The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity Wendy A. Szymczak and George S. Deepe, Jr This information is current as J Immunol 2009; 183:1964-1974; Prepublished online 8 July of September 29, 2021. 2009; doi: 10.4049/jimmunol.0901316 http://www.jimmunol.org/content/183/3/1964 Downloaded from References This article cites 57 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/183/3/1964.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity1 Wendy A. Szymczak*† and George S. Deepe, Jr.2*‡ Expression of the chemokine receptor CCR2 can be detrimental or beneficial for infection resolution. Herein, we examined whether CCR2 was requisite for control of infection by the dimorphic fungus Histoplasma capsulatum. H. capsulatum-infected CCR2؊/؊ mice manifested defects in inflammatory cell recruitment, increased IL-4, and progressive infection. Increased IL-4 in CCR2؊/؊ mice primarily contributed to decreased host resistance as demonstrated by the ability of IL-4-neutralized CCR2؊/؊ mice to resolve infection without altering inflammatory cell recruitment. Surprisingly, numerous alveolar macrophages and dendritic cells contributed to IL-4 production in CCR2؊/؊ mice. IL-4-mediated impairment of immunity in CCR2؊/؊ mice was associated with increased arginase-1 and YM1 transcription and increased transferrin receptor expression by phagocytic cells. Immunity in mice lacking the CCR2 ligand CCL2 was not impaired despite decreased inflammatory cell recruitment. Neu- Downloaded from .tralization of the CCR2 ligand CCL7 in CCL2؊/؊ mice, but not wild type, resulted in increased IL-4 and fungal burden Thus, CCL7 in combination with CCL2 limits IL-4 generation and exerts control of host resistance. Furthermore, increased phagocyte-derived IL-4 in CCR2؊/؊ mice is associated with the presence of alternatively activated phagocytic cells. The Journal of Immunology, 2009, 183: 1964–1974. nfection by the dimorphic fungus Histoplasma capsulatum known receptor for CCL2 that promotes signaling. The latter ap- http://www.jimmunol.org/ occurs when conidia and mycelial fragments from disturbed pears to be more potent in monocyte recruitment than the other I soil are coincidentally inhaled by a host and deposited within ligands that engage CCR2 (16). the lung (1). The lung environment supports conversion of conidia Mice lacking CCR2 exhibit disturbances in monocyte egress to the virulent yeast phase (2). Alveolar macrophages (M␾)3 are a from bone marrow and monocyte recruitment to sites of infections first line of host defense against the invading pathogen, but H. (18–20). Alterations in M␾, dendritic cell (DC), and T cell re- capsulatum yeast cells replicate and disseminate to other organs, cruitment in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice during infection have most likely transported by resting M␾ (3–5). The host must mount been reported (21–26). Recently, CCL7 has been shown to mediate ␾ aTH1 immune response to activate M and resolve infection (6– monocyte egress from bone marrow upon Listeria monocytogenes ϩ 10). CD4 T cells and IFN-␥ must be present in order for mice to infection (27). Aside from its role in inflammation, CCR2 is es- by guest on September 29, 2021 ␣ survive infection (8). Other TH1 cytokines including TNF- and sential for promoting a TH1 response and limiting infection by GM-CSF are also required for resolution of infection (7, 9). several pathogens (24–26, 28, 29). Conversely, lack of CCR2 pro- H. capsulatum infection induces a complex inflammatory re- motes protective immunity by altering the influx of monocyte- sponse that consists of numerous cell populations (11). Recruit- derived cells that generate noxious inflammatory mediators. The ment of immune cells into infected tissues is governed by multiple net result is a decrement in the severity of immunopathology and mediators including chemokines. These soluble factors are small as a consequence improved survival (18). molecules that induce chemotaxis, activate T cells, and direct We have sought to determine whether signaling through the che- or maintain TH1orTH2 immunity during infection upon engage- mokine receptor CCR2 is necessary for resolution of H. capsula- ment with their cognate receptors (12, 13). In mice, the chemokine tum infection. CCR2Ϫ/Ϫ mice manifested higher fungal burdens, receptor CCR2 directs the migration of myeloid lineage cells by diminished inflammatory cell recruitment, and succumbed to in- engaging the chemokine ligands