The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity Wendy A

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

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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 © 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 experiments 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). ␮ ␥ For neutralization of IL-4, mice were injected i.p. with 1 mg of rat anti- Leukocytes were stimulated with 1 g/ml LPS and 100 ng/ml IFN- for mouse IL-4 (clone11B11) or 1 mg of rat IgG1 control Ab dissolved in 1 ml 24 h. Measurement of NO reaction products nitrate and nitrite were deter- of HBSS at the time of infection. For survival studies, mice were injected mined by the nitrate/nitrite assay (Cayman Chemical) according to the additionally at 1-wk intervals. For CCL7 and CCL12 neutralization, 100 manufacturer’s instructions. ␮g of polyclonal goat anti-mouse CCL7 and/or anti-CCL12 (R&D Sys-

tems) or goat IgG in 0.5 ml of HBSS was administered i.p. at the time of IL-4 secretion assay http://www.jimmunol.org/ infection and 3 days after infection. For CD4ϩ T cell depletion, mice were given 500 ␮g of anti-CD4 (clone GK1.5) in 500 ␮l of HBSS at the time of An IL-4 secretion assay and detection kit (Miltenyi Biotec) were used infection and 3 days after infection. This treatment resulted in Ͼ95% de- according to the manufacturer’s instructions. Leukocytes isolated from the ϩ Ϫ Ϫ ϫ 6 pletion of CD4 cells from wild-type (WT) and CCR2 / mice. lung were restimulated ex vivo in a 96-well plate at 1.5 10 leukocytes/ 200 ␮l of medium for3hinthepresence of 20 ng/ml PMA and 1 ␮g/ml Isolation of lung leukocytes ionomycin. Cells were then incubated an additional 45 min with IL-4 cap- ture Ab at 37°C. Leukocytes were incubated with anti-IL-4-PE and the Lungs were homogenized in HBSS using a gentleMACS Dissociator indicated additional Abs before enrichment for IL-4- secreting cells ␮ (Miltenyi Biotec). The solution was filtered through 60- m nylon mesh using anti-PE microbeads and an LS column (Miltenyi Biotec). Un- (Spectrum Laboratories) and washed with HBSS. Leukocytes were isolated stimulated lung leukocytes were used as negative controls. Recovered by Lympholyte M (Cederlane Laboratories) separation. cells were analyzed using a FACSCalibur flow cytometer or sorted with by guest on September 29, 2021 Flow cytometry a FACSVantage cell sorter as described above. The phenotype of cells from mouse lungs was determined by incubating Statistics lung leukocytes with the indicated Abs and CD16/32 to limit nonspecific binding. Leukocytes were stained at 4°C for 15 min in HBSS containing ANOVA was used to compare multiple groups while Student’s t test was 1% BSA and 0.01% sodium azide. Cells were stained with combinations of used to compare two groups. Survival was analyzed using log rank. A the following Abs: FITC-conjugated CD4, CD8, Ly-6C, Mac-3, I-Ab, value of p Ͻ 0.05 was considered statistically significant. CD69, and NK1.1; PE-conjugated CD11c, I-Ab, transferrin receptor, Ly6G, CD3, and Mac3; PerCP-conjugated CD11b and CD4; and allophy- cocyanin-conjugated CD11c, CD3, and CD25 from BD Biosciences. PE- Results Ϫ Ϫ conjugated Dec205 was purchased from Miltenyi Biotec. FC␧R1-FITC Fungal burden and course of infection in CCR2 / and was a gift from Dr. F. Finkelman (Cincinnati Children’s Hospital). Cells CCL2Ϫ/Ϫ mice were washed and resuspended in 1% paraformaldehyde to fix. Appropriate isotype controls were performed in parallel. Fluorescence intensity was To determine whether CCR2 was necessary for control of H. cap- assessed using a FACSCalibur (BD Biosciences) flow cytometer and an- sulatum infection, we infected CCR2Ϫ/Ϫ and WT mice with a alyzed using FCS Express Software. Intracellular staining for IFN-␥ was sublethal number of yeast cells i.n. and assessed fungal burden and ϫ 5 performed subsequent to ex vivo stimulation of 5 10 cells/well in a survival. At day 7 after infection, CCR2Ϫ/Ϫ mice exhibited a 0.85 96-well plate with 20 ng/ml PMA, 1 ␮g/ml ionomycin, and 1 ␮g/ml brefel- din A for 4 h. Surface-stained cells were permeabilized in Cytofix/Cyto- log10 increase in CFU in the lungs compared with WT (Fig. 1A). perm (BD Biosciences) for 20 min, washed in Permwash buffer (BS Bio- The burden in spleens was similar between the two strains (Fig. sciences), and stained with PE-conjugated IFN-␥ for 30 min. 1A). Between days 7 and 14 after infection, fungal burden in the Ϫ/Ϫ Histology lungs and spleens of CCR2 mice increased, whereas the burden diminished in lungs and spleens of WT mice (Fig. 1A). CCR2Ϫ/Ϫ Lungs were inflated, excised, fixed in 10% formalin, and embedded in ␮ mice infected with a sublethal number of yeast cells were unable paraffin blocks. Sections (5 m) were stained with H&E. Analysis of the Ϫ/Ϫ sections was performed in a blinded fashion. to resolve infection; all CCR2 mice succumbed by day 20 (Fig. 1B). Lungs from moribund CCR2Ϫ/Ϫ mice contained Ͼ108 CFU. FACS sorting of lung leukocyte populations CCL2 is a major ligand of CCR2 (16) and impaired immunity is To enrich for specific lung leukocyte populations, cells were stained often ascribed to loss of CCL2 signaling (19, 20, 24). To ascertain with the indicated Abs as described above and sorted in HBSS using a whether CCL2 was requisite for protective immunity to H. cap- FACSVantage (BD Biosciences) cell sorter. Isolated cells exhibited sulatum infection, CCL2Ϫ/Ϫ mice were infected with a sublethal Ͼ95% purity. number of yeast. CCL2Ϫ/Ϫ mice manifested a transient increase in RNA isolation and cDNA synthesis fungal burden in the lungs and spleen only at day 14 (Fig. 1A). By Ϫ/Ϫ Total RNA from whole lungs was isolated from mouse lungs using TRIzol day 21, the number of CFU in lungs of CCL2 mice was similar Ϫ Ϫ (Invitrogen). To isolate RNA from sorted populations containing low cell to that of WT. All CCL2 / mice survived (Fig. 1B). 1966 H. capsulatum INFECTION IN CCR2Ϫ/Ϫ MICE

