Mast Cell-Specific Deletion of Group III Secreted Phospholipase A2 Impairs Mast Cell Maturation and Functions

Tissue-resident mast cells (MCs) have important roles in IgE-associated and -independent allergic reactions. Although microenvironmental alterations in MC phenotypes affect the susceptibility to allergy, understanding of the regulation of MC maturation is still incomplete. We previously reported that group III secreted phospholipase A2 (sPLA2-III) released from immature MCs is functionally coupled with lipocalin-type prostaglandin D2 (PGD2) synthase in neighboring fibroblasts to supply a microenvironmental pool of PGD2, which in turn acts on the PGD2 receptor DP1 on MCs to promote their proper maturation. In the present study, we reevaluated the role of sPLA2-III in MCs using a newly generated MC-specific Pla2g3-deficient mouse strain. Mice lacking sPLA2-III specifically in MCs, like those lacking the enzyme in all tissues, had immature MCs and displayed reduced local and systemic anaphylactic responses. Furthermore, MC-specific Pla2g3-deficient mice, as well as MC-deficient KitW-sh mice reconstituted with MCs prepared from global Pla2g3-null mice, displayed a significant reduction in irritant contact dermatitis (ICD) and an aggravation of contact hypersensitivity (CHS). The increased CHS response by Pla2g3 deficiency depended at least partly on the reduced expression of hematopoietic PGD2 synthase and thereby reduced production of PGD2 due to immaturity of MCs. Overall, our present study has confirmed that MC-secreted sPLA2-III promotes MC maturation, thereby facilitating acute anaphylactic and ICD reactions and limiting delayed CHS response.


Introduction
Mast cells (MCs) promote acute allergic reactions, including anaphylaxis, a severe and potentially fatal immunoglobulin (IgE)-dependent immediate hypersensitivity reaction to apparently harmless antigen (Ag), as well as certain IgE-independent innate and adaptive immune disorders [1][2][3]. In addition to these detrimental functions, MCs also have beneficial functions by regulating both innate and adaptive immune responses against invading microorganisms, venom, and environmental toxins [1][2][3]. Crosslinking of the high-affinity receptor for IgE (FcεRI) on the surface of MCs with IgE and specific Ag initiates signals leading to the release of preformed, granule-stored molecules (degranulation), such as histamine and proteases, newly synthesized lipid mediators, such as the arachidonic acid (AA) metabolites prostaglandin D 2 (PGD 2 ) and leukotriene C 4 , and prestored or newly transcribed cytokines and chemokines such as tumor necrosis factor-α types that potentially express this enzyme, we generated MC-specific Pla2g3 knockout (KO) mice by crossing Pla2g3-floxed (Pla2g3 fl/fl ) mice with Mcpt5-Cre mice, which have been used for MC-specific deletion of target genes [34][35][36]. We provide evidence that MC-specific Pla2g3 deficiency fully recapitulates the MC maturation defects as observed in global Pla2g3 deficiency. Additionally, by using MC-specific Pla2g3 KO mice as well as Kit W-sh mice adoptively transferred with Pla2g3 −/− BMMCs, we addressed the roles of MC-derived sPLA 2 -III in irritant contact dermatitis (ICD), an acute inflammation, and contact hypersensitivity (CHS), a Th1-dependent delayed-type allergic response.