CCL2, CCL7, CCL8, and CCL12 fection. The major defect in immunity in CCR2Ϫ/Ϫ mice was in- (14–17). CCL2 is a major ligand of CCR2 and CCR2 is the only creased production of IL-4 in the lungs. Neutralization of IL-4 facilitated resolution of infection in CCR2Ϫ/Ϫ mice, but it did not alter the defects in inflammatory cell recruitment to the lung. Al- *Division of Infectious Diseases, University of Cincinnati, Cincinnati, OH 45267; though loss of CCL2 or CCL7 alone was not essential for control †Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and ‡Division of Infectious of infection, the combined absence of CCL2 and CCL7 resulted in Diseases, Veterans Affairs Hospital, Cincinnati, OH 45267 increased IL-4 and fungal burden. These data demonstrate that Received for publication April 27, 2009. Accepted for publication May 29, 2009. CCL7 and CCL2 must engage CCR2 for optimal immunity to H. The costs of publication of this article were defrayed in part by the payment of page capsulatum. CCL2 and CCL7-mediated signaling through CCR2 charges. This article must therefore be hereby marked advertisement in accordance is requisite for limiting IL-4 production. Moreover, the elevated with 18 U.S.C. Section 1734 solely to indicate this fact. IL-4 in CCR2Ϫ/Ϫ mice is associated with alternative activation of 1 This work was supported by Grants AI-34361 and AI-70337 and a Merit Review phagocytic populations. from the Veterans Affairs. 2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., University of Cincinnati College of Medicine, 231 Albert Sabin Way, Medical Science Building Materials and Methods 7155, Cincinnati, OH 45267-0560. E-mail address: [email protected] Mice 3 ␾ Abbreviations used in this paper: M , macrophage; Arg-1, arginase 1; DC, dendritic Ϫ/Ϫ Ϫ/Ϫ cell; cDC, conventional dendritic cell; MFI, mean fluorescence intensity; iNOS, in- Male C57BL/6 mice and breeding pairs of CCR2 , CCL2 , and IL- Ϫ/Ϫ ducible NO synthase; TipDC, TNF and iNOS-producing DC; i.n., intranasal(ly); WT, 4 mice on a C57BL/6 background were purchased from The Jackson Ϫ Ϫ Ϫ Ϫ wild type; qRT-PCR, quantitative real-time PCR; RQ, relative quantification. Laboratory. CCR2 / and CCL2 / mice were backcrossed more than www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901316 The Journal of Immunology 1965 nine generations. Animals were housed in isolator cages and were main- numbers, a Micro RNAEasy Kit (Qiagen) was used according to the man- tained by the Department of Laboratory Animal Medicine (University of ufacturer’s instructions. Oligo(dT)-primed cDNA was prepared by using a Cincinnati, Cincinnati, OH), which is accredited by the Association for Reverse Transcriptase System (Promega). Assessment and Accreditation of Laboratory Animal Care. All animal ex- periments were performed in accordance with the Animal Welfare Act Quantitative real-time PCR (qRT-PCR) guidelines of the National Institutes of Health and all protocols were ap- proved by the Institutional Animal Care and Use Committee of the Uni- qRT-PCR for cytokine transcription analysis was performed using TaqMan versity of Cincinnati. Master Mix and primers from Applied Biosystems. Samples were analyzed on an Applied Biosystems Prism 7500. In each experiment, the housekeep- Preparation of H. capsulatum and infection of mice ing gene HPRT was used as an internal control. The conditions for ampli- H. capsulatum yeast strain G217B was grown for 72 h at 37°C as previ- fication were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of ously described (6). To produce infection in mice, animals were inoculated 95°C for 15 s and 60°C for 1 min. intranasally (i.n.) with 2 ϫ 106 H. capsulatum yeast cells in a ϳ30-␮l volume of HBSS (HyClone). Measurement of cytokines by ELISA Organ culture for H. capsulatum Lungs were homogenized in 5 ml of HBSS and centrifuged. Cytokines in homogenates were quantified by ELISA. IL-4, TNF-␣, IFN-␥, IL-1␤, and Organs were homogenized in sterile HBSS and serially diluted and plated GM-CSF ELISAs were purchased from Endogen and IL-10 and IL-12 onto mycosel-agar plates containing 5% sheep blood and 5% glucose. ELISA kits were purchased from R&D Systems. Plates were incubated at 30°C for 1 wk. The limit of detection was 1 ϫ 102 CFU. Measurement of NO In vivo neutralization of IL-4, CCL7, CCL12, and CD4ϩ T cell Isolated lung leukocytes from mice were plated ex vivo at 5 ϫ 105 cells/ Downloaded from depletion well in a 96-well plate in DMEM supplemented with 10% FBS (HyClone).