Research ArticleImmunology

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide–dependent neutrophil recruitment

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Science Signaling  15 Jan 2019:
Vol. 12, Issue 564, eaar5514
DOI: 10.1126/scisignal.aar5514

Antifungal immunity

Zymosan is a yeast cell wall component that is recognized by various pattern recognition receptors. In mouse models, zymosan induces skin inflammation and arthritis. Nitric oxide (NO) and chemoattractants, such as leukotriene B4 (LTB4), are implicated in inducing neutrophil migration to sites of inflammation. Hyperactivation of this inflammatory response underlies some diseases associated with fungal infection. Takahashi et al. found that neutrophil recruitment to zymosan in mice deficient in the pattern recognition receptor CD300b was impaired compared to that in wild-type mice. Furthermore, inflammatory dendritic cells produced NO in response to zymosan and stimulated neutrophil recruitment in a CD300b-dependent manner. The authors identified phytosphingosine, a lipid component of zymosan, as a potential CD300b ligand, which suggests that this interaction may contribute to antifungal immune responses.

Abstract

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b−/− mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

INTRODUCTION

Fungi, which are classified into yeast and filamentous forms, are associated with various human diseases that result from the lack of immune recognition or hyperactivation of inflammatory response. The first step in the development of an immune response to fungal infection is the recognition of invading fungal pathogen-associated molecular patterns by several pattern recognition receptors, including Toll-like receptors (TLRs) and C-type lectin receptors, such as dendritic cell (DC)–associated C-type lectin 1 (dectin-1) and dectin-2 (14). Fungal cell walls contain glycoproteins, mainly mannose in the outer layer and chitin and β-glucan in the inner layer. Recognition of fungal β-glucan by dectin-1 present in monocytes, macrophages, and DCs induces cytokine production and internalization of the fungal pathogen (5, 6). In addition, dectin-1 amplifies signals produced by TLRs that recognize mannan-containing structures to enhance the inflammatory response to fungal infection (7). Zymosan is an insoluble preparation of the cell wall of yeast Saccharomyces cerevisiae and contains polysaccharides (e.g., β-glucan, mannan, and chitin) and lipids (8, 9). Zymosan is commonly used to induce sterile inflammation. Previous studies showed that several immune receptors, including dectin-1 and TLR2, and local nitric oxide (NO) and leukotriene B4 (LTB4) production are implicated in zymosan-induced inflammation in dorsal air pouches, arthritis models, or both (1016). NO is produced by various cells through inducible NO synthase (iNOS), neuronal NOS, and endothelial NOS. In particular, iNOS-derived NO in myeloid cells, such as macrophages, monocytes, DCs, and neutrophils, exerts numerous direct and indirect effects on microorganisms (17). On the other hand, LTB4, a powerful neutrophil chemoattractant, is produced not only by neutrophils but also by other cells, including macrophages, monocytes, mast cells, and epithelial cells (18, 19). However, mechanisms underlying the development of zymosan-induced inflammation involving the production of NO, LTB4, or both are incompletely understood. In this study, we identified cell populations similar to NO-producing inflammatory DCs or tumor necrosis factor (TNF)/iNOS-producing DCs (Tip-DCs) (20) as the main source of NO in zymosan-induced dorsal air pouch and arthritis models. Tip-DCs are characterized as CD11b+Ly-6C+MHC-II+ cells expressing high amounts of iNOS and are recruited in a C-C chemokine receptor type 2 (CCR2)–dependent manner during inflammation after infection (21, 22). Bone marrow (BM)–derived DCs generated in the presence of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (GM/IL-4–DCs) are similar to Tip-DCs (23).

CD300b, also called leukocyte mono-immunoglobulin–like receptor 5 (LMIR5), belongs to paired activating and inhibitory receptor family CD300 (also called LMIR). The CD300 family proteins CD300a (LMIR1) and CD300f (LMIR3) function as inhibitory receptors, whereas other CD300 family proteins, including CD300b (LMIR5), function as activating receptors (2428). Several CD300 family proteins recognize ceramides and sphingolipids and regulate cell type–dependent activation, including chemical mediator release or phagocytosis (2733). CD300b is broadly expressed by various myeloid cells and transduces an activating signal by interacting with 12-kDa DNAX-activating protein (DAP12), an adaptor protein that contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic region (26). T cell immunoglobulin and mucin protein-1 (TIM-1) and phosphatidylserine were identified as CD300b ligands (33, 34). The CD300b–TIM-1 interaction accelerates ischemia/reperfusion-induced acute kidney injury (34), and the CD300b-phosphatidylserine interaction induces the engulfment of apoptotic cells by macrophages (33). CD300b promotes lipopolysaccharide (LPS)–induced peritoneal inflammation through several mechanisms (35, 36). However, we found that CD300b deficiency did not affect LPS-stimulated neutrophil accumulation in a dorsal air pouch model (34) but profoundly decreased neutrophil accumulation in response to zymosan. In addition, we found that equivalent amounts of CD300b are expressed in neutrophils, inflammatory DCs, macrophages, and eosinophils in the dorsal air pouch exudates before treatment. Therefore, CD300b is suggested to spatially and temporally regulate immune cell activation and subsequent inflammation by recognizing specific ligands present in zymosan.

Sphingolipids contain a long-chain base, a fatty acid, and a polar head group. S. cerevisiae produces two types of long-chain bases, namely, dihydrosphingosine and its 4-hydroxy derivative phytosphingosine, which generates phytoceramide. In contrast, mammals contain only small amounts of dihydrosphingosine and phytosphingosine (9, 3739). Yeast sphingolipids play various roles in yeast cell function (9). However, the effect of yeast-derived sphingolipids on mammalian immune responses is unclear. In the present study, we identified phytosphingosine, a lipid component of zymosan, as a previously uncharacterized ligand of CD300b and found that the binding of phytosphingosine to CD300b in NO-producing inflammatory DCs induced zymosan-dependent neutrophil accumulation in dorsal air pouch and arthritis models.

