Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15( S)-: Identification of a Novel Pathway for Regulating Allergic This information is current as of October 2, 2021. Yeping Cai, Rakesh K. Kumar, Jiansheng Zhou, Paul S. Foster and Dianne C. Webb J Immunol 2009; 182:5393-5399; ; doi: 10.4049/jimmunol.0803874

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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

Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15(S)-Lipoxygenase: Identification of a Novel Pathway for Regulating Allergic Inflammation1

Yeping Cai,* Rakesh K. Kumar,† Jiansheng Zhou,* Paul S. Foster,‡ and Dianne C. Webb2*

The Ym1/2 lectin is expressed abundantly in the allergic mouse lung in an IL-13-dependent manner. However, the role of Ym1/2 in the development of allergic airways disease is largely unknown. In this investigation, we show that treatment of mice with anti-Ym1/2 Ab during induction of allergic airways disease attenuated mediastinal lymph node production of IL-5 and IL-13. Ym1/2 was found to be expressed by dendritic cells (DCs) in an IL-13-dependent manner and supplementation of DC/CD4؉ T cell cocultures with Ym1/2 enhanced the ability of IL-13؊/؊ DCs to stimulate the secretion of IL-5 and IL-13. Affinity chromatography identified 12/15(S)-lipoxygenase (12/15-LOX) as a Ym1/2-interacting protein and functional studies suggested that Ym1/2 pro- moted the ability of DCs to stimulate cytokine production by inhibiting 12/15-LOX-mediated catalysis of 12-hydroxyeicosatetra- Downloaded from enoic acid (12(S)-HETE). Treatment of DC/CD4؉ T cell cultures with the 12/15-LOX inhibitor baicalein enhanced, whereas 12(S)-HETE inhibited the production of Th2 cytokines. Notably, delivery of 12(S)-HETE to the airways of mice significantly attenuated the development of allergic airways inflammation and the production of IL-5 and IL-13. In summary, our results suggest that production of Ym1/2 in response to IL-13 promotes Th2 cytokine production and allergic airways inflammation by inhibiting the production of 12(S)-HETE by 12/15-LOX. The Journal of Immunology, 2009, 182: 5393–5399. http://www.jimmunol.org/ f the Th2 cytokines, IL-13 is a particularly potent and Notably, since T cells do not bind IL-13 or express an IL-13 re- important mediator of asthma. In human airways, ex- ceptor (10), the influence of IL-13 on T cell function is likely O pression of IL-13 is elevated in the bronchial mucosa of through an intermediary rather than as a direct effect on T cells. To atopic asthmatics compared with nonasthmatics (1) and further investigate intermediaries that may function downstream of IL-13, up-regulated in response to allergen provocation (2). Studies in we previously used a protein-profiling approach to demonstrate that mouse models indicate that IL-13 plays a key role in eosinophil the Ym2 chitinase-like lectin was progressively and abundantly up- recruitment, goblet cell hyperplasia, airway hyperresponsiveness, regulated in the lung during the development of allergic inflammation and some aspects of airway remodeling (3–8). Although involve- by a process that was dependent on CD4ϩ T lymphocytes and sig- ment of IL-13 in pathways regulating allergic airways disease is naling by IL-13 and IL-4 (7, 11). Notably, instillation of IL-13 into the by guest on October 2, 2021 well characterized, recent work in our laboratory has focused on lungs of naive mice was sufficient to induce high-level expression of the way in which IL-4 and IL-13 interact to modulate initiation of Ym2 (11). The closely related Ym1 isomer (91% identity with Ym2) immune processes in the allergic lung. We demonstrated that IL-4 was constitutively expressed in the mouse lung, but not up-regulated and IL-13 play integrated roles in the maturation of dendritic cells by allergic responses to the same extent as Ym2 (11). Because they (DCs)3 following allergen exposure and the ability of these cells to can only be distinguished by gene sequence analysis, many studies do ϩ regulate Th2 cytokine production by memory CD4 T cells (9). not specifically differentiate between these isomers and refer to these proteins collectively as Ym1/2. The observation that Ym1/2 is abundantly expressed during the *John Curtin School of Medical Research, Australian National University, Canberra, Australia; †Department of Pathology, School of Medical Sciences, University of New development of allergic airways disease has resulted in escalating South Wales, Sydney, Australia; and ‡Priority Research Centre for Asthma and Re- use of this protein as a convenient marker of allergic airway in- spiratory Disease, Faculty of Health and Hunter Medical Research Institute, Univer- flammation and alternatively activated macrophages (12–17). Im- sity of Newcastle, Newcastle, Australia portantly, human YKL-40, which is highly homologous to mouse Received for publication November 18, 2008. Accepted for publication February 14, 2009. Ym1/2, is similarly elevated in asthmatics and is correlated with The costs of publication of this article were defrayed in part by the payment of page airway remodeling, reduced forced expiratory volume in 1s, and charges. This article must therefore be hereby marked advertisement in accordance greater patient dependence on asthma medications (18). However, with 18 U.S.C. Section 1734 solely to indicate this fact. although the expression of YKL-40 parallels the severity of asth- 1 This work was supported by National Health and Medical Research Peter Doherty matic responses, it is not known whether “recovery of YKL-40 Training Fellowship 179841 (to D.C.W.), National Health and Medical Research Council Project Grant 366765 (to D.C.W. and R.K.), and National Health and Med- from these patients indicates either a causative or a sentinel role for ical Research Council Program Grant 224207 (to P.S.F.). this molecule in asthma” (18). 2 Address correspondence and reprint requests to Dr. Dianne C. Webb, Division of Some insight into the function of Ym1/2 has been generated by Molecular Bioscience, John Curtin School of Medical Research, Australian National studies showing that Ym1 was expressed in DCs from the draining University, Canberra, ACT, Australia 0200. E-mail: [email protected] lymph nodes of Brugia malayi-infected mice in a Th2-dependent 3 Abbreviations used in this paper: DC, dendritic cell; BALF, bronchoalveolar lavage fluid; 12(S)-HETE, 12-hydroxyeicosatetraenoic acid; 12/15-LOX, 12/15-lipoxygen- manner (13) and that treatment of DCs with the lipid-lowering , ase; MLN, mediastinal lymph node; WT, wild type; 15(S)-HETE, 15-hydroxyeico- simvastatin, stimulated the ability of DCs to promote Th2 biasing in satetraenoic acid; HpETE, hydroxyperoxyeicosatetraenoic acid; LDL, low-density li- cocultured CD4ϩ T cells by a mechanism dependent on Ym1 (19). poprotein; PPAR␥, peroxisome proliferator-activated nuclear ␥. Because these studies suggested that Ym1/2 plays a role in DC func- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 tion and as we have shown that IL-13 regulates both expression of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803874 5394 Ym1/2 REGULATES 12/15-LOX AND Th2 CYTOKINES

