Intranasal administration of ceramide liposome suppresses allergic rhinitis by targeting CD300f in murine models

Allergic rhinitis (AR) is caused by type I hypersensitivity reaction in the nasal tissues. The interaction between CD300f and its ligand ceramide suppresses immunoglobulin E (IgE)-mediated mast cell activation. However, whether CD300f inhibits the development of allergic rhinitis (AR) remains elusive. We aimed to investigate the roles of CD300f in the development of AR and the effectiveness of intranasal administration of ceramide liposomes on AR in murine models. We used ragweed pollen-induced AR models in mice. Notably, CD300f deficiency did not significantly influence the ragweed-specific IgE production, but increased the frequency of mast cell-dependent sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in our models. Similar results were also obtained for MCPT5-exprssing mast cell-specific loss of CD300f. Importantly, intranasal administration of ceramide liposomes reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in AR models. Thus, CD300f–ceramide interaction, predominantly in mast cells, alleviates the symptoms and progression of AR. Therefore, intranasal administration of ceramide liposomes may be a promising therapeutic approach against AR by targeting CD300f.

www.nature.com/scientificreports/ the prevalence of AR has been increasing worldwide, there is an urgent need to identify potential therapeutic targets and develop more effective and safer therapeutic strategies against AR 1,2 .
In the present study, we used murine models 3,4,30,31 of AR induced by RW pollen in wild-type (WT) and CD300f −/− mice and demonstrated that CD300f suppresses the symptoms of AR as well as mast cell degranulation and eosinophil accumulation in the nasal tissues.Importantly, the intranasal administration of ceramide liposomes improves the symptoms of AR by targeting CD300f, which may provide a novel therapeutic strategy against AR.

Ethical approval
All experiments using mice were approved by the Institutional Review Committee of Juntendo University (approval no 2020136, 2021196, 2022107) and carried out in accordance with relevant guidelines and regulations.All methods are reported in accordance with ARRIVE guidelines.
The liposomes were prepared as follows.The reactor vessel charged with distilled water and sample material was connected to the system with 1/16 inch tubing and placed horizontally in the water bath controlled at 50 °C.Carbon dioxide from a siphon-type cylinder was pumped through the chiller tubing to maintain the contents in the liquid phase.The contents were then heated in the water bath to the supercritical state before introducion into the reactor vessel.The pressure was controlled by the BPR at 20 MPa.When the temperature and pressure in the reactor vessel attained the desired condition, ultrasonication was started at 45 kHz and 600 W. After a

RW pollen-induced murine models of AR
The murine models of AR were used with some modifications 3,4,30,31,35 .Mice were sensitized twice within a 2-week interval by intradermal injection with 100 μg RW pollen plus 1 mg alum on day 0 and intraperitoneal injection with 100 μg RW pollen on day 14, and then intranasal injection with 1 mg RW pollen or phosphate buffer saline (PBS) for four consecutive days (on days 28-31).Alternatively, mice were given intradermal injection with PBS plus 1 mg alum on day 0 and intraperitoneal injection with PBS on day 14, and then intranasal injection with PBS for four consecutive days (on days 28-31) as controls.The frequency of freezing was counted for 10 min immediately after nasal challenge with RW pollen.Sampling of tissues or blood was performed one day after the final challenge with RW pollen.

Treatment with liposomes in mouse models of AR
For intravenous treatment with antibody or liposomes in AR models, mice were intravenously injected with 5 μg of anti-ceramide Ab or control Ab or 2 μg of ceramide liposomes or distilled water as a control on day 27 and days 28, 29, 30, and 31 at 3 h before each intranasal challenge with RW pollen.For intranasal treatment with liposomes, mice were intranasally injected with 2 μg of ceramide liposomes, PS liposomes, or distilled water as a control or 5 mg of fluticasone propionate (GlaxoSmithKline, Tokyo, Japan) or 0.6% dimethyl sulfoxide (DMSO) as a control on day 27 and days 28, 29, 30, and 31 at 5 h before each intranasal challenge with RW pollen 36 .In Fig. 5f, mice were intranasally injected with 0.2, 0.6, or 2 μg of ceramide liposomes or distilled water as a control.In Fig. S6b, mice were intranasally injected with 0.4 or 2 μg of ceramide liposomes or distilled water as a control.

