CD300f:IL-5 cross-talk inhibits adipose tissue eosinophil homing and subsequent IL-4 production

Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5 Tg /Cd300f −/− mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5 Tg /Cd300f −/− mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f −/− mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5 Tg /CD300f −/− were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.

receptors that are capable of delivering co-activating or inhibitory signals by binding adaptor molecules such as DNAX-associated protein 10 (DAP10) or phosphatases, respectively 18 . Specifically, CD300a and CD300f have long cytoplasmic tails containing immunoreceptor tyrosine based inhibitory motifs (ITIM) that enable them to suppress cellular activation by recruitment of phosphatases such as SHP-1 and SHP-2 17,18 . Signaling via CD300f is not confined to phosphatases as CD300f can recruit the p85α subunit of the PI3K signaling pathways as well 19,20 . Intriguingly, under baseline conditions in the adipose tissue, CD300f is highly expressed by eosinophils, whereas other resident cells such as macrophages and dendritic cells express low levels of CD300f 21 . The findings reported in this manuscript were compelled by our unexpected finding of a gross morphological feature seen in genetically engineered hypereosinophilic mice deficient in Cd300f; these mice displayed a prominent rigid humpback, composed of a massive accumulation of eosinophil in the adipose tissue. Accordingly, we aimed to define molecular pathways that regulate the activities of adipose tissue eosinophils with specific emphasis on the cross-talk between CD300f and IL-5 receptor signaling. We report that CD300f expression is regulated by IL-5 and that increased IL-5 levels resulted in distinct accumulation of eosinophils in white and brown adipose tissue, which was histologically characterized by mixed eosinophilic and macrophage/giant cell foci and accompanied by increased IL-4 levels, macrophage and monocyte proliferation, alternatively activated macrophage markers and macrophage-derived CCL24 (eotaxin-2). Cd300f −/− mice showed an age-dependent increase in adipose tissue eosinophils, which was associated with decreased adipose tissue weight as well as decreased weight gain and glucose intolerance following high fat diet, a phenomenon which was markedly augmented in Il5 Tg /CD300f −/− mice. These data identify a novel checkpoint for regulating adipose tissue eosinophils and adipose tissue homeostasis, and have potential implications in understanding and treating metabolic diseases such as obesity and diabetes.

Results
CD300f is expressed by eosinophil progenitors. We have recently shown that IL-5-induced responses are regulated by ITIM-bearing receptors such as PIR-B 22 . Therefore, we were interested in examining whether bone marrow eosinophil progenitors express additional ITIM-bearing receptors such as those belonging to the CD300 receptor family. To this end, the expression of CD300a and CD300f was determined in hematopoietic stem and progenitor cells including hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), megakaryocyte-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and eosinophil progenitors (EoPs) (see gating strategy in S1) 22,23 . Among all progenitor cells, the expression of CD300f was exclusive to EoPs (Fig. 1A), while CD300a displayed a broad pattern of expression, readily detected even by early HSCs (S2).

Overexpression of IL-5 increases CD300f expression. The eosinophil-associated expression pattern of
CD300f raised the possibility that the expression of CD300f may be affected by eosinophil-associated cytokines such as IL-5. Indeed, assessment of bone marrow eosinophils from Il5 −/− mice demonstrated that the expression of CD300f was decreased in immature (Siglec-F + /CCR3 − ) and mature (Siglec-F + /CCR3 + ) eosinophils (Fig. 1B). Furthermore, the expression of CD300f steadily increased upon addition of IL-5 to in vitro generated bone marrow-derived eosinophils (Fig. 1C, days 1-7 gating on all cells; days 8-14 gating on Siglec-F + cells). Consistent with this finding, the expression of CD300f (but not CD300a, S2) was highly increased on eosinophils that were obtained from IL-5 overexpressing mice (Il5 Tg mice, Fig. 1D). Interestingly, although the expression of CD300f was highest in colonic and white adipose tissue eosinophils in wild type mice, the expression of CD300f was further increased following exposure to IL-5 (Fig. 1D). Importantly, the specificity of our anti-CD300f antibody was recently established using Cd300f −/− cells 21 .