Ym1/2 and the maturation and function of DCs (9), we considered Ym1/2 was coupled for 20 h at 4°C (pH 9.0) by N-alkyl linkage that Ym1/2 could function as an interface between IL-13 and the to Reacti-Gel per the manufacturer’s recommendations (Pierce Biotech- ability of this cytokine to influence the functional interaction between nology). Remaining active sites on the gel were blocked with 1 M Tris (pH ϩ 8.5). The Ym1/2-coupled gel was then washed with lysis buffer: 0.5% DCs and CD4 T cells. Therefore, aims of the current study were to CHAPS detergent in 25 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM 1) characterize the role of Ym1/2 in regulating cytokine production in EDTA, and protease inhibitors (Roche Diagnostics). Two ϫ 108 cells from the lymphoid compartment during allergic airway inflammation, 2) the lungs of allergic BALB/c mice were solubilized in lysis buffer at identify the relevant target protein(s) with which Ym1/2 interacts, and 4°C for 2 h with gentle mixing. After removal of cell debris, solubilized cytosolic and membrane proteins were incubated with the Ym1/2-cou- 3) define the mechanistic pathway by which Ym1/2 may regulate the ϩ pled Reacti-Gel at 4°C with gentle mixing overnight. The mixture was functional interplay between DCs and CD4 T cells. poured into an empty column and then washed with PBS. The Ym1/2- intereacting proteins were eluted from the column using 2% SDS, 0.25 Materials and Methods M Tris, and 0.4 mM EDTA (pH 8.0). After concentrating, fractions Induction of allergic airways disease were analyzed by SDS-PAGE. Allergic airway inflammation was induced in 6- to 8-wk-old BALB/c wild- Identification of Ym1/2-interacting proteins type (WT) and IL-13Ϫ/Ϫ mice (20) by i.p. sensitization (day 0) with 50 ␮g of OVA (fraction V; Sigma-Aldrich) mixed with 20 ␮l of Rehydragel The protein band of interest was excised from the SDS-PAGE gel and (Reheis) made up to a total volume of 200 ␮l with 0.9% sterile saline. On subjected to in-gel tryptic digest for 16 h at 37°C. MALDI mass spec- days 12, 14, 16, and 19, all mice were challenged with an aerosol of 10 trometry was performed on peptides generated by tryptic digest by the mg/ml OVA in 0.9% saline three times for 30 min per day with 30-min Australian Proteome Analysis Facility (Macquarie University, Sydney, breaks between aerosols as previously described (9). In some experiments, New South Wales, Australia). Peptide masses were screened using mice were treated with 1 mg of purified rabbit polyclonal anti-Ym1/2 Ab SWISS-PROT and TREMBL databases. Downloaded from (11) or 1 mg of purified rabbit control Ab injected i.p. during the aerosol ϩ challenge period on days 12, 15, and 17. In other experiments, 40 ␮lof500 Purification of DCs and CD4 T cells for mixed cell cultures ng/ml 12-hydroxyeiosatetraenoic acid (12(S)-HETE; Cayman Chemicals) ϩ or vehicle control was delivered to the nares of fluorothane-anesthetized DCs and CD4 T cells were purified from MLN of 15–25 allergic mice per mice 2 h before each OVA challenge. Mice were sacrificed on day 20 and treatment group as described previously (9). Essentially, MLN were incu- bronchoalveolar lavage fluid (BALF) was obtained by cannulating the tra- bated at 37°C for 15 min in 2.5 ml of collagenase buffer (10 mM HEPES chea and gently flushing the airways with two 1-ml volumes of PBS. Lungs (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) containing 300 U/ml collagenase type IV (Worthington Biochem) and 200 were also dissected from the thoracic cavity for recovery of mediastinal http://www.jimmunol.org/ lymph nodes (MLN). All mice were treated according to Australian Na- U/ml DNase I (Roche Diagnostics). A cell suspension was prepared from tional University Animal Welfare guidelines (Protocol JMB32/07) and the collagenase-digested MLN by gently pushing the tissue through a cell housed in a specific pathogen-free facility. strainer as previously described (9). Cells were washed and incubated with Fc block (Fc␥II/IIIR; BD Biosciences) and then enriched for expression of ϩ ϩ Measurement of cytokine production by MLN CD11c DCs or CD4 T cells using the MiniMACS magnetic bead system according to the manufacturer’s recommendations (Miltenyi Biotec). The Cells from MLN were isolated, washed in MLC medium (9) and stimulated eluate was passed through a second column to enhance purity of the ϩ ϩ 5 ϩ with 1 mg/ml OVA in MLC medium for 68 h at 37°C and 5% CO2 in CD11c or CD4 T cells. For mixed cell cultures, 2 ϫ 10 CD4 T cells 96-well plates with 2 ϫ 106 cells/well. The concentrations of IL-5 and and 4 ϫ 104 DCs in 200 ␮l of MLC medium containing 200 ␮g/ml OVA IL-13 in the cell-free supernatant were measured by ELISA as described were plated per well of round-bottom 96-well plates and then incubated for elsewhere (9). The paired capture and detection IL-5 Abs were from BD 5 days at 37°C and 5% CO2. Negative controls consisted of the same by guest on October 2, 2021 Pharmingen and the IL-13 Abs were from R&D Systems. numbers of either CD4ϩ T cells or DCs. In some experiments, cocultures were treated during the 5-day incubation with either 16 or 32 ng/ml 12(S)- Western blots HETE, 10 ␮M baicalein (BIOMOL), purified Ym1/2 (2 ␮g/ml), or appro- Samples (2.5 ϫ 105 cells/lane) were electrophoresed on 4–12% NU-PAGE priate vehicle controls. gels according to the manufacturer’s recommendations (Invitrogen) and then electrophoretically transferred to a polyvinylidene difluoride mem- C18 extraction of culture supernatants and measurement of brane using a Multiphor Novablot semidry transfer system (Pharmacia Bio- 12(S)-HETE and 15(S)-HETE tech). The membrane was blocked, then probed with a 1/1000 dilution of ϩ rabbit anti-Ym1/2 Ab (11) or 1/2000 anti-12-lipoxygenase (murine leuko- DC/CD4 T cell culture supernatants were extracted by reverse-phase cyte) polyclonal Ab (Cayman Chemicals), and then with anti-rabbit alka- chromatography using SPE (C18) cartridges (Cayman Chemicals) as de- line phosphatase-conjugated Ab (Sigma-Aldrich). Alkaline phosphatase scribed by the manufacturer. Essentially, proteins were precipitated with was detected with stabilized Western blue substrate (Promega). , which was then evaporated. The dried sample was resuspended in 50 mM acetate buffer (pH 4.0) and applied to an activated C-18 column.