Histological analyses
Histological analyses were performed as previously described 3,4,30,31,38 .Briefly, the removed noses were fixed in 4% paraformaldehyde for 3 days and decalcified in 0.12 mol/L ethylenediaminetetraacetic acid (EDTA) solution (pH 6.5) for 2 weeks.After decalcification, paraffin-embedded sections were stained with Giemsa, Congo red, chloroacetate esterase, or toluidine blue.The numbers of mast cells and degranulated mast cells or the numbers of eosinophils were counted in the nasal mucosa lining the nasal cavity in chloroacetate esterase-stained sections or Congo red-stained sections, respectively.Both toluidine blue-and chloroacetate esterase-stained mast cells were counted as connective tissue mast cells 37 .

Induction of passive cutaneous anaphylaxis (PCA)
PCA was performed as previously described 9,16 .Briefly, mice were injected intradermally with 50 ng anti-dinitrophenyl (DNP) IgE (H1-ε-26) into each ear.Twenty-four hours later, the mice administered an intravenous injection of Evans blue dye (Sigma-Aldrich, St Louis, MO) containing 250 μg DNP-human serum albumin.The amounts of extravasated dye at 30 min after antigen challenges were evaluated by measuring the absorbance at 620 nm wave length.

In vitro analysis of mast cell degranulation
To generate bone marrow-derived mast cells (BMMCs), BM cells were cultured in the presence of 10 ng/mL IL-3 for 5 weeks.To analyze mast cell degranulation, the β-hexosaminidase assay 9 was performed for BMMCs that had been sensitized with 0.5 mg/mL anti-trinitrophenyl (TNP) IgE for 12 h and then stimulated with 10 ng/mL TNPbovine serum albumin (BSA) in the presence of 10 mg/mL ceramide liposomes, PS liposomes, or vehicle for 1 h.

Flow cytometry
Single cell suspensions of peritoneal cells, BM cells, peripheral blood (PB) cells, spleen cells, or nasal tissue cells were analyzed by flow cytometry using FACSVerse (BD Biosciences) equipped with FlowJo software (Tree Star) 9,16,21,37 .To prepare nasal tissue cells 28 , noses were minced and digested 2 mg/mL collagenase type I (FUJI-FILM) and 0.1 mg/mL DNase I (Roche) for 1 h at 37 °C.After filtrating and washing, cell suspensions were used.

CD300f deficiency increased the frequency of sneezing by enhancing the nasal mast cell degranulation in murine models of AR
To examine the role of an inhibitory receptor CD300f in the development of AR, we used murine models of AR.
Mice were immunized twice with RW pollen on days 0 and 14 and then were intranasally challenged with RW pollen for four consecutive days on days 28 to 31 (Fig. 1a).Notably, nasal challenges with RW pollen significantly increased the frequency of sneezing during the observation period in CD300f −/− mice compared with that in WT mice (Fig. 1b).Although nasal challenges with RW pollen increased RW-specific IgE levels in serum after the final challenge in both WT and CD300f −/− mice compared with challenge with PBS, CD300f deficiency did not significantly affect RW-specific IgE levels in the serum after the final challenge with RW pollen (Fig. 1c).Staining of nasal tissue sections showed that the numbers of mast cells were comparable between WT and CD300f −/− mice, irrespective of nasal challenges (Fig. 1d).Notably, about 40% to 60% of mast cells in the nasal tissues were connective tissue mast cells stained by both toluidine blue-and chloroacetate esterase (Fig. 1e).In contrast, the percentages of nasal mast cell degranulation were higher in CD300f −/− mice than in WT mice after the last challenge with RW pollen (Fig. 1f; Supplementary Fig. S1).We confirmed that mast cell deficiency profoundly lowered the sneezing frequency in the same models (Fig. 1g,h).These results indicated that CD300f deficiency increased the frequency of sneezing presumably due to enhanced mast cell degranulation in AR models.

