Immunological Determinants of Clinical Outcome in Peruvian Patients with Tegumentary Leishmaniasis Treated with Pentavalent Antimonials

The mechanisms linking the immune response to cutaneous andmucosal leishmaniasis (CL and ML, respectively) lesions andthe response to treatment are incompletely understood. Our aimswere to prospectively assess, by quantitative reverse transcription-PCR,the levels of mRNA for gamma interferon, tumor necrosis factoralpha, interleukin-10 (IL-10), IL-4, and IL-13, as well as thepresence of T cells (CD2) and macrophages (CD68), in CL andML lesions and to follow their changes in response to treatmentwith pentavalent antimonials. The leishmanin skin test (LST)was performed on all CL and ML patients before treatment. Thepatient population included individuals living in areas of Peruwhere the disease is endemic, i.e., 129 with CL and 43 withML. Compared to CL patients, the LST induration size was larger,the levels of all cytokine mRNAs but IL-10 were higher, T-cellmRNA was similar, and macrophage mRNA was lower in ML patients.The proportion of CL patients with an LST induration size of>8 mm was higher among responders to treatment. In CL, thepretreatment levels of cytokine mRNAs did not discriminate betweenresponders and nonresponders; however, treatment was more oftenaccompanied by a reduction in the levels of T-cell and cytokinemRNAs in responders than in nonresponders. Furthermore, theproduction of cytokines per T cell and macrophage decreasedwith treatment but IL-10 production remained high in nonresponders.Overall, these findings point to complex relationships amongNew World Leishmania parasites, skin and mucosal immune responses,and treatment outcome. The persistence of high levels of IL-10in CL is characteristically associated with a poor responseto treatment.

INTRODUCTION

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INTRODUCTION

Members of the genus Leishmania are protozoan intracellularparasites that cause a large spectrum of diseases in humans,including cutaneous leishmaniasis (CL), mucosal leishmaniasis(ML), visceral leishmaniasis (VL), and mixed forms. The clinicalpresentation depends on the infecting species and the host'simmune response (23). American CL usually presents with oneor more ulcerated skin lesions that may persist for months oryears and eventually self-cure, leaving atrophic scars. In CL,treatment is indicated to accelerate the cure rate, reduce thesize of scars, prevent relapses, and prevent dissemination tomucosae (42). In contrast, ML is a debilitating disease thatdoes not self-cure and can be life threatening if not treated(15, 16, 27). Inadequate clinical response of CL and VL patientsto pentavalent antimonials (SbV) is an emerging problem (16,27, 37, 43). For instance, in Peru, where several Leishmaniaspecies coexist (Leishmania braziliensis, L. guyanensis, L.peruviana, and L. lainsoni), a 21% clinical failure rate ofSbV treatment has been reported (4). While the role of parasiteresistance to SbV therapy remains to be defined, several otherdeterminants, such as clinical characteristics, parasite species,parasite virulence factors, and the immunological response ofthe host, may influence the clinical outcome (4, 13, 18, 39,44).

Cellular immunity is thought to be essential for controllingintracellular pathogens. In the paradigmatic mouse model ofleishmaniasis caused by L. major, resistance and susceptibilityto infection are determined by functionally distinct T-cellsubsets (17, 36). A predominant T helper 1 (Th1) response inducedby interleukin-12 (IL-12) with high secretion of gamma interferon(IFN- ${gamma}$ ) and tumor necrosis factor alpha (TNF- ${alpha}$ ) leads to the activationof infected macrophages, an increase in inducible nitric oxidesynthase, and subsequent control of the infection, as observedin C57BL/6 mice. In contrast, a Th2 response mediated by IL-4and IL-13 increases susceptibility to infection and permitsdisease progression, as observed in BALB/c mice. In addition,IL-10 is an important mediator shared by multiple T-cell subsets,including T cells with regulatory functions (T_reg cells), witha role in L. major reactivation (25). Distinct differences,however, have been documented in the relative contributionsof the inflammatory mediators to parasite clearance and thedevelopment of chronic lesions when Old and New World Leishmaniaspecies have been tested in animal models. Paradoxically, IL-4and IL-13 do not participate in exacerbating disease expressionin L. donovani mouse infection and L. amazonensis amastigotesshowed enhanced replication in IFN- ${gamma}$ -stimulated murine macrophages(2, 35, 40). Furthermore, L. braziliensis causes small, nonulcerative,and self-healing skin swelling in both C57BL/6 and BALB/c miceand results in skin ulcers only in IL-12^–/– or STAT4^–/–mice (38).