RT-PCR for Ym1/2, 12(S)-lipoxygenase (12-LOX), and GAPDH After washing with ultra-pure H2O and then hexane, hydrophobic material was eluted from the column with 1% methanol in ethyl acetate. After RNA was purified with TRIzol reagent (Invitrogen) and reverse transcribed evaporating the organic solvent, the HETE-containing sample was resus- using oligo(dT) and Superscript III Reverse Transcriptase (Invitrogen). pended in immunoassay buffer and 12(S)-HETE and 15(S)-HETE PCR specific for ym1/2 was performed using the forward primer 5Ј-CT were assayed using Correlate-enzyme immunoassay 12(S)-HETE or 15(S)- GATCTATGCCTTTGCTGG and the reverse primer 5Ј-CACAGATTCT HETE enzyme immunoassay kits per the manufacturer’s instructions (As- TCCTCAAAAGC for 30 cycles at an annealing temperature of 53°C. say Designs). These primers were designed from GenBank D87757 and generate a 340-bp PCR fragment. PCR specific for arachidonate Alox15 (leukocyte Statistical analysis type) was performed using the forward primer 5Ј-GAGCTGGTGTCAA GAGATCAC and the reverse primer, 5Ј-GGTCTTGATTAAATAAC The significance of differences between experimental groups was analyzed CAATCGAG for 35 cycles at an annealing temperature of 53°C. These using the Student unpaired t test. Values are reported as the mean Ϯ SEM. primers were designed from GenBank entry L34570 and generate a PCR Differences in means were considered significant if p Ͻ 0.05. fragment of 528 bp. PCR for the housekeeping gene, gapdh, was performed with the forward primer 5Ј-GCCAAGGTCATCCATGACAAC and the re- verse primer 5Ј-GTTGTCATTGAGAGCAATGCC for the same number Results of cycles used for either ym1/2 or Alox15 amplification. The primers were Ym1/2 enhances Th2 cytokine production in allergic mice designed from GenBank entry NM_008084 and generate a PCR fragment of 418 bp. To confirm that expression of Ym1/2 was regulated by IL-13 dur- ing the development of allergic airways disease, we compared ex- Preparation of Ym1/2 affinity matrix pression of Ym1/2 in the BALF of WT and IL-13Ϫ/Ϫ mice. Ym1/2 was highly purified from the BALF of allergic mice using anion Whereas Ym1/2 was barely detectable in the lungs of nonallergic exchange and size exclusion HPLC as described previously (11). Purified WT mice, it was highly expressed in allergic WT mice. In marked The Journal of Immunology 5395