and CCL2Ϫ/Ϫ (1.41 Ϯ 0.39, n ϭ 7) mice in comparison to WT (5.56 Ϯ 0.68, n ϭ 11). We next determined the percentage and absolute number of phagocytic cells in the lungs of CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice. At day 7, the percentage and number of inﬂammatory monocytes, TNF, and inducible NO synthase (iNOS)-producing DC (TipDC), and CD8Ϫ conventional DC (cDC) were decreased in CCR2Ϫ/Ϫ mice (Fig. 2, B and C, and Table I). The mean ﬂuorescence intensity (MFI) of I-Ab expression (mean Ϯ SEM) on CD11cϩ cells from CCR2Ϫ/Ϫ mice (568 Ϯ 25, n ϭ 8) was decreased ( p Ͻ 0.001) in comparison to WT (864 Ϯ 36, n ϭ 8). The relative percentage of neutrophils and tissue M␾ was increased in lungs of CCR2Ϫ/Ϫ mice in comparison to WT (Fig. 2C); however, the number was similar to WT (Table I). The relative percentage of alveolar M␾ was similar in WT and CCR2Ϫ/Ϫ mice (Fig. 2C), although the number was decreased in CCR2Ϫ/Ϫ mice (Table I). Assessment of leukocyte populations in the lungs of CCL2Ϫ/Ϫ mice revealed disturbances similar to those of infected CCR2Ϫ/Ϫ

mice. The percentage and number of inflammatory monocytes and Downloaded from TipDC were decreased in CCL2Ϫ/Ϫ mice in comparison to WT, although not as severely as in the lungs of CCR2Ϫ/Ϫ mice (Fig. 2, B and C, and Table I). The number of CD8Ϫ cDC in the lungs of CCL2Ϫ/Ϫ mice was decreased in comparison to WT (Table I). However, the percentage of CD8␣Ϫ cDC (Fig. 2C) and MFI b Ϫ (mean Ϯ SEM) I-A expression of CD8␣ cDC from WT (1331 Ϯ http://www.jimmunol.org/ 151, n ϭ 4) and CCL2Ϫ/Ϫ mice (1205 Ϯ 195, n ϭ 4) were similar ( p Ͼ 0.05). Neutrophil number, which was not affected by loss of CCR2, was reduced in CCL2Ϫ/Ϫ mice (Table I). To determine whether the decrement in inflammatory cell number in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice resulted from global disturbances in chemokine expression, we examined transcription of chemokines in the lungs at day 7 after infection. The generalized impairment in inflammatory cell recruitment was not associated FIGURE 1. Fungal burden and survival of H. capsulatum-infected with a loss of CCL2, CCL3, CCL4, CCL7, or CXCL10. Mean by guest on September 29, 2021 ϫ 6 mice. Mice were infected with 2 10 yeasts and fungal burden in the Ϯ Ϯ ϭ log10 ( SEM) transcription of CCL2 (1.67 0.2, n 6), CCL7 lungs and spleens was assessed at days 7 and 14 after infection in WT and Ϯ ϭ Ϯ ϭ CCR2Ϫ/Ϫ mice or at days 7, 14, and 21 in WT and CCL2Ϫ/Ϫ mice (A; n ϭ (1.27 0.14, n 6), and CXCL10 (2.6 0.21, n 6) in the Ͼ p Ͻ 0.001. Data represent the mean Ϯ SEM of two to three lungs of WT mice relative to uninfected controls was similar ( p ,ءء .(10–8 Ϫ/Ϫ experiments. Survival of infected mice was assessed (B; n ϭ 8–9). 0.05) in CCR2 mice (CCL2, 1.97 Ϯ 0.11; CCL7, 1.59 Ϯ 0.07; CXCL10, 2.75 Ϯ 0.13, n ϭ 8). Transcription of CCL3 (1.99 Ϯ 0.07, n ϭ 7) and CCL4 (2.04 Ϯ 0.06, n ϭ 7) in CCR2Ϫ/Ϫ mice Ͻ Ϫ/Ϫ Ϫ/Ϫ was increased ( p 0.001) in comparison to transcription in WT Inflammatory response in CCR2 and CCL2 mice (CCL3, 1.45 Ϯ 0.1; CCL4, 1.53 Ϯ 0.07, n ϭ 7). CCL5 was the Because CCR2Ϫ/Ϫ mice exhibit disturbed inflammatory cell re- only chemokine reduced ( p Ͻ 0.001) in CCR2Ϫ/Ϫ mice (0.69 Ϯ cruitment to infected tissues (19, 20, 22, 25, 26, 29, 30), we as- 0.03, n ϭ 7) in comparison to WT (1.18 Ϯ 0.09, n ϭ 7). sessed the absolute number and phenotype of inflammatory cells in We asked whether the impaired number in leukocyte popula- the lungs during H. capsulatum infection. At days 3 and 7, fewer tions in the lungs of CCR2Ϫ/Ϫ mice was present earlier than day leukocytes were recovered from the lungs of infected CCR2Ϫ/Ϫ or 7 after infection. At day 3, the percentage of monocytes, TipDC, CCL2Ϫ/Ϫ mice compared with WT (Fig. 2A). Between days 3 and and CD8Ϫ cDC in lungs of CCR2Ϫ/Ϫ mice was decreased as com- 7, the number of leukocytes increased ϳ4-fold in CCR2Ϫ/Ϫ and pared with WT (Fig. 2D). Ϫ/Ϫ CCL2 mice, but this value was still decreased by 4.5-fold com- Ϫ/Ϫ pared with WT. Histology of lungs from infected CCR2 mice The diminished number of inflammatory cells in the lungs of Histopathology of the lungs from WT and CCR2Ϫ/Ϫ mice 7 days CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice prompted us to examine specific after infection indicated similar alterations in lung pathology be- leukocyte populations in lungs. The percentage of CD4ϩ and tween the two groups. WT and CCR2Ϫ/Ϫ mice manifested mod- CD8ϩ T cells was similar among WT, CCR2Ϫ/Ϫ, and CCL2Ϫ/Ϫ erate to severe multifocal peribronchiolar pyogranulomatous pneu- mice (Fig. 2B), but the numbers (mean Ϯ SEM ϫ 104) were de- monia, moderate to severe lymphocyte cuffing, and mild to creased ( p Ͻ 0.05) in CCR2Ϫ/Ϫ (CD4ϩ, 5.8 Ϯ 0.2; CD8ϩ, 6.4 Ϯ moderate edema. ϭ Ϫ/Ϫ ϩ Ϯ ϩ Ϯ 0.1, n 12) and CCL2 mice (CD4 , 4.6 1.0; CD8 , 5.2 Ϫ/Ϫ Ϫ/Ϫ 0.1, n ϭ 7) in comparison to WT (CD4ϩ, 23.4 Ϯ 0.3; CD8ϩ, Cytokine profile in the lungs of CCR2 and CCL2 mice 18.3 Ϯ 0.3, n ϭ 11). We analyzed the number of activated CD4ϩ We analyzed cytokine transcripts in lungs by qRT-PCR to deter- T cells in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice since defects in T cell mine whether the absence of CCR2 or CCL2 affected the polar- activation have been attributed to loss of CCR2 (22, 31). ization of the immune response upon H. capsulatum infection. CD3ϩCD4ϩCD25ϩCD69ϩ T cells (mean ϫ 104 Ϯ SEM) were CCR2Ϫ/Ϫ or CCL2Ϫ/Ϫ mice did not manifest a decrement in tran- decreased ( p Ͻ 0.001) in both CCR2Ϫ/Ϫ (0.64 Ϯ 0.13, n ϭ 10) scription of IL-10, IL-12, IFN-␥, TNF-␣, or GM-CSF over the The Journal of Immunology 1967

