Interleukin-18 Receptor α Modulates the T Cell Response in Food Allergy.

PURPOSE: The prevalence of food allergy, triggered by T-helper type 2 (Th2) cell-mediated inflammation, is increasing worldwide. Interleukin (IL)-18 plays an important role in inflammatory diseases by binding with the IL-18 receptor. IL-18/IL-18 receptor α (IL-18Rα) is a cofactor for immunoglobulin E (IgE) production and Th2 cell development. Studies have not investigated the association between the IL-18/IL-18Rα signaling pathway and food allergy. Here, we investigated the role of IL-18Rα in food allergy induction and development.
METHODS: Wild-type (WT) and IL-18Rα-null mutant (IL-18Rα-/-) C57BL/6 mice were sensitized and challenged using ovalbumin (OVA) for food allergy induction. Food allergy symptoms, T cell-mediated immune responses, and signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways were analyzed in mice.
RESULTS: IL-18Rα expression was increased in WT mouse intestines after OVA treatment. Food allergy-induced IL-18Rα-/- mice showed attenuated systemic food allergic reactions, OVA-specific IgE and mouse mast cell protease-1 production, inflammatory cell infiltration, and T cell activation. Ex vivo experiments showed that cell proliferation and Th2 cytokine production were lower in IL-18Rα-/- mouse splenocytes than in WT mouse splenocytes. IL-18Rα blockade in WT splenocytes attenuated cell proliferation and Th2 cytokine production. Moreover, STAT3 phosphorylation was reduced in IL-18Rα-/- mice, and SOCS3 and SOCS1 activation were diminished in IL-18Rα-/- intestinal T cells.
CONCLUSIONS: IL-18Rα regulates allergic reactions and immune responses by regulating T cell responses in food allergies. Moreover, IL-18Rα is involved in the STAT/SOCS signaling pathways. Targeting IL-18Rα signaling might be a novel therapeutic strategy for food allergy.

INTRODUCTION

Food allergy is an immunoglobulin E (IgE)-mediated adverse hypersensitivity reaction to ingested food and is an increasing public health concern affecting millions of people worldwide over the past few decades.1 Clinical symptoms of food allergy can be mild reactions, such as itching and swelling, to life-threatening systemic anaphylaxis.23 Despite their increased prevalence, current therapeutic strategies are limited by our incomplete understanding of the immunologic events that initiate and propagate type 2 inflammation.4 Thus, a better understanding of the underlying immune mechanisms and signaling pathways of food allergy is warranted to develop more effective and safe therapies that provide long-term protection in patients of various ages and with different responsiveness.567 Typically, when food allergens penetrate the epithelial barrier, naïve T cells differentiate into CD4+ T-helper type 2 (Th2) cells, initiating the transcription of several cytokines, including interleukin (IL)-4, IL-5, and IL-13. Th2 cells promote antigen-specific IgE development through class-switching via B cells, ultimately inducing Th2 cell-mediated effector responses through mast cells during food allergy.89 Furthermore, T cell responses activate signal transduction pathways, including Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. STAT protein activation is regulated by cytokine-induced phosphorylation and suppressors of cytokine signaling (SOCS) proteins.10 SOCS is a direct target gene for STAT, which is not only driven by the cytokine-mediated activation of JAK/STAT signaling but also acts as a negative regulator that inhibits JAK signaling.11 SOCS family proteins also contribute to Th cell differentiation during immune responses.1213

IL-18 is an IL-1 family cytokine produced by various cells such as antigen-presenting cells, T cells, and natural killer cells.141516 IL-18 receptor (IL-18R) is a heterodimeric complex composed of a signaling alpha subunit (IL-18Rα) and a ligand-binding beta subunit (IL-18Rβ). IL-18Rα is an extracellular signaling domain, whereas IL-18Rβ is an adapter molecule.17 In the downstream signaling pathway of IL-18R, myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 trigger the nuclear translocation of nuclear factor-κB and transcription of pro-inflammatory genes.141819 IL-18 and its receptors are pleiotropic molecules involved in several inflammatory disorders, and polymorphisms in the IL-18Rα/IL-18 receptor accessory protein locus are associated with disease susceptibility.20212223 Furthermore, IL-18Rα is regulated during CD4+ T cell differentiation to T-helper type 1 (Th1) or Th2 pathways in a sophisticated manner.24 Although IL-18/IL-18Rα is primarily involved in Th1-associated functions, it also augments Th2 responses.162425 IL-18 is a cofactor in inducing IL-4 and IL-13 production, as well as interferon (IFN)-γ expression in T cells, and IL-18 administration to mice increases IL-4 and serum IgE production and induces Th2 cell development.25 Although IL-18 is thought to be associated with allergy and intestinal barrier function, the role of IL-18/IL-18Rα in food allergy and the major cellular source and downstream consequences of this interaction remain unexplored.

