Received 6 April 2007/Returned for modification 14 May 2007/Accepted 28 July 2007

Transcription

1 INFECTION AND IMMUNITY, Oct. 2007, p Vol. 75, No /07/$ doi: /iai Copyright 2007, American Society for Microbiology. All Rights Reserved. Role of Interleukin-1 in Activating the CD11c high CD45RB Dendritic Cell Subset and Priming Leishmania amazonensis- Specific CD4 T Cells In Vitro and In Vivo Lijun Xin, 1 Yongguo Li, 1,2 and Lynn Soong 1,3 * Department of Microbiology and Immunology 1 and Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, Institute for Human Infections and Immunity, 3 University of Texas Medical Branch Galveston, Texas, and Department of Infectious Diseases, Harbin Medical University, Harbin, Hei Long Jiang Province, China 2 Received 6 April 2007/Returned for modification 14 May 2007/Accepted 28 July 2007 Cutaneous leishmaniasis associated with Leishmania amazonensis infection is characterized by uncontrolled parasite replication and profound immunosuppression; however, the underlying mechanisms remain largely unclear. One possibility is that the L. amazonensis parasite modulates antigen-presenting cells, favoring the generation of pathogenic Th cells that are capable of recruiting leukocytes but insufficient to fully activate their microbicidal activities. To test this possibility, we infected bone marrow-derived dendritic cells (DCs) of C57BL/6 mice with L. amazonensis or Leishmania major promastigotes and assessed the activation of DC subsets and their capacity in priming CD4 T cells in vitro. In comparison to L. major controls, L. amazonensisinfected DCs secreted lower levels of interleukin-1 (IL-1 ) and IL-1, were less potent in activating the IL-12p40-producing CD11c high CD45RB CD83 CD40 DC subset, and preferentially activated CD4 T cells with a IFN- low IL-10 high IL-17 high phenotype. Although the addition of IL-1 at the time of infection markedly enhanced DC activation and T-cell priming, it did not skew the cytokine profile of DCs and pathogenic Th cells, as local injection of IL-1 following L. amazonensis infection accelerated Th cell activation and disease progression. This study suggests that intrinsic defects at the level of DC activation are responsible for the susceptible phenotype in L. amazonensis-infected hosts and that this parasite may have evolved unique mechanisms to interfere with innate and adaptive immunity. Cutaneous leishmaniasis is mostly caused by Leishmania major infection in the Old World and Leishmania mexicana, Leishmania braziliensis, orleishmania amazonensis infection in the New World. In experimental cutaneous leishmaniasis models, the susceptibility of BALB/c mice to L. major infection is attributable to the selective expansion of Th2 cells, whereas resistance seen in other mouse strains, such as C57BL/6 and C3H mice, is associated with predominant Th1 responses (30). Most inbred mouse strains, however, are susceptible to L. amazonensis infection. Accumulating evidence has revealed that a weak Th1/Th2 mixed response rather than a biased Th2 response is responsible for nonhealing lesions in L. amazonensis-infected hosts (1, 15). Decreased expression of the interleukin-12 receptor (IL-12R) 2 chain in CD4 T cells of L. amazonensis-infected C3H mice and IL-4 / C57BL/6 mice suggests that an IL-4-independent mechanism is responsible for reduced IL-12 responsiveness and an impaired Th1 response in L. amazonensis-infected hosts (17). However, intralesional treatment of L. amazonensis antigen-pulsed dendritic cells (DCs) with IL-12 failed to promote healing (36), and in * Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Medical Branch, Medical Research Building 3.142, 301 University Blvd., Galveston, TX Phone: (409) Fax: (409) Supplemental material for this article may be found at Published ahead of print on 6 August vivo antigen stimulation in the presence of exogenous IL-12 also failed to enhance gamma interferon (IFN- ) production in CD4 T cells from L. amazonensis-infected mice (29). Therefore, while deficiency in IL-12 production/responsiveness is a hallmark in L. amazonensis-infected hosts, it may not be the sole contributing factor for susceptibility. Our recent study of an array of cytokines, chemokines, and their receptors in tissues of L. major-and L. amazonensis-infected C57BL/6 mice has indicated that the deficient Th1 response in L. amazonensis-infected mice is largely due to profound impairments in multiple immune responses at an early stage of the infection (16). Of particular relevance to this study is our finding that, while IL-1, IL-1, and IL-1R antagonist are rapidly produced and adequately down-regulated during the course of L. major infection, there is a significant delay and reduced magnitude for the production of all three molecules during L. amazonensis infection (16). IL-1 is an important proinflammatory cytokine in inducing and maintaining innate and adoptive immunity (25). Studies have demonstrated the protective role of IL-1 in mice against infections with intracellular pathogens, including Mycobacterium avium, Toxoplasma gondii, and Listeria monocytogenes (5, 7, 13, 19). Also, IL-1 treatment at the time of T-cell differentiation can inhibit disease progression in L. major-susceptible BALB/c mice (40). In this study, we investigated the activation and maturation of different DC subsets after exposure to L. amazonensis and L. major parasites, the impact of these DC subsets on T-cell 5018

