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. 2021 Jun 20;205(3):306–315. doi: 10.1111/cei.13619

A novel mast cell‐dependent allergic peritonitis model

Hadas Pahima 1, Pier Giorgio Puzzovio 1, Francesca Levi‐Schaffer 1,✉
PMCID: PMC8374212  PMID: 33999404

Abstract

Typical murine models of allergic inflammation are induced by the combination of ovalbumin and aluminum hydroxide. However, accumulating evidence indicates that, in models of asthma and atopic dermatitis, allergic inflammation can be generated in the absence of aluminum hydroxide. Moreover, co‐administration of Staphylococcus aureus enterotoxin B with ovalbumin can enhance inflammation. The objective of this study was to establish a rapid and mast cell‐dependent murine model of allergic inflammation by inducing allergic peritonitis using ovalbumin and S. aureus enterotoxin B. Allergic peritonitis was induced in C57BL/6 mice by subcutaneous sensitization and intraperitoneal challenge with ovalbumin and S. aureus enterotoxin B. Disease characteristics were assessed by flow cytometry, enzyme‐linked immunosorbent assay (ELISA), trypan blue exclusion and colorimetric assays. The time–course of the allergic peritonitis revealed a peak of peritoneal inflammation 48 h after challenge, as assessed by total cells and eosinophil counts. The decrease of cell numbers started 96 h post‐challenge, with complete clearance within 168 h. Moreover, significantly higher levels of tryptase and increased vascular permeability were found 30 min following challenge. Allergic inflammation induction by ovalbumin and S. aureus enterotoxin B was impaired in mast cell‐deficient mice and partially restored by mice reconstitution with bone marrow‐derived mast cells, indicating the mast cell role in this model. We present a novel model of allergic peritonitis that is mast cell‐dependent, simple and robust. Moreover, the use of S. aureus enterotoxin B better resembles human allergic inflammation, which is known to be characterized by the colonization of S. aureus.

Keywords: allergic inflammation, allergic peritonitis, animal models, eosinophils, mast cells

Novel murine, mast cells‐dependent model of allergic peritonitis, induced by ovalbumin and Staphylococcus aureus enterotoxin B. This model displays rapid mast cell activation, as detected by the release of tryptase and TNFα 30min after challenge. Thereafter, the peak of inflammation occurs 48h later, as demonstrated by eosinophil recruitment and increase of cell numbers in the peritoneal cavity.

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INTRODUCTION

Allergic diseases such as asthma, atopic dermatitis, allergic rhinitis and food allergy affect ~20% of the population worldwide [1]. The most prominent cells involved in allergy are the mast cells (MCs), considered the primum movens of the reaction upon their activation by immunoglobulin (Ig)E‐allergen, followed by eosinophils (Eos), that heavily contribute to the inflammatory outcome. Moreover, their soluble and physical crosstalk, that we identified and named the allergic effector unit, is a key factor, increasing the proinflammatory potential of the two cells and hence the allergic inflammation [2, 3]. In addition, several reports have shown the almost exclusive presence of Staphylococcus aureus and its exotoxins, such as S. aureus enterotoxin B (SEB) and others in asthma, allergic rhinitis, food allergy and atopic dermatitis [4, 5, 6]. Interestingly, S. aureus was found to directly bind and activate mast cells (MCs) [7] and Eos [8].

Until recently, the most common protocol used in the investigation of allergic inflammation in mice involved the use of the antigen ovalbumin (OVA), together with aluminum hydroxide (Alum). However, it was shown that Alum can activate the innate immune response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome through increasing uric acid levels [9]. Furthermore, Alum was found to induce allergic inflammation via activation of dendritic cells (DCs) independently of MCs [10]. Indeed, it was shown that 24 h following intraperitoneal (i.p.) injection, DCs and neutrophils were detected and increased during the inflammation in the peritoneal cavity, while MCs and macrophages, typical resident cells, decreased [10]. In addition, Alum co‐administration with an antigen was demonstrated not to be necessary for the induction of acute allergic airway inflammation [11] and AD [12].

