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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 References This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/174/5/2934.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 non- healing 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 regula- tory 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 Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2935 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.
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