Ym1/2 interacts with arachidonate 12-LOX To investigate molecular mechanisms underlying the ability of Ym1/2 to potentiate production of IL-5 and IL-13, we used affinity chroma- tography to identify proteins that interact with Ym1/2. Purified Ym1/2 (Fig. 2A) was covalently coupled to a solid-phase matrix and incu- bated with combined cytoplasmic and membrane fractions of deter- gent-solubilized lung cells from allergic mice. Nonbinding proteins were removed by washing the matrix with PBS (Fig. 2B, lanes 1–4) and Ym1/2-interacting proteins were then dissociated from the matrix with the strong anionic detergent SDS. A protein band of ϳ70 kDa was identified in the SDS eluate (Fig. 2B, lane 5) and excised from the gel for in-gel digestion with trypsin to generate peptide fragments. MALDI mass spectrometry was used to determine the size of these peptides and to generate a “peptide fingerprint” (Fig. 2C) that enabled protein identification using analysis with the SWISS-PROT and FIGURE 1. Ym1/2 enhances Th2 cytokine production in allergic mice. A, Ym1/2 expression in BALF of WT and IL-13Ϫ/Ϫ mice detected by TREMBL databases. Analysis of the molecular masses of the trypsin- Western blot with anti-Ym1/2 Ab. Lane 1, Nonallergic WT BALF; lane 2, generated peptides putatively identified the protein as arachidonate allergic WT BALF; lane 3, nonallergic IL-13Ϫ/Ϫ BALF; lane 4, allergic 12-LOX: murine leukocyte type (molecular mass, 75.4 kDa), also Ϫ/Ϫ

IL-13 BALF; and lane 5, purified Ym1/2. B, Th2 cytokine production known as 12/15(S)-lipoxygenase (12/15-LOX). Western blot con- Downloaded from by cultures of MLN from allergic mice treated with anti-Ym1/2 or control firmed the identity of the Ym1/2-interacting protein as 12/15-LOX Ab. Values are the mean Ϯ SEM of three assays per group. u, Control (Fig. 2D). -p Ͻ 0.05 for anti ,ء .Ab-treated mice and f, anti-Ym1/2 Ab-treated mice Ym1/2 Ab-treated mice compared with control Ab-treated mice. Data are DCs and CD4ϩ T cells differentially express Ym1/2 and 12-LOX representative of at least two independent experiments. Having identified 12/15-LOX as a Ym1/2-interacting protein,

we next investigated whether these proteins formed a functional http://www.jimmunol.org/ contrast, levels were extremely low in BALF from both nonaller- interaction that mediated the ability of Ym1/2 to influence Th2 gic and allergic IL-13Ϫ/Ϫ mice (Fig. 1A). Since Ym1/2 is a se- cytokine production by MLN cells. RT-PCR showed that both creted protein and known to be expressed by DCs in the draining ym1/2 and Alox15 were expressed in MLN from allergic mice lymph nodes of Brugia malayi-infected mice (13), we investigated (Fig. 3A), suggesting that Ym1/2 could both interact with 12/ whether treating mice with anti-Ym1/2 Ab during the development 15-LOX and exert its Th2-promoting effect in a localized man- of allergic airways disease influenced cytokine production by ner in the MLN. Analysis of ym1/2 and Alox15 expression re- MLN. We particularly focused on IL-5 and IL-13 because these vealed a distinct pattern in DCs and CD4ϩ T cells. In purified Th2 cytokines are recognized as important mediators of eosino- DCs, both ym1/2 and Alox15 were expressed in an IL-13-de- philic inflammation and allergic responses. When allergic mice pendent manner (Fig. 3, B and C). In contrast, CD4ϩ T cells by guest on October 2, 2021 were treated with anti-Ym1/2 Ab, the production of IL-5 and IL-13 expressed high levels of Alox15, although ym1/2 was barely were attenuated by 31 and 40%, respectively, when compared with detectable (Fig. 3, B and C). These findings suggested that a allergic mice treated with control Ab (Fig. 1B). These observations functional interaction between these proteins could occur either establish the enhanced secretion of Ym1/2 in the allergic state and in an autocrine fashion in WT DCs or in a paracrine fashion that the secreted form of Ym1/2 is able to potentiate Th2 cytokine such that Ym1/2 is secreted from DCs to influence the function production by MLN cells. of 12/15-LOX in CD4ϩ T cells.