It is noteworthy that BALB/C mice are often used in allergic inflammation studies, as they are prone to T helper type 2 (Th2) responses. Nevertheless, C57BL6 are employed more often because of the availability of transgenic and knock‐out (KO) strains based on their background [13, 14].

Therefore, our aim was to develop a mast cell‐dependent, rapid and reproducible model of allergic inflammation with the closest features to human Th2 allergic diseases in C57BL6 mice. Hence, we established an allergic peritonitis (AP) model by employing sensitization and challenge with OVA and SEB.

MATERIALS AND METHODS

Mice

Mast cell‐deficient KitW‐sh/W‐sh (Sash) mice (on a C57BL/6 background, purchased from Jackson Laboratories, Cambridge, Massachusetts, USA) were housed and bred in the pathogen‐free animal facilities of The Medical School of the Hebrew University of Jerusalem. C57BL/6 wild type (WT) mice were purchased from Harlan Laboratories Inc., Ramla, Israel.

All murine experiments were approved by the Animal Experimentation Ethics Committee of the Hebrew University of Jerusalem and performed in accordance with the guidelines of the committee.

Allergic peritonitis (AP)

Eight‐ to 9‐week‐old C57BL/6 (WT) females and/or males were sensitized on days −14 and −7 with subcutaneous (s.c.) injection of 100 μg of OVA (A5378‐10G; Sigma‐Aldrich, St Louis, Missouri, USA) + 1 μg of SEB (S4881‐5 mg; Sigma‐Aldrich) in 200 μl phosphate‐buffered saline (PBS) (no. 1927718; Biological Industries, Beit‐Haemek, Israel) per mouse [OVA (500 μg/ml)/SEB (5 μg/ml)] and challenged at day 0 with i.p. injection of 10 μg OVA + 0.1 μg SEB in 200 μl of PBS per mouse [OVA (50 µg/ml)/SEB (0.5 µg/ml)] or PBS only. Mice were euthanized by CO2 inhalation on days 0 (0 h), 2 (48 h), 4 (96 h), 7 (168 h) and 10 (240 h) and peritoneal lavage was performed with 3 ml PBSX1 + 3% FCS. Peritoneal cells were counted in a hemocytometer by Trypan blue (no. 03‐102‐1B; Biological Industries) staining. Peritoneal lavages were centrifuged at 150 g for 5 min at 4°C and supernatants were collected and stored or immediately used for further analysis. Cell pellets were resuspended in flow cytometry (FC) buffer [0.5% bovine serum albumin (BSA) + 2% FCS in PBSX1] for FC analysis.

FC analysis

Cells were resuspended in a concentration of 3 × 106 cells/ml and 3 × 105 cells/100 µl were seeded per well of a 96 U‐bottomed plate. Cells were centrifuged (150 g for 5 min at 4°C), resuspended and incubated in 100 µl blocking buffer (FC buffer + 5% goat serum) (15 min on ice). Thereafter, cells were centrifuged, resuspended and incubated with the following antibodies for 40 min: CD117 (c‐Kit) monoclonal antibody (2B8), allophycocyanin (APC) (no. 17‐1171‐82; Thermo Fisher Scientific, Waltham, Massachusetts, USA), phycoerythrin (PE) anti‐mouse FcεRIα antibody (no. 134308; BioLegend, Inc., San Diego, California, USA), PE rat anti‐mouse CD170 (Siglec‐F) (no. 552126; BD Biosciences, San Jose, California, USA), APC anti‐mouse CD193 (CCR3) antibody (144512; BioLegend, Inc., San Diego, California, USA), PE anti‐mouse F4/80 antibody (no. 123109; BioLegend, Inc.), CD11b monoclonal antibody (M1/70), APC (no. 17‐0112‐82; Thermo Fisher Scientific). All antibodies were matched with their corresponding isotype control: APC rat IgG2b, κ isotype control antibody (no. 400612; BioLegend, Inc.), Armenian hamster IgG isotype control (eBio299Arm), PE (no. 12‐4888‐83; Thermo Fisher Scientific), PE rat IgG2a, κ isotype control antibody (400508; BioLegend, Inc.), APC rat IgG2a, κ isotype control antibody (400512; BioLegend, Inc.). After incubation, cells were washed twice with FC buffer, centrifuged and resuspended in 200 µl FC buffer and acquired in a BD™ LSR II flow cytometer.