FIGURE 2. Inflammatory response in the lungs of infected CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice. Lung leukocytes were isolated 3 and 7 days after infection and enumerated (A; n ϭ 12–20). Representative FACS plots of leukocytes from the lungs of WT, CCL2Ϫ/Ϫ, and CCR2Ϫ/Ϫ mice 7 days after infection to demonstrate gating for monocyte, M␾, and DC populations. The majority of CD11cϩ cells in the lungs of infected mice were DC at day 7 as demonstrated by I-Ab high expression (18, 55). Downloaded from Two distinct populations of CD11cϩ cells were identi- fied by the level of Ly6C expression. TipDC were high Ly6C CD11cintI-Ab high (23, 56). CD8Ϫ cDC were int Ly6C CD11chighI-Ab high (18, 21). Inflammatory monocytes were defined as CD11cϪLy6Chigh (20, 57). Mono- cyte and DC populations were CD11bhigh, CD8Ϫ cDC Ϫ ␣ were CD8 , and many TipDC produced TNF- when http://www.jimmunol.org/ stained intracellularly (data not shown). Alveolar M␾ were CD11cϩI-Ab low/intMac3high (33, 58) and tissue M␾ were Mac3ϩCD11cϪ (33). Whereas alveolar M␾ were CD11blow/int, tissue M␾ were CD11bint/high (data not shown) (B). Percentages of examined leukocyte populations 7 days after infection in the lungs of WT, CCR2Ϫ/Ϫ, and CCL2Ϫ/Ϫ mice were determined by flow cytometry. CD4ϩ and CD8ϩ T cells also expressed CD3, neutrophils were CD11bhighCD11cϪLy6Ghigh (C; n ϭ 8–12). Leukocyte populations 3 days after infection by guest on September 29, 2021 p Ͻ ,ءء .(in WT and CCR2Ϫ/Ϫ mice (D; n ϭ 8–12 0.001 vs WT; ##, p Ͻ 0.001 vs CCL2Ϫ/Ϫ. Data represent the mean Ϯ SEM of two to three experiments.

course of infection as compared with WT (Fig. 3). Transcription of IL-1␤, a proinflammatory cytokine that contributes to host immu- Table I. Absolute number of myeloid populationsa nity to H. capsulatum infection (32), was not different between CCR2Ϫ/Ϫ mice and WT. In contrast, an elevation in IL-4 was Ϫ Ϫ Mean Cell No. Ϯ SEM ϫ 105 observed at days 3, 5, and 7 in CCR2 / mice (Fig. 3). GM-CSF Ϫ/Ϫ Phenotype WT CCL2Ϫ/Ϫ CCR2Ϫ/Ϫ transcription was also significantly increased in CCR2 mice at day 3 (Fig. 3). Increased IL-4 in the lungs of CCR2Ϫ/Ϫ mice was b c Neutrophil 1.67 Ϯ 0.12 1.11 Ϯ 0.13 1.78 Ϯ 0.23 not a result of a preexisting bias in expression of this cytokine. Ϯ Ϯ b Ͻ b,c Monocyte 0.59 0.67 0.23 0.05 0.01 Ϯ TipDC 1.63 Ϯ 0.10 0.54 Ϯ 0.09b 0.10 Ϯ 0.01b,c Mean log10 ( SEM) transcription of IL-4 within the lungs of un- Ϫ/Ϫ CD8Ϫ cDC 1.56 Ϯ 0.29 0.71 Ϯ 0.10b 0.15 Ϯ 0.03b,c infected CCR2 mice was only 0.07 Ϯ 0.1 (n ϭ 4) greater than Alveolar M␾ 0.40 Ϯ 0.07 0.21 Ϯ 0.03b 0.12 Ϯ 0.02b,c that of WT mice and not statistically significant ( p Ͼ 0.05). ␾ Ϯ Ϯ Ϯ Tissue M 1.57 0.20 1.07 0.20 1.57 0.21 One explanation for the increase in IL-4 may be the elevated a Absolute numbers of leukocytes 7 days after infection. Data represent the fungal burden in the lungs. Accordingly, we infected WT mice mean Ϯ SEM of two to three experiments (n ϭ 8–12). ϫ 7 ϳ b p Ͻ 0.001 vs WT. with 1 10 yeast i.n., which results in a 10-fold increase in c p Ͻ 0.001 vs CCL2Ϫ/Ϫ. fungal burden in the lungs of WT mice 7 days after infection. IL-4 1968 H. capsulatum INFECTION IN CCR2Ϫ/Ϫ MICE

FIGURE 3. Cytokine transcription in the lungs of infected CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice. Whole lung RNA was prepared at the indicated time points after infection for use in subsequent qRT-PCR analysis. Transcription is expressed as log10 relative quantiﬁca- tion (RQ) relative to uninfected WT ,ءء p Ͻ 0.01 and ,ء .(lung. (n ϭ 5–8 p Ͻ 0.001. Data represent the mean Ϯ SEM of two to three experiments.