2 VOL. 75, 2007 ROLE OF IL-1 IN LEISHMANIA AMAZONENSIS INFECTION 5019 responses, and the specific role of IL-1 in L. amazonensis infection in vitro and in vivo. Compared with L. major counterparts, L. amazonensis promastigotes are less potent in stimulating DC maturation/activation, and L. amazonensis-infected DCs preferentially induce CD4 T cells with an IFN- low IL- 10 high IL-17 high phenotype. Furthermore, in contrast to a previous study with L. major infection (40), although the addition of IL-1 at the time of infection markedly enhanced DC activation and T-cell priming, it cannot skew the cytokine profile of DCs and pathogenic Th cells. MATERIALS AND METHODS Mice. Female C57BL/6, BALB/c (Harlan Sprague Dawley, Indianapolis, IN), and OT-II mice (obtained from Chen Dong from the University of Texas MD Anderson Cancer Center) were used in this study. Mice were maintained under specific-pathogen-free conditions and used for experimentation at 6 to 8 weeks of age, according to protocols approved by the institutional animal care and use committees. Parasite culture and antigen preparation. Infectivity of L. amazonensis (MHOM/BR/77/LTB0016) and L. major (MRHO/SU/59/P/LV39) was maintained by regular passage through BALB/c mice. Promastigotes were cultured at 23 C in Schneider s Drosophila medium (Invitrogen, Carlsbad, CA), ph 7.0, supplemented with 20% fetal bovine serum (Sigma, St. Louis, MO), 2 mm L-glutamine, and 50 g/ml gentamicin. Stationary promastigote cultures of less than five passages were used for DC or animal infection. To prepare promastigote lysates, parasites ( /ml) were subjected to six freeze-thaw cycles and a 15-min sonication. DC generation and infection. DCs were grown from C57BL/6 bone marrow in complete Iscove modified Dulbecco medium containing 10% fetal bovine serum, supplemented with 20 ng/ml recombinant granulocyte-macrophage colony-stimulating factor (ebioscience, San Diego, CA) or 6% culture supernatants of J558L cells that were stably transfected with the murine granulocyte-macrophage colony-stimulating factor gene (28). At day 8, bone marrow-derived DCs were harvested and adjusted to /well in 12-well plates. Cells were incubated with parasites (8:1 parasite-to-cell ratio) in the presence or absence of 100 ng/ml mouse recombinant IL-1 (ebioscience) at 33 C for 12 h and then at 37 C for another 12 h. Lipopolysaccharide (LPS) (100 ng/ml) of Salmonella enterica serovar Typhimurium (Sigma) was added as a positive control. At 24 h postinfection, cells were collected for fluorescence-activated cell sorting (FACS) analysis or RNA extraction, and the supernatants were harvested for cytokine detection. T-cell priming in vitro. Naïve CD4 T cells were purified from the spleens of C57BL/6 or OT-II mice by negative selection using magnetic beads (Miltenyi Biotec, Auburn, CA), and their purity was routinely around 95%. Purified CD4 T cells ( ) were cocultured with parasite-infected, mitomycin C-pretreated DCs at a T-cell-to-DC ratio of 10:1 in 96-well plates for 3 days. Supernatants were harvested for cytokine detection. To assess T-cell proliferation, 1 Ci of [ 3 H]thymidine was added at 18 h before harvest, and incorporated radioactivity was determined on a microplate scintillation and luminescence counter (Packard Instrument Company, Downers Grove, IL). Intracellular staining and FACS. The following monoclonal antibodies were purchased from ebioscience unless stated otherwise: fluorescein isothiocyanateconjugated anti-cd45rb (C363.16A); anti-ifn- (XMG1.2), anti-cd44 (IM7), and anti-cd25 (PC61.5); phycoerythrin (PE)-conjugated anti-cd83 (Michel- 17), anti-cd40 (3/23), anti-il-17 (TC11-18H10), anti-il-12p40 (C17.8), anti- CD62L (MEL-14), and anti-cd69 (H1.2F3); PE-Cy5-conjugated anti-cd11c (N418), and anti-cd4 (GK1.5), as well as isotype control antibodies, including fluorescein isothiocyanate-conjugated rat immunoglobulin G2a (IgG2a); PEconjugated rat IgG1, IgG2a, and IgG2b; and PE-Cy5-conjugated hamster IgG. Briefly, cells were washed, blocked with 1 g/ml Fc receptor blocker, stained for specific surface antigen, fixed/permeabilized with a Cytofix/Cytoperm kit, and then stained for specific cytokines. For staining intracellular cytokines, DCs were infected with parasites