Mice
The targeting vector for the Pla2g3 gene was obtained from the Knockout Mouse Project (KOMP) Repository (clone PRPGS00113_A_H09, Project No. 33384; The Knockout Mouse Project, Mouse Biology Program, University of California, Davis, CA, USA; www.KOMP.org (accessed on 4 April 2011)) [37]. The Pla2g3 conditional-ready mutant allele was generated by KOMP through insertion of the promoter-driven L1L2_Bact_P cassette into the mouse Pla2g3 gene at chromosome 11. The functional gene product is composed of 2 FRT sites flanking an IRES:lacZ-trapping cassette and a floxed human β-actin promoter-driven neo cassette inserted into the intron 1 of the Pla2g3 gene and an additional third loxP site downstream of exon 3. The targeting vector was transferred into C57BL/6 mouse-derived embryonic stem (ES) cells (RENKA; Niigata University, Niigata, Japan) [38] via electroporation, and the cells that contained the correctly targeted Pla2g3 locus were identified by PCR and confirmed by Southern blot analysis. Chimeric mice were generated with the recombinant ES cells using an aggregation method. Two chimeras with higher thañ 70% coat color chimerism were mated with C57BL/6N mice (Japan SLC, Shizuoka, Japan) to achieve germline transmission. We then generated a conditional KO mouse strain by crossing heterozygous mice with CAG-Flpe mice (The Jackson Laboratory, Bar Harbor, ME, USA) expressing the flippase recombinase under the control of the actin promoter. This resulted in the excision of the IRES:lacZ and neo cassettes and the generation of a floxed allele. We further crossed conditional KO mice with Mcpt5-Cre mice expressing the Cre recombinase under the control of the MC-specific protease Mcpt5 promoter, generously provided by Dr. Axel Roers (Institute for Immunology, Medical Facility Carl Gustav Carus, University of Technology Dresden, Dresden, Germany) [39], to obtain a null allele. MC-specific Pla2g3 KO (Pla2g3 fl/fl Mcpt5-Cre) mice and control littermates (Pla2g3 fl/fl ) were used for all animal experiments. Mouse genotypes were determined by PCR of tail-snip DNA using GeneAmp Fast PCR Master Mix (Thermo Fisher Scientific-Applied Biosystems, Waltham, MA, USA) and genotyping primers as follows: forward, 5 -GCGCCATTGCTCGAACTGTGGTTG-3 ; reverse, 5 -AGGCCAGGCACAGTCTTTCCTCT-3 .
Global Pla2g3 −/− mice were described previously [16,40]. Hpgds −/− mice [41] were provided by Dr. Yoshihiro Urade (Osaka Bioscience Institute, Osaka, Japan). MC-deficient Kit W-sh mice (Kit W-sh HNihrLaeBsmJ, Stock No. 005051) were purchased from the Jackson Laboratory. Age-matched male mice (8-12 weeks of age) were used in each experiment. Mice were maintained in animal facilities in the Tokyo Metropolitan Institute of Medical Science and the University of Tokyo under specific pathogen-free conditions. All animal experiments were approved by the institution and conformed to the Japanese Guide for the Care and Use of Laboratory Animals.
The fibroblast-directed maturation of immature BMMCs toward connective tissuetype MC (CTMC)-like cells was described previously [16,[43][44][45]. Briefly, BMMCs were seeded onto the monolayer of Swiss 3T3 fibroblasts (Japanese Cancer Research Resource Bank, Osaka, Japan) and cocultured for appropriate periods (typically 4 days) in the presence of 100 ng/mL mouse SCF (Peprotech, Cranbury, NJ, USA). The cells were trypsinized and reseeded in culture dishes, and adherent fibroblasts and nonadherent MCs were collected, or pure MCs were isolated using CD117 Microbeads and autoMACS Pro Separator (Miltenyi Biotec, Tokyo, Japan). The maturation of BMMCs into CTMC-like cells was verified by staining of their granules with alcian blue and counterstaining with safranin O (Muto Pure Chemicals, Tokyo, Japan), as described previously [44].
Before or after coculture with 3T3 fibroblasts, 10 6 BMMCs were preloaded for 2 h with 1 µg/mL anti-dinitrophenyl (DNP) IgE (clone SPE-7, Sigma-Aldrich, St. Louis, MO, USA) in Tyrode's buffer. After removal of the excess antibody, the cells were stimulated with 100 ng/mL human serum albumin (HSA) conjugated with DNP (DNP-HSA; Sigma-Aldrich) as an Ag for 10 min at 37 • C. As required for experiments, the cells were directly activated by 10 µg/mL Compound 48/80 (C48/80; Sigma-Aldrich), an IgE-independent MC secretagogue. The degree of degranulation was determined by measuring the release of β-hexosaminidase (β-HEX), as described previously [43]. The levels of PGD 2 were determined by ELISA in accordance with the manufacturer's instructions (PGD 2 -MOX ELISA Kit, Cayman Chemical, Ann Arbor, MI, USA).