RESULTS

CD300b deficiency reduces zymosan-induced neutrophil accumulation in the dorsal air pouches of mice

We previously found that CD300b deficiency did not affect LPS-induced neutrophil recruitment in the mouse model of dorsal air pouch inflammation (34). However, here, we found that injection of zymosan, an insoluble preparation of S. cerevisiae cell wall, into air pouches decreased the number of CD11b+Ly-6G+ neutrophils in air pouch exudates at both 1.5 and 4 hours after the injection in CD300b−/− mice compared with that in wild-type (WT) mice (Fig. 1A). However, the lack of DAP12, an adaptor protein for CD300b, did not influence zymosan-induced neutrophil recruitment in the same model (fig. S1), suggesting that several DAP12-coupled receptors may positively or negatively regulate this response. Because zymosan, a well-known TLR2 agonist, also contains β-glucan and mannan, we compared the number of neutrophils recruited to the air pouches in WT and CD300b−/− mice in response to Pam3CSK4 (a TLR1/2 agonist), FSL-1 (a TLR2/6 agonist), curdlan (a dectin-1 agonist), or mannan (a dectin-2 agonist). We observed that CD300b deficiency did not affect neutrophil recruitment to the air pouches in response to stimulation with any of these reagents (Fig. 1, B and C). However, injection of a heat-killed preparation of S. cerevisiae (HKSC) or the opportunistic yeast pathogen Candida glabrata (HKCG) into the air pouches decreased the number of neutrophils recruited to these air pouches in CD300b−/− mice compared with that in WT mice (Fig. 1D). LPS stimulation results in the cleavage of CD300b present on the surface of neutrophils, leading to the release of soluble CD300b (sCD300b) (35). However, we observed that zymosan did not induce the release of sCD300b either in vitro or in vivo (fig. S2, A to C), indicating that sCD300b was not involved in zymosan-induced neutrophil accumulation. Therefore, we hypothesized that the interaction between CD300b and its unknown ligands, including those present in zymosan, induced neutrophil accumulation in this animal model. Because LTB4, iNOS-dependent NO production, or both contribute to zymosan-induced inflammation in several animal models (1016), we measured the amounts of NO and LTB4 in air pouch exudates of WT and CD300b−/− mice injected with zymosan. We found that the amounts of NO (NO2 and NO3) and LTB4 levels were reduced in the air pouch exudates of CD300b−/− mice compared to those in WT mice at 1.5 hours after zymosan treatment (Fig. 1, E and F). These findings indicate that CD300b deficiency decreases neutrophil recruitment to zymosan-treated air pouches, which might be associated with decreased production of NO, LTB4, or both.

Fig. 1 CD300b deficiency reduces zymosan-induced neutrophil accumulation in the mouse model of dorsal air pouch inflammation.

(A to D) Numbers of neutrophils recruited into the dorsal air pouches of WT or CD300b−/− [knockout (KO)] mice before or at the indicated times after treatment with zymosan (n = 5 mice) (A); 4 hours after treatment with LPS (n = 4 mice), Pam3CSK4 (n = 6 mice), or FSL-1 (n = 6 mice) (B); 4 hours after treatment with curdlan or mannan (n = 6 mice per group) (C); or 4 hours after treatment with HKSC or HKCG (n = 6 mice per group) (D). (E and F) The amounts of NO2 + NO3 (n = 6 mice per group) (E) or LTB4 (n = 5 mice per group) (F) in the dorsal air pouch exudates of WT or CD300b−/− mice before or after the indicated times of zymosan treatment. Data are means ± SD and are representative of two independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test (A to E) or t test with Welch’s correction (D).

Reduced neutrophil accumulation in zymosan-treated air pouches of CD300b−/− mice is associated with decreased NO production

To clarify the exact role of NO in zymosan-induced neutrophil accumulation in the dorsal air pouches of mice, we pretreated WT or CD300b−/− mice with NG-nitro-l-arginine methyl ester (l-NAME), an NO synthesis inhibitor, or 1400 W dihydrochloride, a selective iNOS inhibitor, before treatment with zymosan. We found that pretreatment with l-NAME or 1400 W dihydrochloride decreased the number of neutrophils in the air pouch exudates of WT mice at 4 hours after zymosan treatment to levels comparable to those in CD300b−/− mice (Fig. 2A and fig. S3A). In addition, treatment with l-NAME or 1400 W dihydrochloride decreased the amounts of NO, LTB4, KC (keratinocyte chemoattractant), and MIP-2 (macrophage inflammatory protein 2) in the air pouch exudates of WT mice at 1.5 hours after zymosan treatment such that they were comparable to those in CD300b−/− mice (Fig. 2, B and C, and fig. S3, B to G). However, neither l-NAME nor 1400 W dihydrochloride statistically significantly affected neutrophil recruitment or production of NO, LTB4, KC, and MIP-2 in zymosan-treated CD300b−/− mice (Fig. 2, A to C, and fig. S3, A to G). In contrast, treatment with sodium nitroprusside (SNP), an NO donor, increased the number of neutrophils in the air pouch exudates of zymosan-treated CD300b−/− mice such that they were comparable to those in zymosan-treated WT mice; however, SNP treatment alone tended to induce neutrophil recruitment in CD300b−/− mice (Fig. 2D).

Fig. 2 Reduced neutrophil accumulation results from the decreased NO production in zymosan-treated air pouches of CD300b−/− mice.

(A) Numbers of neutrophils recruited to the dorsal air pouches of WT and CD300b−/− KO mice that were pretreated with vehicle or l-NAME for 30 min and then treated with zymosan for 4 hours (n = 5 mice per group). (B and C) The amounts of NO2 and NO3 (n = 6 mice per group) (B) and LTB4 (n = 5 mice per group) (C) in the pouch exudates of the indicated mice after 30-min pretreatment with vehicle or l-NAME and then 1.5 hours after injection with zymosan (n = 5 mice per group). (D) Numbers of neutrophils recruited to the dorsal air pouches of the indicated mice 4 hours after treatment with SNP alone, zymosan alone, SNP and zymosan, or vehicle (n = 5 mice per group). (E and F) The amounts of NO2 and NO3 (E) and LTB4 (F) in the pouch exudates of the indicated mice that were pretreated with an Ab against Gr-1 (anti–Gr-1) or a control Ab and then treated with zymosan for 1.5 hours (n = 4 mice per group). Data are means ± SD and are representative of two independent experiments. *P < 0.05 and **P < 0.01 by analysis of variance (ANOVA) with Holm-Šídák multiple comparison test (A to F). ns, not significant.