for 24 h, and 1 l GolgiStop (BD Biosciences) was added 6 h before harvest. OT-II CD4 T cells were cocultured with DCs for 6 days and then restimulated with phorbol myristate acetate (PMA) (100 ng/ml; Sigma) and ionomycin (1 g/ml; Sigma) for 6hinthepresence of GolgiStop. Cells were analyzed on the FACScan (BD Biosciences) using FlowJo software (TreeStar, Ashland, OR). In vivo priming and evaluation of infection. C57BL/6 mice (five mice/group) were infected subcutaneously (s.c.) in the right hind foot with or stationary promastigotes of L. amazonensis. IL-1 (100 ng/mouse) or phosphatebuffered saline (PBS) was injected at the infection site at 1, 3, and 7 days postinfection or once per week for a total of 8 weeks. Lesion size was monitored with digital calipers (Control Company, Friendswood, TX), and parasite burdens were measured via a limiting dilution assay (16). After 3 weeks, popliteal draining lymph node (LN) cells were collected from individual mice and stained immediately for the expression of activation markers on CD4 and CD8 T cells. LN cells ( /well/ml) were stimulated with PMA/ionomycin for 2 h, and then GolgiPlug (BD Biosciences) was added for 4 h. Intracellular cytokine staining for IFN-, IL-10, and IL-17 was gated on CD4 T cells. In some cases, LN cells ( /well/ml) were restimulated with parasite lysates (equivalent to parasites). Supernatants were harvested at 72 h for cytokine measurement by an enzyme-linked immunosorbent assay (ELISA). Cytokine ELISA. The levels of cytokines in supernatants were measured using ELISA kits purchased from BD Biosciences or ebioscience (for IL-17), and their detection limits were 15 pg/ml for IL-1 and IFN- ; 4 pg/ml for IL-1, IL-6, and IL-10; 10 pg/ml for IL-12p40, and 8 pg/ml for IL-17. Reverse transcription-pcr. Total RNA was extracted from to DCs using the RNeasy system (QIAGEN, Valencia, CA), and cdna was synthesized from 1 g of total RNA using Superscript II reverse transcriptase (Invitrogen Life Technologies) primed with oligo(dt). PCR was performed using the RedTaq PCR reagents (Sigma) with the following primers: for IL-12p40, 5 -GGAGACCCTGCCCATTGAACT-3 (sense) and 5 -CAACGTTGCATCC TAGGATCG-3 (antisense); for IL-23p19, 5 -TGCTGGATTGCAGAGCAGTA A-3 (sense) and 5 -CTGGAGGAGTTGGCTGAGTC-3 (antisense); for IL- 12p35, 5 -CTACACAAGAACGAGAGTTGC-3 (sense) and 5 -ATCACCCTG TTGATGGTCACG-3 (antisense); and for -actin, 5 -CCAGCCTTCCTTCTT GGGTA-3 (sense) and 5 -CTAGAAGCACTTGCGGTGCA-3 (antisense). PCR products were analyzed on 1% agarose gels. Statistical analysis. The differences between the values for two different groups were determined by Student s test. A one-way analysis of variance test was used for multiple group comparisons. The tests were performed using GraphPad Prism, version 4.00, for Windows (GraphPad Software, San Diego, CA). P values of 0.05 and 0.01 were considered statistically significant. RESULTS Low levels of IL-1 production in L. amazonensis-infected DCs. We have previously shown that L. amazonensis-infected C57BL/6 mice had marked defects in expression of multiple inflammatory cytokines and chemokines, including IL-1 and IL-1, in draining LN cells and foot tissues (16). To evaluate the biological function of IL-1 in a nonhealing model of Leishmania infection, we first examined IL-1 production in LPSstimulated DCs that were exposed to different doses of promastigotes (from 1:1 to 8:1 parasite-to-cell ratios). We consistently observed that L. amazonensis-infected DCs produced significantly lower levels of IL-1 (Fig. 1A) and IL-1 (Fig. 1B) than L. major-infected DCs in an infection dosedependent fashion. To validate this observation, we also infected cells in the absence of LPS and measured the levels of IL-1 and IL-1 in cell extracts. As shown in Fig. 1C, L. amazonensis-infected DCs contained significantly lower levels of IL-1 than those in L. major-infected DCs. These results suggest an impaired production of IL-1 in L. amazonensisinfected DCs. Impaired activation of the CD11c high CD45RB DC subset after L. amazonensis infection. Since the maturation status of DC and distinct DC subsets can influence Th cell differentiation and the subsequent immune responses to pathogens (3, 41), we next examined the activation of different DC subsets after Leishmania infection. At 24 h postinfection, there were relatively high frequencies (42%) of the CD11c high CD45RB DC subset but low frequencies (9.8%) of the CD11c low CD45RB DC subset in L. major-infected DCs; the frequencies of these two DC subsets were intermediary (33.8% and 20.2%, respectively) in L. amazonensis-infected DCs in com-