Ϯ Downloaded from transcription (mean log10 SEM) at day 7 in WT mice infected mokine receptor. The absence of IL-4 caused a modest and tran- with 1 ϫ 107 yeast cells was increased (0.21 Ϯ 0.81, n ϭ 4) sient alteration in fungal burden in lungs as compared with WT compared with uninfected controls but not signiﬁcantly different (Fig. 5E). ( p Ͼ 0.05) as compared with WT mice infected with 2 ϫ 106 yeast ϩ Ϫ/Ϫ cells (0.46 Ϯ 0.07, n ϭ 3). These results indicate that fungal bur- Characterization of IL-4 cells in CCR2 mice den alone cannot account for increases in IL-4. IL-4-secreting cells were isolated from pools of infected lungs at

The elevation in IL-4 was confined to the lungs. Mean log10 day 7 of infection and analyzed for surface phenotype (Fig. 6A). http://www.jimmunol.org/ (ϮSEM) IL-4 transcription in spleens of infected WT mice Approximately 60% of the IL-4ϩ cells in CCR2Ϫ/Ϫ mice were (0.13 Ϯ 0.1, n ϭ 4) did not differ ( p Ͼ 0.05) from that of CD11cϩ and ϳ50% of those were Mac3high (Fig. 6, A and B). The Ϫ Ϫ CCR2 / mice (0.095 Ϯ 0.03, n ϭ 4) 7 days postinfection. IL-4-secreting CD11cϩ cells also expressed intermediate to high We assessed protein concentrations of several cytokines to con- levels of CD11b and I-Ab. demonstrating that these cells were firm the findings with qRT-PCR. IL-4 was increased within the lungs of CCR2Ϫ/Ϫ mice at day 7 postinfection (Fig. 4). IL-10, IL-12, IFN-␥, TNF-␣, GM-CSF, and IL-1␤ did not differ between CCR2Ϫ/Ϫ and WT mice (Fig. 4). by guest on September 29, 2021 To ensure that a defect in TH1 polarization and subsequent IFN-␥ production did not occur in the lungs of CCR2Ϫ/Ϫ mice, we performed intracellular staining to detect IFN-␥- producing CD4ϩ T cells 7 days postinfection. The number of IFN-␥-producing CD4ϩ T cells (mean Ϯ SEM ϫ 104) did not differ ( p Ͼ 0.05) between WT (7.9 Ϯ 0.1, n ϭ 4) and CCR2Ϫ/Ϫ mice (5.1 Ϯ 0.3, n ϭ 4). We examined IFN-␥ transcription from FACS sorted CD3ϩ T cells from the lungs at day 7. Transcription of IFN-␥ Ϯ ϩ Ϫ/Ϫ (log10 SEM) in CD3 T cells from CCR2 mice was only 0.013 Ϯ 0.007 lower relative to WT (n ϭ 8 pooled mice per group).

Neutralization of IL-4 restores protective immunity in CCR2Ϫ/Ϫ mice Neutralization of IL-4 produced a decrement in fungal burden and restored protective immunity in CCR2Ϫ/Ϫ mice (Fig. 5, A and B). All CCR2Ϫ/Ϫ mice given mAb to IL-4 and WT mice were sacri- ﬁced after 42 days and lungs were cultured for the presence of H. capsulatum. CFU was Ͻ102 in WT mice and ranged from Ͻ102 to 103 in CCR2Ϫ/Ϫ mice. The data indicate that neutralization of IL-4 promoted clearance of the fungus. We ascertained whether the salutary effect of IL-4 neutralization was a consequence of an improvement in the inﬂammatory response to H. capsulatum. Treatment with mAb to IL-4 did not increase the number of leukocytes in the lungs of CCR2Ϫ/Ϫ mice at day 7 (Fig. 5C). The percentages of CD8␣Ϫ cDC, TipDC, and monocytes in IL-4-neutralized CCR2Ϫ/Ϫ mice were similar to those of infected controls (Fig. 5D). Ϫ/Ϫ We assessed fungal burden in the lungs and spleens of IL-4 FIGURE 4. Cytokine protein levels in lung homogenates 7 days post Ϫ/Ϫ mice to determine whether neutralization of IL-4 in CCR2 infection. Protein concentrations were determined by ELISA. (n ϭ 6–12). could result in effects on immunity unrelated to lack of the che- *, p Ͻ 0.05. Data represent the mean Ϯ SEM of 2–3 experiments. The Journal of Immunology 1969

FIGURE 5. Neutralization of IL-4 in CCR2Ϫ/Ϫ mice prevents mortality from H. capsulatum infection. CCR2Ϫ/Ϫ mice treated with 1 mg of anti-IL-4 or 1 mg of rat IgG and WT mice were infected with 2 ϫ 106 yeast cells and fungal burden was assessed 7 days after infection (A; n ϭ 7–8). Sur- vival of infected mice was monitored (B; n ϭ 6). At day 7, inﬂammatory cell

recruitment was evaluated. Absolute Downloaded from numbers of leukocytes within the lungs were enumerated (C) and relative percentages of leukocyte populations were analyzed by ﬂow cytometry (D). Fungal burden in the lungs and spleens of WT and IL-4Ϫ/Ϫ mice over the course of infection (E; n ϭ 8–12). http://www.jimmunol.org/ p Ͻ 0.05. Data ,ء p Ͻ 0.001 and ,ءء represent the mean Ϯ SEM of two to three experiments. by guest on September 29, 2021