CD300f deficiency increased nasal eosinophils in murine models of AR
Histological analysis showed that eosinophil numbers in the nasal tissues under steady conditions were comparable between WT and CD300f −/− mice.Intranasal challenges with RW pollen increased eosinophils in the nasal tissues from both mice; however, the latter exhibited higher numbers of eosinophils and higher percentages of degranulated eosinophils in the nasal tissues than did the former (Fig. 2a-c; Supplementary Fig. S2).These results indicated that CD300f deficiency enhanced RW-induced eosinophil accumulation and degranulation in murine models of AR.Besides, flow cytometric analysis showed that CD300f was highly expressed in mast cells, eosinophils, and neutrophils in the nasal tissues under steady conditions.Intranasal challenges with RW pollen slightly increased surface expression levels of CD300f in mast cells and eosinophils (Fig. 2d).Taken together, these results indicated that CD300f deficiency accelerated the symptoms and development of AR in murine models.

Treatment with ceramide antibody increased the frequency of sneezing in murine models of AR
To examine the role of the interaction between CD300f and its ligand ceramide in the development of AR, we intravenously injected ceramide antibody 9 into the sensitized WT mice on days 27 to 31 before intranasal challenges in AR models (Fig. 3a).Notably, the frequency of sneezing was higher in the mice administered ceramide antibody than in those administered control antibody (Fig. 3b).Treatment with the ceramide antibody failed to influence serum levels of RW-specific IgE in WT mice (Fig. 3c).Besides, WT mice given an injection of ceramide antibody exhibited remarkable increase in degranulated mast cell number, but not in mast cell and eosinophil number, in the nasal tissues after the final challenge with RW pollen compared with those given control antibody (Fig. 3d-f).These results indicated that treatment with the ceramide antibody disrupted the in vivo CD300fceramide binding 9 , and increased the frequency of sneezing by enhancing mast cell degranulation.

Intranasal administration of ceramide liposomes reduced the symptoms of AR in murine models
We next investigate if treatment with ceramide liposomes improved the development of acute AR.We confirmed that ceramide liposomes inhibited IgE-mediated degranulation of WT BMMCs, but not of CD300f-deficient BMMCs (Supplementary Fig. S3).Then, WT mice were intravenously administered ceramide liposomes on days 27-31 before intranasal challenges in the same models (Supplementary Fig. S4a).In contrast to the ceramide antibody, an intravenous administration of ceramide liposomes reduced the percentages of nasal mast cell degranulation in the nasal tissues.The same administration neither affected the serum levels of RW-specific IgE nor affected the nasal mast cell and eosinophil numbers (Fig. S4b-f).
We then asked if an intranasal administration of ceramide liposomes is effective for the improvement of AR.Importantly, this treatment on days 27-31 reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal mucosa (Fig. 4a,b,e,f).However, the intranasal administration of ceramide liposomes did not significantly alter RW-specific IgE levels in serum and nasal mast cell numbers (Fig. 4c,d).Similarly to the case of ceramide liposomes, intranasally administration of corticosteroid (fluticasone propionate) reduced the frequency of sneezing and the numbers of degranulated mast cells and eosinophils in the nasal tissues 38 ; however, the same treatment neither significantly influenced serum levels of RW-specific IgE nor the nasal mast cell numbers (Supplementary Fig. S5).Thus, intranasal administration of ceramide liposomes reduced the symptoms of AR in murine models.