IL-5-driven adipose tissue eosinophil accumulation is negatively regulated by CD300f. The finding that CD300f expression was regulated by IL-5 levels raised the hypothesis that CD300f regulates IL-5-induced responses in eosinophils. To assess this possibility, we generated Il5 Tg mice that lack Cd300f (Il5 Tg / Cd300f −/− mice). Strikingly, six to eight week old Il5 Tg /Cd300f −/− mice developed an obvious rigid humpback, which was anatomically located in the proximity of the interscapular brown adipose tissue (BAT) 24 . Thus, we were interested in determining whether Il5 Tg /Cd300f −/− mice display increased eosinophil accumulation in the BAT. Eosinophils were readily detected in the BAT of Il5 Tg mice. Nonetheless, Il5 Tg /Cd300f −/− mice displayed substantially elevated BAT eosinophil levels (~5-fold increase) in comparison with Il5 Tg mice ( Fig. 2A). To assess whether increased eosinophilia in Il5 Tg /Cd300f −/− mice was restricted to the BAT or evident in the white adipose tissue (WAT) as well, the inguinal white adipose tissues of Il5 Tg /Cd300f −/− mice were excised and eosinophil levels assessed. The WAT of Il5 Tg /Cd300f −/− mice displayed a ~7.5-fold increase in eosinophil levels in comparison with Il5 Tg mice ( Fig. 2A). Of even greater interest was the finding that elevated eosinophilia was specific to the adipose tissue since bone marrow, splenic and gastrointestinal eosinophil levels were comparable between Il5 Tg and Il5 Tg / Cd300f −/− mice (Fig. 2B). The increased eosinophil levels in adipose tissue was associated with decreased peripheral blood eosinophilia (Fig. 2C), consistent with a recruitment from the blood into the adipose tissue 21 .
Histological assessment of WAT and BAT revealed marked cellular infiltrate in the adipose tissue of Il5 Tg / Cd300f −/− mice (Fig. 2D,E). In fact, the adipose tissue of Il5 Tg /Cd300f −/− mice displayed numerous cellular foci that consisted of cells with eosinophil, macrophage and giant cell morphology (Fig. 2D,E). Indeed, anti-eosinophil major basic (MBP) protein staining, which specifically stains eosinophils 25 , demonstrated the abundance of eosinophils in these cellular foci (Fig. 2F).
CD300f negatively regulates eosinophil-dependent adipose tissue IL-4 production. The unique structural change in the adipose tissue with the presence of macrophages and giant cells suggested that Il5 Tg / Cd300f −/− mice would display increased IL-4 expression 26 . While IL-4 mRNA expression was nearly undetectable in the WAT and BAT of wild type and Cd300f −/− mice in comparison with Hprt (0.000076 ± 0.000087 and 0.00017 ± 0.00086, respectively) the expression of IL-4 was markedly increased in the WAT and BAT of Il5 Tg mice (~69-and ~122-fold increase in comparison with wild type mice). IL-4 mRNA expression was further elevated in the WAT and BAT of Il5 Tg /Cd300f −/− mice by ~17-and ~10-fold compared to Il5 Tg mice, respectively (Fig. 3A). Elevated IL-4 expression in Il5 Tg /Cd300f −/− mice was observed in adipose tissue protein extracts as well (Fig. 3B).