The precise role of Th1 and Th2 polarized responses in the healingand persistence of human leishmaniasis remains unclear (23).When semiquantitative PCR detection methods have been used,type I cytokine and TNF- ${alpha}$ mRNAs were detected not only in localizedand recent but also in diffuse and chronic CL lesions. In contrast,type 2 cytokine mRNAs were preferentially identified in thelatter (9, 20, 24, 34). Further, a correlation was found betweenhigh intralesional levels of IL-10 and a poor response to treatmentin L. guyanensis infection (7). The mucosal forms are characterizedby an exacerbated cell-mediated immune response and high intralesionallevels of TNF- ${alpha}$ , IFN- ${gamma}$ , and IL-4, leading to extensive tissuedestruction but not to parasite clearance (3, 5, 34, 47). Adefect in IL-10 receptor expression was shown to correlate withhigh inflammatory responses in ML (14). Finally, T_reg cellsand high IL-10 production potential have been described in theskin of individuals infected with L. braziliensis (10).

Within the framework of Leishnatdrug-R, a multicenter studyon SbV treatment failure in leishmaniasis, we prospectivelysearched for immunological determinants of treatment outcomeamong Peruvian patients. We asked whether the leishmanin skintest (LST) and the pattern of cytokines present in the skinand mucosal lesions could predict treatment outcome. We usedreal-time quantitative PCR (Q-PCR)-based methods to detect thelevels of prototypic type 1 and 2 cytokine mRNAs, as well asof CD2 and CD68 mRNAs as surrogates for the presence of T cellsand macrophages, respectively, before and 10 days after treatmentinitiation.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Patients. Patients were recruited at the Institute of Tropical MedicineAlexander von Humboldt (ITMAvH) in Lima, Peru. The diagnosisof CL or ML was confirmed by direct microscopic observationof parasites on stained smears, by positive culture, or by positivePCR-based detection. The Leishnatdrug-R study protocol was approvedby the ethical committees of ITMAvH and the Institute of TropicalMedicine, Antwerp, Belgium. An addendum to the Leishnatdrug-Rprotocol for immunological investigations was approved by theITMAvH ethical committee. Written informed consent was obtainedfrom all individuals. The evolution of lesions was clinicallyassessed at the end of treatment and during subsequent follow-upvisits for 12 months as described previously (4). Only skinor mucosal biopsy samples from patients with a clear clinicaloutcome upon the completion of a course of SbV were eligiblefor quantitative reverse transcription-PCR analysis. First-linetreatment was based on 20 mg/kg/day of SbV (sodium stibogluconateor meglumine antimoniate) given intravenously for 20 days inCL and 30 days in ML. Skin biopsy samples were also taken from10 healthy individuals (healthy control group) attending a plasticsurgery clinic in Geneva, Switzerland.

LST. Leishmania antigens were prepared at ITMAvH as described previously(26). Forty-eight hours after intradermal injection of the antigen(0.1 ml), induration size was measured. The test was consideredpositive when the diameter of the induration was $≥$ 5 mm.

