent the critical difference between the V1 and Sd inflammatory abrogates natural resistance of C3H/HeN mice to infection with Leishmania ma- sites. Autocrine IL-10 produced by infected macrophages is apt, jor. J. Immunol. 143:266. 7. Wang, Z. E., S. L. Reiner, S. Zheng, D. K. Dalton, and R. M. Locksley. 1994. for example, to be far more effective at deactivating infected cells ϩ CD4 effector cells default to the Th2 pathway in interferon-␥-deficient mice than IL-10 produced by Treg. And although the quantitation of infected with Leishmania major. J. Exp. Med. 179:1367. total iNOS in the lesion indicates a highly activated tissue envi- 8. Swihart, K., U. Fruth, N. Messmer, K. Hug, R. Behin, S. Huang, G. Del Giudice, M. Aguet, and J. A. Louis. 1995. Mice from a genetically resistant background ronment, the deactivated tissue might be confined to the infected lacking the interferon-␥ receptor are susceptible to infection with Leishmania macrophages or other parasitized cells. major but mount a polarized T helper cell 1-type CD4ϩ T cell response. J. Exp. The nonhealing infections produced by L. major Sd in B6 mice Med. 181:961. 9. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, mimics in some respects the pattern of susceptibility displayed by M. K. Gately, J. A. Louis, and G. Alber. 1996. Genetically resistant mice lacking “resistant” mice infected with new world cutaneous Leishmania interleukin-12 are susceptible to infection with Leishmania major and mount a amazonensis strains and their related subspecies Leishmania mexi- polarized Th2 cell response. Eur. J. Immunol. 26:1553. 10. Ghalib, H. W., M. R. Piuvezam, Y. A. Skeiky, M. Siddig, F. A. Hashim, cana (34, 35). In fact, the L. amazonensis studies in resistant mice A. M. el-Hassan, D. M. Russo, and S. G. Reed. 1993. Interleukin 10 production (C3H and C57BL/6) provided an early challenge to the role of IL-4 correlates with pathology in human Leishmania donovani infections. J. Clin. Invest. 92:324. and Th2 polarization as a necessary condition for the evolution of 11. Karp, C. L., S. H. el-Safi, T. A. Wynn, M. M. Satti, A. M. Kordofani, nonhealing disseminating forms of leishmaniasis, as L. amazonen- F. A. Hashim, M. Hag-Ali, F. A. Neva, T. B. Nutman, and D. L. Sacks. 1993. In sis-infected IL-4 knockout mice and anti-IL-4-treated mice still vivo cytokine profiles in patients with kala-azar: marked elevation of both interleukin-10 and interferon-␥. J. Clin. Invest. 91:1644. developed nonhealing lesions (35). In contrast, results from two 12. Kenney, R. T., D. L. Sacks, A. A. Gam, H. W. Murray, and S. Sundar. 1998. studies involving IL-10 knockout mice indicated a role for IL-10 in Splenic cytokine responses in Indian kala-azar before and after treatment. J. In- promoting parasite growth, since 1–2 log reductions were observed fect. Dis. 177:815. 13. Gasim, S., A. M. Elhassan, E. A. Khalil, A. Ismail, A. M. Kadaru, A. Kharazmi, in the deficient mice (36, 37). A critical difference between these and T. G. Theander. 1998. High levels of plasma IL-10 and expression of IL-10 non-cure phenotypes and the non-cure Sd infections reported here by keratinocytes during visceral leishmaniasis predict subsequent development of ␥ post-kala-azar dermal leishmaniasis. [Published erratum appears in 1998 Clin. is that whereas the new world strains induced only low level IFN- Exp. Immunol. 112:547.] Clin. Exp. Immunol. 111:64. responses throughout infection, Sd elicits a strong sustained Th1 14. Ribeiro-de-Jesus, A., R. P. Almeida, H. Lessa, O. Bacellar, and E. M. Carvalho. response. 1998. Cytokine profile and pathology in human leishmaniasis. Braz. J. Med. Biol. Res. 31:143. We observed in our studies of L. major Sd infection in B6 mice 15. Holaday, B. J., M. M. Pompeu, S. Jeronimo, M. J. Texeira, A. Sousa Ade, that despite their inability to heal their primary lesion, when the A. W. Vasconcelos, R. D. Pearson, J. S. Abrams, and R. M. Locksley. 1993. The Journal of Immunology 2941