Since eosinophils are the major source for IL-4 in the adipose tissue 3, 5 , increased IL-4 expression in Il5 Tg / Cd300f −/− mice led us to hypothesize that CD300f may act as a negative regulator of eosinophil-derived IL-4 production. To assess this hypothesis, primary eosinophils were purified from the WAT of Il5 Tg and Il5 Tg /Cd300f −/− mice and baseline secretion of IL-4 was assessed (Fig. 3C). In agreement with previous studies showing that adipose tissue eosinophils secrete IL-4 2-4, 6, 27 , baseline IL-4 secretion was detected in the supernatants of primary adipose tissue eosinophils that were obtained from Il5 Tg mice (Fig. 3C). Notably, eosinophils from Il5 Tg /Cd300f −/− mice displayed enhanced secretion of IL-4 under baseline conditions (Fig. 3C). Furthermore, activation of eosinophils from Il5 Tg /Cd300f −/− mice with phorbol 12-myristate 13-acetate (PMA), a potent activator of eosinophils, resulted in substantially increased IL-4 secretion in comparison with PMA-stimulated eosinophils from Il5 Tg mice and untreated eosinophils (Fig. 3C). Increased eosinophil levels ( Fig. 2) and IL-4 production ( Fig. 3A-C) by eosinophils raised the possibility that CD300f may regulate eosinophil differentiation and/or maturation in the bone marrow. To address this possibility, GeneChip ® microarray analysis of highly purified primary bone marrow eosinophils that were sorted from Il5 Tg and Il5 Tg /Cd300f −/− mice was performed. This analysis revealed that with the exception of Cd300f (p < 0.0001), no other differences were apparent between eosinophils from Il5 Tg and Il5 Tg /Cd300f −/− mice including no difference in Il4 mRNA expression ( Figure S3). Furthermore, no developmental differences were observed in the absence of Cd300f since eosinophils from bone marrow-derived cultures did not show any differences in overall cell counts, cell proliferation or apoptosis (S4). Collectively, this data suggests that increased IL-4 production in the adipose tissue of Il5 Tg /Cd300f −/− mice may be due to increased eosinophil accumulation and/or increased production of IL-4 in Cd300f −/− eosinophils in response to local activating factors.
IL-5 and phosphorylation of ERK was determined using PhosphoFlow as we previously published [29][30][31][32] . IL-5 stimulated I.29 cells containing an empty vector displayed elevated ERK phosphorylation (Fig. 3E,F). Overexpression of CD300f blocked IL-5-induced ERK activation (Fig. 3E,F). Importantly, decreased IL-5-induced responses in CD300f-expressing I.29 cells were not due to alterations in IL-5Rα expression (Fig. 3G). CD300f has been shown to associate with PI3K 19,20 . Thus, we were interested in determining whether CD300f could regulate the activation of AKT, a downstream mediator of PI3K signaling 33 . To this end, CD300f-expressing and empty vector infected control cells were stimulated with IL-5 and the phosphorylation of AKT was determined. In response to stimulation with IL-5, CD300f-expressing I.29 cells showed elevated AKT activation in comparison with control cells (Fig. 3H).

CD300f-IL-5 receptor cross talk regulates alternatively activated macrophage formation in adipose tissue.
To determine whether increased IL-4 production in the adipose tissue of Il5 Tg /Cd300f −/− mice resulted in increased alternatively activated macrophage formation, the expression of various alternatively activated markers was examined 34   Increased IL-4 production in the WAT Il5 Tg /Cd300f −/− mice was associated with enhanced monocyte (defined as: 4H) and to lesser extent macrophages (defined as Fig. 4H). While ~15% of the monocytes from the WAT of Il5 Tg mice stained positive for EdU, ~37% of the monocytes from the WAT of Il5 Tg /Cd300f −/− mice were EdU + (more than 2-fold increase, p < 0.05). Subsequently, a ~15-and ~10-fold increase in total macrophage numbers was observed in the WAT and BAT of Il5 Tg /Cd300f −/− mice, respectively (Fig. 4I). Since increased numbers of adipose tissue macrophages could result from increased chemotaxis of monocytes and their subsequent differentiation into macrophages, we determined the levels of CCL2, a major monocyte chemoattractant, which may be produced by multiple cell types including eosinophils and fibroblasts in response to GM-CSF 35,36 . Interestingly, Il5 Tg / Cd300f −/− mice displayed increased Ccl2 expression in the BAT but not WAT (S5).