3 5020 XIN ET AL. INFECT. IMMUN. Downloaded from FIG. 1. Deficient production of IL-1 and IL-1 in L. amazonensisinfected DCs. Bone marrow-derived DCs from C57BL/6 mice were infected with promastigotes of L. amazonensis (La) or L. major (Lm) at the indicated parasite-to-cell ratios in the presence of LPS (A and B) (20 ng/ml) or absence of LPS (C) (at a parasite-to-cell ratio of 8:1). Culture supernatants (A and B) and cell lysates (C) were collected at 24 h postinfection for measuring the levels of IL-1 and IL-1 by ELISAs. Shown are representative results from three independent experiments for panels A and B and two independent experiments for panel C. Statistically significant differences between the two infection groups are indicated as follows:, P 0.05;, P parison to medium controls (25.3% and 25.6%, respectively, Fig. 2A). Intracellular staining revealed that IL-12p40 was predominantly secreted by the CD11c high CD45RB DC subset. In comparison to the medium controls, L. major-infected DCs produced fourfold-more IL-12p40, whereas L. amazonensisinfected DCs produced only twofold-more IL-12p40 (Fig. 2A and C). To evaluate the maturation/activation status of parasite-infected DCs, we used different cell surface makers, including CD83, CD40, CD80, CD86, and major histocompatibility complex class II molecules. We found that both L. amazonensisand L. major-infected DCs expressed high percentages of FIG. 2. Differential activation of DC subsets after Leishmania infection in vitro. Bone marrow-derived DCs were left untreated (control) or infected with L. amazonensis (La) or L. major (Lm) promastigotes (8:1 parasite-to-cell ratio) at 33 C for 12 h and then at 37 C for an additional 12 h. GolgiStop was added 6 h prior to cell harvest. (A) DCs were stained for surface markers (CD11c and CD45RB) and intracellular IL-12p40. (B) Surface expression of CD40 and CD83 was measured for DC maturation. Shown are representative data from at least six independent experiments. The numbers in panels A and B indicate percentages. (C) The percentages of CD11c DCs that also expressed IL-12p40, CD40, or CD83 were shown as means plus standard deviations (error bars) from six independent experiments. Statistically significant differences (P 0.01) between the two infection groups are indicated by two asterisks. CD80, CD86, and major histocompatibility complex class II molecules (data not shown). However, the frequencies of CD40 and CD83 expression were increased about twofold in L. amazonensis-infected DCs, while their expression levels were on July 3, 2018 by guest

4 VOL. 75, 2007 ROLE OF IL-1 IN LEISHMANIA AMAZONENSIS INFECTION 5021 FIG. 3. Exogenous IL-1 enhances the activation of L. amazonensis-infected DCs. (A) Bone marrow-derived DCs were infected with L. amazonensis (La) promastigotes in the absence or presence of IL-1 (100 ng/ml) for 24 h. DCs were stained for surface expression of CD11c, CD45RB, CD40, and CD83 and then for intracellular IL-12p40. The numbers indicate percentages; shown are representative data from four independent experiments. (B) The percentages of CD11c DCs that also expressed IL-12p40, CD40, or CD83 were shown as means plus standard deviations (error bars) from four independent experiments. Statistically significant differences (P 0.05) between the two infection groups are indicated by one asterisk. (C) Bone marrow-derived DCs were infected with promastigotes in the absence (open bars) or presence of different doses of IL-1 (50, 100, and 200 ng/ml) (solid bars). Results are presented as percentages of IL-12p40-producing DCs, and we present one of three independent experiments with a similar trend. increased about four- and threefold, respectively, in L. majorinfected DCs compared with uninfected controls (Fig. 2B and C). Furthermore, CD40 and CD83 were mainly expressed on the CD11c high CD45RB DC subset (Fig. 2B and 3A). Together, these results suggest that L. amazonensis-infected DCs displayed a semiactivated profile and contained a lower frequency of the IL-12-producing CD11c high CD45RB CD83 CD40 DC subset. Exogenous IL-1 enhances the activation and maturation of L. amazonensis-infected DCs. Given the low potential of L. amazonensis parasites to activate DCs to produce cytokines, we asked whether exogenous IL-1 could overcome immune deficiencies in L. amazonensis-infected DCs. As shown in Fig. 3A, treatment with IL-1 alone promoted DC activation, and parasite infection in the presence of IL-1 further increased the frequency of the CD11c high CD45RB DC subset from 35.8% (parasite alone) to 41.9%. Accordingly, the frequency of the CD11c low CD45RB DC subset in the parasite plus IL-1 group decreased from 20.4% (parasite alone) to 14.3%. Likewise, exogenous IL-1 promoted the activation status of L. amazonensis-infected DCs, as judged by high frequencies of IL-12p40-producing cells (17.0%) and increased frequencies of CD40 (20.2%) and CD83 (24.9%) DCs. The enhancement of these molecules by IL-1 was statistically significant (Fig. 3B). Of note, although IL-1 treatment resulted in a dosedependent enhancement of