Quantitative RT-PCR
The reagents and instrument required for quantitative RT-PCR were purchased from Thermo Fischer Scientific. Total RNA was isolated from mouse BMMCs, fibroblasts, splenocytes, or ear skin using TRIzol reagent in accordance with the manufacturer's instruments. First-strand cDNA synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed with a predesigned primer-probe set (TaqMan Gene Expression Assay) and TaqMan Gene Expression Master Mix on a StepOnePlus real-time PCR system. TaqMan Gene Expression Assays for , and Ifng (Mm01168134_m1) were used. Expression levels of the transcripts were normalized to Gapdh (Mouse GAPD Endogenous Control) or Kit and fold changes were calculated by the ∆∆Ct method. In essence, genes expressed in MCs were normalized with Kit, while those expressed in fibroblasts were normalized with Gapdh, in accordance with our previous paper [16].

Anaphylaxis
IgE-mediated or C48/80-induced anaphylactic responses were examined as described previously [16,45]. Briefly, in a model of passive cutaneous anaphylaxis (PCA), mouse ears were passively sensitized by subcutaneous injection with 30 ng of anti-DNP IgE monoclonal antibody. On the next day, the mice were challenged by intravenous injection of a mixture of 60 µg of DNP-HSA and 1 mg Evans blue (Fujifilm Wako). In a model of IgE-independent anaphylaxis, mice were intradermally administrated with 250 ng of C48/80 followed by an immediate intravenous injection with Evans blue. Vascular permeability in the mice were Cells 2021, 10, 1691 5 of 15 measured 30 min after the Ag challenge. The ear tissues were collected and lysed, and amounts of the dye were determined. In a model of passive systemic anaphylaxis (PSA), mice were passively sensitized by intravenous injection with 16.5 µg of anti-DNP IgE. On the next day, the mice were challenged intravenously with 500 µg of DNP-HSA. The rectal temperature was measured over time after Ag challenge with an electronic thermometer (Physitemp Instruments, Clifton, NJ, USA).

Dermatitis
For an ICD model, mice were directly challenged with 20 µL of 0.3% (v/v) 2,4dinitrofluorobenzene (DNFB; Sigma-Aldrich) in a vehicle of acetone/olive oil (4:1) to the ear (10 µL to each side). For a CHS model, mice were sensitized on the shaved abdomen with 50 µL of 0.5% (v/v) DNFB. At 5 days after sensitization, mice were challenged with 20 µL of 0.3% (v/v) DNFB to the ear (10 µL to each side), as described previously [46,47]. Ear thickness was measured before and at 4 h in ICD and at several intervals after hapten challenge in CHS with a micrometer (Mitsutoyo, Kanagawa, Japan).

MC Reconstitution
BMMCs (10 6 ) were reconstituted for 6 weeks by intradermal injection into 6-week-old MC-deficient Kit W-sh mice [16,30]. The mice were subjected to CHS, as described above. Alternatively, MCs from the base to the tip of the ears from these mice were evaluated histologically by toluidine blue staining, as described below.

Histology
Ear pinnae from mice were fixed with 10% (v/v) formalin solution and embedded in paraffin, and 4 µm sections were cut and then stained with toluidine blue or hematoxylin and eosin (Merck Millipore, Burlington, MA, USA). For MC quantification, diffuse toluidine blue + cells with no clearly defined cell membrane indicated MCs.

Statistical Analysis
Results are presented as box plots with Tukey whiskers or mean ± SEM. Statistical analysis was performed with Prism 9 (GraphPad, San Diego, CA, USA) software. Twotailed Mann-Whitney test and ordinary one-way or two-way ANOVA with post hoc Tukey multiple comparisons test were performed as noted in the respective figure legends.