sCD48 levels

sCD48 levels were determined using mouse CD48 quantification kit (Cat. no. K4‐003‐096), according to the manufacturer’s instructions.

Vascular permeability

The OVA/SEB activator protein (AP) model was induced and 1 h before the challenge mice were injected intravenously (i.v.) with fluorescein isothiocyanate (FITC)‐dextran (no. 46945‐100 mg; Sigma‐Aldrich), 0.3 mg/150 µl in PBS per mouse. Challenge was then performed, and mice were euthanized 30 min later and peritoneal lavage (3 ml) was performed. Cells were separated from supernatants by centrifugation and supernatants were analyzed for FITC‐dextran levels in Cytation 3 Cell Imaging Multi‐Mode Reader (BioTek Instruments, Inc., Winooski, Vermont, USA) using the following parameters: excitation: 490 nm, emission: 520 nm.

Tryptase and cytokines levels in peritoneal lavage

For assessing tryptase and cytokine levels in the peritonealcavity, OVA/SEB AP‐induced and challenged mice were euthanized 30 min or 48 h after challenge, respectively, and peritoneal lavages (1 ml) were collected.Cells were separated from supernatants by centrifugation and supernatants werecleared by any remaining cell by a second centrifugation step. Tryptase levelswere assayed as described before [15]. Cytokines levels were determined byusing interleukin (IL)‐4 (900‐K49; PeproTech, Rehovot, Israel), interferon (IFN)‐γ (900‐K98; PeproTech) andIL‐17A (900‐K392; PeproTech) enzyme‐linked immunosorbent assay (ELISA) kits, according to the manufacturer'sinstructions.

OVA‐specific IgE levels

OVA‐specific IgE levels were determined using the Legend MaxTM mouse OVA‐specific IgE ELISA kit (Cat. no. 4398071), according to the manufacturer’s instructions.

Bone marrow‐derived mast cell (BMMC) sensitization with serum from immune OVA/SEB mice and activation with either OVA or SEB

BMMCs were prepared as previously described [16]. Thereafter, BMMCs were sensitized on with 1:5 diluted serum harvested from OVA/SEB‐challenged mice 48 h after the challenge. The next day, sensitized BMMCs were washed and resuspended in BMMCs culture medium and activated for 24 h at 37°C with either 0.5 µg/ml OVA or 10 µg/ml SEB. After incubation supernatants were collected and analyzed for tumor necrosis factor (TNF)‐α levels.

Sash mice intraperitoneal reconstitution with BMMCs

The OVA/SEB AP model was induced in 6–12‐week‐old female Sash mice, as described above. Twenty‐four h before challenge with OVA/SEB, mice were reconstituted i.p. with 2 × 106 BMMCs/200 μl PBS obtained from C57BL/6. Mice were euthanized 48 h after challenge and peritoneal lavages (3 ml) were processed as described above.

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between experimental groups were performed using one‐way analysis of variance (ANOVA) and post‐hoc Tukey’s or Dunnett’s multiple comparison tests. For fewer than three experimental groups, Student’s unpaired two‐tailed t‐test was employed. Data were analyzed with Microsoft Excel (Microsoft, Inc., Redmond, Washington, USA) and Prism version 6.0 (GraphPad Software, San Diego, California, USA). A p‐value less than 0.05 was considered statistically significant for all analyses.

