Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy

Abstract

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

Figure 1

Lin⁻IL-9-producing cell frequency correlates positively with susceptibility to experimental food allergy. (A) Flow cytometric analysis of intracellular IL-9 and IFN-γ production by CD3⁺CD4⁺ T or Lin⁻ cells or (H, I) frequencies of IL-9-producing cells in LP of small intestine of indicated mouse strains (H) or in the Lin⁻ fraction in the indicated tissues from BALB/c mice (I) after six intragastric OVA challenges. (B–G) Indicated mouse strains were sensitized and challenged six times intragastrically with OVA before measuring the indicated features of experimental food allergy, including; Incidences of allergic diarrhea (B) and hypothermia (C), intestinal mast cell numbers (D), MCPt-1 production (E), and concentration of OVA-specific IgE (F) and OVA-specific IgG (G). Data in A–I are representative of three independent experiments (n = 4 mice per group). Fraction indicates incidences of allergic diarrhea (B) and hypothermia (C). Error bars denote mean ± S.E.M. *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001. NS, not significant. See also Figure S1.

Figure 2

Lin⁻GFP^hiIL-17Rb⁻c-Kit⁺ST2⁺β7integrin^lo cells are the multi-functional IL-9-producing mucosal mast cells (MMC9s). (A, B) Detection of (A) and measurements of indicated cytokines secreted by (B) two dominant Lin⁻GFP^hi cell fractions that are IL-17RB⁻ (A) and IL-17RB⁺ (B), and GFP^hiIL-17RB⁺ and GFP⁻IL-17RB⁻ subsets of CD3⁺CD4⁺ T cells in LP of small intestine of IL-4-eGFP (4GET) mice after six intragastric OVA challenges. (C–H) Detection of (C) and flow cytometric analysis of indicated intracellular cytokines by (D) β7integrin^lo (β7^lo) and β7integrin^hi (β7^hi) subsets of Lin⁻GFP^hiIL-17RB⁻c-Kit⁺ST2⁺ cells. (E) Expression of the indicated genes by purified intestinal β7integrin^lo and β7integrin^hi subsets of Lin⁻GFP^hiIL-17RB⁻c-Kit⁺ST2⁺, in vitro-generated bone marrow-derived mast cells (BMMCs), ILC2, and CD4⁺GFP^hiIL-17RB⁺Th2 cells from mice with active allergic diarrhea, was analyzed by quantitative real-time PCR using primers referenced in the methods. (F) Measurement of indicated cytokine production by purified intestinal β7integrin^lo and β7integrin^hi subsets of Lin⁻GFP^hiIL-17RB⁻c-Kit⁺ST2⁺ after 3 days culture with SCF plus IL-3 in the presence or absence of IL-33. (G) Measurement of intracellular MCPt-1 within purified Lin⁻IL-17RB⁻c-Kit⁺ST2⁺β7integrin^lo or bone marrow-derived mast cells (BMMCs) and their MCPt-1 secretion after IgE crosslinking. Giemsa staining (H) and electron microscopic analysis (I) of purified Lin⁻IL-17RB⁻c-Kit⁺ST2⁺β7integrin^lo cells. Gene expression data are expressed as relative fold difference (E). Scale bars are 20 μm (H) or 2 μm (I). Data in A–G are representative of three independent experiments. Error bars denote mean ± S.E.M (B, F, G). **p≤ 0.01. NS, not significant. See also Figure S2 and S3.

Figure 3

Blockade of intestinal β7^hiMCP recruitment from bone marrow results in the failure of MMC9 induction. (A–F) Detection and frequency of donor-derived MMC9s (Lin⁻GFP⁺ST2⁺c-Kit⁺ β7integrin^lo) and MCPs (Lin⁻GFP⁺ST2⁺c-Kit⁺β7integrin^hi), and mast cell (MC) number (A–B and D–E), and serum MCPt-1 and OVA-IgE titers, and diarrhea incidence (C, F) of irradiated recipient BALB/c mice reconstituted with sorted bone marrow mast cell progenitors (Lin⁻ Ly-6c⁻FcεR⁻CD41⁻CD71⁻FLK2⁻CD150⁻c-Kit⁺ β7integrin⁺ cells) from IL-4-eGFP (4GET) mice (A–C) or received anti-α4β7integrin (LPAM-1) or isotype-matched mAb and reconstituted with wild type bone marrow progenitors from IL-4-eGFP (4GET) mice (D–F) one day before second sensitization and six intragastric OVA challenges. Data represent one of three independent experiments (n=6 mice per group) (A–C) or one of two independent experiments (n=4 mice per group) (D–F). LP, laminar propria. * p<0.05, ** p<0.01. NS, not significant. See also Figure S4.