from the myeloid lineage (Fig. 6A). The percentage of IL-4-se- populations, lung leukocytes were sorted and transcription was creting cells expressing CD11c and Mac3high or Mac3low/int was assessed by qRT-PCR. Because sorting of phagocytic cells similar in WT and CCR2Ϫ/Ϫ mice (Fig. 6B). CD11cϪMac3ϩ tis- could result in DC-T cell clusters, cells expressing CD3 were sue M␾ (33), CD3ϩCD4ϩ T cells, NK1.1ϩCD3Ϫ NK cells, excluded to ensure that T cell contamination did not account for NK1.1ϩCD3ϩ NKT cells, and CD11cϪFC␧R1ϩFSC/SSClow ba- IL-4 transcription in sorted phagocytic cell populations. Sorted sophils secreted IL-4 in the lungs of both WT and CCR2Ϫ/Ϫ mice, CD11cϩMac3high alveolar M␾, CD11cϩMac3low/int DC, and although these populations comprised a lesser proportion of the Mac3ϩCD11cϪ tissue M␾, as well as CD4ϩCD3ϩ T cells from total IL-4ϩ cells than those expressing CD11c (Fig. 6B). CCR2Ϫ/Ϫ mice 7 days after infection exhibited increased IL-4 We sorted IL-4-secreting populations that were CD11cϩ transcription in comparison to the respective WT population Mac3high or CD11cϩMac3low/int from CCR2Ϫ/Ϫ mice and per- (Fig. 6D). formed cytospins to conﬁrm the morphology of both populations ϩ Ϫ/Ϫ (Fig. 6C). The IL-4-secreting, CD11cϩMac3high cells were highly Depletion of CD4 T cells from CCR2 mice vacuolated and manifested a high cytoplasm:nuclei ratio charac- To determine whether IL-4 production by CD4ϩ T cells contrib- teristic of alveolar M␾. Many yeast cells were associated with uted to the demise of CCR2Ϫ/Ϫ mice, we depleted CD4ϩ T cells ϩ high Ϯ CD11c Mac3 cells (Fig. 6C, arrows). Approximately 45% of and assessed fungal burden in the lungs. CFU (mean log10 the Mac3ϩCD11chigh cells appeared to contain H. capsulatum SEM) in the lungs of CD4ϩ T cell-depleted CCR2Ϫ/Ϫ mice 7 days yeast cells. after infection (8.6 Ϯ 0.08, n ϭ 8) was similar ( p Ͼ 0.05) to The IL-4-secreting CD11cϩMac3low/int population exhibited a infected CCR2Ϫ/Ϫ mice given rat IgG (8.2 Ϯ 0.2, n ϭ 8). typical DC morphology. CD11cϩMac3low/int cells were smaller, Expression of IL-4-regulated genes and proteins in lungs of not as highly vacuolated, the cytoplasm:nuclei ratio was lower, and Ϫ/Ϫ characteristic dendrites were observed. Approximately 23% of CCR2 mice these cells were infected when we analyzed the cytospins. Slides We determined the impact of IL-4 by evaluating other downstream were examined for eosinophils, which express CD11c (34), but no targets of this cytokine. Arginase-1 (Arg-1) is induced by IL-4 (35) cells with characteristic granules were detected. and promotes survival of intracellular organisms (36, 37). Because The above experiments deﬁned by phenotype the populations Arg-1 utilizes the same substrate as iNOS, L-arginine, increased that generated IL-4. To quantify IL-4 production by the various Arg-1 can deplete cells of L-arginine, resulting in decreased NO 1970 H. capsulatum INFECTION IN CCR2Ϫ/Ϫ MICE

FIGURE 6. Phenotype of IL-4-secreting lung leukocytes. Representa- tive plots of IL-4-secreting cells from the lungs of WT and CCR2Ϫ/Ϫ mice 7 days after infection. IL-4ϭpositive cells were gated on to determine phe- ϩ notype (A). Percentage of IL-4 leu- low kocytes expressing the indicated surface markers (B; n ϭ 3–5, 2–4 lungs pooled/group). Magniﬁcation (ϫ100) of FACS-sorted populations from the lungs of 10 pooled CCR2Ϫ/Ϫ mice 7 days after infection. Arrows indicate

H. capsulatum yeast (C). Log10 RQ IL-4 transcription from FACS-sorted Downloaded from populations. Data are expressed relative to WT lung RNA from uninfected mice (D; n ϭ 2–4, 8–12 pooled lungs/ p Ͻ 0.05 ,ء p Ͻ 0.001 and ,ءء .(group Data represent the mean Ϯ the SEM of three to ﬁve experiments. http://www.jimmunol.org/

production (35, 38, 39). Increased Arg-1 can also be beneficial to CCL7 and CCL12 concomitantly in WT mice or neutralization of intracellular organisms because it contributes to polyamine syn- CCL12 in CCL2Ϫ/Ϫ mice did not impair immunity (Fig. 8, A–C) by guest on September 29, 2021 thesis (37). Transcription of Arg-1 was increased in lung leuko- despite a decrement in the absolute number of inflammatory lung Ϫ Ϫ cytes from CCR2 / mice in comparison to WT 7 days after in- leukocytes compared with WT mice (Fig. 8D). In contrast, neu- fection, although not earlier (Fig. 7A). IL-4 can inhibit iNOS tralization of CCL7 in CCL2Ϫ/Ϫ mice resulted in increased IL-4 generation (38), but iNOS transcription was similar in leukocytes transcription and fungal burden compared with goat IgG-treated Ϫ/Ϫ from the lungs of WT and CCR2 mice at days 3, 5, and 7 after WT or CCL2Ϫ/Ϫ mice (Fig. 8, A and B). In addition, the relative infection (Fig. 7A). Examination of NO production revealed no percentage of CD8Ϫ cDC was decreased and the relative percent- Ϫ/Ϫ disturbances from lung leukocytes from CCR2 mice in com- age of neutrophils increased in CCL7-neutralized CCL2Ϫ/Ϫ mice parison to WT (Fig. 7B). in comparison to controls (Fig. 8C). IL-4 induces alternative activation of M␾ and these cells are defective in their ability to degrade intracellular organisms (40, 41). Elevated Arg-1 expression often accompanies alternative ac- Discussion tivation of M␾ (41, 42). To determine whether phagocytic cells Ϫ Ϫ These data demonstrate that CCR2, but not CCL2 alone, was es- were alternatively activated in CCR2 / mice, we analyzed tran- sential for resolution of H. capsulatum infection. Although scription of YM1 and FIZZ1, two markers of alternative activation Ϫ/Ϫ Ϫ/Ϫ in sorted phagocytic populations, as well as Arg-1 (43). YM1 and CCR2 and CCL2 mice displayed severe reductions in the Arg-1 were increased in CD11cϩMac3high alveolar M␾ and number of inflammatory cells in lungs, only the former developed Mac3ϩCD11cϪ tissue M␾ from CCR2Ϫ/Ϫ mice in comparison to a progressive infection associated with elevated IL-4 and alterna- the respective WT populations (Fig. 7C). YM1 and Arg-1 were tive activation of phagocytic cells. The deleterious effect of IL-4 Ϫ/Ϫ increased in CD11cϩMac3low/int DC from CCR2Ϫ/Ϫ mice in com- was evidenced by the ability of CCR2 mice to survive infection parison to WT DC (Fig. 7C). There was no difference in FIZZ1 upon IL-4 neutralization independent of an effect on inflammatory transcription between WT or CCR2Ϫ/Ϫ tissue M␾ (Fig. 7C). cell recruitment. Disruption of CCL7 and CCL2 signaling mim- Transferrin receptor expression (mean MFI Ϯ SEM), another icked the absence of CCR2. These results demonstrate that CCL7 marker of alternative activation (40), was increased on alveolar in conjunction with CCL2 are involved in the regulation of IL-4 M␾ as well as DC from CCR2Ϫ/Ϫ mice in comparison to WT, and immunity to H. capsulatum infection. although expression was similar on tissue M␾ (Fig. 7D). Reduced recruitment of myeloid lineage cells, disturbed cytokine production, and exacerbated infection observed in CCR2Ϫ/Ϫ Regulation of IL-4 by CCR2 ligands mice have been at least partially attributed to loss of CCL2 sig- To ascertain which ligands mediated control of IL-4 production naling (19, 20, 24). An intact CCR2-CCL2 axis is obligate for and immunity to H. capsulatum infection, we neutralized CCR2 resolution of Toxoplasma gondii infection. Lack of CCR2 or CCL2 ligands in WT or CCL2Ϫ/Ϫ mice. Neutralization of CCL7 or decreases recruitment of Gr-1ϩ monocytes in association with an The Journal of Immunology 1971