Intranasal administration of ceramide liposomes, but not of PS liposomes, reduced the frequency of sneezing by targeting CD300f in murine models of AR
We found that the intranasal administration of ceramide liposomes failed to influence the frequency of sneezing in CD300f-deficient mice (Fig. 5a,b).Besides, we confirmed that the intranasal administration of phosphatidylserine neither affect the frequency of sneezing nor the numbers of degranulated mast cells and eosinophils in the nasal tissues (Fig. 5c-e).In addition, the intranasal administration of ceramide liposomes (0.2, 0.6, 2 mg) dose-dependently reduced the frequency of sneezing (Fig. 5f).Although treatment with ceramide antibody increased the frequency of sneezing in WT mice in AR models, the increased frequency was reduced by the intranasal administration of ceramide liposomes (Supplementary Fig. S6).Collectively, these results indicated that intranasal administration of ceramide liposomes increased the local levels of CD300f ligands and inhibited the IgE-mediated mast cell degranulation in the nasal tissues by targeting CD300f, thereby improving the clinical symptoms of AR.
Vol:.( 1234567890 We then used murine models of AR in CD300f flox/flox MCPT5-Cre +/− and CD300f flox/flox mice (Fig. 7a).Consistent with our result (Fig. 1f), flow cytometric analysis showed that CD300f expression was lost in approximately 50% of nasal mast cells, which were supposed to be connective tissue mast cells, in CD300f flox/flox MCPT5-Cre +/− mice (Fig. 7b).The frequency of sneezing after the last challenge with RW pollen was significantly higher in CD300fflox/flox MCPT5-Cre +/− mice than in CD300f flox/flox mice (Fig. 7c).Serum levels of RW-specific IgE and nasal mast cell numbers were comparable between these two types of mice (Fig. 7d,e).Importantly, MCPT5-expressing mast cell-specific loss of CD300f enhanced mast cell degranulation and increased eosinophil numbers in the nasal tissues (Fig. 7f,g).These results suggested that nasal mast cell-expressing CD300f plays critical roles in inhibiting the development of AR in murine models.