To investigate the involvement of neutrophils in LTB4 production, we intravenously treated the mice in different groups with an antibody (Ab) against Gr-1, a neutrophil-depleting Ab, before injecting zymosan. Flow cytometry analysis confirmed substantial depletion of neutrophils in air pouch exudates after zymosan treatment (fig. S4). Moreover, anti–Gr-1–induced neutrophil depletion reduced the amount of LTB4 in the air pouch exudates of zymosan-treated WT mice to amounts comparable to those in CD300b−/− mice but did not statistically significantly affect NO production in these mice (Fig. 2, E and F), indicating that neutrophils are the major source of LTB4, but not of NO, in air pouches. Thus, these results suggest that reduced neutrophil recruitment to the dorsal air pouches of zymosan-treated CD300b−/− mice results from decreased NO production.

NO-producing inflammatory DCs are responsible for the CD300b-dependent recruitment of neutrophils to zymosan-treated air pouches

To identify NO-secreting cells in zymosan-treated air pouches, we stained tissue sections of the skin covering the dorsal air pouches in zymosan-treated WT or CD300b−/− mice with Abs against CD300b, iNOS, and F4/80, which is expressed by macrophages and DCs. Skin sections obtained from zymosan-treated CD300b−/− mice contained few iNOS+ cells compared to those obtained from zymosan-treated WT mice (Fig. 3A), which was consistent with decreased NO production in the air pouch exudates of CD300b−/− mice. Note that iNOS+ cells also stained positively for both CD300b and F4/80 (Fig. 3A), suggesting that NO was produced by CD300b+F4/80+ cells in air pouches. Next, we stained cells present in the air pouch exudates of zymosan-treated WT or CD300b−/− mice with Abs against F4/80 and Ly-6C and found that F4/80Ly-6Cint cells, the major populations of air pouch exudate cells, were CD300b+ neutrophils (Fig. 3, B and C). In addition, we found that F4/80+ cells were subdivided into two distinct populations, namely, F4/80+Ly-6Chigh and F4/80+Ly-6Clow cells. The former cell populations were CD11c+CD80+CD86+MHC-II+/highCCR2+CD300b+, whereas the latter were CD11clow/+CD80+CD86low/+MHC-IIlow/highCCR2CD300b, which included MHC-IIlow eosinophils and MHC-IIhigh macrophages (Fig. 3, B to D). Note that the surface expression of CD300b was undetectable in F4/80+Ly-6Clow cells (eosinophils and macrophages) in the air pouch exudates at 4 hours after zymosan treatment, although we found similar amounts of CD300b in eosinophils, macrophages, neutrophils, and inflammatory DCs present in the air pouch exudates before treatment with zymosan (fig. S5, A and B). Together, these observations suggest that F4/80+Ly-6ChighCD300b+ cells correspond to iNOS+ NO-producing inflammatory DCs (20), which were the main source of NO in zymosan-treated air pouches. Note that the air pouch exudates of CD300b−/− mice contained fewer F4/80+Ly-6Chigh inflammatory DCs at 1.5 and 4 hours after zymosan treatment than those of WT mice (Fig. 3E). Moreover, we found that pretreatment with clodronate liposomes decreased the number of F4/80+Ly-6Chigh cells in the air pouch exudates of WT mice treated with or without zymosan (Fig. 3F and fig. S5C). By contrast, clodronate liposome pretreatment did not statistically significantly decrease the number of macrophages in the air pouch exudates of mice before zymosan treatment (fig. S5C). Clodronate liposome pretreatment reduced the number of neutrophils and the amounts of NO, LTB4, KC, and MIP-2 in the air pouch exudates of WT mice such that they were comparable to those in CD300b−/− mice (Fig. 3, G to I, and fig. S6, A and B). Therefore, it is possible that F4/80+Ly-6ChighCD300b+ inflammatory DCs produce increased amounts of NO in response to zymosan, leading to neutrophil accumulation partly through the increased production of the neutrophil chemoattractants LTB4, KC, and MIP-2 in the mouse model of dorsal air pouch inflammation.

Fig. 3 NO-producing inflammatory DCs are responsible for the CD300b-dependent recruitment of neutrophils to zymosan-treated air pouches.

(A) The dorsal air pouches of WT and CD300b−/− mice were treated for 4 hours with zymosan, and then, frozen sections of the skin covering the pouches were treated with Abs against the indicated targets or with 4′,6-diamidino-2-phenylindole (DAPI) and then were analyzed by confocal microscopy. Scale bars, 20 μm. The images are representative of three experiments. (B to D) WT and CD300b−/− mice were treated for 4 hours with zymosan. (B) Cells in air pouch exudates were analyzed by flow cytometry with Abs against the indicated markers and were classified as F4/80Ly-6Cint (R1), F4/80+Ly-6Chigh(R2), or F4/80+Ly-6Clow (R3). (C and D) Cells of the indicated groups from WT mice were stained with Abs against the indicated markers and were analyzed by flow cytometry. (A to D) Data are representative of four independent experiments. (E) Number of F4/80+Ly-6Chigh cells in the pouches (n = 6). (F to I) WT and CD300b−/− mice were pretreated with control liposomes or clodronate liposomes before being treated for 4 hours (F and G) or 1.5 hours (H and I) with zymosan. (F) Percentages of cells in the air pouch exudates of WT mice that were stained with indicated Abs and analyzed by flow cytometry. Data are representative of four independent experiments. (G to I) Numbers of neutrophils (n = 5 mice per group) (G) and amounts of NO2 and NO3 (n = 4 mice per group) (H) and LTB4 (n = 6 mice per group) (I) in the dorsal air pouches of the indicated mice. Data are means ± SD and are representative of two independent experiments (E and G to I). **P < 0.01 by Mann-Whitney test (E) or by ANOVA with Holm-Šídák multiple comparison test (G to I).