recruitment or the TH1toTH2 shift or both contribute to increased fungal burden remains unresolved. We found that the absence of CCL2 did not induce a polarized cytokine response, a ﬁnding quite different from that in experimental cryptococcosis. Similar to C. neoformans and Leishmania major infection of CCR2Ϫ/Ϫ mice, H. capsulatum infection resulted in increased generation of IL-4. However, elevated IL-4 in H. capsulatum-infected CCR2Ϫ/Ϫ mice was not accompanied by decreased generation of ␥ TH1 cytokines, including IFN- as occurs during C. neoformans and L. major infection (24, 25, 29). Thus, a reduction in these mediators is not the reason for exacerbation of infection. The data suggest that IL-4 is a highly potent inﬂuence on immunity even Ϫ/Ϫ when TH1 cytokine levels are maintained. However, CCR2 mice do not succumb to infection as quickly as mice lacking IFN-␥ ␣ or TNF- (6, 7), suggesting that TH1 cytokines partially impair fungal replication in the absence of CCR2. Infection of CCR2Ϫ/Ϫ mice with H. capsulatum also differs from Mycobacterium bovis infection of CCR2Ϫ/Ϫ mice in which a more aggressive infection

is attributable to a decrease in IFN-␥ without a concomitant in- Downloaded from crease in IL-4. The absence of CCR2 or CCL2 during H. capsulatum infection did not impair the generation of the proinﬂammatory mediators IL-12, IFN-␥, TNF-␣, GM-CSF, or IL-1␤. Therefore, the impairment in leukocyte migration and/or retention in lungs cannot be

attributed to the loss of these molecules. Moreover, production of http://www.jimmunol.org/ several chemokines that may contribute to the inflammatory cell recruitment was not diminished. Decreased DC number in lungs may contribute to defects in T cell activation and expansion observed in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice. Immature DC kill yeast cells (45), and the severe reduction in the number of TipDC and CD8␣Ϫ cDC in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice may limit their capacity to exert this action and thereby reduce the quantity of Ag that can be presented to T cells. Thus, the paucity of T cells in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice may by guest on September 29, 2021 FIGURE 7. Analysis of downstream targets of IL-4 signaling. Log10 RQ transcription of Arg-1 and iNOS from isolated lung leukocytes from result from a decreased number of DC-T cell interactions ensuing WT and CCR2Ϫ/Ϫ mice days 3, 5, and 7 after infection (A; n ϭ 6–8). in decreased expansion and not only decreased T cell emigration. Measurement of NO products nitrate and nitrite produced by ex vivo-stim- Disturbances in cell recruitment were not directly responsible Ϫ Ϫ ulated lung leukocytes isolated from WT and CCR2Ϫ/Ϫ mice 7 days after for the accelerated mortality in infected CCR2 / mice. The in- ϭ infection (B; n 5). Log10 RQ Arg-1, iNOS, YM1, and FIZZ1 transcrip- flammatory response both from a qualitative as well as quantitative tion from FACS-sorted populations 7 days after infection relative to whole assessment was unchanged in mice that received mAb to IL-4. lung transcription from uninfected WT mice (C; n ϭ 3–4, 8–12 pooled Additional support for the argument that the substandard inflam- lungs/group). MFI transferrin receptor (TR) expression of phagocytic pop- Ϫ/Ϫ matory response did not contribute greatly to impaired immunity is ulations from WT and CCR2 lungs 7 days after infection (D; n ϭ 8). Ϫ Ϫ that CCL2 / mice manifested many of the same alterations in p Ͻ 0.05. Data represent the mean Ϯ SEM of two to ,ء p Ͻ 0.001 and ,ءء four experiments. inflammatory cell recruitment yet did not succumb to infection. Nevertheless, a decrease in the numbers of cells concurrent with increased IL-4 may contribute to the failure to resolve infection. inability to control infection (19). Monocyte recruitment is de- More IL-4 molecules may be available to interact with fewer increased in CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice infected with L. mono- flammatory cells, therefore increasing the amount of cytokine en- cytogenes, but it is the deficit in TipDC that results in decreased gaging receptors on individual cells and modulating their effector TNF-␣ and iNOS production and impaired immunity (20, 23). In function. The above argument is supported by the finding that contrast, the absence of CCL2 was not a major influence on the transgenic mice overexpressing IL-4 only in the lungs resolve H. course of histoplasmosis. CCL2Ϫ/Ϫ mice exhibited a transient in- capsulatum infection despite exhibiting higher fungal burden in crease in fungal burden in the lungs and spleens but they cleared comparison to controls. These animals do not manifest a deficit in infection and survived. Elimination of yeast cells transpired even inflammatory cells (46). though phagocyte recruitment, including monocytes and TipDC, IL-4 is detrimental to resolution of Histoplasma infection when was profoundly disturbed. it is aberrantly increased as observed in infected CCR2Ϫ/Ϫ mice or In experimental Cryptococcus neoformans infection, the ab- when increased in combination with IL-10 as occurs in the absence sence of CCR2 or CCL2 is accompanied by a poor TH1 response of GM-CSF (9). Our data indicate that improved immunity in IL- Ϫ/Ϫ and a heightened TH2 response in addition to decreased recruit- 4-neutralized CCR2 mice does not result from effects on im- ment of M␾, DC, and CD8ϩ T cells in CCR2Ϫ/Ϫ mice or CD4ϩ munity that are independent of loss of CCR2, since IL-4Ϫ/Ϫ mice

T cells in CCL2-neutralized mice (25, 44). Trapping of TH1, but did not exhibit accelerated clearance of yeast. Early increase in Ϫ/Ϫ not TH2 cells in lymph nodes causes a skewing to type 2 immunity transcription of GM-CSF in CCR2 mice is unlikely to contrib- in lungs of CCL2-neutralized mice, whereas CCR2Ϫ/Ϫ mice lack ute to the inability of CCR2Ϫ/Ϫ mice to control infection. GM-

