Stem Cell Factor Neutralization Protects From Severe Anaphylaxis in a Murine Model of Food Allergy

Abstract

Food allergy is a growing public health problem with ~15 million people affected in the United States. In allergic food disease, IgE on mast cells bind to ingested antigens leading to the activation and degranulation of mast cells. Stem cell factor (SCF) is mast cell growth and activation factor that is required for peripheral tissue mast cells. We targeted a specific isoform of SCF, the larger 248 amino acid form, that drives peripheral tissue mast cell differentiation using a specific monoclonal antibody in a model of food allergy. Ovalbumin sensitized and intragastrically challenged mice were monitored for symptoms of anaphylaxis including respiratory distress, diarrhea, and a reduction in body temperature. During the second week of challenges, allergic mice were injected with an antibody to block SCF248 or given IgG control. Mice treated with α-SCF248 had a decreased incidence of diarrhea and no reduction in body temperature suggesting a reduction in anaphylaxis compared to IgG control treated animals. Re-stimulated mesenteric lymph nodes indicated that α-SCF248 treated mice had decreased OVA-specific Th2 cytokine production compared to IgG control treated allergic animals. The reduction of food induced anaphylaxis was accompanied by a significant reduction in gut leak. The mesenteric lymph node cells were analyzed by flow cytometry and showed a decrease in the number of type 2 innate lymphoid cells in mice injected with α-SCF248. Morphometric enumeration of esterase+ mast cells demonstrated a significant reduction throughout the small intestine. Using a more chronic model of persistent food-induced anaphylaxis, short term therapeutic treatment with α-SCF248 during established disease effectively blocked food induced anaphylaxis. Together, these data suggest that therapeutically blocking SCF248 in food allergic animals can reduce the severity of food allergy by reducing mast cell mediated disease activation.

Keywords: anaphylaxis; food allergy; innate lymphoid cell; mast cell; stem cell factor.

Figure 1

Neutralizing SCF248 reduces anaphylactic symptoms in allergic mice. (A) Model to induce anaphylaxis. Mice were sensitized with OVA and alum on day 0, then challenged with OVA by oral gavage on days 14, 16, 18, 21, 23, 25, and 28. SCF248 was neutralized by intraperitoneal injection on days 21, 23, and 25. Animals were euthanized 60 min after final challenge. (B) Prior to SCF248 neutralization (day 21, challenge four), temperatures were recorded at 15, 30, and 60 min following oral challenge. Symptoms were continuously monitored for 60 min, and mice were assigned as score on a scale of one to five based on symptom presentation. *p < 0.05, **p < 0.01 for OVA + IgG and OVA + αSCF248 compared to PBS. (C) Following SCF248 neutralization (day 28, challenge seven), temperatures were monitored for 60 min, and animals were euthanized. Mice were assigned a clinical score based on symptoms. Results are from five mice per group. Date show mean ± SEM, and are from one of three independent experiments. *p < 0.05, **p < 0.01 for OVA + IgG compared to PBS and OVA + αSCF248.

Figure 2

Anti-SCF248 treatment reduces mast cells, eosinophils, and ILC2s in the intestines of allergic mice. (A) Representative sections of the small intestine stained with chloroacetate esterase to visualize mast cells. Details are shown in the inset boxes. Scale bar = 50 μm. Mast cells are indicated by arrows. (B) Mast cells were counted in the duodenum, jejunum, and ileum. Numbers represent the mean of five high-powered fields per section. (C) Mast cells, eosinophils, and ILC2s in the small intestine lamina propria were quantified by flow cytometry. Results are from four to six mice per group and represent one of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

The Th2 response is decreased in the lymph nodes of mice treated with α-SCF248. (A) Mesenteric lymph nodes were removed and single cell suspensions were restimulated with OVA. Th2 cytokines were measured in the supernatant by Bioplex assay after 48 h of culture. (B) The number of ILC2s in the mesenteric lymph nodes were measured by flow cytometry. Five mice were used in each group, and data represent one of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Mast cell activation and intestinal permeability are decreased in mice treated with α-SCF248. (A) Mast cell activation was assessed by measuring murine mast cell protease concentration in the serum 30 min after final challenge. Protease concentration was measured by ELISA. (B) Intestinal permeability was analyzed by delivering 4 kDa FITC-dextran by oral gavage, then measuring FITC in the plasma by fluorescence 4 h later. (C) Western blot of JAM-A was performed from the small intestine, with b-actin as a control. Densitometry analysis of JAM-A results are shown as a percentage of b-actin control. Each lane represents a different tissue sample, and one of two representative blots is shown. (D) OVA-specific IgE was measured in the serum of allergic mice by ELISA. Results represent one of three independent experiments with four to five mice per group. ELISA samples were run in duplicate, in addition to the biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Anti-SCF248 treatment protects mice in a model of established food allergy. (A) Food allergy was induced by sensitizing mice with OVA and alum, followed by seven challenges with OVA, ending on day 28. SCF248 was blocked by injection with a monoclonal antibody on days 28, 31, and 35. Mice were given one additional challenge with OVA on day 35, following SCF248 neutralization. (B) Rectal temperatures were recorded for at 15, 30, and 60 min following challenge at day 35. (C) Mice were monitored for 60 min following final challenge, and assigned a score based on clinical symptoms. Results are from five mice per group and represent one of three independent experiments. *p < 0.05, ***p < 0.001.

Figure 6

Anti-SCF248 treatment decreases intestinal mast cells and Th2 inflammation in model of established food allergy. (A) Mast cells were enumerated following staining of sections with chloroacetate esterase to visualize cells. Details are shown in the inset boxes. Scale bar = 50 μm. Arrows point to mast cells. (B) Mast cell numbers per high powered field were determined by counting five sections each in the duodenum, jejunum, and ileum. (C) Murine mast cell protease was measured by ELISA to determine the extent of mast cell activation. (D) Mesenteric lymph nodes were dissociated and single cell suspensions restimulated with OVA. After 48 h of culture, Th2 cytokines were measured in the supernatants by Bioplex assay. Five mice were used in each group, and data represent one of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.