Phytosphingosine, a component of zymosan, is a potential ligand of CD300b

To identify potential CD300b ligands from zymosan-derived components, we used 2B4-GFP reporter cells in which activation of the transcription factor NFAT (nuclear factor of activated T cells) drives the expression of green fluorescent protein (GFP) (40, 41). 2B4-GFP cells were transduced with a retrovirus expressing the chimeric receptor CD300b-CD3ζ, which contains the N-terminal extracellular and transmembrane domains of CD300b and an intracellular domain of human CD3ζ containing an ITAM, to generate the CD300b–2B4-GFP reporter cell line. Binding of CD300b ligands to this chimeric receptor would be expected to induce GFP expression in the CD300b–2B4-GFP cells. Although zymosan did not induce GFP expression in either 2B4-GFP or CD300b–2B4-GFP cells, plate-coated total lipids extracted from zymosan induced GFP expression in CD300b–2B4-GFP cells but not in 2B4-GFP cells (Fig. 4A). These results suggested that a certain lipid component of zymosan acted as a CD300b ligand. Because zymosan is a cell wall component of S. cerevisiae, we focused on yeast sphingolipids, such as phytoceramide, dihydrosphingosine, and phytosphingosine (9). Results of functional reporter assays showed that, among the yeast lipids tested, plate-coated phytosphingosine (C-18) induced GFP expression in CD300b–2B4-GFP cells but not in 2B4-GFP cells (Fig. 4B). In addition, neither plate-coated LPS nor phosphatidylserine induced GFP expression in the CD300b–2B4-GFP cells. Moreover, we observed that phytosphingosine-induced GFP expression decreased after pretreatment with a soluble Ab against CD300b (Fig. 4C). Results of mass spectrometry (MS) analysis showed that phytosphingosine (C-18 and C-20) was a lipid component of zymosan (fig. S7, A to E) (36). Results of a physical binding assay confirmed an interaction between phytosphingosine and CD300b-Fc, which is an extracellular domain of CD300b fused to the Fc portion of human immunoglobulin G1 (IgG1) (27), but not between phytosphingosine and control Fc, in a concentration-dependent manner (Fig. 4D). Collectively, these results suggest that phytosphingosine is a potential ligand of CD300b.

Fig. 4 Phytosphingosine, a component of zymosan, is a potential ligand of CD300b.

(A and B) 2B4-GFP or CD300b–2B4-GFP cells were incubated in the presence of zymosan, or phorbol 12-myristate 13-acetate (PMA) and ionomycin, or on plates coated with either zymosan-derived lipids (A) or the indicated lipids (B) for 24 hours. DHS, dihydrosphingosine; PHS, phytosphingosine; PS, phosphatidylserine. The cells were then analyzed by flow cytometry to detect GFP. (C) CD300b–2B4-GFP cells were incubated for 24 hours on plates coated with phytosphingosine in the presence of anti-CD300b or control Ab (each at 10 μg/ml). The cells were then analyzed by flow cytometry to detect GFP. (A to C) Data are representative of three independent experiments. (D) Plates coated with the indicated concentrations of phytosphingosine (C-18) were incubated with CD300b-Fc or control Fc (each at 10 μg/ml). The amount of either CD300b-Fc or control Fc that was bound to the wells was quantified by enzyme-linked immunosorbent assay (ELISA). Data are means ± SD of three biological replicates.

Phytosphingosine acts as a ligand of CD300b both in vitro and in vivo

To determine whether phytosphingosine activated NO-producing inflammatory DCs through CD300b, we cultured BM cells with both GM-CSF and IL-4 to generate GM/IL-4–DCs, which are similar to Tip-DCs (23). We found that GM/IL-4–DCs contained two subsets of cells, namely, CD11c+MHC-IIhigh cells and CD11c+MHC-II+ cells. Moreover, CD11c+MHC-IIhigh cells expressed more CD300b than CD11c+MHC-II+ cells (Fig. 5A). Therefore, we used CD11c+MHC-IIhigh cells sorted from GM/IL-4–DCs (MHC-IIhigh GM/IL-4–DCs) for performing subsequent experiments. Next, we stimulated MHC-IIhigh GM/IL-4–DCs derived from WT or CD300b−/− mice with phytosphingosine, zymosan, or LPS. We observed that CD300b deficiency reduced TNF-α and NO production by MHC-IIhigh GM/IL-4–DCs in response to zymosan, but not LPS, and almost abrogated plate-coated phytosphingosine-induced TNF-α and NO production (Fig. 5B). Moreover, pretreatment with a soluble Ab against CD300b reduced TNF-α production by CD300b-expressing MHC-IIhigh GM/IL-4–DCs in response to zymosan or phytosphingosine to levels comparable to those of CD300b−/− cells (Fig. 5C). These results suggest that the binding of phytosphingosine to CD300b activates NO-producing inflammatory DCs. However, other immune receptors besides CD300b may also contribute to zymosan-induced NO production. On the other hand, CD300b deficiency did not substantially affect the phagocytosis of fluorescein isothiocyanate (FITC)–labeled zymosan particles, suggesting that the phytosphingosine-CD300b interaction was dispensable for zymosan phagocytosis (fig. S8). To further determine the in vivo role of phytosphingosine in zymosan-induced neutrophil recruitment, we injected phytosphingosine-containing vesicles into the dorsal air pouches of WT or CD300b−/− mice (34, 42). Our results showed that treatment with phytosphingosine-containing vesicles induced substantial neutrophil recruitment to the air pouches in WT mice but not in CD300b−/− mice (Fig. 5D). In addition, delipidation of zymosan substantially inhibited its ability to induce neutrophil recruitment to the air pouches (fig. S9). These results indicate that the interaction between phytosphingosine and CD300b in NO-producing inflammatory DCs contributes to zymosan-induced neutrophil accumulation in this mouse model.

Fig. 5 Phytosphingosine acts as a potential ligand of CD300b both in vitro and in vivo.

(A) Left: GM/IL-4–DCs were stained with anti-CD11c and anti–major histocompatibility complex II (MHC-II) Abs and then analyzed by flow cytometry. R1 and R2 indicate populations of cells characterized as CD11c+MHC-IIhigh and CD11c+MHC-II+ cells, respectively. Middle and right: GM/IL-4–DCs in the R1 or R2 gates were stained with an Ab against CD300b and analyzed by flow cytometry. Data are representative of three independent experiments. (B) CD11c+MHC-IIhigh cells sorted from WT or CD300b−/− GM/IL-4–DCs were treated with zymosan or LPS or were cultured on phytosphingosine-coated plates for 12 hours. The amounts TNF-α (left) and NO2 and NO3 (right) were determined by ELISA and colorimetric NO2/NO3 assay, respectively. (C) CD11c+MHC-IIhigh cells sorted from WT or CD300b−/− GM/IL-4–DCs were treated with zymosan or were cultured on phytosphingosine-coated plates for 12 hours in the presence of an Ab against CD300b (anti-CD300b) or a control Ab. The amounts of TNF-α produced by the cells were determined by ELISA. (B and C) Data are representative of three independent experiments. Data are means ± SD of three biological replicates and are representative of three independent experiments. (D) Numbers of neutrophils recruited to the dorsal air pouches of WT and CD300b−/− mice 4 hours after treatment with phytosphingosine-containing vesicles (n = 5 mice per group). Data means ± SD and are representative of three independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test (B and D) or by ANOVA with Holm-Šídák multiple comparison test (C).