IL-12p40-producing DCs following infection with both L. amazonensis and L. major parasites, even the highest concentration of IL-1 (200 ng/ml) was still incapable of stimulating L. amazonensis-infected DCs to a level that was comparable to that of L. major alone (Fig. 3C). Collectively, these results suggest that intrinsic defects in L. amazonensis-infected DCs cannot be fully overcome by exogenous IL-1. Given that IL-12p40 is the common subunit for IL-12 and IL-23, which correspond to IFN- -producing Th1 cells and IL-17-producing Th17 cells, respectively (11, 12), we examined gene expression of the counterpart subunits, IL-12p35 and IL-23p19. Consistent with our FACS results, L. amazonensisinfected DCs apparently expressed lower levels of IL-12p40 mrna than did L. major-infected DCs. There were no major changes in expression levels for the IL-12p35 and IL-23p19 genes in parasite-infected DCs, unless exogenous IL-1 was added (Fig. 4A). Furthermore, although low levels of IL-12p40 and IL-10 were detected in the supernatants of L. amazonensis-infected DCs, production of these two cytokines was significantly increased by IL-1 (Fig. 4B). In the absence of other stimuli, the levels of IL-12p70 and IL-23 in infected DCs were below the detection limits of ELISAs (data not shown). IL-1 treatment promotes the activation of L. amazonensisspecific CD4 T cells. To assess the antigen-presenting function of DCs, we determined the effect of parasite-infected DCs on priming naïve CD4 T cells. Consistently, L. amazonensisinfected DCs induced relatively low levels of CD4 T-cell proliferation, and IL-1 treatment significantly enhanced the antigen presentation potential of these DCs, as judged by the levels of CD4 T-cell proliferation (Fig. 5A). L. amazonensis-

5 5022 XIN ET AL. INFECT. IMMUN. FIG. 4. Cytokine expression from parasite-infected DCs in the presence of IL-1. Bone marrow-derived DCs were infected with L. amazonensis (La) orl. major (Lm) promastigotes in the presence ( ) or absence ( ) of IL-1 (100 ng/ml). (A) At 24 h postinfection, DCs were collected for RNA isolation. Reverse transcription-pcr analyses were performed with primers specific to the indicated genes. (B) The levels of IL-12p40 and IL-10 in the supernatants were assayed by ELISAs. The levels of IL-12p70 and IL-23 were below detection (data not shown). Results are shown as means plus standard deviations (error bars) from a total of five independent experiments performed in duplicate. Statistically significant differences between the two infection groups are indicated as follows:, P 0.05;, P infected DCs preferentially induced a novel phenotype of IFN- low IL-10 high IL-17 high Th cells, which was characteristically different from that of L. major-inducing Th1 cells (Fig. 5B). Despite IL-1 treatment having endowed parasite-infected DCs to produce more cytokines in naïve CD4 T cells, it did not skew the cytokine profile of parasite-specific Th cells, as judged by intracellular cytokine staining (Fig. 5C). Therefore, IL-1 treatment can amplify, but not alter, the signal pathways initiated in L. amazonensis-infected DCs. To further assess the potential of L. amazonensis-specific Th cells to produce IL-17, a cytokine known to be crucial in neutrophil recruitment and inflammatory diseases, we purified CD4 T cells from naïve, syngeneic OT-II mice and cocultured these cells with parasite-infected, OVA protein-pulsed DCs. As shown in Fig. 5D, L. amazonensis-infected DCs induced a higher percentage of IL-17-producing CD4 T cells than L. major-infected DCs did (18.3% versus 9.4% in the absence of IL-1 ; 19.8% versus 15.3% in the presence of IL-1, respectively). In agreement with previous reports (6, 33), our in vitro studies with L. amazonensis parasites also revealed that the frequencies of IL-17-producing cells were inversely correlated with those of IFN- -producing cells (Fig. 5D) and that IL-1 -treated DCs enhanced IL-17-producing CD4 T cells. Lesional injection with IL-1 accelerates disease progression in L. amazonensis-infected mice. To confirm our in vitro findings regarding the effects of IL-1 on DCs and T cells, we further explored the in vivo function of IL-1. Mice were infected s.c. with L. amazonensis promastigotes in the hind foot and then injected in the same foot with 100 ng of IL-1 at 1, 3, and 7 days of infection or weekly for a total of 8 weeks. Surprisingly, IL-1 injection at the time of T-cell priming (Fig. 6A) or during the course of T-cell expansion (Fig. 6B) resulted in an earlier onset of and more progressive lesions that contained significantly high loads of parasites. Since the pace of lesion development in L. amazonensis-infected mice is regulated by a fine balance between lesional CD4 CD25 regulatory T cells (Treg) and pathogenic CD4 effector T cells (14), we examined the activation status and cytokine