RNA preparation from skin and mucosal biopsy samples. Biopsies were performed by punch (4 mm) at the margins of skinor mucosal lesions at day 0 (before treatment) and at day 10of treatment. The samples were stabilized in 1 ml of RNAlaterreagent (Qiagen) and stored at –80°C until processing.Total RNA was isolated with TRIzol reagent (Life Technologies)according to the manufacturer's protocol and suspended in 30µl RNase-free water. All samples were DNase treated withthe DNA-free kit (Ambion) to remove possible contaminating genomicDNA. The quality of the resulting RNA was determined with theRNA 6000 Nano Labchip kit on a Bioanalyzer 2100 (version A02.12SI 292; Agilent Technologies).

Real-time Q-PCR. One microgram of total RNA was reverse transcribed with SuperScriptII (Invitrogen) and random primers. The cDNA obtained was diluted1:24 and subsequently amplified with TaqMan assay reagents (UniversalPCR Master Mix Buffer) in a final volume of 10 µl withprimer sets for the human IFN- ${gamma}$ , IL-4, IL-10, IL-13, TNF- ${alpha}$ , CD2,CD68, glyceraldehyde-3-phosphate dehydrogenase, and 18S rRNAfrom Applied Biosystems (Foster City, CA) and human TATA bindingprotein (TBP), glucuronidase B (GusB), and 5-aminolevulinicacid synthase 1 (ALAS-1) from the Genomic Platform (NCCR Frontiersin Genetics, University of Geneva). Reactions were run on theABI PRISM 7900HT sequence detection system (Applied Biosystems)with the following thermal profile: initial denaturation at95°C for 10 min, followed by 45 cycles of denaturation at95°C for 15 s and annealing at 60°C for 1 min. Negativecontrols for cDNA synthesis (i.e., without reverse transcriptase)were included for each gene. Q-PCR efficiencies for all geneswere higher than 90%, as assessed by standard curves based oncDNA serial dilutions. All reactions were done in triplicate,and the arithmetic average cycle threshold (C_T) was used fordata analysis.

Data analysis and statistics. The interassay variability of the internal standards was 0.7%for IFN- ${gamma}$ , 0.8% for IL-4, 0.9% for IL-10, 0.5% for IL-13, 1.1%for TNF- ${alpha}$ , 1.4% for CD2, 2.0% for CD68, 1.7% for TBP, 0.9% forGusB, and 0.5% for ALAS-1. The (nonnormalized) relative quantitieswere calculated with the formula E^–C^T, with E equalingthe Q-PCR efficiencies determined and ${Delta}$ C_T corresponding to theC_T of the standard sample minus the C_T of the test sample. Thestandard sample was prepared by mixing equal amounts of cDNAoriginating from 15 different skin biopsy samples. These raw(nonnormalized) relative quantities for all genes were processedwith the geNorm VBA applet for MS Excel to determine the threemost stably expressed genes (those for TBP, GusB, and ALAS-1),which were subsequently used to determine the normalizationfactor for each sample as described by Vandesompele et al. (46).

The normalized relative quantity (RQ) or expression level wasassessed for each cytokine, CD2, and CD68 in biopsy samplestaken at days 0 and 10 of treatment. For each skin sample, theratios of the RQs of the various cytokines and the RQ of CD2or CD68 were computed to estimate the production of cytokineper T cell or macrophage, respectively.

Statistical analysis was performed with the SPSS software, version11.0, and GraphPad Prism, version 4. Categorical variables werecompared by using cross-tabulations and chi-square tests, whereasnumerical variables (means) were compared by using analysisof variance or the Student t test at a critical ${alpha}$ level of 0.05.All P values were two sided.