CD300b deficiency ameliorates zymosan-induced arthritis by reducing NO production

To further determine the role of CD300b in zymosan-induced inflammation, we used WT and CD300b−/− mice with zymosan-induced arthritis (10, 12, 43, 44). Zymosan injection into the knee joint cavities increased knee thickness and caused inflammatory cell infiltration, bone erosion, and cartilage destruction in WT mice (Fig. 6, A to C). CD300b−/− mice showed milder knee thickness throughout the observation period compared to that of WT mice (Fig. 6A). Histological evaluation provided remarkably lower inflammation scores (on days 3, 7, and 28), bone erosion scores (on day 28), and cartilage destruction scores (on day 28) for CD300b−/− mice when compared to WT mice (Fig. 6, B and C) (19, 45). In addition, CD300b deficiency reduced neutrophil recruitment to the knee joint cavities at 6 and 24 hours after zymosan treatment similar to that observed in the mouse model of dorsal air pouch inflammation (Fig. 6D). Flow cytometry analysis identified F4/80+Ly-6Chigh cells in the lavage of zymosan-stimulated knee joint cavities of WT mice (Fig. 6E), similar to those detected in the air pouch exudates of zymosan-treated mice. Next, WT and CD300b−/− mice were pretreated with clodronate liposomes or control liposomes before zymosan injection into the knee joint cavities. Our results showed that pretreatment with clodronate liposomes, which substantially decreased the number of F4/80+Ly-6Chigh cells but not that of F4/80+Ly-6Clow cells (Fig. 6E), decreased neutrophil recruitment to the knee joint cavities of WT mice at 6 hours after zymosan injection such that the numbers were equivalent to those in CD300b−/− mice (Fig. 6F). Similarly, pretreatment with l-NAME decreased the number of neutrophils recruited to the knee joint cavities of WT mice so that they were comparable to those in CD300b−/− mice (Fig. 6G). These results suggest that NO-producing inflammatory DCs are critical for the CD300b-dependent recruitment of neutrophils to zymosan-treated joint cavities and for the subsequent induction of arthritis.

Fig. 6 CD300b deficiency ameliorates zymosan-induced arthritis by reducing NO production.

(A to G) WT and CD300b−/− (KO) mice were intra-articularly injected with zymosan. (A) The change in knee thickness of the indicated mice was monitored over time (n = 8 mice per group). (B) Knee sections from zymosan-treated WT and KO mice were taken at the indicated times and stained with hematoxylin and eosin. Scale bars, 20 μm. Data are representative of three independent experiments. (C) The indicated histological scores were determined on days 3 (n = 7 mice per group), 7 (n = 12 mice per group), or 28 (n = 12 mice per group). Data are means ± SD and are pooled from two independent experiments. (D) Numbers of neutrophils in the knee joint cavities of zymosan-treated WT and KO mice at the indicated times (n = 4 to 6 mice per group). (E) Cells isolated from the synovial tissues of clodronate liposome– or control liposome–pretreated WT mice after zymosan treatment were stained with Abs against the indicated marker and analyzed by flow cytometry. The percentages of F4/80Ly-6Cint cells (R1), F4/80+Ly-6Chigh cells (R2), and F4/80+Ly-6Clow cells (R3) in CD11b+ cell populations were determined. Data are representative of five independent experiments. (F and G) Numbers of neutrophils isolated from the knee joint cavities of WT and KO mice that were pretreated with clodronate liposomes or control liposomes (F) or with vehicle or l-NAME (G) and then treated with zymosan (n = 5 mice per group). Data are means ± SD and are representative of two independent experiments (A, D, F, and G). *P < 0.05 and **P < 0.01 by Student’s t test (C and D) or by ANOVA with Holm-Šídák multiple comparison test (F and G).

DISCUSSION

The present study provides insights on the role of the CD300b-NO axis in zymosan-induced neutrophil recruitment to dorsal air pouches and joint cavities. The first and most important finding of the present study was the potential identification of a previously uncharacterized CD300b ligand. Our experimental data showed that CD300b deficiency in the mouse model of dorsal air pouch inflammation impaired neutrophil recruitment in response to zymosan (a cell wall extract of S. cerevisiae) but not in response to agonists of TLR1/2, TLR2/6, TLR4, dectin-1, or dectin-2, suggesting that zymosan contained unknown ligands of CD300b. On the basis of finding that several CD300 family proteins recognize ceramides and sphingolipids (2733), we focused on lipids specific to or abundant in fungi and identified phytosphingosine as a candidate CD300b ligand by performing physical binding and functional reporter assays (8, 9). Because CD300b appeared to recognize phytosphingosine, but not dihydrosphingosine, it seems that the hydroxyl group at the C4 position of phytosphingosine is pivotal for the recognition of phytosphingosine by CD300b. Moreover, phytosphingosine-containing vesicles, albeit to a lesser extent than zymosan itself, induced neutrophil recruitment to the air pouches of WT mice. However, CD300b deficiency abrogated the phytosphingosine-induced accumulation of neutrophils. Therefore, phytosphingosine may act as a CD300b ligand in vivo and play an important role in zymosan-induced neutrophil recruitment. Because the removal of lipids from zymosan led to substantial, but not complete, suppression of zymosan-induced neutrophil recruitment in the same model, the most convincing explanation is that phytosphingosine-mediated CD300b signals collaborate with other signals, such as TLR2 and dectin-1 signals, to induce maximal neutrophil recruitment in response to zymosan. Alternatively, other phytosphingosine-like lipids present in zymosan may also act as CD300b ligands, which should be explored in further studies. However, the present study showed that specific recognition of zymosan-derived lipids by CD300b promotes neutrophil accumulation in the mouse model of dorsal air pouch inflammation.