profiles of CD4 T cells in the draining LN. At 3 weeks postinfection when IL-1 -treated mice displayed significant lesions with high parasite loads (Fig. 7A and B), LN cells of these mice showed higher percentages of CD44 high CD62L low CD69 CD4 effector T cells but comparable percentages of CD25 CD4 Treg cells compared to cells of L. amazonensis-infected mice (Fig. 7C). Also, cytokine production of IFN-, IL-10, and IL-17 from CD4 T cells was dramatically enhanced by IL-1 treatment (Fig. 7D). In vitro recall studies further confirmed that LN cells derived from L. amazonensis-infected, IL-1 -treated mice produced significantly higher levels of IFN-, IL-10, IL-6, and IL-17 than did the infection controls (Fig. 7E), suggesting that the disease-enhancing effects of IL-1 were due to promoted activation of effector, rather than regulatory, CD4 T cells. There was no significant expansion of CD8 effector T cells (data not shown), confirming a limited role of CD8 T cells in primary L. amazonensis infection (32). Together, these results indicate that IL-1 treatment promotes the activation and expansion of L. amazonensis-specific pathogenic Th cells in vivo and accelerates cutaneous lesions. DISCUSSION The mechanisms responsible for the impaired immune response during L. amazonensis infection remain largely unclear. In this study, we first examined whether DC subsets responded to L. amazonensis and L. major parasites differently and how these parasite-exposed DCs influence CD4 T-cell activation. Using an in vitro DC infection and T-cell priming system, we have provided evidence that L. amazonensis infection induces relatively low frequencies of the IL-12-producing CD11c high CD45RB CD40 CD83 DC subset (Fig. 2) and that these DCs preferentially induce a novel subset of Th cells with an IFN- low IL-10 high IL-17 high phenotype (Fig. 5). Due to the deficient expression of IL-1 family molecules in tissues of L.

6 VOL. 75, 2007 ROLE OF IL-1 IN LEISHMANIA AMAZONENSIS INFECTION 5023 FIG. 5. IL-1 treatment promotes L. amazonensis-infected DCs to prime CD4 T cells. Bone marrow-derived DCs were infected with L. amazonensis (La)orL. major (Lm) promastigotes for 24 h in the absence or presence of IL-1 (100 ng/ml) and then cocultured with spleen-derived naïve CD4 T cells at a 1:10 DC-to-T-cell ratio. (A) After 4 days of cultivation, T-cell proliferation was measured by a 3 H uptake assay. (B) Culture supernatants were collected at 72 h for measuring the levels of cytokines by ELISAs. Shown are representative data from one of three independent experiments. Statistically significant differences between the two infection groups are indicated as follows:, P 0.05;, P (C) Intracellular IFN- and IL-10 from naïve CD4 T cells cocultured with parasite-infected DCs for 6 days were measured by flow cytometry. (D) Bone marrow-derived DCs infected with L. amazonensis or L. major promastigotes were pulsed overnight with OVA protein (200 g/ml) and then cultured with purified OT-II CD4 T cells for 6 days. PMA and ionomycin were added together with GolgiStop 6 h prior to cell harvest. Cells were stained for intracellular IFN- and IL-17 and analyzed by FACS. In panels C and D, the numbers indicate percentages; shown are representative data from three independent experiments. In the absence of OVA, the percentages of IFN- - and IL-17-producing cells were less than 2% and 1%, respectively (data not shown). amazonensis-infected mice (16) and low levels of IL-1 and IL-1 in L. amazonensis-infected DCs (Fig. 1), we sought to examine whether exogenous IL-1 would intervene in the susceptibility of mice to L. amazonensis infection, as previously reported for IL-1 treatment in L. major-infected mice (40). We found that although exogenous IL-1 markedly promoted the activation of the parasite-infected DC subset and significantly enhanced CD4 T-cell responses in vitro and in vivo (Fig. 4 to 6), the intrinsic defects in L. amazonensis-infected DCs could not be overcome by exogenous IL-1, because the levels of DC activation and cytokine production in IL-1 -treated, L. amazonensis-infected DCs remain much lower than those of L. major-infected DCs (Fig. 3 and 4). Surprisingly, local injection of IL-1 not only failed to reduce susceptibility but dramatically accelerated cutaneous disease following L. amazonensis infection (Fig. 6 and 7). These findings reemphasize our view that complex pathogenic mechanisms are associated with nonhealing New World leishmaniasis (14, 32). It is well demonstrated that murine DCs can internalize L. major promastigotes or amastigotes and then release IL-12p40 for the initiation of anti-leishmania immunity (21, 38, 39). However, weakly/suboptimally matured DCs (26) and IFN- activated macrophages (27) appear to be favored for enabling the sustained survival and growth of L. amazonensis parasites. We have