RESULTS

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RESULTS

Clinical outcome and patient group classification. Two hundred twenty-one patients with CL, 155 males and 66 females,aged 3 months to 85 (mean ± standard deviation, 29 ±19) years, were included in the Leishnatdrug-R study betweenApril 2003 and December 2004. Of the 221 enrolled patients withCL, 129 (58%) had a biopsy sample of a skin lesion taken beforetreatment. No skin biopsy was performed on 92 (42%) patientsfor various reasons (e.g., patient's refusal, lesion on theface). Exclusion criteria for performing mRNA studies on skinbiopsy samples were incomplete treatment or treatment with drugsother than SbV (n = 9), unclear clinical outcome (n = 34), andnonavailability of biopsy material (n = 22; 7 before treatment,15 at day 10 of treatment). CL patients were subdivided intothree groups. Group A included patients definitely cured (n= 62), group B included patients with treatment failure (n =11), and group C included patients with previous failure torespond to antimonial therapy (n = 8). In group A, 37 randomsamples were tested at day 0, of which 21 were tested at day10 of treatment. In group B, all 11 samples were tested at day0 and 7 were tested at day 10 (no biopsy material was availableat day 10 for 4 of them).

Forty-three patients with ML, 40 males and 3 females aged 9to 72 (mean ± standard deviation, 39 ± 15) years,were enrolled in the study. Twenty-two (51%) had a biopsy ofa mucosal lesion done at baseline. After the exclusion of 11patients with incomplete or non-SbV-based treatment (n = 7)or with an unclear clinical outcome (n = 4), patients subsequentlycured formed group D (n = 8) and patients with subsequent treatmentfailure formed group E (n = 3). All but one had a previous historyof untreated CL. We tested biopsy samples taken at day 10 oftreatment from 10 patients, 7 in group D and 3 in group E. Skinbiopsy samples from healthy individuals undergoing abdominoplasticsurgery for esthetic reasons and living in Geneva (Switzerland)were included as normal controls (group H, n = 10).

LST. The LST was performed before treatment on 188 patients withCL (85%). After the exclusion of patients with a prior historyof leishmaniasis, with treatment differing from SbV, or withan unclear clinical outcome, 163 patients were included in theanalysis. The proportions of positive LST results were 89.5and 96.7% (P = 0.31) of 133 patients definitely cured and 30patients with treatment failure, respectively. The proportionof patients with an LST induration size of >8 mm was significantlyhigher (P = 0.008) in patients definitely cured (66.2%) thanin patients with treatment failure (40%). The LST was performedon 36 patients with ML (84%). A positive reaction was foundin 34 (94.4%) of the 36 patients. In patients with a positiveLST result, the induration size was significantly larger inML patients (median, 13 mm; range, 5 to 35 mm) than in CL patients(median, 9 mm; range, 5 to 30 mm; P < 0.001). Thus, in CLpatients, LST positivity does not predict the response to treatmentbut induration size may do so.

Detection and quantification of intralesional cytokine mRNA before treatment. To identify immunological parameters capable of predicting responseto treatment, we prospectively compared the relative mRNA levelsof inflammatory cytokines in the skin and mucosal lesions acrossdifferent groups of leishmaniasis patients before treatmentand in healthy controls. In CL patients at baseline, the skinlesion levels of IFN- ${gamma}$ , IL-4, IL-10, IL-13, and TNF- ${alpha}$ mRNAs weresimilar in groups A and B. However, TNF- ${alpha}$ (difference of normalizedRQ = 0.301; 95% confidence interval [CI], 0.069 to 0.533 [P= 0.02]) and IL-13 (difference of normalized RQ = 1.7; 95% CI,0.856 to 2.544 [P < 0.001]) mRNA levels were significantlyhigher in patients with previous treatment failure (group C)than in group A (Fig. 1). All CL lesional tissues were positivefor these cytokines. In contrast, cytokines were inconstantlydetected in healthy skin, of which 40% of the samples were positivefor IFN- ${gamma}$ , 10% were positive for IL-4, 80% were positive forIL-10, 80% were positive for IL-13, and 90% were positive forTNF- ${alpha}$ . When detected, IFN- ${gamma}$ , IL-4, IL-10, and TNF- ${alpha}$ mRNA levelswere significantly lower in healthy skin than in CL lesions(IFN- ${gamma}$ , IL-4, and IL-10, P < 0.001; TNF- ${alpha}$ , P = 0.001) (Fig.1).