Discussion
In the present study, we used murine models of RW pollen-induced AR 3,4,30,31,35 .The engagement of RW-specific IgE-bound FcεRI with RW pollen has been reported to trigger nasal mast cell degranulation, resulting in sneezing, a major symptom of AR, by the local release of chemical mediators in murine models [1][2][3][4]30,31 . Interstingly, we demonstrated that CD300f deficiency increased the frequency of sneezing as well as the percentages of nasal mast cell degranulation in mice, whereas it did not significantly influence the RW-specific IgE production or nasal mast cell numbers.Mast cell deficiency strongly mitigated the sneezing symptom in AR models.Besides, CD300f was highly expressed in mast cells, but not in basophils, in murine nasal tissues.Considering the inhibitory functions of CD300f in mast cells 9,16 , it is therefore plausible that CD300f suppresses the FcεRI -mediated mast cell degranulation in nasal tissues, thereby alleviating the AR symptoms in murine models.Moreover, per our previous results that CD300f-ceramide binding inhibits the IgE-driven mast cell activation 9,16,17,21,22 , intravenous administration of ceramide antibody or ceramide liposomes increased or decreased the sneezing symptom presumably by interfering with the CD300f-ceramide interaction or increasing the levels of ceramide surrounded by nasal mast cells, respectively.Intriguingly, loss of CD300f also enhanced eosinophil infiltration and degranulation in the nasal tissues in AR models: activated eosinophils are thought to induce the damage of the nasal tissues, which is associated with nasal congestion 1,2 .Importantly, CD300f deficiency only in MCPT5expressing mast cells, corresponding to connective tissue mast cells, increased the frequency of sneezing, the percentages of nasal mast cell degranulation, and the numbers of eosinophils infiltrating into the nasal tissues in AR models.It should be noted that comparable numbers of connective tissue mast cells and mucosal mast cells distribute in the nasal tissues.In addition, it was recently reported that eotaxin (CCL11) and its receptor CCR3 are critical in nasal eosinophil accumulation in AR 39,40 .Histamine released from degranulated mast cells induces local production of eotaxin in endothelial cells 41 .Moreover, histamine, adenosine, or tryptase released from degranulated mast cells directly activates eosinophils 42 .Accordingly, it is reasonable to conclude that the mast cell-expressing CD300f inhibited the FcεRI -mediated mast cell degranulation in the nasal tissues, thereby suppressing local infiltration of eosinophils in AR models.Given that CD300f inhibits eotaxin-induced eosinophil activation 20 , it seems possible that eosinophil-expressing CD300f also suppressed eosinophil accumulation to a certain degree in AR models.
Most importantly, we demonstrated that the intranasal administration of ceramide liposomes, but not PS liposomes, suppressed the sneezing symptoms as well as mast cell degranulation and eosinophil infiltration in the nasal tissues in AR models.Taken together, the results suggest that ceramide liposomes inhibit nasal mast cell degranulation predominantly by targeting the nasal mast cell-expressing CD300f, which in turn suppresses eosinophil infiltration.Because CD300f was highly expressed in nasal eosinophils, the interaction between eosinophil-expressing CD300f and ceramide may also play a certain role in inhibiting eosinophil accumulation.Consistent with enhanced expression of CD300f in PB eosinophils from AR patients 20 , we found increased expression of CD300f in nasal mast cells and eosinophils from mice with AR, implying that CD300f down-regulates the development of AR.In any case, intranasal administration of ceramide liposomes suppressed the development of AR in murine models by targeting CD300f.However, we should consider that cellular and extracellular ceramides have a variety of biological functions in various tissues that are associated with the length and saturation of fatty acyl chains in the ceramides 43,44 .Endogenous intracellular ceramides appear to be different from exogenous ceramides, such as ceramide (d18:1/24:0) liposomes used in this study.In contrast, endogenous extracelluar ceramides such as those present in lipoproteins may act as CD300f ligands, similarly to ceramide liposomes.Cell-permeable short-chain ceramides have various functions [43][44][45][46] .For example, C6-ceramide suppresses ERK activation in FceRI-stimulated RBL-2H3 cells by activating a proteins tyrosine phosphatase 45 .Additionally, C2-ceramide inhibits the degranulation in FceRI-stimulated RBL-2H3 cells by suppressing phospholipase D activation 46 .We cannot rule out the possibility that these actions are also involved in the inhibition of mast cell activation by ceramide (d18:1/24:0) liposomes; however, cell-permeable short-chain ceramides have biological properties different from those of natural species 43,44 .
In the present study, we prepared ceramide liposomes by using ultrasonic supercritical fluid system 33,34 .
Accordingly, the improvement of this method will be critical for the clinical application of ceramide liposomes as a therapeutic strategy against AR.This treatment is expected to have an immediate effect on the AR symptoms.Notably, the intranasal administration of corticosteroid or ceramide liposomes showed similar treatment effects on AR in murine models.Although considerable controversy exists over whether glucocorticoids inhibit histamine release from mast cells 47 , it was reported that glucocorticoids rapidly inhibit IgE-mediated exocytosis of mast cells by reducing [Ca 2+ ] i elevation 48 .Because these two treatments are effective against AR through different molecular mechanisms, the combination therapy with ceramide liposomes and corticosteroid may be a promising treatment against AR 1,2 .Nonetheless, we should measure comprehensively ceramide levels in the nasal It should be noted that in the present study, CD300f seemed to play a negligible role in antigen-specific IgE production in our models of AR.The route of RW administration and/or the presence of alum adjuvant in RW administration may affect Th2/ T follicular helper (Tfh) responses via CD300f.Analysis of mice deficient in CD300f specifically in immune cells (e.g., dendritic cells) will be required for further investigation.
In conclusion, CD300f inhibits the symptoms and progression of AR in murine models.Intranasal administration of ceramide liposomes might be an attractive therapeutic strategy against AR.