TH1 polarization (24). Whether the decreased inﬂammatory cell CSF acts as a protective cytokine during H. capsulatum infection 1972 H. capsulatum INFECTION IN CCR2Ϫ/Ϫ MICE

FIGURE 8. CCL7 in combination with CCL2 is necessary for immunity to H. capsulatum infection. Mice were infected with 2 ϫ 106 yeast cells and treated with the indicated neutralizing Abs or goat IgG. Fungal burden was assessed 7 days after ϭ infection (A; n 3–6). Log10 RQ IL-4 transcription from whole lungs 7 days after infection (B; n ϭ 3–6). Relative percentages of lung leukocyte populations 7 days after infection (C; n ϭ 3–6). Absolute number of lung leukocytes 7 days after in- p Ͻ 0.001 vs ,ءء .(fection (D; n ϭ 3–6 WT ϩ goat IgG and CCL2Ϫ/Ϫ ϩ goat IgG; p Ͻ 0.05 vs WT ϩ goat IgG and ,ء CCL2Ϫ/Ϫ ϩ goat IgG; and #, p Ͻ 0.05 vs WT ϩ goat IgG. Data represent the mean Ϯ SEM of two experiments (n ϭ 5–6 per group) with the exception of WT ϩ Downloaded from anti-CCL7 and WT ϩ anti-CCL7 and anti- CCL12 groups (n ϭ 3, one experiment).