Viable adipose tissue endothelial cells express a CD300f ligand. The finding that CD300f specifically regulated the accumulation of eosinophils into the adipose tissue suggested that CD300f ligands may be expressed and perhaps restricted to adipose tissue and that their expression may be augmented by IL-5. To this end, enzymatically digested adipose tissue cells were stained with CD300f-Fc fusion protein. Among all viable cells, CD300f-Fc fusion protein was found to bind endothelial cells (CD45 − /CD31 + /CD34 + /DAPI − , Fig. 5A). Notably, binding of CD300f-Fc fusion protein to viable endothelial cells was further augmented in endothelial cells obtained from Il5 Tg mice (Fig. 5B,C). Importantly, CD300f-Fc fusion protein was specific to viable endothelial cells in the adipose tissue since viable lung endothelial cells displayed nearly no binding (Fig. 5C). Cd300f −/− mice display age-related accumulation of eosinophils and macrophages. Since CD300f regulated eosinophil homing to the adipose tissue, we were interested in determining whether Cd300f −/− mice displayed increased eosinophil and macrophage levels under baseline conditions. Cd300f −/− mice displayed age-associated accumulation of eosinophils in the adipose tissue (Fig. 6A). Eosinophil levels in Cd300f −/− mice were significantly higher in mice as they achieved 5 months of age. Consistently, increased eosinophil numbers in aged Cd300f −/− mice were associated with increased macrophage numbers (Fig. 6B), as well as decreased WAT weight (Fig. 6C) and size (Fig. 6D).
Cd300f −/− mice display decreased diet-induced weight gain and glucose intolerance. To assess the physiological role of CD300f in obesity and glucose metabolism, eight-week old Cd300f −/− mice were fed with normal chow or high fat diet and their weight as well as sensitivity to glucose challenges were monitored. Cd300f −/− mice displayed decreased weight gain in response to high fat diet that was evident at later (starting at week 13) time points of the diet (Fig. 6E). Decreased weight gain in Cd300f −/− mice was not due to decreased food intake since Cd300f −/− mice that were assessed in metabolic cages displayed slightly but significantly increased appetite (S6). Moreover, following 18 weeks (i.e. when the mice were 26 weeks old) of high fat diet, Cd300f −/− mice displayed increased glucose tolerance (Fig. 6F). No differences were observed in weight gain or glucose sensitivity in response to normal chow diet (S7).
Il5 Tg /CD300f −/− are protected from diet-induced weight gain and glucose intolerance. To further explore the association between increased eosinophilia in Cd300f −/− mice and decreased weight gain and glucose metabolism, we subjected eight-week old Il5 Tg and Il5 Tg /Cd300f −/− mice to a normal chow and high fat diet. Notably, glucose tolerance test following normal chow diet revealed no significant differences between Il5 Tg and Il5 Tg /Cd300f −/− mice ( Figure S8). Although Il5 Tg mice were relatively protected from diet-induced obesity and glucose intolerance they still exhibited significantly increased weight gain and glucose intolerance in comparison with normal chow-fed mice or day 0 (Fig. 7A,B). In contrast, Il5 Tg /Cd300f − /mice displayed markedly decreased weight accumulation in response to high fat diet (Fig. 7C). Protection from diet-induced weight gain was associated with substantially improved glucose tolerance testing (Fig. 7D). Moreover, WAT size (Fig. 7E) and weight (Fig. 7F) were substantially smaller in Il5 Tg /Cd300f −/− mice. Assessment of Il5 Tg /Cd300f −/− mice in metabolic cages showed that decreased weight gain in Il5 Tg /Cd300f −/− mice was not due to decreased food/water uptake, appetite or increased urination (S9). Importantly, although no differences were observed in weight gain between Il5 Tg and Il5 Tg /Cd300f −/− mice in response to normal chow diet (S10), decreased adipose tissue weight and size was still observed (Fig. 7F). Consistent with decreased WAT size and weight, adipocyte size was smaller in the WAT of Il5 Tg /Cd300f −/− mice in comparison with Il5 Tg mice (Fig. 7G,H).