RESULTS

OVA/SEB allergic peritonitis: cellular and soluble parameters

AP was induced in female C57BL/6 mice by OVA/SEB s.c sensitization (days −14 and −7) and challenge by i.p. injection with lower concentrations of OVA/SEB on day 0 (Figure 1a). As shown in Figure 1b,c, in this model the peak of inflammation, as detected by the increase of total cells and Eos (CD170+/CD193+) numbers, was found 48 h following challenge. Total cells and Eos numbers began to decrease 96 h after challenge, returning to baseline levels (time‐point 0 h) within 168 h. MCs (CD117+/FCεRI+) numbers did not display significant fluctuations with a trend of increase 48 h after challenge (Figure 1d). It is noteworthy that macrophage (F4/80+/CD11b+) numbers had already increased significantly at 48 h, reaching a peak at 168 h, and their number remained significantly higher than time‐point 0 h until 240 h (Figure 1e). Additionally, macrophages, as assessed at 48 h, showed increased mRNA expression levels of the M1 marker inducible nitric oxide synthase (iNOS) [17], indicating a proinflammatory phenotype at this time‐point (Supporting information, Figure S1A). Moreover, sCD48 levels peaked 48 h after challenge and returned to baseline levels at 168 h (Figure 1f). C57BL/6 males showed a similar inflammation pattern, albeit less strong than in female mice (Supporting information, Figure S2).

FIGURE 1.

FIGURE 1

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Time–course of inflammation in the ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP). (a) OVA/SEB AP was carried out in 8–9‐week‐old C57BL/6 wild‐type (WT) female mice. Inflammation was measured, at different time‐points, by (b) peritoneal total cells (trypan blue), (c) eosinophils, (d) mast cells (MCs), (e) macrophages (FC analysis) cell numbers and (f) sCD48 levels [enzyme‐linked immunosorbent assay (ELISA)]. Data are the mean ± standard error of the mean (SEM), n = 4–5 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001

OVA/SEB allergic peritonitis induces a mixed inflammation response

To further characterize our model, we analyzed peritoneal levels of IL‐4, IFN‐γ and IL‐17A and cytokine prototypes from Th1, Th2 and Th17 immune responses. Only IL‐4 levels were significantly increased 48 h following challenge (Figure 2a), while IFN‐γ and IL‐17A levels increased, but not significantly (Figure 2b,c). These results indicate that the OVA/SEB AP might induce a mixed inflammatory response shifted towards Th2 features.

FIGURE 2.

FIGURE 2

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Cytokine levels from total cells in the peritoneal lavage of C57BL/6 mice following the activator protein (AP) model. (a) Interleukin (IL)‐4, (b) IL‐17A and (c) interferon (IFN)‐γ levels in the peritoneal lavage of phosphate‐buffered saline (PBS)‐ or ovalbumin/Staphylococcus aureus enterotoxin B (OVA/SEB)‐challenged mice 48 h after challenge. Data are the mean ± standard error of the mean (SEM), n = 4 mice/group, *p < 0.05

OVA/SEB allergic peritonitis induces production of OVA‐ and SEB‐specific IgE

Next, we evaluated levels of OVA‐specific IgE in the serum of AP induced WT mice 48 h after challenge. We found these antibodies to be increased, although not significantly, in WT OVA/SEB‐challenged mice in comparison to PBS‐challenged mice (Figure 3a). In order to check OVA‐specific IgE together with SEB‐specific IgE, we analyzed WT BMMCs sensitized with serum retrieved from OVA/SEB‐challenged mice and activated them with either OVA or SEB. Our results show that under these conditions BMMCs were specifically activated by either OVA (significantly) or SEB (not significantly) to release TNF‐α (Figure 3b). These data reconfirm that OVA/SEB mice produce IgE specific for both the allergens.

FIGURE 3.