previously reported that DCs infected with L. amazonensis amastigotes even failed to induce CD40-dependent IL-12 production (28). In this study, we have provided additional evidence to support and expand previous reports, showing that L. amazonensis promastigotes are indeed less potent in stimulating DC maturation and cytokine production in comparison to L. major promastigotes. Does L. amazonensis infection induce aberrant DC function (due to gained regulatory mechanisms) or just partial DC activation? We favor the latter possibility. First, L. amazonensis infection triggers low frequencies of IL-12-producing DCs among CD11c high CD45RB CD40 CD83 cells, a special DC subset known to be a major source of IL-12 (41), as well as low levels of IL-1. Second, L. amazonensis infection induces low levels of IL-10 in DCs (Fig. 4). Since CD11c low CD45RB high DCs have been recently defined as a novel regulatory DC subset that secretes high levels of IL-10 following activation and induces Tr1 cells in vivo and in vitro (9, 34, 41), we tested whether L. amazonensis parasites can activate regulatory DCs and, therefore, suppress protective immune responses. Using purified CD11c high CD45RB CD40 CD83 and CD11c low CD45RB high cells from L. amazonensis-infected DCs and coculturing them with naïve CD4 T cells, we found that only L. amazonensis-infected CD11c high CD45RB CD40 CD83 DCs were capable of inducing

7 5024 XIN ET AL. INFECT. IMMUN. FIG. 6. Local injection of IL-1 accelerates lesion development. C57BL/6 mice were infected s.c. with stationary L. amazonensis (La) promastigotes and then injected with IL-1 (100 ng/mouse) or PBS at the local infection site at 1, 3, and 7 days postinfection (A) or once per week for 8 weeks (B). Lesion sizes were monitored weekly. Parasite burdens from these groups of mice at 8 weeks were determined by limited dilution assay (16). Shown are the representative data from two independent experiments. Statistically significant differences between the two infection groups are indicated as follows:, P 0.05;, P strong CD4 T-cell proliferation and cytokine production (e.g., IL-12, IFN-, and IL-10, as shown in supplemental Fig. S1). Therefore, the involvement of regulatory DCs and Treg cells is less likely to be the mechanism responsible for the impaired Th1 cell activation in L. amazonensis-infected hosts. Third, we explored the possible involvement of some negative regulators in the B7 family (e.g., B7-H1, B7-DC, and B7-H3) (10) and found no major differences at the mrna level for these molecules between L. amazonensis- and L. major-infected DCs (data not shown). Taken together, these observations suggest intrinsic defects in DC activation following L. amazonensis infection. This view is further supported by our IL-1 treatment studies, in which exogenous IL-1 can partially overcome defects in L. amazonensis-infected DCs via increasing the number of IL-12-producing cells and DC activation status (Fig. 3C). IL-1 treatment by itself, however, cannot alter the phenotype and functional features of L. amazonensis-infected DCs, as judged by T-cell cytokine profiles following in vitro priming and in vivo infection. Adequately activated DCs can prime naïve T cells toward a Th1 or Th2 phenotype (24), whereas partially activated DCs provide insufficient costimulation and preferentially prime IL- 10-producing regulatory T cells (e.g., Tr1) that suppress the antigen-specific T-cell response (8, 18). We have shown herein that L. amazonensis-infected DCs preferentially prime a novel CD4 effector T-cell population with an IFN- low IL-10 high IL-17 high phenotype (without detectable IL-4 by intracellular staining). This population of Th1-like cells most likely consists of pathogenic T cells responsible for immunopathogenesis in L. amazonensis-infected mice, acting similarly to the pathogenic Th1 cells described in a nonhealing model of L. majorinfected C57BL/6 mice (2). IL-1 treatment greatly enhanced the antigen-presenting function of L. amazonensis-infected DCs via increasing priming/expansion of these pathogenic Th cells and, therefore, promoting lesion progression. The lack of strong Th1 cell activation, presumably due to the deficient expression of T-bet (29), and the expansion of Th1-like pathogenic cells are general features in L. amazonensis-susceptible BALB/c, C57BL/6, and C3H mice. We infer that the activation of these Th1-like pathogenic cells (IFN- low IL-10 high IL-17 high CD4 T cells) facilitates the recruitment of monocytes and immature macrophages (14, 16), which creates a unique microenvironment for enhanced parasite growth and persistence (27). Currently, there is limited information on the role of IL-17 in Leishmania infection. IL-17 is involved in regulating tissue inflammation and development of autoimmune disorders and infectious diseases (20). Given the chronic