Generation of MC-Specific Pla2g3-Deficient Mice
We successfully generated Pla2g3-floxed (Pla2g3 fl/fl ) mice, in which the exons encompassing the catalytic domain of the Pla2g3 gene were replaced with the IRES:lacZ and neo cassettes and flanked by FRT or loxP sites, for the purpose of conditional inactivation of the gene in a cell-and tissue-specific manner ( Figure 1A). To examine the function of Pla2g3 in MCs, we crossed Pla2g3 fl/fl mice with Mcpt5-Cre mice [39], which express Cre recombinase selectively in MCs. Mice with conditional deletion of Pla2g3 (Pla2g3 fl/fl Mcpt5-Cre) were screened by genotyping PCR (Figure 1B,C).
( Figure 1D), whereas Pla2g3 was expressed normally in other cells such as splenocytes ( Figure 1E), confirming that Cre-mediated recombination efficiently ablated Pla2g3 in MCs.

MC-Specific Pla2g3 Ablation Impairs MC Maturation
BMMCs prepared from global Pla2g3 −/− mice exhibit impaired fibroblast-driven maturation and thereby IgE-dependent and -independent activation in ex vitro culture [16]. To confirm our findings in global Pla2g3 −/− mice, we investigated the effects of MC-specific ablation of Pla2g3 on the maturation and activation of BMMCs before and after coculture with Swiss 3T3 fibroblasts. Pla2g3 deficiency in IL-3-maintained BMMCs affected neither proliferation (data not shown), cell surface expression of c-Kit and FcεRIα as assessed by flow cytometry (Figure 2A

MC-Specific Pla2g3 Ablation Impairs MC Maturation
BMMCs prepared from global Pla2g3 −/− mice exhibit impaired fibroblast-driven maturation and thereby IgE-dependent and -independent activation in ex vitro culture [16]. To confirm our findings in global Pla2g3 −/− mice, we investigated the effects of MC-specific ablation of Pla2g3 on the maturation and activation of BMMCs before and after coculture with Swiss 3T3 fibroblasts. Pla2g3 deficiency in IL-3-maintained BMMCs affected neither proliferation (data not shown), cell surface expression of c-Kit and FcεRIα as assessed by flow cytometry (Figure 2A), nor granule staining with alcian blue ( Figure 2B). While Pla2g3 fl/fl BMMCs after coculture contained more safranin-positive granules than did the cells before coculture, Pla2g3 fl/fl Mcpt5-Cre BMMCs were stained with safranin only weakly even after coculture ( Figure 2B), suggesting immaturity of the granules. The maturation of Pla2g3 fl/fl BMMCs to CTMC-like cells resulted in marked induction of Hdc (encoding histidine decarboxylase, a histamine-biosynthetic enzyme), MC-specific proteases Mcpt4 and Mcpt6 (encoding chymase and tryptase, respectively), Hpgds (encoding H-PGDS, which is responsible for PGD 2 production in BMMCs but not in fibroblasts), and Ptgdr (encoding DP1, a PGD 2 receptor that provides an MC maturation signal) [16]. The induction of these MC maturation markers was markedly impaired, whereas the constitutive expression of Kit was unaffected, in Pla2g3 fl/fl Mcpt5-Cre BMMCs ( Figure 2C). In addition, the induction of Ptgds (encoding L-PGDS, which is responsible for PGD 2 production in fibroblasts but not in BMMCs) ( Figure 2D) and PGD 2 production ( Figure 2E) were lower in fibroblasts cocultured with Pla2g3 fl/fl Mcpt5-Cre BMMCs than in those cocultured with Pla2g3 fl/fl BMMCs, verifying a bidirectional interaction between MCs and fibroblasts.  After but not before coculture, FcεRI-dependent release of β-HEX (a degranulation marker) following sensitization with DNP-specific IgE and challenge with DNP-HSA as an Ag was significantly lower in Pla2g3 fl/fl Mcpt5-Cre BMMCs than in Pla2g3 fl/fl BMMCs ( Figure 2F). Because Hdc expression was markedly reduced in Pla2g3 fl/fl Mcpt5-Cre BMMCs after coculture ( Figure 2C), it is likely that histamine synthesis and release were concomitantly reduced in these cells, as seen in global Pla2g3 −/− mice [16]. The maturation of Pla2g3 fl/fl BMMCs to CTMC-like cells increased IgE-dependent PGD 2 synthesis ( Figure 2G), with a concomitant increase in Hpgds expression ( Figure 2C). However, the coculture-driven elevation of IgE-mediated PGD 2 generation occurred only modestly in Pla2g3 fl/fl Mcpt5-Cre cells ( Figure 2G), most likely due to the reduced induction of Hpgds ( Figure 2C). Although cocultured Pla2g3 fl/fl CTMC-like cells acquired sensitivity to C48/80, which acts on mouse Mas-related G protein-coupled receptor B2 (Mrgprb2; the ortholog of human MRGPRX2 [48]), C48/80-induced degranulation and PGD 2 production were markedly lower in cocultured Pla2g3 fl/fl Mcpt5-Cre cells ( Figure 2H,I). Thus, as in the case of global Pla2g3 deletion [16], MC-specific Pla2g3 deletion impairs fibroblast-directed maturation and thereby IgE-mediated and even -independent activation of BMMCs in ex vitro culture.