FIGURE 2. Ym1/2 is a 12/15- LOX . A, SDS-PAGE of Ym1/ 2 purified by anion exchange and HPLC size-exclusion chromatogra- phy and used for preparing the Ym1/2 affinity matrix. B, SDS-PAGE of washes and eluate from solubilized lung cells applied to the Ym1/2 affin- ity column. Lanes 1–4, PBS washes and lane 5, SDS eluate. Arrow indi- cates the protein band excised for fur- ther analysis. C, MALDI spectrum of peptides derived from tryptic digest of the protein indicated in B. D, West- ern blot of the SDS eluate using anti- 12/15-LOX Ab to confirm identity of the protein band in B, lane 5. 5396 Ym1/2 REGULATES 12/15-LOX AND Th2 CYTOKINES

DC morphology (data not shown) was consistent with that previ- ously observed for MLN DCs purified using this method (9). Some low levels of contaminating B cells and macrophages were ob- served, but this was consistently below 5% of viable cells (Fig. 4A). Cocultures of WT DCs with WT CD4ϩ T cells exhibited significantly increased production of IL-5 (9-fold) and IL-13 (8- fold) compared with CD4ϩ T cells cultured alone and cytokine production was undetectable in DCs cultured without T cells (data not shown). IL-13Ϫ/Ϫ DCs were significantly less efficient at stim- ulating IL-13 production by T cells than WT DCs (Fig. 4C). How- ever, the addition of Ym1/2 to IL-13Ϫ/Ϫ DCs significantly en- hanced their ability to stimulate IL-5 and IL-13 production by cocultured T cells (Fig. 4, B and C). Because our data indicated that Ym1/2 interacted with 12/15-LOX (Fig. 2), we also investi- gated whether Ym1/2 influenced the function of 12/15-LOX in this FIGURE 3. Expression of ym1/2 and Alox15 in total MLN cells, MLN ϩ culture system. 12/15-LOX inserts molecular oxygen into arachi- CD4 T cells, and MLN DCs from allergic mice determined by RT-PCR. A, RT-PCR of ym1/2 and Alox15 in total MLN cells. Lane 1, ym1/2; lane 2, donic acid, resulting in the formation of 12(S)-HETE and 15(S)- 12/15-LOX; and lane 3, gapdh. B, RT-PCR of ym1/2 in MLN DCs and CD4ϩ HETE. Notably, mouse leukocyte 12/15-LOX produces three Ϫ/Ϫ times more 12(S)-HETE than 15(S)-HETE (21). Although 12(S)- T cells. Lane 1, ym1/2 WT DCs; lane 2, ym1/2 IL-13 DCs; lane 3, ym1/2 Downloaded from WT CD4ϩ T cells; lane 4, gapdh WT DCs; lane 5, GAPDH IL-13Ϫ/Ϫ DCs; HETE was detected in the supernatants of WT DC/WT CD4 co- and lane 6, gapdh WT CD4ϩ T cells. C, RT-PCR of Alox15 in MLN DC and cultures, these levels were amplified in cocultures with IL-13Ϫ/Ϫ ϩ CD4 T cells. Samples are the same as in B. Data are representative of at least DCs (Fig. 4D). Of particular interest was the observation that ex- two independent experiments. ogenous Ym1/2 suppressed 12(S)-HETE production in cocultures with IL-13Ϫ/Ϫ DCs (Fig. 4D). When cultured in the absence of DCs, WT CD4ϩ T cells produced high levels of 12(S)-HETE Ym1/2 modulates Th2 cytokine and 12(S)-HETE production (11.48 Ϯ 0.93 ng/ml), suggesting that the predominant source of http://www.jimmunol.org/ Because treatment of allergic mice with anti-Ym1/2 Ab attenuated 12/15-LOX in this system is the T cell and that coculture with DCs Th2 cytokine production and because we had shown that ym1/2 inhibits T cell production of 12(S)-HETE. Because 12/15-LOX can was expressed in DCs derived from MLN of allergic mice, we next also produce 15(S)-HETE, we determined whether Ym1/2 also in- investigated whether Ym1/2 potentiated the ability of DCs to stim- fluenced production of this eicosanoid. 15(S)-HETE was detected ulate Th2 cytokine production by CD4ϩ T cells. Because IL-13Ϫ/Ϫ in the supernatants of cocultures of WT or IL-13Ϫ/Ϫ DCs with WT DCs produced barely detectable levels of ym1/2 (Fig. 3B), we were CD4 T cells, but 15(S)-HETE was not significantly higher in cul- particularly interested in whether exogenous Ym1/2 could modify tures with IL-13Ϫ/Ϫ DCs (Fig. 4E). Although exogenous Ym1/2 Ϫ/Ϫ ϩ the ability of IL-13 DCs to stimulate CD4 T cells. DCs en- significantly suppressed production of 15(S)-HETE, it was only re- by guest on October 2, 2021 riched from MLN of allergic WT and IL-13Ϫ/Ϫ mice typically duced by ϳ30%, whereas 12(S)-HETE was suppressed by ϳ95%. showed high-level expression of I-Ad MHC class II on 63% of Collectively, these data show that Ym1/2 can enhance Th2 cytokine cells and the endocytic marker CD205 on 19% of cells (Fig. 4A). production, particularly IL-5, by CD4ϩ T cells. Additionally,