Figure 4

MMC9s and CD4⁺Th2 cell frequencies correlate positively with susceptibility to experimental food allergy. (A–F) Detection (A) and frequency of MMC9s (A–F), MCPs (A, B), and CD4⁺IL-17RB⁺ Th2 cells (A, B) in LP of small intestine of naïve, sensitized, or BALB/c mice after indicated times of intragastric OVA challenge. Serum samples (C, E, F) or intestinal tissue (D) were collected for measurement of indicated features of experimental food allergy, including; allergic diarrhea incidence (B), MCPt-1 production (C), intestinal mast cell numbers (D), and titers of OVA-specific IgE (E) and OVA-specific IgG (F). (G) Frequency of indicated cell populations in LP of small intestine of indicated mouse strains after six intragastric OVA challenges. Spearman’s rank coefficients and two-tailed P values were used to quantify the correlations between the indicated features of experimental food allergy and frequency of MMC9s (C–F). Fractions indicate incidence of allergic diarrhea (B). Data in A–G are representative of three independent experiments (n=4 mice per group). Error bars denote mean ± S.E.M. *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001. NS, not significant. See also Figure S5.

Figure 5

STAT6 signaling and T cells are required for MMC9 induction that promotes experimental food allergy. (A–F) Detection (A, D) and frequency (B, E) of MMC9s (Lin⁻c-Kit⁺ST2⁺ β7integrin^lo) and MCPs (Lin⁻c-Kit⁺ST2⁺β7integrin^hi) and measurement of indicated features of experimental food allergy after six intragastric OVA challenges (B, C, E, F) in wild type BALB/c or indicated gene deficient murine strains (A–C) or sensitized wild type BALB/c treated with anti-CD4 or isotype control mAbs one day before first and fourth intragastric OVA challenges (D–F). (G–I) Detection (G) and frequency (H) of MMC9s, MCPs, and CD4⁺Th2 cells, and measurement of indicated features of experimental food allergy (H, I) in indicated sub-lethally irradiated recipient strains reconstituted with BM progenitors from WT 4GET mice. Data in A–I represent one of three independent experiments (n=4 mice per group). Fractions indicate incidence of allergic diarrhea (C, F, I). Error bars denote mean ± S.E.M. *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001. NS, not significant.

Figure 6

MMC9s can be induced in IL-9 or IL-9R-deficient mice, but fail to promote experimental food allergy. (A–E) Detection of Lin⁻IL-17RB⁻c-Kit⁺ST2⁺ cells (A) and flow cytometric analysis of indicated intracellular cytokine production by Lin⁻IL-17RB⁻ cells (B), and staining for intestinal mastocytosis (C), and frequency of MMC9s (Lin⁻c-Kit⁺ST2⁺β7integrin^lo) and MCPs (Lin⁻c-Kit⁺ ST2⁺β7integrin^hi) (D) and measurement of indicated features of experimental food allergy (D, E) in wild type BALB/c or indicated gene deficient murine strains after six intragastric OVA challenges. (F–I) Detection (F, G) and frequency (H) of donor-derived MMC9s (Lin⁻c-Kit⁺ ST2⁺ β7integrin^loGFP⁻) and MCPs (Lin⁻c-Kit⁺ST2⁺β7integrin^hiGFP⁻), and measurement of indicated features of experimental food allergy (H, I) in sub-lethally irradiated 4GET recipients reconstituted with BM progenitors from indicated murine strains. Data in A–I represent one of three independent experiments (n=4 or 8 mice per group). Scale bars are 20 μm (C). Error bars denote mean ± S.E.M. **p≤ 0.01. NS, not significant. See also Figure S6.

Figure 7

MMC9s drive intestinal mastocytosis and Il9 transcript expression is increased in food allergy patients. (A–D) Detection (A) and frequency (B) of MMC9s and MCPs, measurement of indicated features of experimental food allergy (B, C), staining of intestinal mastocytosis and goblet cell hyperplasia (D), from mice treated with anti-FcεRIα or isotype control mAbs. (E, F) Numbers of intestinal mast cells (E) and Incidence of allergic diarrhea (F) in anti-FcεRIα antibody-treated mice reconstituted with purified MMC9s or saline only, or treated with saline plus anti-FcεRIα before re-challenge with OVA intragastrically twice (E) or indicated times (F) (12 mice per group). (G) Expression of the indicated genes by duodenal biopsies from control and food allergy subjects was analyzed by quantitative real-time PCR using primers referenced in the methods. Data in A–D represent one of three independent experiments (n=4 mice per group). Gene expression data are expressed as relative fold difference as described in method (G). Error bars denote mean ± S.E.M. Scale bars are 20 μm (D) and 100 μm (D, the corner insets). *p≤ 0.05; **p≤ 0.01. See also Figure S7.