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FIG. 1. Relative expression levels of the genes for IFN- ${gamma}$ , TNF- ${alpha}$ , IL-4, IL-13, and IL-10 in patients with tegumentary leishmaniasis lesions before treatment. Box plots represent 25th, 50th, and 75th percentiles. Statistically significant differences determined by analysis of variance were as follows: 1, higher levels of IFN- ${gamma}$ , TNF- ${alpha}$ , IL-4, and IL-13 in group D than in group A, all P < 0.001, and lower levels of IL-10, P = 0.03; 2, higher levels of TNF- ${alpha}$ and IL-13 in group C than in group A, P = 0.02 and P < 0.001, respectively; 3, higher levels of TNF- ${alpha}$ in group E than in group D, P = 0.02; 4, higher levels of IFN- ${gamma}$ , IL-4, and IL-10 in group A than in group H, P < 0.001, and higher levels of TNF- ${alpha}$ , P = 0.001. Group A, definitive CL cure; group B, CL treatment failure; group C, previous CL treatment failure; group D, definitive ML cure; group E, ML treatment failure; group H, healthy controls.

Interestingly, pretreatment levels of IFN- ${gamma}$ , IL-4, IL-13, andTNF- ${alpha}$ mRNAs were higher in ML than in CL lesions (P $≤$ 0.001),whereas IL-10 levels were lower (P = 0.01). Among ML patients,those who would fail treatment (group E) had significantly higherlevels of TNF- ${alpha}$ (difference of normalized RQ = 0.57; 95% CI,0.05 to 1.09 [P = 0.03]) than did those subsequently cured (groupD) (Fig. 1).

Overall, these data indicate that there is a large degree ofheterogeneity in the levels of cytokines detected in mucocutaneouslesions of leishmaniasis patients which reflect the tissue siteof the lesion and, at least in part, previous and future responsesto treatment.

Detection and quantification of intralesional cytokine mRNA at day 10 of treatment. We then assessed the impact of 10 days of treatment with SbVon cytokine mRNA levels in skin and mucosal lesions. As expected,the mRNA levels of all cytokines decreased with treatment; however,interesting differences were observed between CL individualssubsequently cured (group A) and those with subsequent treatmentfailure (group B) since all cytokine levels decreased more ingroup A than in group B, a difference that was statisticallysignificant for IFN- ${gamma}$ (P = 0.021) and IL-13 (P = 0.032) (Fig.2). As observed in patients with CL, also in ML patients allcytokine mRNA levels decreased with treatment, but within thelimited number of individuals tested, no differences betweenresponders and nonresponders were found (not shown). The sharperdecline in the IL-13 mRNA levels of CL individuals subsequentlycured, together with higher baseline levels of IL-13 mRNA inCL individuals with previous treatment failures, indicates apossible role for IL-13 in lesion persistence.

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FIG. 2. Changes with treatment in the relative expression levels of IFN- ${gamma}$ , TNF- ${alpha}$ , IL-4, IL-13, and IL-10 in patients with cutaneous leishmaniasis. Samples were collected prospectively and assessed at days 0 and 10 of treatment. Samples are separated according to the response to treatment. Group A, CL patients definitively cured upon treatment; group B, CL patients with treatment failure. *, P < 0.021 for IFN- ${gamma}$ and P < 0.032 for IL-13.

T cells and macrophages in CL and ML patients and healthy controls. To assess the presence and amounts of T cells and macrophagesin the skin, we quantified CD2 and CD68 gene expression levelsas surrogate markers for T cells and macrophages, respectively.Relative expression levels of CD2 at day 0 of treatment weresimilar across all of the groups of leishmaniasis patients,while in healthy skin they were much lower (P < 0.001). Ofinterest, the relative expression levels of CD2 decreased significantly(P = 0.025) after 10 days of treatment only in group A and notin the others (Fig. 3A). In contrast to CD2, the relative expressionlevels of CD68 at day 0 were lower in ML than in CL patients(Fig. 3B); however, the CD68 levels did not significantly changewith treatment, whatever the outcome was (Fig. 3C).