Discussion
The ability of eosinophils to migrate into the adipose tissue and modulate the activities of macrophages by secreting IL-4, has become a paradigm in adipose tissue homeostasis 37 . Nonetheless, pathways that regulate the recruitment and subsequent activation of eosinophils in the adipose tissue are largely unknown. In this study, we demonstrate that: a) CD300f is uniquely expressed by eosinophil progenitors; b) overexpression of IL-5 increases CD300f expression and baseline CD300f expression is partially dependent on IL-5; c) overexpression of IL-5 in the absence of CD300f results in a marked and specific increase in adipose tissue eosinophils, which were located in cellular foci and were accompanied by the accumulation of giant cells and macrophages; d) Specific accumulation of eosinophils in the adipose tissues of Il5 Tg /Cd300f −/− was associated with increased expression of a CD300f ligand in viable adipose tissue endothelial cells and elevated production of IL-4; e) Subsequently, increased IL-4 expression resulted in increased monocyte and macrophage proliferation consequently resulting in increased macrophage levels and induction of hallmark alternatively activated genes; f) CD300f regulated eosinophil and macrophage accumulation in the adipose tissue in an age-dependent fashion and was associated with decreased white adipose tissue size and weight as well as decreased weight gain and glucose intolerance after feeding with high fat diet. Finally, g) Cd300f −/− mice displayed a dramatic reduction in diet-induced weight gain and glucose intolerance in the presence of elevated IL-5 levels. Collectively, our data illustrate CD300f as a novel intrinsic counter-regulator of IL-5-driven adipose tissue eosinophil accumulation and IL-4 production, which governs diet-induced adipose tissue homeostasis and subsequent weight gain and glucose metabolism.
Notably, the finding that Il5 Tg /Cd300f −/− mice had increased alternatively activated macrophage accumulation in the adipose tissue, which was associated with decreased diet-induced weight gain and insulin resistance is likely an eosinophil-dependent phenomenon. This notion is based on several observations. We have recently shown that ILC2 cells (which have key roles in the adipose tissue) 4, 6 and adipose tissue macrophages do not express CD300f 21,32 . Furthermore, murine IL-5 receptor is uniquely expressed by eosinophils and B cells 38,39 , and eosinophil-derived IL-4 has been recently shown to regulate macrophage polarization and subsequent glucose metabolism 3 . One of our striking observations was the specific increase in accumulation of eosinophils into the WAT and BAT of Il5 Tg /Cd300f −/− mice. This finding was of particular interest since the back of Il5 Tg /Cd300f −/− mice was extremely rigid (likely due to stiffness of the BAT) to a state that we could differentiate between Il5 Tg / Cd300f −/− and Il5 Tg mice simply by identifying a dorsal patch of induration (100% prediction). Directly related, many obese individuals develop a humpback (commonly termed: Buffalo hump). Although the mechanisms for hump formation are unclear, excessive cortisol levels and excessive fat deposits have been implicated in this abnormal anatomical formation, at least in part. It is interesting that the mice developed fat accumulation which clinically resembled a "buffalo hump" but this cannot be simply explained as these same mice were protected from obesity and the presence of eosinophils in buffalo humps has not been examined. Nevertheless, our data suggest that IL-5, CD300f and eosinophils are integrated component of adipose tissue formation and may have implications for the type of dysregulated processes that involved in humpback formation.