FIGURE 3

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Ovalbumin and Staphylococcus aureus enterotoxin B (OVA/SEB)‐specific immunoglobulin (Ig)E production in the OVA/SEB activator protein (AP) model. (a) OVA‐specific IgE levels in the sera of wild‐type (WT) OVA/SEB‐ or phosphate‐buffered saline (PBS)‐challenged mice 48 h following challenge. (b) Tumor necrosis factor (TNF)‐α release from serum‐sensitized, OVA‐ or SEB‐activated WT bone marrow‐derived mast cells. (BMMCs). Data are the mean ± standard error of the mean (SEM), n = 3; *p < 0.05

OVA/SEB allergic peritonitis is a MC‐dependent model

As one of our main aims was to establish a MC‐dependent model, we examined, 30 min post‐challenge in the OVA/SEB AP, tryptase and TNF‐α levels in the peritoneal lavage as markers of MC degranulation. Both these preformed mediators were found to be significantly increased in the OVA/SEB‐challenged mice in comparison to PBS‐challenged mice (Figure 4a,b). Similarly, vascular permeability in the peritoneum was significantly increased at the 30 min time‐point, further bolstering the MC role in this model (Figure 4c).

FIGURE 4.

FIGURE 4

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Vascular permeability and soluble mediators release in the peritoneal lavage of C57BL/6 mice following the activator protein (AP) model. (a) Tryptase, (b) tumor necrosis factor (TNF)‐α levels in peritoneal lavage and (c) vascular permeability in the peritoneal cavity of C57BL/6 mice 30 min following challenge as measured by fluorescein isothiocyanate (FITC)‐dextran levels in peritoneal lavage. Data are the mean ± standard error of the mean (SEM), n = 5–7 mice/group; *p < 0.05, **p < 0.01

Moreover, we induced the OVA/SEB AP model in MC‐deficient Sash mice. Forty‐eight h post‐challenge, total cell infiltration into the peritoneal cavity was increased both in AP‐induced Sash mice and WT mice, although Sash mice presented slightly lower cell infiltration than WT mice (1.4‐ and 1.8‐fold increase, respectively) (Figure 5a). However, Eos numbers in the peritoneal cavity were significantly reduced in Sash OVA/SEB‐challenged mice in comparison to the WT group (Figure 5b). sCD48 levels in the peritoneal cavity were significantly augmented in the WT OVA/SEB group, but not significantly in the Sash OVA/SEB group (Figure 5e). Macrophage numbers showed a small, albeit not significant, increase in OVA/SEB‐challenged WT mice, but no difference in the corresponding group of Sash mice (Figure 5c]. It is important to mention that in the OVA/SEB Sash mice group, two mice were found to have MCs in the peritoneal cavity and developed inflammation as WT mice. Therefore, they were excluded from the experiment (data not shown). As expected, all the other Sash mice presented with no MCs in the peritoneal cavity (Figure 5d). To further dissect the role of MCs in our peritonitis model, Sash mice were reconstituted with BMMCs in the peritoneum in an ‘overshoot fashion’ (2 × 106 cells) 24 h before challenge (Supporting information, Figure S3). Forty‐eight h post‐challenge, when MC numbers were found to be ~106 (Figure 6a), inflammation was partially restored, as demonstrated by increments in total cell and in Eos numbers in comparison to non‐reconstituted Sash mice (Figure 6a,b).

FIGURE 5.

FIGURE 5

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Ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP) model in mast cell (MC)‐deficient mice. OVA/SEB AP was carried out in 8–9‐week‐old C57BL wild‐type (WT) and KitW‐sh/W‐sh (Sash) female mice. Inflammation was measured at the 48 h time‐point by (a) peritoneal total cells (trypan blue), (b) eosinophils, (c) mast cells (MCs), (d) macrophages [fluorescence activated cell sorter (FACS) analysis] cell numbers, n = 6–8 mice per group and (e) sCD48 levels [enzyme‐linked immunosorbent assay (ELISA)], n = 6–8 mice per group. Data are the mean ± standard error of the mean (SEM); *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 6.