MC-Specific Pla2g3 Ablation Ameliorates MC-Associated Anaphylaxis and Irritant Dermatitis
We then evaluated the impacts of MC-specific Pla2g3 deficiency on anaphylactic responses in vivo. In agreement with the ex vivo experiments (Figure 2 It has recently been shown that ICD requires CTMC activation in a manner dependent on substance P released from nociceptive neurons and MrgprB2 expressed on MCs [49]. MrgprB2 functions as an activating receptor for IgE-independent CTMC degranulation in response to multiple exogenous and endogenous ligands such as C48/80 and substance P [48][49][50][51]. We observed that the IgE-independent, C48/80-induced anaphylactic response was markedly lower in Pla2g3 fl/fl Mcpt5-Cre mice than in Pla2g3 fl/fl mice ( Figure 3F). Upon ICD, acute ear swelling in response to a single challenge with DNFB was significantly attenuated in Pla2g3 fl/fl Mcpt5-Cre mice compared with Pla2g3 fl/fl mice ( Figure 3G) as well as in MC-deficient Kit W-sh mice compared with Kit +/+ mice ( Figure 3H). Collectively, these results indicate that the ablation of Pla2g3 specifically in MCs leads to impaired MC maturation, accompanied by reduced MC-associated anaphylaxis and irritant dermatitis, in vivo.

MC-Specific Pla2g3 Ablation Excacerbates CHS
The contribution of MCs to CHS, a delayed-type allergic response that depends on Th1 immunity, remains controversial. Indeed, the use of different MC-deficient animals has suggested a positive immunostimulatory role [52][53][54][55][56], a negative immunomodulatory

MC-Specific Pla2g3 Ablation Excacerbates CHS
The contribution of MCs to CHS, a delayed-type allergic response that depends on Th1 immunity, remains controversial. Indeed, the use of different MC-deficient animals has suggested a positive immunostimulatory role [52][53][54][55][56], a negative immunomodulatory role [35,[57][58][59], or no role [60][61][62] of MCs in several CHS models depending on the experimental conditions used. In this study, to evaluate how MC-derived sPLA 2 -III would contribute to CHS, we subjected MC-specific Pla2g3 fl/fl -deficient mice to a model of DNFB-induced CHS.
After the second challenge (elicitation) with DNFB, ear swelling was markedly increased in Pla2g3 fl/fl Mcpt5-Cre mice compared to their Pla2g3 fl/fl counterparts ( Figure 4A). Histological examination of tissue sections revealed that DNFB-induced epidermal and dermal hyperplasia and immune cell recruitment were considerably greater in Pla2g3 fl/fl Mcpt5-Cre mice than in Pla2g3 fl/fl mice ( Figure 4B). Consistent with the dependence of CHS on IFN-γ-producing CD8 + T cells [63], the DNFB-induced induction of Ifng was higher in the ear of Pla2g3 fl/fl Mcpt5-Cre mice than in that of Pla2g3 fl/fl mice ( Figure 4C).