Il5 Tg mice display elevated eosinophilia in various organs such as the spleen, peritoneal cavity and GI tract 40 , which were not affected by the lack of Cd300f. This organ-specific phenomenon, is likely explained by two complementary findings. First, we demonstrate that the adipose tissue of Il5 Tg /Cd300f −/− mice displayed substantially increased expression of monocyte/macrophage-derived CCL24 (but not CCL11), which has been previously shown to regulate eosinophil migration to the gut and adipose tissue in an integrin-dependent fashion 3,41,42 . Moreover, our findings demonstrate that baseline IL-4 secretion was increased in Cd300f −/− eosinophils and that IL-4 secretion was further increased in Cd300f −/− eosinophils following activation with PMA. Whether CD300f negatively regulates IL-4 production from eosinophils in response to local factors in the adipose tissue is yet to be defined. Since IL-4 stimulates CCL24 production 43 , increased CCL24 production by macrophages is likely caused by the elevated levels of IL-4, which were present in the adipose tissue of Il5 Tg /Cd300f −/− mice. Second, we show that adipose tissue endothelial cells distinctly express a CD300f ligand, and that its expression is amplified in the presence of IL-5. We have recently shown that CD300f acts as a negative regulator of eosinophil chemotaxis towards CCL24 and CCL11 but not towards other chemo attractants such as LTB 4 21 . Therefore, the finding that adipose tissue endothelial cells express ligands for CD300f places them at an interface to deliver inhibitory signals via CD300f to suppress eosinophil accumulation in response to adipose derived CCL24. Collectively, integration of these findings suggests a model where eosinophil accumulation in the adipose tissue is suppressed by CD300f upon binding to its ligand. In the absence of CD300f, the inhibitory signal towards IL-5-driven and CCL24-mediated accumulation of eosinophils is impaired. Thus, more eosinophils enter the adipose tissue where they secrete increased levels of IL-4. Subsequently, elevated IL-4 activates macrophages to secrete increased levels of CCL24, which further enhances eosinophil migration in a "vicious cycle"-like fashion.
Although Cd300f −/− mice displayed decreased weight gain and increased glucose uptake, these parameters were relatively modest especially in comparison with Il5 Tg /Cd300f −/− mice. The reason for this modest baseline effect is possibly due to additional functions that CD300f has in the regulation of immune responses by type 2 cytokines such as IL-4 and IL-33 32,44 . We have recently shown that CD300f is physically associated with IL-4Rα and that it is necessary for IL-4-induced activation of multiple cells including macrophages and eosinophils. In fact, IL-4 stimulated Cd300f −/− macrophages display decreased activation of STAT6 and subsequently decreased induction of hallmark alternatively activated markers such as Relm-α and arginase1 44 . Thus, on one hand Cd300f −/− mice display elevated eosinophilia which secrete increased levels of IL-4 and on the other hand, Cd300f −/− macrophages may be less responsive to IL-4, which may cause them to promote obesity and glucose intolerance. This balance is substantially altered in the presence of IL-5, which enhances eosinophil accumulation and results in markedly elevated levels of IL-4 that can overcome the requirement for CD300f in IL-4-induced responses. In support of this notion, dose response experiments stimulating macrophages with IL-4 revealed that CD300f is required for IL-4-induced macrophage stimulation at low doses (1-20 ng/ml) but not at high doses (i.e. 50-100 ng/ml) (Munitz et al. data not shown). Therefore, increased levels of IL-4 in the adipose tissue of Il5 Tg /Cd300f −/− mice is likely capable of overcoming the requirement of CD300f in IL-4 induced responses. Hence, macrophages (which express low levels of CD300f) in Il5 Tg /Cd300f −/− mice can readily respond to IL-4 and are polarized into an alternatively activated state. This results in substantial protection from diet-induced weight gain and insulin resistance in Il5 Tg /Cd300f −/− mice in comparison with CD300f −/− mice. While we cannot exclude regulatory effects of CD300f on macrophages and/or monocytes it is important to note that under baseline conditions the expression of CD300f in the adipose tissue is exclusively confined to eosinophils 21 . Furthermore, macrophages and monocytes do not respond to IL-5. Thus, if such an effect is present, it is likely a secondary one.