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FIG. 3. Relative T-cell CD2 (A, days 0 and 10) and macrophage CD68 (B, day 0; C, days 0 and 10) gene expression in individuals with tegumentary leishmaniasis and their changes with treatment (A and C). Box plots represent 25th, 50th, and 75th percentiles. P values were calculated with paired (A and C) and unpaired (B) t tests. Groups are defined in the legend to Fig. 1.

Relationship between T-cell and macrophage numbers and cytokines in lesional tissues. We then addressed the question of whether cytokine levels inthe skin samples were dependent on T-cell and/or macrophagenumbers, making the simplified assumption that only CD2- and/orCD68-positive cells contribute to the production of the cytokineswe studied. This was the case for IFN- ${gamma}$ (r² = 0.617) TNF- ${alpha}$ (r²= 0.793), and IL-10 (r² = 0.677), which correlated significantlywith relative CD2 quantities, and for TNF- ${alpha}$ (r² = 0.650) andIL-10 (r² = 0.678), which correlated with relative CD68 quantities.The correlation was poor, however, for IL-4 and IL-13. Moreinterestingly, by assessing the amount of cytokines producedper cell, we observed that the production of IL-10 per T celldecreased significantly in CL responders but not in CL nonresponders(Fig. 4). This was in sharp contrast to IFN- ${gamma}$ and TNF- ${alpha}$ , whichdecreased or tended to decrease in both groups of CL patients(Fig. 4). Similarly, the production of IL-10 per macrophagedecreased significantly with treatment only in CL responders,while it did not in CL nonresponders (Fig. 5). Overall, thesedata indicate that in CL, the failure to respond to treatmentis preceded by a failure to reduce the number of T cells, whichfurthermore continue to produce high levels of IL-10.

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FIG. 4. Changes in cytokine production per T cell during treatment from day 0 to day 10. Cytokine production per T cell was assessed as the ratio of the relative gene expression of a cytokine versus the relative gene expression of CD2, a marker for T cells, only for the cytokines IL-10, IFN- ${gamma}$ , and TNF- ${alpha}$ , which show a correlation with CD2 expression of >0.5. Group A, CL patients definitively cured by treatment; group B, CL patients with treatment failure.

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FIG. 5. Changes in cytokine production per macrophage from day 0 to day 10 of treatment. Cytokine production per macrophage was assessed as the ratio of the relative gene expression of a cytokine versus the relative gene expression of CD68, a marker for macrophages, only for the cytokines IL-10 and TNF- ${alpha}$ , which show a correlation with CD68 expression of >0.5. Group A, CL patients definitively cured by treatment; group B, CL patients with treatment failure.

DISCUSSION

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DISCUSSION

The objective of the present work was to assess whether thepattern of cytokines detected in the skin and mucosal lesionsand the LST could predict clinical responses to antimonial treatmentamong Peruvian patients with tegumentary leishmaniasis. Altogether,we assessed whether CL and ML are characterized by differentpatterns of cytokine expression. While no single assessed parametercould definitively predict the response to treatment, we foundthat the proportion of CL individuals with an LST indurationsize of >8 mm was higher among patients subsequently curedby treatment. Furthermore, the levels of the cytokines studieddecreased or tended to decrease more in CL patients who wouldrespond to treatment than in those who would not. In this respect,the persistence in the lesions of T cells producing high levelsof IL-10 was distinctively characteristic of CL patients nonresponsiveto treatment. Additional findings included higher levels ofTNF- ${alpha}$ and IL-13 mRNAs in CL patients with previous treatmentfailure and higher levels of IFN- ${gamma}$ , TNF- ${alpha}$ , IL-13, and IL-4 mRNAsand lower levels of macrophages (CD68) and IL-10 mRNA in MLpatients than in CL patients.