(47). Thus, the early increase in IL-4 most likely contributes in- A great deal of literature regarding the biological activity of http://www.jimmunol.org/ ϩ dependently to the negative impact on immunity observed in IL-4 stems from one of its principal sources, the CD4 TH2 cell. CCR2Ϫ/Ϫ mice. In Histoplasma-infected CCR2Ϫ/Ϫ mice, CD4ϩ cells were a Our data suggest that CCR2Ϫ/Ϫ mice succumbed to infection source of IL-4 during H. capsulatum infection; however, loss of due to increased IL-4 in the lungs resulting in an inability to con- these cells in CCR2Ϫ/Ϫ mice did not diminish the fungal burden. trol yeast replication, not dissemination. Although lung histopa- Thus, it appears that IL-4 produced by other leukocyte populations thology was similar in CCR2Ϫ/Ϫ and WT mice, increased IL-4 contributes to impaired immunity. was specific to lungs, not spleens, and although fungal burden IL-4 induces alternatively activated M␾ that are advantageous increased in lungs over the course of infection, burden in spleens for the clearance of helminths, but promote survival of intracellular remained stationary. CCR2Ϫ/Ϫ mice did not succumb as a result of pathogens (40, 41, 51). In addition, in vitro evidence demonstrates by guest on September 29, 2021 excessive lung inflammation. CCR2Ϫ/Ϫ mice exhibited decreased that H. capsulatum replication is enhanced in bone marrow-de- numbers of inflammatory lung leukocytes and did not exhibit an rived M␾ treated with IL-4 (M. Winters and G. S. Deepe, Jr., exacerbated TH1orTH17 response (data not shown) as demon- unpublished observation). Alternatively activated cells manifest a strated by cytokine production. profile of phenotypic and genotypic characteristics that discrimi- The primary sources of IL-4 are T cells, basophils, mast cells, nate them from classically activated M␾. Among these are en- and eosinophils in TH2-eliciting infection (34). However, we hanced expression of Arg-1, although this is not an exclusive prop- found that CD11cϩMac3high and CD11cϩMac3low/int cells con- erty of alternative activation (35). Elevated expression of Arg-1 tributed to IL-4 production in H. capsulatum-infected WT and may enhance permissiveness of M␾ for intracellular pathogens CCR2Ϫ/Ϫ mice. Others have reported Mac3ϩCD11cϩ leukocytes including Francisella tularensis and C. neoformans (41, 42). The in the lung as alveolar M␾ (33), although Mac3 surface expression importance of Arg-1 expression in host defenses to intracellular has been reported on DC (48). By morphology the pathogens has been reinforced by studies demonstrating that mice Mac3ϩCD11chigh cells were characteristic of M␾ while the lacking Arg-1 in M␾ are more efficient in clearing T. gondii or Mac3ϪCD11clo/int cells appeared to be DC. The finding that alve- Mycobacterium tuberculosis (52). The observed increase in YM1, olar M␾ and DC were generators of IL-4 was unanticipated. Our Arg-1, and expression of transferrin receptor in M␾ from H. cap- results extend and expand other reports indicating that M␾ and DC sulatum-infected CCR2Ϫ/Ϫ mice suggest alternative activation that can generate IL-4 (41, 49, 50). Conventional sources of IL-4 such is deleterious to control of H. capsulatum replication. as basophils contributed to IL-4 production. Because we did not CCL7 is considered a crucial molecular effector in host resis- determine whether the population we termed basophils expressed tance depending on the model of infection. The functional at- c-kit, a mast cell marker, it is possible that mast cells also contributes of this chemokine are diverse. In murine listeriosis, CCL7 tributed to IL-4 production. promotes immunity by attracting monocytes and TipDC to in- Increased IL-4 production by CCR2Ϫ/Ϫ leukocytes most likely fected tissues. This activity is similar to that observed with CCL2. results from an increment in each cell’s capacity to generate this The absence of either CCL2 or CCL7 is associated with an ele- cytokine. The number of IL-4-generating leukocytes was not sig- vated bacterial burden compared with controls, but the magnitude nificantly increased in the lungs of CCR2Ϫ/Ϫ mice, yet all phago- is not the same as detected in CCR2Ϫ/Ϫ mice (27). Alternatively, cytic populations and CD4ϩ T cells transcribed more IL-4 than CCL7 production at the site of Leishmania infection promotes per- ϩ respective WT populations. The number of phagocytic cells se- sistence of infection through recruitment of IL-4 TH2 leukocytes creting IL-4 in CCR2Ϫ/Ϫ mice was greater than nonphagocytic (53). The findings herein differ considerably from those aforemen- populations, but the quantity produced does not appear to be tioned studies. In contrast to L. monocytogenes infection, CCL7 greater since all leukocyte populations transcribed equivalent was dispensable for protective immunity in murine histoplasmosis amounts of IL-4. as assessed by fungal burden. The inability to control infection The Journal of Immunology 1973 required the combined absence of CCL2 and CCL7. Furthermore, 8. Allendorfer, R., G. D. Brunner, and G. S. Deepe, Jr. 1999. Complex requirements the impairment in immunity could not be directly attributed to for nascent and memory immunity in pulmonary histoplasmosis. J. Immunol. 162: 7389–7396. altered monocyte or TipDC trafficking. Our results differed con- 9. Deepe, G. S., Jr., R. Gibbons, and E. Woodward. 1999. Neutralization of endog- siderably from those in experimental leishmaniasis. In histoplas- enous granulocyte-macrophage colony-stimulating factor subverts the protective mosis, CCL7 in conjunction with CCL2 was responsible for re- immune response to Histoplasma capsulatum. J. Immunol. 163: 4985–4993. 10. Zhou, P., M. C. Sieve, J. Bennett, K. J. Kwon-Chung, R. P. Tewari, straint, not production, of IL-4. To our knowledge, these pivotal R. T. Gazzinelli, A. Sher, and R. A. Seder. 1995. IL-12 prevents mortality in mice findings are the first demonstration of a cooperative role of CCL2 infected with Histoplasma capsulatum through induction of IFN-␥. J. Immunol. 155: 785–795. and CCL7 in regulating IL-4 production and, consequently, control 11. Cain, J. A., and G. S. Deepe, Jr. 1998. Evolution of the primary immune response of an intracellular infection. to Histoplasma capsulatum in murine lung. Infect. Immun. 66: 1473–1481. CCL2 and CCL7 may regulate IL-4 directly through engage- 12. Luther, S. A., and J. G. Cyster. 2001. Chemokines as regulators of T cell differ- entiation. Nat. Immunol. 2: 102–107. ment of CCR2 on DC. Bone marrow-derived DC lacking CCR2 13. Wong, M. M., and E. N. Fish. 2003. Chemokines: attractive mediators of the produce IL-4 and stimulate T cell IL-4 production to a greater immune response. Semin. Immunol. 15: 5–14. extent than WT bone marrow-derived DC (49), suggesting that 14. Kurihara, T., and R. Bravo. 1996. Cloning and functional expression of mCCR2, a murine receptor for the C-C chemokines JE and FIC. J. Biol. Chem. 271: signaling through CCR2 is necessary for limiting IL-4 production. 11603–11607. Alternatively, IL-4 production may be limited indirectly through 15. Sarafi, M. N., E. A. Garcia-Zepeda, J. A. MacLean, I. F. Charo, and A. D. Luster. CCR2ϩ cells recruited by CCL2 and CCL7. In support of the latter 1997. Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemo- Ϫ kine that is a structural and functional homologue of human MCP-1. J. Exp. Med. hypothesis, the relative percentage and number of CD8 cDC in 185: 99–109. Ϫ/Ϫ Ϫ/Ϫ the lungs of CCR2 and CCL7-neutralized CCL2 mice are 16. Sozzani, S., D. Zhou, M. Locati, M. Rieppi, P. Proost, M. Magazin, N. Vita, similarly depressed to a greater extent than that observed in J. van Damme, and A. Mantovani. 1994. Receptors and transduction pathways for Downloaded from Ϫ/Ϫ monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similari- CCL2 mice. ties and differences with MCP-1. J. Immunol. 152: 3615–3622. The role of chemokines in mediating protective immunity to H. 17. Traynor, T. R., and G. B. Huffnagle. 2001. Role of chemokines in fungal infec- capsulatum in humans is unknown. The importance of study of tions. Med. Mycol. 39: 41–50. 18. Lin, K. L., Y. Suzuki, H. Nakano, E. Ramsburg, and M. D. Gunn. 2008. CCR2ϩ chemokines in this infectious disease is highlighted by the recent monocyte-derived dendritic cells and exudate macrophages produce Influ- wave of cases of disseminated histoplasmosis in those receiving enza-induced pulmonary immune pathology and mortality. J. Immunol. 180: 2562–2572. TNF antagonists. Since TNF can modulate chemokine generation http://www.jimmunol.org/ 19. Robben, P. M., M. LaRegina, W. A. Kuziel, and L. D. Sibley. 2005. Recruitment (54), perhaps one of the underlying immune deficiencies is an al- of Gr-1ϩ monocytes is essential for control of acute toxoplasmosis. J. Exp. Med. teration in chemokine generation rather than a direct effect of TNF. 201: 1761–1769. The role of chemokines in controlling infection begins with an 20. Serbina, N. V., and E. G. Pamer. 2006. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor understanding of the mouse. In that way, we can eventually move CCR2. Nat. Immunol. 7: 311–317. to ask clinically relevant questions regarding the human condition. 21. Osterholzer, J. J., J. L. Curtis, T. Polak, T. Ames, G. H. Chen, R. McDonald, Our studies contribute to the understanding of the complexity of G. B. Huffnagle, and G. B. Toews. 2008. CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates host control of intracellular organisms and highlight the necessity in the lungs of mice infected with Cryptococcus neoformans. J. Immunol. 181: of regulating IL-4 during H. capsulatum infection. Downstream 610–620. effects of loss of signaling through CCR2 vary depending on what 22. Peters, W., H. M. Scott, H. F. Chambers, J. L. Flynn, I. F. Charo, and J. D. Ernst. by guest on September 29, 2021 2001. Chemokine receptor 2 serves an early and essential role in resistance to organism is eliciting infection, the dose of organism, and the route Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 98: 7958–7963. of infection, suggesting a multifunctional role of CCR2. Further 23. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, and E. G. Pamer. 2003. TNF/iNOS-producing dendritic cells mediate innate immune defense studies examining the initial stimulus that results in increased IL-4 against bacterial infection. Immunity 19: 59–70. Ϫ/Ϫ production in CCR2 mice upon H. capsulatum infection 24. Traynor, T. R., A. C. Herring, M. E. Dorf, W. A. Kuziel, G. B. Toews, and may identify previously unknown roles of CCR2 in controlling G. B. Huffnagle. 2002. Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity. J. Immu- infection. nol. 168: 4659–4666. 25. Traynor, T. R., W. A. Kuziel, G. B. Toews, and G. B. Huffnagle. 2000. CCR2 Acknowlegments expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection. J. Immunol. 164: 2021–2027. 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dendritic cell activity against Histoplasma capsulatum is mediated via phagoly- of alveolar macrophages but not pulmonary dendritic cells after pneumococcal by guest on September 29, 2021 sosomal fusion. Infect. Immun. 73: 6803–6811. challenge. J. Infect. Dis. 193: 205–213.