FIGURE 4. Treatment of DC/CD4ϩ T cell cultures with exogenous Ym1/2 amplifies Th2 cytokine production and suppresses 12(S)-HETE production. A, Expression of high levels of I-Ad and CD205 and low levels of the B cell marker CD19 and the macrophage marker F4/80 in DCs purified from MLN of allergic mice. B–D, Production of IL-5 (B), IL-13 (C), 12(S)-HETE (D), or 15(S)-HETE (E) by cocultures of either WT or IL-13Ϫ/Ϫ DCs with purified WT CD4ϩ T cells from MLN of allergic mice. Ym1/2 was also added to cocultures with IL-13Ϫ/Ϫ DCs. Values are the mean Ϯ SEM of three assays. f, WT DCs/WT CD4ϩ T cells with vehicle control; Ⅺ, IL-13Ϫ/Ϫ DCs/WT CD4ϩ T cells with vehicle control; and u, IL-13Ϫ/Ϫ DCs/WT CD4ϩ T cells with p Ͻ 0.05 for Ym1/2 treatment compared with vehicle control treatment of cocultures with IL-13Ϫ/Ϫ DCs. #, p Ͻ 0.05 for ,ء .␮g/ml purified Ym1/2 2 cocultures with IL-13Ϫ/Ϫ DCs compared with those with WT DCs. Data are representative of at least two independent experiments. The Journal of Immunology 5397

tures containing WT DC (Fig. 4D), these cells were used to de- termine the effects of exogenous 12(S)-HETE. We found that in- creasing the levels of 12(S)-HETE in cultures significantly suppressed production of IL-5 and IL-13 (Fig. 5A). We then used the 12/15 LOX inhibitor baicalein to investigate the impact of 12/15-LOX on cytokine production. For this experiment, we used IL-13Ϫ/Ϫ DCs to stimulate WT CD4ϩ T cells since these cultures had produced the highest levels of 12(S)-HETE (Fig. 4D) and the effects of the 12/15-LOX inhibitor might be more pronounced un- der these conditions. In direct contrast to treatment with exogenous 12(S)-HETE, baicalein enhanced the ability of IL-13Ϫ/Ϫ DCs to ϩ FIGURE 5. 12(S)-HETE suppresses and the 12/15-LOX inhibitor ba- stimulate IL-5 and IL-13 production by WT CD4 T cells (Fig. icalein amplifies cytokine production by DC/CD4ϩ T cell cocultures. WT 5B). Although we cannot exclude the possibility that baicalein may and IL-13Ϫ/Ϫ DCs were cultured with WT CD4ϩ T cells. WT DC cultures influence other lipoxygenase-related , treatment of cul- were treated with 12(S)-HETE (A) and IL-13Ϫ/Ϫ DC cultures were treated tures with baicalein significantly inhibited 12(S)-HETE production with baicalein (B). u, Vehicle control; f, 16 ng/ml 12(S)-HETE (A)or (vehicle, 5.65 Ϯ 0.25; baicalein, 2.35 Ϯ 0.15 ng/ml), but, inter- baicalein (B)-treated cells; and Ⅺ, 32 ng/ml 12(S)-HETE. Values are the estingly, not 15(S)-HETE production (vehicle, 3.56 Ϯ 0.26; ba- p Ͻ 0.05 for treatment groups icalein, 3.50 Ϯ 0.14 ng/ml). These data demonstrate that 12/15- ,ء .mean Ϯ SEM of three assays per group compared with vehicle control groups. Data are representative of at least LOX activity suppresses Th2 cytokine production in the MLN of two independent experiments. mice with allergic airways disease. Downloaded from

12(S)-HETE production is amplified in the absence of both DC-de- 12(S)-HETE inhibits eosinophilia and Th2 cytokine production rived IL-13 and reduced endogenous Ym1/2 and is potently sup- in vivo pressed by exogenous Ym1/2. Because 12(S)-HETE suppressed Th2 cytokine production in vitro,