Potential role for interleukin-10 in the immunosuppression associated with kala 26. Spath, G. F., and S. M. Beverley. 2001. A lipophosphoglycan-independent azar. J. Clin. Invest. 92:2626. method for isolation of infective Leishmania metacyclic promastigotes by density 16. Ismail, A., A. M. El Hassan, K. Kemp, S. Gasim, A. E. Kadaru, T. Moller, gradient centrifugation. Exp. Parasitol. 99:97. A. Kharazmi, and T. G. Theander. 1999. Immunopathology of post kala-azar 27. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile. J. Pathol. G. Schuler. 1999. An advanced culture method for generating large quantities of 189:615. highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 17. Gasim, S., A. M. Elhassan, A. Kharazmi, E. A. Khalil, A. Ismail, and 223:77. T. G. Theander. 2000. The development of post-kala-azar dermal leishmaniasis 28. Carrera, L., R. T. Gazzinelli, R. Badolato, S. Hieny, W. Muller, R. Kuhn, and (PKDL) is associated with acquisition of Leishmania reactivity by peripheral D. L. Sacks. 1996. Leishmania promastigotes selectively inhibit interleukin 12 blood mononuclear cells (PBMC). Clin. Exp. Immunol. 119:523. induction in bone marrow-derived macrophages from susceptible and resistant 18. Ajdary, S., M. H. Alimohammadian, M. B. Eslami, K. Kemp, and A. Kharazmi. mice. J. Exp. Med. 183:515. 2000. Comparison of the immune profile of nonhealing cutaneous Leishmaniasis 29. Mendez, S., S. K. Reckling, C. A. Piccirillo, D. Sacks, and Y. Belkaid. 2004. Role patients with those with active lesions and those who have recovered from in- for CD4ϩCD25ϩ regulatory T cells in reactivation of persistent leishmaniasis and fection. Infect. Immun. 68:1760. control of concomitant immunity. J. Exp. Med. 200:201. 19. Louzir, H., P. C. Melby, A. Ben Salah, H. Marrakchi, K. Aoun, R. Ben Ismail, 30. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the and K. Dellagi. 1998. Immunologic determinants of disease evolution in localized development and function of CD4ϩCD25ϩ regulatory T cells. Nat. Immunol. cutaneous leishmaniasis due to Leishmania major. J. Infect. Dis. 177:1687. 4:330. 20. Akuffo, H., K. Maasho, M. Blostedt, B. Hojeberg, S. Britton, and M. Bakhiet. 31. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell de- 1997. Leishmania aethiopica derived from diffuse leishmaniasis patients prefer- velopment by the transcription factor Foxp3. Science 299:1057. entially induce mRNA for interleukin-10 while those from localized leishmani- 32. Guizani-Tabbane, L., K. Ben-Aissa, M. Belghith, A. Sassi, and K. Dellagi. 2004. asis patients induce interferon-␥. J. Infect. Dis. 175:737. Leishmania major amastigotes induce p50/c-Rel NF-␬B transcription factor in 21. Melby, P. C., F. J. Andrade-Narvaez, B. J. Darnell, G. Valencia-Pacheco, human macrophages: involvement in cytokine synthesis. Infect. Immun. 72:2582. V. V. Tryon, and A. Palomo-Cetina. 1994. Increased expression of proinflam- 33. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. In- matory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect. terleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683. Immun. 62:837. 34. Roberts, M., J. Alexander, and J. Blackwell. 1990. Genetic analysis of Leishma- 22. Kane, M. M., and D. M. Mosser. 2001. The role of IL-10 in promoting disease nia mexicana infection in mice single gene Scl-2 controlled predisposition to Downloaded from progression in leishmaniasis. J. Immunol. 166:1141. cutaneous lesion development. J. Immunogenet. 17:89. 23. Noben-Trauth, N., R. Lira, H. Nagase, W. E. Paul, and D. L. Sacks. 2003. The 35. Afonso, L., and P. Scott. 1993. Immune responses associated with susceptibility relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to of C57BL/10 mice to Leishmania amazonensis. Infect. Immun. 61:2952. Leishmania major. J. Immunol. 170:5152. 36. Padigel, U. M., J. Alexander, and J. P. Farrell. 2003. The role of interleukin-10 24. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. in susceptibility of BALB/c mice to infection with Leishmania mexicana and CD4ϩCD25ϩ regulatory T cells control Leishmania major persistence and im- Leishmania amazonensis. J. Immunol. 171:3705. munity. Nature 420:502. 37. Jones, D. E., M. R. Ackermann, U. Wille, C. A. Hunter, and P. Scott. 2002. Early

25. Neva, F. A., D. Wyler, and T. Nash. 1979. Cutaneous leishmaniasis—a case with enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 in- http://www.jimmunol.org/ persistent organisms after treatment in presence of normal immune response. terleukin-10-deﬁcient mice does not lead to resolution of infection. Infect. Im- Am. J. Trop. Med. Hyg. 28:467. mun. 70:2151. by guest on September 29, 2021