The second most important finding of the present study was the determination of the molecular mechanisms through which CD300b promoted neutrophil accumulation in the mouse model of zymosan-induced inflammation. Consistent with the results of previous studies that highlighted the involvement of iNOS, NO, and LTB4 in zymosan-induced inflammation (1016), our results showed reduced amounts of NO and LTB4 and reduced neutrophil recruitment in the air pouches of zymosan-treated CD300b−/− mice compared to those of WT mice. In addition, in vivo experiments involving either an NO synthesis inhibitor or an NO donor showed that CD300b-dependent NO production promoted neutrophil accumulation. Results of both confocal microscopy and flow cytometry analysis of immune cells in the air pouches of zymosan-treated mice identified F4/80+Ly-6ChighCD11C+CCR2+CD300b+iNOS+ cells as the main source of NO, which corresponded to NO-producing inflammatory DCs or Tip-DCs reported in a previous study (20). In addition, pretreatment with clodronate liposomes efficiently decreased the number of these cells before and after zymosan treatment, which is consistent with the notion that Tip-DCs are derived from CCR2+ inflammatory monocytes (21, 22). Furthermore, pretreatment with clodronate liposomes reduced the amounts of LTB4 and NO and suppressed neutrophil accumulation in a CD300b-dependent manner. On the other hand, pretreatment with an Ab against Gr-1 decreased neutrophil number and LTB4 production but did not substantially change NO production in the air pouches in zymosan-treated mice. These results suggest that neutrophils are the main source of LTB4, but not of NO, in this mouse model. Because LTB4 released from neutrophils is critical for neutrophil accumulation in mouse models of inflammation (19), zymosan administration may stimulate CD300b-dependent NO production by F4/80+Ly-6ChighCD11C+ inflammatory DCs in the dorsal air pouches, resulting in LTB4-dependent neutrophil accumulation. However, the mechanisms through which NO promotes neutrophil recruitment are unclear. One possibility is that CD300b-mediated NO production might directly or indirectly increase local vascular permeability in this mouse model. Alternatively, NO might promote zymosan-induced production of the neutrophil chemoattractants KC and MIP-2 by various cells. Because phytosphingosine, a component of zymosan, stimulated NO production by GM/IL-4–DCs, which are similar to Tip-DCs (23), in a CD300b-dependent manner, the interaction between CD300b and phytosphingosine may trigger NO-dependent neutrophil accumulation in the air pouches of zymosan-treated mice. This may be the mechanism underlying zymosan-induced neutrophil accumulation in joint cavities, although the relevant cell populations involved are not the same between dorsal air pouches and joint cavities. Because zymosan activated inflammatory DCs through CD300b, although it did not activate 2B4 reporter cells expressing CD300b, we speculate that inflammatory DCs, but not reporter T cells, break down or digest zymosan into several components so that the exposed lipid, phytosphingosine, can potentially bind to and act as a ligand for CD300b in inflammatory DCs. Because TLR signals increase the abundance of iNOS through activation of the transcription factors nuclear factor κB and activating protein 1 (17), CD300b signals may augment the TLR2-mediated increase in iNOS abundance in zymosan-stimulated inflammatory DCs. In addition, CD300b seems to function differently from dectin-1 in skewing NO production in response to zymosan, although both CD300b-coupled DAP12 and dectin-1 contain ITAMs in the cytoplasmic region. Further studies are needed to completely understand the relevant molecular mechanisms.

Determining the cell types that are the earliest responders to zymosan is difficult. We found that CD300b was expressed in macrophages and eosinophils, as well as neutrophils and inflammatory DCs, in the dorsal air pouch exudates of WT mice before treatment with zymosan; however, cell surface CD300b was undetectable in macrophages and eosinophils recruited or activated in response to zymosan. The underlying mechanisms should be resolved in further studies. However, it is important to note that pretreatment with clodronate liposomes depleted inflammatory DCs, but not macrophages, in the air pouches before and after zymosan treatment and dampened NO-dependent neutrophil accumulation. Accordingly, it is plausible to conclude that inflammatory DCs, one of the earliest responders, are responsible for CD300b-mediated, NO-dependent neutrophil accumulation. Nonetheless, we cannot rule out the possibility that neutrophils, macrophages, and eosinophils all play a contributory role as one to promote neutrophil recruitment during early response.

NO increases or decreases the abundance of LTB4 and the extent of neutrophil influx into joint cavities and peritoneal cavities, respectively, in response to zymosan (13). Tissue-dependent opposing effects of NO on neutrophil recruitment may be due to the different structure of tissues with or without a basement membrane (peritoneal cavities versus joint cavities) or differential cell populations in these tissues (13, 17). Because NO performs multiple functions that exert proinflammatory or anti-inflammatory effects in the body in a dose- and tissue-dependent manner (17), it is reasonable to conclude that CD300b-mediated, NO-dependent neutrophil accumulation is induced in a tissue-specific manner in response to zymosan. Given that phytosphingosine is present abundantly in fungi (9, 3739) and that CD300b deficiency impaired neutrophil recruitment in response to HKSC or HKCG, the interaction between CD300b and phytosphingosine may induce NO-dependent neutrophil recruitment to sites of fungal infections, thus conferring resistance to these infections or inducing persistent inflammatory diseases. Moreover, this interaction is expected to induce adaptive and innate immunity by activating NO-producing inflammatory DCs. To establish this hypothesis, comparative analysis of fungal infection models between WT and CD300b−/− mice will be indispensable.

In conclusion, zymosan-induced neutrophil accumulation in mouse models of dorsal air pouch inflammation and arthritis depended on the activation of NO-producing inflammatory DCs triggered by the recognition of phytosphingosine, a lipid component of zymosan, by CD300b. Therefore, CD300b-targeted therapies may be effective for treating inflammatory diseases associated with some fungal infections.

MATERIALS AND METHODS

Mice

This study included C57BL/6 J mice and the previously described CD300b−/− (also called LMIR5−/−) mice (34) and DAP12−/− mice (46). All animal experiments were approved by the ethical committee of The University of Tokyo (approval no. 20–8) and Juntendo University (approval no. 1212).

Cells

GM/IL-4–DCs were generated by culturing mouse BM-derived cells in the presence of GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) for 7 days, as described previously (23). Neutrophils were isolated from mouse BM by performing three-layer gradient centrifugation, as described previously (25, 47). 2B4-GFP cells (27, 40, 41) were gifts from T. Saito (RIKEN Research Center for Allergy and Immunology).