FIGURE 6

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Ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP) inflammation after bone marrow‐derived mast cells (BMMC) reconstitution (overshoot) in mast cell (MC)‐deficient mice. (a) Total cell and (b) eosinophils in peritoneum lavage of wild‐type (WT) mice or KitW‐sh/W‐sh (Sash) mice, with or without reconstitution of WT bone marrow‐derived mast cells BMMCs 48 h after challenge. Data are the mean ± standard error of the mean (SEM), n = 4–6 mice/group; *p < 0.05

DISCUSSION

In this paper we propose a new murine AP model induced by two antigens, OVA and SEB, for studying MCs‐dependent AI. OVA, a typical experimentally used antigen and allergen, was administered together with SEB (that itself is a superantigen), used as adjuvant. The SEB adjuvant‐like activity was investigated previously in an asthma model [18]. It was shown that repeated sensitization with a combination of SEB together with OVA increased total cell and Eos numbers in bronchoalveolar lavage compared to sensitization with either OVA or SEB alone [18]. Considering these data, we sought to use SEB to avoid the MC‐independent inflammation induction by Alum [10]. Moreover, we aimed to use a model that would include the typical S. aureus/exotoxins co‐existence with the allergen taking place in allergy diseased patients. In our model we observed an inflammation pattern similar to that previously reported for OVA/Alum AP [19]. A difference was found, however, in the timing of the peak of inflammation that occurred in the OVA/Alum model at 72–96 h after challenge in BALB/C mice [19, 20] and also in C57BL/6 (unpublished data). In the present OVA/SEB model the peak of inflammation is 48 h after challenge as indicated by the increase of Eos numbers a main characteristic of AI. This is in accordance with the results of Zuany‐Amorim et al., showing that OVA/Alum AP induction in BALB/C mice displayed a peak of inflammation at 48 h [21]. An additional explanation to this early peak could be provided by the superantigen role of SEB, as we previously found that SEB‐induced peritonitis presented with increased total cell and Eos numbers 48 h after challenge [22]. In our new model we also detected an increase in macrophages infiltration. This is not surprising, as it was previously reported that both SEB and OVA, respectively, increased macrophages numbers on the site of inflammation [23, 24]. As expected, Eos, and consequently total cell numbers, decreased in the course of AP, while macrophages remained stable from 48 h after challenge. This might be the effect of the contribution of macrophages in both inflammation and resolution [25, 26]. Indeed, we observed that the macrophage phenotype in our model shifted towards M2, as shown by Arg1 [17] expression increasing during the course of AP (Supporting information, Figure S1B). To further emphasize the induction of allergic inflammation using OVA/SEB AP, we analyzed sCD48 levels in peritoneal lavage at the detected peak of inflammation. It was previously published by our group that sCD48 levels correlate with Eos numbers in SEB‐induced peritonitis [22]. Furthermore, sCD48 levels in serum of mild asthma patients were significantly higher than in the control group and correlated with the Eos numbers [27]. In this model we found consistently high levels of sCD48 in the peritoneal lavage at 48 h that returned to basal levels 168 h after challenge.

Many allergic diseases are characterized by the involvement of a mixed Th2 and Th1 [28, 29, 30, 31] response and, as demonstrated by more recent evidence, of a Th17 response [32]. For example, an atopic dermatitis murine model induced by OVA/SEB displayed increased mRNA levels of the Th2 cytokines IL‐4 and IL‐13, together with the Th1‐related cytokines IFN‐γ and 12p40 [12]. Moreover, Bui et al. showed, in a murine OVA/Alum‐induced asthma model, induction of IL‐4 but not IFN‐γ release in mice bronchoalveolar lavage fluid [33]. In addition, Bui et al. showed an increase in the release of the Th17‐related cytokine IL‐17A [33]. Significant levels of IL‐17A, both at mRNA and protein levels, were found in asthma patients [32]. IL‐17A was detected in the skin of OVA/SEB‐induced AD mice [31]. Corresponding with the aforementioned evidence, our AP model showed significantly elevated levels of IL‐4 simultaneously with a trend of increased levels of both IL‐17A and IFN‐γ. These findings indicate that our model might induce a mixed Th1/Th2/Th17 response skewed towards Th2. After characterizing the inflammatory features of the OVA/SEB‐induced AP, our next aim was to understand the involvement of MCs. We therefore evaluated MC degranulation by measuring the levels of the MC preformed mediators, tryptase and TNF‐α [34, 35] in the peritoneal lavages. Indeed, we detected significantly higher levels of both mediators shortly after challenge (30 min), indicating that MCs are directly activated by OVA/SEB, possibly via IgE‐dependent activation, as we detected an increase in OVA‐specific IgE levels in the mice serum and SEB‐specific IgE, as shown by TNF‐α release from serum‐sensitized BMMCs following activation with SEB. This is in accordance with previous reports showing that both OVA and SEB induce production of specific IgE during murine allergic inflammation [12]. In addition, it was shown that children suffering from atopic dermatitis presented with high levels of SEB‐specific IgE in their blood [36].