Figure 1 .
Figure 1.CD300f deficiency exacerbated RW pollen-induced AR in murine models.(a) A schematic representation of murine models of RW-induced AR. "RW" or "PBS" indicates mice given the intranasal injection of RW or PBS, respectively, after sensitization with RW. "Control" indicates mice given the intranasal injection of PBS after subcutaneous injection of alum alone and intraperitoneal injection of PBS.(b) The frequency of sneezing in WT and CD300f −/− (KO) mice after each challenge with RW pollen on days 28, 29, 30, and 31.n = 6 per group; ± SD.Data are pooled from two independent experiments.(c-g) The levels of RW-specific IgE in serum (c), the numbers of mast cells (d), the percentages of connective tissue mast cells (e), and the percentages of degranulated mast cells (f).(c,e) n = 3-5 per group; ± SD.Data are representative of two independent experiments.(d,f)n = 6-10 per group; ± SD.Data are pooled from two independent experiments.(g) The frequency of sneezing in WT and Kit W−sh/W−sh mice after the last challenge with RW pollen on day 32.(h) The numbers of mast cells in the nasal tissues from WT and Kit W−sh/W−sh mice after the last challenge with RW pollen on day 32.n = 6 per group; ± SD.Data are pooled from two independent experiments.*P < 0.05 or **P < 0.01.ns not significant.

Figure 2 .Figure 3 .
Figure 2. CD300f deficiency enhanced eosinophil infiltration in the nasal tissues in murine models of AR.(a) The numbers of eosinophils in the nasal tissues from WT and CD300f −/− mice after the last challenge with RW pollen or PBS on day 32.n = 6-9 per group; ± SD.Data are pooled from two independent experiments.*P < 0.05.(b,c) Congo red staining (b) and Giemsa staining (c) of the nasal sections of WT mice and CD300f −/− mice after the last challenge with RW pollen on day 32.(b,c) Scale bars, 50 μm.Data are representative of three independent experiments.(d) Surface expression levels of CD300f in FcεRI + c-Kit + mast cells, FcεRI + c-Kit − CD49b + basophils, CD11b + Ly-6G + neutrophils, CD11b + Ly-6G − Siglec-F + eosinophils, CD11b + Ly-6G − Siglec-F − CD11c + dendritic cells, and CD11b + Ly-6G − Siglec-F − CD11c − Mf/monocytes in the nasal tissues of WT mice under steady conditions or on day 31 in AR models.White and gray histograms indicate staining with anti-CD300f Ab and control Ab, respectively.Data are representative of three independent experiments.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Intranasal administration of ceramide liposomes reduced the symptoms of AR in murine models.(a) A schematic representation of intranasal treatment with ceramide liposomes or vehicle in AR models.(b) The frequency of sneezing in WT mice treated with ceramide liposomes or vehicle after each challenge with RW pollen on days 28, 29, 30, and 31.(c-g) The levels of RW-specific IgE in serum (c) and the numbers of mast cells (d) and eosinophils (f) and the percentages of degranulated mast cells (e) in the nasal tissues from ceramide liposome-or vehicle-treated WT mice after the last challenge with RW pollen on day 32.Data are representative of two independent experiments.n = 6 per group; ± SD. *P < 0.05 or **P < 0.01.ns not significant.

*Figure 7 .
Figure 7. Loss of CD300f in MCPT5-expressing mast cells exacerbated the RW pollen-induced AR in murine models.(a) A schematic representation of RW-induced AR models in CD300f flox/flox MCPT5-Cre +/− and CD300 flox/flox mice in the C57BL/6J background.(b) The percentages of CD300f-positive nasal mast cells in CD300f flox/flox MCPT5-Cre +/− and CD300 flox/flox mice after the last challenge with RW pollen on day 31.(c) The frequency of sneezing in CD300f flox/flox MCPT5-Cre +/− and CD300 flox/flox mice after the last challenge with RW pollen on day 31.(d-g) The levels of RW-specific IgE in serum (d) and the numbers of mast cells (e) and eosinophils (g) and the percentages of degranulated mast cells (f) in the nasal tissues from CD300f flox/flox MCPT5-Cre +/− and CD300 flox/flox mice after the last challenge with RW pollen on day 32.Data are representative of two independent experiments.n = 3-5 per group; ± SD. *P < 0.05.ns not significant.
https://doi.org/10.1038/s41598-024-58923-wwww.nature.com/scientificreports/tissues before and after the intranasal administration of ceramide liposomes in the next step of our research, thereby clarifying how ceramide liposomes are absorbed into and retained on the nasal tissues.

Loss of CD300f in MCPT5-expressing mast cells in mice increased the frequency of sneezing in AR models
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