Antibodies and other reagents

Goat polyclonal Ab against CD300b (LMIR5) (AF2580) was purchased from R&D Systems. Soluble LMIR5 was detected using rat monoclonal Ab against CD300b (LMIR5) (2C12) (ACTGen) and goat biotinylated polyclonal Ab against CD300b (LMIR5) as capture and detection Abs, respectively, as described previously (35). Goat IgG, SNP dihydrate (71778), LPS (Escherichia coli 0111:B4), and S. cerevisiae mannan were obtained from Sigma-Aldrich. Zymosan, Pam3CSK4, FSL-1, and HKSC were obtained from InvivoGen. HKCG was obtained from the National Institute of Infectious Diseases (Tokyo, Japan). Rat IgG2b was purchased from eBioscience. An Ab against Gr-1; FITC-conjugated Abs against mouse Ly-6G, F4/80, and CD11c; phycoerythrin (PE)–conjugated Abs against mouse Ly-6C, CD11b, CD11c, CD80, CD86, and MHC-II; allophycocyanin (APC)–conjugated Ab against mouse Ly-6C; and peridinin chlorophyll protein–conjugated streptavidin were obtained from BioLegend. R-PE–conjugated AffiniPure F(ab′)2 fragment donkey Ab against goat IgG (H + L) was purchased from Jackson ImmunoResearch Laboratories Inc. APC-conjugated Ab against CCR2 and ELISA kits to measure TNF-α, LTB4, KC, and MIP-2 were obtained from R&D Systems. Colorimetric NO2/NO3 Assay Kit-C II was obtained from Dojindo. Clodronate liposomes (Clophosome) and control liposomes were obtained from FormuMax Scientific Inc. C2-phytoceramide was obtained from Abcam Biochemicals. dl-Dihydrosphingosine was obtained from Santa Cruz Biotechnology, and phytosphingosine (C18H39NO3) was obtained from Tokyo Chemical Industry Co. S. cerevisiae–derived d-ribo-phytosphingosine (C18H39NO3) was obtained from Avanti Polar Lipids Inc., and 1,2-dipalmitoyl-sn-glycero-3-phosphoserine was obtained from Echelon Biosciences Inc. Zymosan A (S. cerevisiae) bioparticles and fluorescein conjugates were obtained from Thermo Fisher Scientific.

Extraction of lipids from zymosan

Lipids were extracted from zymosan by the Bligh and Dyer method (48). Briefly, 3.5 mg of zymosan was vortexed for 1 min in 112.5 μl of chloroform:methanol [1:2 (v/v)], and 37.5 μl of chloroform and 37.5 μl of phosphate-buffered saline (PBS) were added to the mixture before mixing. The mixture was centrifuged at 2000g for 5 min, and the lower layer (organic phase) was collected.

Binding assay by performing solid-phase ELISA

Fc fusion protein CD300b (LMIR5)–Fc and control Fc were purified as described previously (34), and solid-phase ELISA was performed, as described previously (27). Briefly, the indicated lipids in methanol (MeOH) (2.5, 5, or 10 μg/ml) or MeOH alone (control) were added to ELISA plates, and the plates were air dried. Next, the plates were washed and incubated with CD300b-Fc (10 μg/ml) or control Fc in the presence of 0.5 mM CaCl2 for 2 hours, which was followed by incubation with peroxidase-conjugated anti-human IgG (Sigma-Aldrich). Absorbance was measured at 450 nm.

DNA constructs

DNA sequences encoding the extracellular and transmembrane domains of FLAG-tagged mouse CD300b (LMIR5) fused with the intracellular domain of human CD3ζ (N. Matsumoto, University of Tokyo) were inserted into pMXs–internal ribosomal entry site–puror (pMXs-IP) to generate pMXs-FLAG-CD300b-CD3ζ-IP (27).

Transfection and infection

Retroviral transduction was performed as described previously (27, 49, 50). Briefly, cells were infected with retroviruses generated by transiently transfecting PLAT-E packaging cells.

Preparation of lipid-containing vesicles

One milligram of dry lipid (phytosphingosine) was hydrated in 1 ml of PBS, and lipid-containing vesicles were generated using an Avanti mini extruder (Avanti Polar Lipids Inc.), according to the manufacturer’s instructions, as described previously (27, 51).

Cell stimulation

GM/IL-4–DCs were stimulated with zymosan (50 μg/ml) (InvivoGen) or LPS (100 ng/ml). Alternatively, the indicated lipids in MeOH (10 μg/ml) or MeOH alone (control) were added to MaxiSorp 96-well plates (Nunc; catalog no. 430341), and the plates were air dried. GM/IL-4–DCs were stimulated for 12 hours on plates coated with phytosphingosine. 2B4-GFP or CD300b–2B4-GFP cells were stimulated with PMA (100 ng/ml) and ionomycin (control) (25 ng/ml) or were cultured for 24 hours on plates coated with zymosan-extracted lipids or the indicated lipids as described previously (27, 30, 51).

Mouse model of dorsal air pouch inflammation

Air pouches were produced on the dorsum of mice, as described previously (34, 42). Briefly, 7 ml of sterile air was injected subcutaneously into the backs of the mice on days 0 and 3. On day 6, 50 μg of zymosan dissolved in PBS, 10 μg of LPS, 10 μg of Pam3CSK4, 1 μg of FSL-1, 200 μg of curdlan, 400 μg of mannan, or 1 × 107 HKSC or HKCG was injected into the air pouches. The air pouches were lavaged with PBS containing 5 mM EDTA at 1.5 or 4 hours after the injection. The total numbers of cells in the lavage fluids were counted, and the percentages of CD11b+Ly-6G+ neutrophils were estimated by performing flow cytometry analysis. In some experiments, l-NAME (25 mg/kg) (Sigma-Aldrich) or 1400 W dihydrochloride (10 mg/kg) (Sigma-Aldrich) was intravenously injected at 30 min before zymosan injection, 100 μl of clodronate liposomes or control liposomes were intravenously injected at 24 hours before zymosan injection, or 250 μg of Ab against Gr-1 or control Ab was intravenously injected at 4 and 24 hours before zymosan injection. Alternatively, 10 μg of SNP alone, 50 μg of zymosan alone, or a combination of SNP and zymosan was injected into the dorsal air pouches of CD300b−/− mice.