MC activation in vivo has been linked to increased vascular permeability [37]. Indeed, this effect was also found in our model, as 30 min after challenge we detected a significant increase in this parameter. To bolster the important role of MCs we performed the OVA/SEB AP in MC‐deficient (Sash) mice. Due to a mutation in the c‐kit regulatory element, Sash mice have MC deficiency in the peritoneal cavity and in additional sites at a young age, and in the skin as they age [38]. Therefore, they have been extensively employed to assess the MC role in different pathological conditions. Sash mice sensitized and challenged with OVA/SEB presented attenuated inflammatory features such as decreased recruitment of Eos and reduced release of sCD48. Reconstitution with BMMCs in the peritoneum in an overshoot protocol partly restored the inflammatory phenotype, as shown by the increase of peritoneal total cells and Eos numbers. The partial, but not complete, recovery of the inflammation might be due to the much lower inflammatory mediator content in BMMCs in comparison to tissue MCs (specifically peritoneal MCs) [39, 40] Notably, BMMCs overshoot in the peritoneal cavity of PBS‐challenged WT mice did not elicit Eos recruitment, demonstrating that the injected BMMCs did not induce inflammation by themselves (unpublished data).

In conclusion, the OVA/SEB‐induced AP model is a Th2‐skewed, MC‐dependent allergic inflammation model. This model is also closer to human pathology due to the presence of the SEB together with a potential allergen, such as OVA, that is relevant to human allergic diseases. Therefore, we suggest this model as an additional, useful tool to study allergic inflammation.

CONFLICT OF INTERESTS

The authors report no conflicts of interest.

AUTHOR CONTRIBUTIONS

Hadas Pahima planned and performed experiments, analyzed the data, prepared the figures and wrote the manuscript. Pier Giorgio Puzzovio performed macrophage‐related experiments, assisted with in vivo experiments, analyzed the data and edited the manuscript. Francesca Levi‐Schaffer designed and supervised the study, analyzed the data, advised, reviewed and edited the manuscript.

Supporting information

Supplementary Material

Click here for additional data file. (59.1KB, docx)

ACKNOWLEDGEMENTS

This work was supported by grants from the Israel Science Foundation (ISF, 472/15), Vigevani Foundation, Rosetrees Trust (UK), Aimwell Trust (UK) to Francesca Levi‐Schaffer. Francesca Levi‐Schaffer is affiliated with the Adolph and Klara Brettler Center for Molecular Pharmacology and Therapeutics at the School of Pharmacy of The Hebrew University of Jerusalem. The authors thank Dr Micha Ben‐Zimra for helpful scientific discussions, Dr Mansour Seaf for technical assistance and Ms Alexandra Eliassaf (The Core Research Facility, The Faculty of Medicine, The Hebrew University of Jerusalem) who provided advice on the FC analysis and technical help.

Pahima H, Puzzovio PG, Levi‐Schaffer F. A novel mast cell‐dependent allergic peritonitis model. Clin Exp Immunol. 2021;205:306–315. 10.1111/cei.13619

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Supplementary Materials

Supplementary Material

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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