We found a high rate of LST positivity in both responder andnonresponder patients, and in contrast to a previous study conductedin Brazil (32), a negative LST was not associated with an increasedrisk of treatment failure. Thus, despite the significantly higherproportion of strong reactions (induration diameter of >8mm) found in CL patients subsequently cured, the LST is notsufficiently discriminative to reliably predict treatment outcome.

To the best of our knowledge, this is the first study in whichQ-PCR was used to prospectively assess the levels of inflammatorycytokines and T-cell and macrophage numbers in New World Leishmaniainfection in relation to the response to treatment. The assumptionwe made that CD2 and CD68 mRNA levels directly reflect the numberof T cells and macrophages may be criticized insofar as in vivomodulation of these markers has not yet been investigated. However,in an in vitro assessment, we observed an excellent correlationbetween CD2 and CD68 mRNA levels and the numbers of purifiedT cells and macrophages, respectively (not shown). Previouswork in French Guyana, taking advantage of semiquantitativecytokine determination, pointed to high intralesional IL-10mRNA expression in CL patients as a predictor of unresponsivenessto treatment (7). Indeed, IL-10 has been associated with Leishmaniasusceptibility for its capacity to downregulate Th1 responses(7, 28, 29, 31). While we did not find higher levels of IL-10in lesions of CL patients with previous treatment failure (groupC) than in those who would respond to treatment (group A), thepersistence of IL-10 production by T cells and macrophages wascharacteristic of nonresponders among CL patients. In addition,IL-10 levels in diseased skin were found to be much higher thanin healthy skin. IL-10 can be secreted by T lymphocytes, macrophages,and stromal cells (14), and our findings suggest that, in vivo,IL-10 may indeed play a major role in CL pathology. Our dataare in agreement with high levels of IL-10 produced by peripheralblood mononuclear cells in L. braziliensis-infected individualswith active skin lesions (41). Furthermore, it has been reportedthat splenic T cells from individuals with Indian visceral leishmaniasishave a high potential for IL-10 production and appear to beresponsible for the high levels of IL-10 detected in the serumof infected individuals. Of interest, phenotypic characterizationindicated that IL-10-producing T cells belong to a subset distinctfrom that of T_reg cells (30). In contrast, T_reg cells (CD4⁺CD25⁺ FoxP3⁺) with high IL-10 production have been identifiedin the skin of individuals infected with L. amazonensis (10).Our experimental approach was not meant to differentiate amongT-cell subsets in CL patients. Considering the similarity betweenour findings and those of others, it is, however, tempting tospeculate that distinct Old and New World Leishmania speciesmay all specifically induce T cells with high IL-10 production,which would reduce the effectiveness of the immune response,thus favoring parasite survival (33, 45). Accordingly, in experimentalVL, a complete cure with amphotericin B in liposomes involvesdownregulation of IL-10 (6).

Our data show that IL-13 mRNA levels were higher in individualswith previous treatment failure (group C) and decreased significantlyin responders (group A) but not in nonresponders (group B).Others have suggested that IL-13 may participate in inducingIL-12 unresponsiveness in T cells and inhibiting the productionof proinflammatory cytokines by macrophages, which would subsequentlyresult in increased susceptibility to Leishmania parasites (1,8, 22). To somehow attenuate the unique role of IL-13 in resistanceto treatment, we found also that IFN- ${gamma}$ mRNA levels decreasedsignificantly in treatment responders but not in nonresponders.Thus, based on our data, it is difficult to point at a preferentialTh2 response as a unique element causing resistance to treatmentin New World CL. Rather, it would appear that both Th1 and Th2cytokines contribute to pathology. In this respect, it is importantto stress that IFN- ${gamma}$ may favor L. amazonensis amastigote replicationin macrophages (35). Thus, subtle immunological characteristicsmay be operationally responsible for treatment outcome. It should,however, be stressed that the observed number of nonrespondersto treatment was lower than expected, thus leading to a lackof power of our study for this group of individuals. Withinthe Leishnatdrug-R study, in the same cohort of Peruvian patients,other parasite and host determinants were found to be of importancefor treatment outcome. These included the parasite species butnot the in vitro sensitivity of parasites to SbV (4, 19, 39,48). Furthermore, a short stay (<72 months) in the area ofdisease transmission was a risk factor for treatment failure,indicating that protective immunity increases with the timeof residence in areas where the disease is endemic (19). Similarly,treatment failure was strongly associated with a short (<5weeks) disease duration, as previously reported (21).