Mouse model of zymosan-induced arthritis

Mice were intra-articularly injected with 150 μg of zymosan into their right knee joints, as described previously (43, 44). Knee joint swelling was measured using a digital caliper (Mitutoyo Corporation, Japan). Histological scoring systems were used, as described previously (19, 45). Knee joints were fixed overnight in 4% paraformaldehyde, defatted in 70% ethanol on the following day, and demineralized in 10% EDTA for 2 weeks. Histological scores of inflammation, cartilage destruction, and bone erosion were determined using a scale of 0 to 5 as follows: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. The mice were euthanized at different time points after zymosan injection. The synovial cavities of the knee joints were washed twice with 300 μl of PBS, and synovial lavage fluids were collected at 6 hours after zymosan treatment. Total cells in the synovial lavage fluids were counted, and the percentages of CD11b+Ly-6G+ neutrophils were estimated by performing flow cytometry analysis (Fig. 6, D, F, and G). Alternatively, synovial tissues isolated from knee joints were digested with collagenase type 1 (1 mg/ml) (Wako Chemicals) and Dispase II (1 mg/ml) (GIBCO) for 1 hour to collect the cells required for flow cytometry analysis (Fig. 6E). In some experiments, the mice were intravenously injected with l-NAME (25 mg/kg) at 30 min before zymosan injection or were intravenously injected with 100 μl of Clophosome or control liposomes twice 24 hours before zymosan injection.

Quantification of TNF-α, LTB4, KC, or MIP-2 and NO abundances

The amounts of TNF-α, LTB4, KC, and MIP-2 in air pouch lavage fluids or in the culture medium of GM/IL-4–DCs were measured by performing ELISA as previously described (27, 51). Nitrate and nitrite abundances in air pouch lavage fluids or in the culture medium of GM/IL-4–DCs were measured using the colorimetric NO2/NO3 Assay Kit-C II, according to the manufacturer’s protocol.

Immunohistochemical analysis

Cryosections of the skin covering the dorsal air pouches were stained with goat Ab against mouse CD300b (LMIR5), purified mouse Ab against iNOS/NOS type II (BD Transduction Laboratories), and rat Ab against mouse F4/80 (Serotec), which was followed by staining with Alexa Fluor 555–conjugated donkey Ab against goat IgG (H + L), Alexa Fluor 488–conjugated goat Ab against mouse IgG (H + L) (Invitrogen), and Alexa Fluor 647–conjugated goat Ab against rat IgG Ab (H + L) (Invitrogen). All the sections were counterstained with DAPI (Invitrogen). Fluorescent images were obtained and analyzed using a confocal microscope (FLUOVIEW FV300 laser scanning biological microscope JX70 system; Olympus, Albertslund, Denmark) equipped with a SenSys/0 L cold charge–coupled device camera (Olympus) (27).

Flow cytometry analysis

Flow cytometry analysis was performed with a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest software (BD Biosciences) and FlowJo software (Tree Star Inc.), as described previously (27).

Phytosphingosine analysis

Phytosphingosine analysis was performed as described previously (52) with some modifications. Briefly, crude lipids were extracted from zymosan using CHCl3-MeOH (2:1, vol.) and were hydrolyzed with 5% HCl in MeOH. The reaction mixture was extracted with n-hexane to remove any fatty acid methyl ester. The MeOH layer was concentrated in vacuo and was converted to a trimethylsilyl (TMS) derivative with the TMS-HT Kit (Tokyo Chemical Industry, Tokyo, Japan), according to the supplier’s instructions. Gas chromatography–MS was performed using a QP-5050 instrument (Shimadzu, Kyoto, Japan) fitted with a fused silica capillary column (DB-1, 30 m by 0.25 mm ϕ; Agilent Technologies, Palo Alto, CA, USA). Alternatively, delipidated zymosan was washed with PBS before injection into dorsal air pouches.

Phagocytosis assay

To perform phagocytosis assays, GM/IL-4–DCs were cultured with FITC-conjugated zymosan particles (50 μg/ml) for the times indicated in the figure legend, and the percentages of FITC-positive cells were measured by performing flow cytometry analysis.

Statistical analyses

All results are expressed as means ± SD. GraphPad Prism 6 (GraphPad software) was used for statistical calculations. An unpaired two-tailed Student’s t test, a t test with Welch’s correction, or a Mann-Whitney test was used to compare two independent groups. ANOVA with Holm-Šídák multiple comparison test was used to compare three or more groups. *P < 0.05 or **P < 0.01 was considered to be statistically significant.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/12/564/eaar5514/DC1

Fig. S1. DAP12 deficiency does not influence zymosan-induced neutrophil recruitment in the mouse model of dorsal air pouch inflammation.

Fig. S2. sCD300b is not involved in zymosan-induced neutrophil accumulation in the mouse model of dorsal air pouch inflammation.

Fig. S3. Both l-NAME and 1400 W dihydrochloride reduce neutrophil recruitment and the amounts of NO, LTB4, KC, and MIP-2 in the air pouches of zymosan-treated WT mice.

Fig. S4. An Ab against Gr-1 decreases the number of neutrophils recruited to air pouches in response to zymosan.

Fig. S5. Pretreatment with clodronate liposomes decreases the number of inflammatory DCs, but not of macrophages, in the air pouch exudates of zymosan-treated WT mice.

Fig. S6. Pretreatment with clodronate liposomes reduces the amounts of KC and MIP-2 in the air pouch exudates of WT mice to levels comparable to those in CD300b−/− mice.

Fig. S7. Phytosphingosine is present in zymosan.

Fig. S8. CD300b deficiency does not affect the phagocytosis of zymosan by GM/IL-4–DCs.

Fig. S9. Delipidation of zymosan decreases the number of neutrophils recruited to the air pouches of zymosan-treated WT mice.

REFERENCES AND NOTES

Acknowledgments: We thank H. Arase (Osaka University) and N. Matsumoto (University of Tokyo) for providing the plasmids. Funding: This study was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture, Japan (JSPS KAKENHI grant numbers 23390257 and 26293231) and from the Kato Memorial Bioscience Foundation (28-B). Author contributions: M.T. performed all of the experiments and participated in writing the manuscript. K.I., Y.Y., A.M., M.I., T.M., A.K., A.T., S.U., H.Y., M.N., T.A., and T.S. assisted in performing the experiments. M.U. and Y.K. analyzed the lipids present in zymosan. H.O. and K.O. analyzed all of the data. T.K. and J.K. conceived the study, analyzed the data, and actively participated in writing the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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