Our study also highlights a key role for CD300f in IL-5-driven accumulation of adipose tissue eosinophils. This finding has therapeutic implications as the IL-5:IL-5Rα signaling pathway is now an entry point for a new class of emerging medicines 45 . Given the importance of IL-5-induced effects on eosinophils, it is prone to tight negative regulation in order to avoid hypereosinophilic responses. In this study, we show specific and restricted regulation of IL-5-driven response by CD300f in adipose tissue eosinophils. Nonetheless, CD300f can regulate the activities of various cells and has been implicated in multiple diseases including allergic diseases, colitis, brain injury and experimental autoimmune encephalomyelitis [46][47][48][49] . Specifically, the expression of CD300f was increased in various clinical conditions. For example, CD300f mRNA expression was increased in biopsies from pediatric Crohn's disease patients. Furthermore, peripheral blood eosinophils from atopic individuals displayed elevated CD300f. In fact, CD300f regulated the development of experimental colitis and allergic diseases 21,47,49,50 . Thus, our findings regarding the role of CD300f in the adipose tissue are likely translatable to human disease as well.
The regulatory function of CD300f on IL-5 appears to be relatively specific in regards to homing of eosinophils into the adipose tissue. CD300f likely acts by counter-regulation of specific IL-5 signaling events such as ERK and pAKT. It is important to note that our signaling studies bear the limitation that they were conducted in B cells that express IL-5 receptors and not eosinophils. Thus, it is possible that in eosinophils, CD300f may govern different pathways. Nonetheless, these studies support our overall observation that CD300f is capable of regulating IL-5-induced effects in the signaling level as well. The finding that CD300f can regulate distinct IL-5-driven responses and signaling cascades is consistent with findings regarding additional receptors expressed by eosinophils, which can regulate only a fraction of IL-5-induced responses in eosinophils. For example, eosinophils that were isolated from mice that lack the signaling domain of SIRPα (Sirp1a Cyto −/− mice) showed no developmental defects in eosinophils and only displayed reduced viability and degranulation, with specific effects in the small intestine 51 . Furthermore, the inhibitory receptor PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils in the bone marrow by suppressing the pro-apoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway 22 . These effects were exclusive to the bone marrow but not peripheral organs. Finally, CD300a, the additional ITIM-bearing receptor belonging to the CD300-Ig superfamily negatively regulates human eosinophil survival in response to GM-CSF and IL-5 52 . Thus, it appears that IL-5-induced eosinophil responses are differentially regulated by ITIM-bearing receptors in a tissue-dependent fashion that is likely based on the availability of ligands and or receptor expression. As anti-IL-5 therapy is now in the clinic, it will be interesting to assess the expression and function of ITIM-bearing receptors in eosinophils from human patients before and after treatment and to assess whether any metabolic (e.g. obesity, diabetes) alterations occur in individuals chronically treated with anti-IL-5 therapy.
Despite its inhibitory activity towards IL-5 induced responses in vivo, the in vitro experiments show that CD300f differentially regulates IL-5-induced signaling cascades. On one hand, CD300f was capable of suppressing IL-5-dependent ERK activation and on the other hand it induced phosphorylation of AKT. The finding that CD300f can elicit both inhibitory and activating signals is consistent with the presence of intracellular domains that contain two ITIM motifs (which mediate inhibition), one immunoreceptor tyrosine-base switch motif (ITSM) 53 and a p85α phosphoinositide 3-kinase-binding motif (which mediates co-activation). Indeed, various studies have demonstrated dual and opposing functions for CD300f. For instance, CD300f acts as a functional binding partner and co-receptor for IL-4Rα, can co-localize with ST2 and can promote phagocytosis 20,32,44 . In contrast, CD300f can suppress cellular chemotaxis and toll-like receptor signaling 21,54,55 . Furthermore, CD300f has been shown to have opposing activation and inhibitory functions in mast cell activation 48,49 . Interestingly, our data demonstrate a novel finding with respect to CD300f signaling. To date, the co-activating or inhibitory activities of CD300f were shown in response to different stimuli, which either trigger co-activation of inhibition. We show that in response to the same stimulus (i.e. IL-5), CD300f is capable of inhibitory (e.g. ERK suppression) and co-activating (e.g. AKT activation) signals simultaneously. Consequently, the overall inhibitory effect of CD300f in respect to IL-5, is likely due to an intracellular balance between differential signaling cascades.