ϩ we tested the possibility that 12(S)-HETE may also attenuate Th2 http://www.jimmunol.org/ 12/15-LOX regulates the function of CD4 memory Th2 cells cytokine production and the severity of allergic responses in vivo. Because the levels of 12(S)-HETE in DC/CD4ϩ T cell cocultures When 12(S)-HETE was delivered intranasally to WT mice during correlated inversely with IL-5 and IL-13 production (Fig. 4), we aeroallergen challenge, the resulting blood neutrophilia and eosin- tested whether exogenous 12(S)-HETE could directly influence the ophilia, airway (cells in BALF) and peribronchial eosinophilia ability of DCs to stimulate CD4ϩ memory Th 2 cells. Because (Figs. 6, A–D), and production of IL-5 and IL-13 by MLN (Fig. lower levels of endogenous 12(S)-HETE were produced in cocul- 6E) were significantly attenuated when compared with vehicle by guest on October 2, 2021

FIGURE 6. Allergic inflammation and Th2 cytokine production are suppressed by 12(S)-HETE in vivo. The percentages of blood leukocytes (A) were determined by morphological criteria of May-Gru¨nwald-Giemsa-stained smears and the numbers of airway leukocytes (B) were quantified by correlating differential counts of May-Gru¨nwald-Giemsa-stained cytospins with the total cell counts in the BALF. Lung sections (C and D) were stained with Carbol’s chromotrope and the numbers of eosinophils (Eos) in the peribronchial region of 10 similar fields of ϫ1000 magnification per mouse were counted. Th2 cytokine production (E) was determined in MLN cells from 12(S)-HETE or vehicle-treated mice. A–C, Values are the mean Ϯ SEM five to six mice per p Ͻ 0.05 ,ء .group. D, Values are the mean Ϯ SEM of three assays per group. u, Vehicle-treated allergic mice and f, 12(S)-HETE-treated allergic mice for 12(S)-HETE-treated mice compared with vehicle-treated mice. Data are representative of at least three independent experiments with different con- centrations of 12(S)-HETE. Lymph, Lymphocyte; Neut, neutrophil. 5398 Ym1/2 REGULATES 12/15-LOX AND Th2 CYTOKINES control-treated mice. Collectively, these data identify 12(S)-HETE 12/15-LOX in or at the surface of the T cell. The mechanism by as a novel and potent inhibitor of allergy-driven eosinophilia and which secreted Ym1/2 inhibits 12/15-LOX, a predominantly cy- production of the Th2 cytokines IL-5 and IL-13 in vivo. toplasmic protein at first seems incongruous. However, 12/15- LOX has been extensively studied for its ability to oxidize low- Discussion density lipoproteins (LDLs), a critical step in the formation of In this study, we have identified Ym1/2 as an inhibitor of a pre- atherosclerotic plaques. Although 12/15-LOX predominantly lo- viously undisclosed 12/15-LOX/12(S)-HETE-dependent regula- calizes to the cytoplasm, treatment of macrophages with LDLs tory pathway that attenuates both the ability of DCs to stimulate induced a rapid translocation of 12/15-LOX to the plasma mem- Th2 cytokine production by CD4ϩ T cells and the development of brane (25). Additionally, 12/15-LOX expressed in J774A cells was allergic airways inflammation. capable of stereo-specific oxygenation of LDLs in the extracel- First, our data established that Ym1/2, which occurs in the al- lular medium, suggesting that the enzyme directly interacts with lergic state in response to IL-13, potentiates Th2 cytokine produc- extracellular LDL particles that make contact with the cell tion by MLN cells. To understand the molecular mechanisms membrane (26). 12/15-LOX also translocates to the cell mem- underlying this observation, we used an affinity chromatography- brane of emerging filopodia when macrophages are incubated based approach, which showed that Ym1/2 interacted with 12/15- with apoptotic cells (27). Considering these data, we think it LOX, the ortholog of human 15-LOX1 (22). Because inhibiting feasible that on appropriate activation of CD4 T cells, 12/15- Ym1/2 in vivo reduced Th2 cytokine production by MLN, we also LOX translocates to the plasma membrane, allowing its acces- investigated whether the interaction between Ym1/2 and 12/15- sibility to DC-derived Ym1/2. LOX regulated the function of MLN-derived DCs and CD4ϩ T In addition to its production of 12(S)-HETE, 12/15-LOX is cells, which are key players in initiating and maintaining Th2 cy- known to also produce 15(S)-HETE, but does so less efficiently to Downloaded from tokine production. Coexpression studies showed that ym1/2 and generate a 12(S)-HETE: 15(S)-HETE ratio of 3:1 (21). However, Alox15 transcripts were generated in WT DCs in an IL-13-depen- our data showed that in DC/CD4ϩ T cell cultures, 15(S)-HETE dent manner, whereas WT CD4ϩ T cells expressed Alox15, but not was produced more efficiently in relation to 12(S)-HETE than pre- ym1/2 transcripts. viously observed for 12/15-LOX. Additionally, 15(S)-HETE pro- Murine 12/15-LOX generates a variety of products including duction was not inhibited by the 12/15-LOX inhibitor baicalein 12(S)- and 