Discussion
Global Pla2g3 −/− mice display reduced anaphylactic responses, which could be attributed to impairment of MC maturation [16]. sPLA2-III, which is likely to be secreted from MCs, acts on adjacent fibroblasts as a paracrine factor to promote the biosynthesis of PGD2, an AA-derived lipid mediator that in turn acts on its receptor DP1 on MCs to coordinate proper MC maturation. In this study, using a conditional KO strain in which the Pla2g3 gene is disrupted specifically in MCs under the Mcpt5 promoter, we have provided In agreement with previous studies demonstrating the exacerbation of DNFB-induced CHS in MC-deficient Kit W-sh mice [55,57,59], Kit W-sh mice exhibited greater ear swelling than did MC-sufficient Kit +/+ mice in our CHS settings ( Figure 4D). When Kit W-sh mice had been intradermally engrafted with BMMCs from global Pla2g3 −/− mice or those from WT littermates, the numbers of reconstituted MCs in the ear skin were similar between the two groups ( Figure 4E). After reconstitution with WT BMMCs, DNFB-increased ear swelling and Ifng expression in Kit W-sh mice were reduced to levels similar to those in Kit +/+ mice, whereas these ameliorating effects were scarcely seen in replicate Kit W-sh mice reconstituted with Pla2g3 −/− BMMCs ( Figure 4F,G). Thus, our present study using two types of MC-specific Pla2g3 deletion models indicates that Pla2g3 in MCs can substantially limit the magnitude of CHS responses.
We have previously shown that sPLA 2 -III secreted from MCs regulates the spatiotemporal mobilization of distinct PGD 2 pools in tissue microenvironments; it is directly coupled with L-PGDS in adjacent fibroblasts to generate a pool of PGD 2 that participates in MC maturation, and indirectly affects H-PGDS-driven production of another pool of PGD 2 in MCs that counteracts the anaphylactic response [16]. PGD 2 can suppress inflammation by limiting neutrophil infiltration, dendritic cell activation, or other mechanisms [24][25][26][27]64]. To assess the possibility that the increased CHS by MC-specific depletion of Pla2g3 might be due to the reduced generation of PGD 2 , we tested the CHS response in Kit W-sh mice engrafted with BMMCs from Hpgds-deficient mice. Although a similar number of MCs was present in the ear of Kit W-sh mice that had been reconstituted with Hpgds-sufficient or -deficient BMMCs (data not shown), Kit W-sh mice transferred with Hpgds −/− BMMCs, like those transferred with Pla2g3 −/− BMMCs ( Figure 4F,G), displayed more severe DNFBinduced ear swelling and Ifng induction than replicate Kit W-sh mice transferred with WT BMMCs (Figure 4H,I). These results suggest that MC-derived sPLA 2 -III limits the CHS response at least in part by a mechanism that depends on the promotion of MC maturation and thereby H-PGDS-driven production of anti-inflammatory PGD 2 .