inflammation and neutrophil/macrophage migration during Leishmania infection (31), it is logical to speculate that IL-17 may play a role in leishmaniasis. Although we consistently observed higher expression of IL-17 following L. amazonensis infection than we did with L. major infection (Fig. 5), the specific condition that induces IL-17 expression remains unclear. The lack of detectable IL-23 expression in L. amazonensis-infected DCs (Fig. 4 and data not shown) suggests an IL-23-independent regulation for IL-17 induction. Given that transforming growth factor (TGF- ), IL-6, and IL-1 have been identified as crucial factors for the differentiation of the Th17 subset (4, 37) and that the blockage of TGF- in L. amazonensis- and L. major-infected BALB/c mice can facilitate lesion healing (23), it will be interesting to further examine the influence of TGF- in IL-17 production and the biological function of IL-17 in Leishmania infection. The effects of IL-1-based therapy in murine cutaneous leishmaniasis vary greatly, depending on the time of treatment and parasite species involved. While IL-1 injection at the stage of T-cell priming (first 3 days of infection) promotes Th1 activation and the control of L. major infection in BALB/c mice (40), prolonged treatment (for 3 weeks) exacerbates the disease (22). Conversely, IL-1R type I-deficient mice developed smaller lesions following infection with L. major (22), and treatment with anti-il-1r monoclonal antibody inhibited le-

8 VOL. 75, 2007 ROLE OF IL-1 IN LEISHMANIA AMAZONENSIS INFECTION 5025 Downloaded from FIG. 7. IL-1 treatment enhances L. amazonensis (La) infection and CD4 T-cell activation in vivo. C57BL/6 mice (five mice/group) were infected s.c. with stationary promastigotes. At 1, 3, and 7 days postinfection, mice were injected in the same foot with IL-1 (100 ng/mouse) or PBS. At 3 weeks postinfection, lesion sizes (A) and tissue parasite burdens (B) were monitored. (C) Draining LN cells were collected from individual mice and stained for CD4 and T-cell activation markers. Numbers are the percentages of positively stained cells gated on CD4 cells. (D) Draining LN cells were stimulated with PMA and ionomycin in the presence of GolgiPlug. Intracellular staining for IFN-, IL-10, and IL-17 were gated on CD4 T cells. The numbers indicate percentages; the pooled data from one of two independent experiments were shown as means standard deviations (n 3). (E) Pooled LN cells were restimulated with parasite lysates for 72 h, and supernatants were harvested for cytokine assays. Shown are representative results from two independent experiments. Statistically significant differences between the two infection groups are indicated as follows:, P 0.05;, P sion development in L. major-infected susceptible and resistant mice (35), suggesting a negative role for IL-1 in developing protective immunity against parasite infection. These seemingly conflicting results may be explained by the distinct roles of IL-1 at different stages of Th cell priming/activation. In the present study, local treatment with IL-1 either at the time of T-cell priming or during the course of T-cell expansion accelerates L. amazonensis infection, with a correlated expansion of IFN- low IL-10 high IL-17 high Th cells. Similar results were observed when infection was initiated with a low dose ( ) of parasites (data not shown). The enhanced activation of effector Th cells (CD44 high CD62L low CD69 ) and their cytokine profiles following IL-1 treatment indicate that IL-1 alone cannot convert L. amazonensis-specific, pathogenic T cells to protective ones. Therefore, an IL-1-based therapy may be beneficial in some Leishmania infection models (e.g., L. major) but be detrimental in others (e.g., L. amazonensis). In summary, our findings indicate that infection with L. amazonensis parasites induces semiactivated DCs, which prime and activate a novel phenotype of pathogenic Th1-like cells. Although exogenous IL-1 can promote activation of parasite-specific CD4 effector T cells by enhancing the antigen-presenting functions of DCs, the intrinsic defects in L. amazonensis-infected DC cannot be overcome solely by the addition of IL-1. This study provides new information on the nature of pathogenic T cells, as well as specific defects at the level of DC activation that are responsible for generation of these effector Th cells during L. amazonensis infection. We are currently examining the activation of signaling pathways in DCs following infection with different Leishmania species. Additional study at the level of innate immunity will further illustrate the complex regulation of host immune responses to Leishmania parasites and mechanisms of pathogenesis responsible for nonhealing cutaneous leishmaniasis. on July 3, 2018 by guest

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