15(S)-hydroxyperoxyeicosatetraenoic acid (HpETE) and Ym1/2, which we showed interacted with and inhibited 12/ http://www.jimmunol.org/ from . These HpETE derivatives are readily oxi- 15-LOX, was less efficient at inhibiting 15(S)-HETE than 12(S)- dized in vivo by cytosolic glutathione-dependent peroxidases to HETE production. Considering that 15(S)-HETE production per- the more stable 12(S)-and 15(S)-HETE derivatives (23), which are sists in 12/15-LOXϪ/Ϫ mice (Fig. E1 in Ref. 24), these data important components in biochemical pathways that have been suggest that 15(S)-HETE is also partially produced from non-12/ linked to a number of disease processes (22). However, because 15-LOX sources in our culture systems. the production of 12(S)-HETE by 12/15-LOX is three times more The observation that Ym1/2 potentiated Th2 cytokine produc- efficient than its production of 15(S)-HETE (21) and because it tion and concomitantly suppressed the production of 12(S)-HETE appears that 15(S)-HETE is also generated from non-12/15-LOX indicated that 12/15-LOX activity might directly modulate Th2 by guest on October 2, 2021 sources in the allergic mouse lung (Fig. E1 in Ref. 24), for the cytokine production. Indeed, when applied to DC/CD4ϩ T cell current study, we elected to focus specifically on the role of 12(S)- cocultures, exogenous 12(S)-HETE attenuated Th2 cytokine pro- HETE in regulating Th2- immune responses. A culture model was duction and, conversely, treatment with the 12/15-LOX inhibitor established using DCs and CD4ϩ T cells purified from the MLN of baicalein stimulated Th2 cytokine production. We then extended allergic mice and, to mirror the effects seen with Ab-mediated this observation to determine whether 12(S)-HETE influenced the neutralization of Ym1/2 in vivo, purified Ym1/2 was added to development of allergic airways disease in vivo. Compared with cocultures of these cells. Ym1/2 significantly potentiated the abil- vehicle control, intranasal delivery of 12(S)-HETE during aeroal- ity of IL-13Ϫ/Ϫ DCs to stimulate IL-5 production, in particular, in lergen challenge potently attenuated the severity of blood, lung, cocultured T cells, although IL-13 production was also enhanced. and airway eosinophilia and, consistent with our observations that Interestingly, the concentration of 12(S)-HETE was significantly 12(S)-HETE influenced T cell function in vitro, significantly in- higher in cocultures with IL-13Ϫ/Ϫ DCs than those with WT DCs, hibited Th2 cytokine production. suggesting an inverse relationship between Ym1/2 expression and To date, it is unclear how 12(S)-HETE regulates Th2 cytokine 12(S)-HETE production. Furthermore, addition of purified Ym1/2 production. However, a candidate pathway may be through regu- to cocultures with IL-13Ϫ/Ϫ DCs significantly suppressed the pro- lation of the peroxisome proliferator-activated nuclear receptor duction of 12(S)-HETE. When cultured without DCs, CD4ϩ T (PPAR)␥. 12/15-LOX is known to interfere with T cell prolifera- cells produced higher levels of 12(S)-HETE than when these cells tion by generating lipid metabolites, including 12(S)-HETE, that were cultured in the presence of either WT or IL-13Ϫ/Ϫ DCs, can function as ligands for PPAR␥ (28–30). Notably, treatment of ϩ ␥ ⌬ suggesting that the interaction between CD4 T cells and DCs mice with the endogenous PPAR ligand 15-deoxy- 12,14-PGJ2 suppressed production of T cell-derived 12(S)-HETE and that this or with synthetic ligand significantly reduces lung inflammation, could be mediated by the Ym1/2 secreted by DCs. mucus production, and remodeling following induction of allergic Since Ym1/2 and 12/15-LOX are coexpressed in WT DCs, it is airways disease (31, 32). Although further studies are required, the possible that these proteins interact within these cells. However, levels of 12(S)-HETE produced in the allergic lung would make the observation that cocultures containing IL-13Ϫ/Ϫ DCs, which it a more likely endogenous ligand of PPAR␥ than 15-deoxy- ϩ ⌬ express negligible levels of 12/15-LOX and CD4 T cells, pro- 12,14-PGJ2, which some have suggested is produced at too low duce high levels of 12(S)-HETE suggests that the 12(S)-HETE is a level to be considered a physiological ligand for PPAR␥ (33). generated predominantly by the T cell. This notion is compounded In summary, we have provided evidence for a mechanism by by our observation that CD4ϩ T cells cultured in the absence of which Ym1/2 produced by DCs down-regulates the activity of 12/ DCs produce even higher levels of 12(S)-HETE than when cul- 15-LOX and the resulting production of 12(S)-HETE, leading to tured with DCs. Because Ym1/2 is not expressed by the CD4ϩ T potentiation of the production of IL-5 and IL-13 by CD4ϩ T cells. cell, it seems likely that Ym1/2 secreted by DCs interacts with Although recent studies have suggested that products of the 12/ The Journal of Immunology 5399

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