In summary, we provide evidence that CD300f is an intrinsic regulator of IL-5-induced eosinophil responses with specific effects in the adipose tissue. These findings provide new insights regarding the molecular pathways fine tuning the activities of adipose tissue eosinophilia and have significant implications in promoting metabolic homeostasis through the eosinophil-macrophage axis.

Methods
Mice. WT C57BL/6 mice were obtained from Harlan Laboratories (Rehovot, IL) and maintained in-house.
Generation of Cd300f −/− and Il5 −/− mice has been previously described 46,56 . Cd300a −/− mice (B6N.129S5-Cd300atm1Lex/Mmcd, ID 032182-UCD) were obtained from the Mutant Mouse Regional Resource Center (MMRC, University of California, Davis, CA; originally donated by Genentech) and backcrossed to C57BL/6 mice for 10 generations 21 . CD3-IL-5 transgenic mice (NJ.1638, Il5 Tg ) mice were kindly provided by Dr. Jamie Lee (Mayo Clinic, Scottsdale, AZ) 40 . Il5 Tg were crossed with Cd300f −/− mice for two generations to form the Il5 Tg /Cd300f −/− line (both on the C57Bl/6 background). All experiments were reviewed and approved by the Animal Care Committee of Tel Aviv University (Number M-13-029, M-13-30), and were performed in accordance with its regulations and guidelines regarding the care and use of animals for experimental procedures. All of the experiments were conducted in the specific pathogen free facilities of the Tel Aviv University. In all experiments, age-, weight-, and sex-matched mice were used.
Preparation of tissue single cell suspension. Abdominal and neck adipose tissue were excised and incubated in 1.5 mg/ml collagenase, 0.75 mg/ml hyaluronidase and 0.1 mg/ml DNase I (Sigma, Rehovot, IL) containing DMEM (Gibco, Invitrogen, Grand Island, NY) with 2% fetal bovine serum (FBS) for 1 hour at 37 °C. The suspension was vortexed and strained. Colon and jejunum was washed with phosphate-buffered saline (PBS), cut into small pieces and incubated in Hank's Balanced Salt Solution (HBSS, Biological industries, IL) supplemented with 5% FBS, 2 mM EDTA and 1 mM DTT, for 40 minutes at 37 °C. Afterwards, the colon pieces were incubated in PBS with calcium chloride and magnesium chloride, supplemented with 5% FBS, 1 mg/ml collagenase A (Roche, Germany) and 0.1 mg/ml DNase I (Sigma, Rehovot, IL), for 40 minutes at 37 °C. The suspension was vortexed, strained and centrifuged to receive single cell suspension of lamina propria cells. Peritoneal lavage was extracted by penetrating and flushing the peritoneal cavity with a 21G needle joined to a 10 ml PBS containing syringe and drawing back the cell containing fluid. Spleen was crushed against a 70 μm cell strainer sunk in PBS. BM cells were extracted as described above. Blood sample were collected from the eye using cannula and heparin-coated tube while the mouse is sedated with a non-lethal dose to isoflurane 99.9%. Tissue eosinophil collection. Single cell suspension was obtained from abdominal adipose tissue or bone marrow as described above. The cells were then enriched for eosinophils using positive selection with magnetic beads (Miltenyi Biotech, Germany) linked to anti-Siglec-F antibody according to manufacturer's instructions.