Discussion
Global Pla2g3 −/− mice display reduced anaphylactic responses, which could be attributed to impairment of MC maturation [16]. sPLA 2 -III, which is likely to be secreted from MCs, acts on adjacent fibroblasts as a paracrine factor to promote the biosynthesis of PGD 2 , an AA-derived lipid mediator that in turn acts on its receptor DP1 on MCs to coordinate proper MC maturation. In this study, using a conditional KO strain in which the Pla2g3 gene is disrupted specifically in MCs under the Mcpt5 promoter, we have provided compelling evidence that MC-derived sPLA 2 -III is indeed a critical regulator of MC maturation.
Although several studies have reported that Mcpt5 is not expressed in all MC populations and recommended to use Cpa3-Cre mice rather than Mcpt5-Cre mice [39,65,66], our present study shows that, as in the case of global Pla2g3 deletion [16], MC-specific Pla2g3 deletion in Pla2g3 fl/fl Mcpt5-Cre mice leads to reduced upregulation of several MC maturation markers, accompanied by decreased degranulation, PGD 2 generation, C48/80 sensitivity, and safranin staining, in BMMCs after coculture with fibroblasts ex vivo, as well as reduced IgE-dependent and -independent systemic and local anaphylactic responses in vivo. These results confirm the crucial role of MC-expressed sPLA 2 -III in the proper maturation of MCs and underline the future use of Pla2g3 fl/fl mice to clarify the cell/tissue-specific functions of sPLA 2 -III in various pathophysiological circumstances.
Our present study has also provided additional insight into the role of MCs in CHS. On the basis of recent research, the roles of MCs in the sensitization and elicitation phases of CHS have been considered as follows: First, during the sensitization phase (which is equivalent to the condition of ICD), MCs are activated directly or indirectly by haptens to release a diverse spectrum of mediators, including histamine and TNF-α, which induce vasodilatation and neutrophil recruitment [53,55]. Consistently, ICD-induced ear swelling, which depends on Mrgprb2 activation [49], is substantially reduced in MC-specific Pla2g3deficient mice. Interactions between MCs and dendritic cells (DCs) by direct contact or MC-secreted TNF-α can amplify DC migration into the draining lymph nodes, where DCs prime naïve T cells to become effector T cells via Ag presentation [67]. Second, during moderate CHS responses, MCs amplify ear swelling, epidermal hyperplasia, and recruitment of neutrophils and CD8 + T cells through releasing TNF-α [34]. Third, during more severe CHS responses, MCs represent an early source of IL-10, which amplifies subsequent recruitment of regulatory T (T reg ) cells and limits ear swelling and epidermal hyperplasia [35,57]. Additionally, MCs can migrate into lymphoid organs, where they produce IL-2, which contributes to maintaining T reg cells and thereby ameliorating CHS [58]. The CHS model employed in the present study fits with the third case, where MCs limit the severity of CHS inflammation. Importantly, the absence of sPLA 2 -III in MCs dampens the suppressive effect of these cells on ear swelling and Ifng induction in CHS, revealing a novel role of this particular sPLA 2 in a specific pathological event. This function appears to depend at least partly on the sPLA 2 -III-driven, indirect mobilization of PGD 2 by MCs, where MC-derived sPLA 2 -III is coupled with L-PGDS-dependent production of the first pool of PGD 2 in fibroblasts, which then facilitates the proper maturation of MCs and thereby H-PGDS-dependent production of the second pool of PGD 2 by these cells. Indeed, Hpgds deficiency in MCs also abrogates the suppressive effect of MCs on CHS, highlighting that, in addition to the cytokines IL-2 and IL-10 [33,35,57,58], the lipid mediator PGD 2 acts as another negative modulator of the CHS responses. In support of this view, mice lacking DP1 (Ptgdr −/− ) also display an exacerbation of CHS by affecting the expression of IL-10 in DCs [26].
Individual sPLA 2 s exhibit unique tissue or cellular distributions and enzymatic properties and exert their specific functions by producing lipid mediators; by altering membrane phospholipid composition; by degrading foreign phospholipids from microorganisms or diet; or by modifying extracellular noncellular lipid compounds such as lipoproteins, pulmonary surfactant, or extracellular vesicles in response to given microenvironmental cues [68][69][70][71]. Currently, two sPLA 2 isoforms have been reported to participate in the regulation of CHS. sPLA 2 -IID, which is expressed in lymph node DCs, protects against CHS by putting a brake on Th1 immunity through mobilization of ω3 polyunsaturated fatty acids and their anti-inflammatory/proresolving metabolites [46]. sPLA 2 -IIF, which is expressed in keratinocytes, exacerbates CHS by facilitating epidermal hyperplasia through the generation of a unique lysophospholipid [47]. The present study showing that sPLA 2 -III in MCs plays a role in limiting CHS by mobilizing anti-inflammatory PGD 2 in a feed-forward loop of MC-fibroblast communication represents the third example of the sPLA 2 -mediated regulation of CHS, further highlighting the diverse functions of individual sPLA 2 isoforms in mobilizing distinct lipids in different cells and tissues.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.