In our study, the mRNA levels of IFN- ${gamma}$ , TNF- ${alpha}$ , IL-4, and IL-13,but not those of IL-10, were much higher in ML than in CL. Ofinterest, macrophages (CD68) were less abundant in ML than inCL, pointing to a higher T-cell-to-macrophage ratio in ML. Inkeeping with these findings, the LST induration size was significantlylarger in ML than in CL. These data are consistent with thehypothesis that in ML a strong uncontrolled inflammatory immuneresponse eventually leads to intense destruction of host tissues(5, 14). Indeed, mucosal disease has been associated with T-cellhyperresponsiveness (12) and high intralesional levels of TNF- ${alpha}$ and IFN- ${gamma}$ have been reported (3, 11, 14). To extend these findings,we found that TNF- ${alpha}$ mRNA levels were particularly high in MLlesions that would not respond to treatment. The other observationwe made of lower levels of IL-10 mRNA in mucosal compared tocutaneous lesions complements the finding by others of decreasedIL-10 receptor expression (14). In this respect, it is possibleto hypothesize that ML may differ from CL as far as the immuneresponse is concerned because both production of and responsivenessto IL-10 are partially defective in ML, thus unleashing thesynthesis of proinflammatory mediators. Why such intense productionof TNF- ${alpha}$ and IFN- ${gamma}$ results not in parasite control but ratherin tissue destruction remains to be ascertained.

In conclusion, our work highlights the complex links betweenNew World Leishmania infection and immune responses in the skinand mucosae. This is made evident by LST responses that showedbigger induration sizes in ML than in CL, consistent with higherlevels of inflammatory cytokine mRNAs found in ML. However,CL responders to treatment segregated in a subgroup with a strongerLST response. Furthermore, the data indicate that IL-10 maybe particularly abundant in CL lesions and that resistance totreatment may correlate with failure to control IL-10 production.

ACKNOWLEDGMENTS

We are grateful to Patrick Descombes and Mylène Docquier(NCCR Frontiers in Genetics, Genomic Platform, CMU, Geneva,Switzerland) for quantitative reverse transcription-PCR support.We deeply thank Pascal Launois in Lausanne (Switzerland) forhis helpful suggestions at the start of the project and JorisMenten in Antwerp (Belgium) for his strong support with statisticalanalysis.

The Leishnatdrug-R study, including all immunological investigations,was supported by the Fifth Framework Program of the EuropeanCommission Community Research (contract ICA-CT-2001-10076) andby the Federal Office for Education and Sciences, Bern, Switzerland.C.C. was, in part, supported by grant 310000-112180 from theSwiss National Science Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Immunology & Allergy, Geneva University Hospital, 1211 Geneva 14, Switzerland. Phone: 41 22 372 9370. Fax: 41 22 372 9418. E-mail: carlo.chizzolini{at}unige.ch

Published ahead of print on 23 February 2009.

Anne Maurer-Cecchini passed away in March 2006. Her enthusiasmand dedication were the soul and principal force behind thispaper.

Editor: W. A. Petri, Jr.

REFERENCES

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