Based on previous studies, we hypothesized that IL-18/IL-18Rα signaling is associated with Th2 cell-mediated food allergy. Here, we aimed to establish an ovalbumin (OVA)-induced food allergy mouse model and compare immune responses between wild-type (WT) and IL-18Rα-null mutant (IL-18Rα−/−) mice. Our results could provide novel insights into the pathogenesis of food allergy and lead to the development of new therapeutic strategies for food allergy.

MATERIALS AND METHODS

Mice

WT female C57BL/6 mice at 5 to 6 weeks of age were purchased from Orient Bio Inc. (Seongnam, Korea). IL-18Rα−/− mice were obtained from The Jackson Laboratory (B6. 129P2-Il18r1/J; Bar Harbor, ME, USA). The mice were housed in an air-conditioned room (23°C ± 2°C) with a 12 hours/12 hours light/dark cycle and allowed free access to food and tap water. Age-, sex-, and weight-matched mice were used in all experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at Yonsei University (Seoul, Korea; #2020-0266).

Experimental food allergy

Mice were sensitized intraperitoneally with 50 μg OVA (grade V; Sigma-Aldrich, Munich, Germany) plus 10 μg cholera toxin (CT; 100B; List Biological Laboratories, Los Angeles, CA, USA) in 150 mL phosphate-buffered saline (PBS) on days 0 and 14. Two weeks after the second sensitization, mice were challenged intragastrically with 100 mg OVA in 200 mL PBS 6 times within 2 weeks. Control mice were sensitized and challenged using only PBS. The rectal temperature was measured before and 30 minutes after the last oral OVA challenge. Mice showing profuse liquid stool within 60 minutes after the final challenge were recorded as diarrhea-positive. Intestinal tissue, spleen, and blood samples were collected from mice 1 day after the last challenge.

Quantitative real-time polymerase chain reaction (PCR)

Total RNA was isolated from the small intestine and intestinal T cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using a qPCR RT master mix kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Real-time PCR was performed with a StepOnePlus™ Real-Time PCR System using Power SYBR green PCR master mix (both from Applied Biosystems, Foster City, CA, USA). β-actin was used as the housekeeping gene, and results were quantified using the 2−ΔΔCT method.

Western blot analysis

Total intestinal proteins were extracted using radioimmunoprecipitation assay buffer containing proteinase inhibitor cocktail (both from Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed as previously described with 20 µg of the quantified protein samples.26 Membranes were incubated overnight at 4°C with primary antibodies against IL-18Rα (Invitrogen) and glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA, USA), followed by incubation for 1 hour at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

IL-18Rα protein levels in the small intestine were determined using an ELISA kit (Cusabio, Waltham, MA, USA) according to the manufacturer’s instructions. To determine anti-OVA IgE serum levels, 96-well plates were coated with 20 μg/mL OVA, and subsequently, the IgE ELISA kit (BD Biosciences, San Diego, CA, USA) was used as previously described.27 Mouse mast cell protease-1 (MCPT-1) serum levels were measured using the ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instruction. IL-5 and IL-13 levels in the supernatant of splenocytes were determined using an ELISA kit (R&D Systems, Minneapolis, MN, USA). ELISAs for phosphorylated STAT3 (Tyr705) and total STAT3 (Abcam, Cambridge, MA, USA) were performed with intestinal lysates according to the manufacturer’s instructions. Analyte values in intestinal lysates were normalized to the total protein concentration.

Histological analysis

The small intestine was fixed with 4% paraformaldehyde and subsequently embedded in paraffin. Paraffin sections (4-μm-thick) were stained with toluidine blue for mast cell staining and hematoxylin and eosin (H&E) for eosinophil staining. Mast cell and eosinophil numbers in the small intestine were evaluated in 3 sections from at least ten mice. Representative microscopic images were obtained using a BX43 Upright Microscope (Olympus, Tokyo, Japan) at ×400 magnification based on a high-power field (HPF).

Isolation of leukocytes

Leukocytes were isolated from the intestinal lamina propria and spleen according to previously described procedures with slight modifications.262829 In brief, the small intestine was cut into 1 cm segments with a rotary incubator for 20 minutes in Hanks’ Balanced Salt Solution (HBSS) medium (Thermo Fisher Scientific) containing 5% fetal bovine serum (FBS) and 2 mM ethylenediaminetetraacetic acid. This process was repeated twice. The remaining tissue was chopped finely and digested with 1 mg/mL collagenase type 4 (Worthington Biochemical, Lakewood, NJ, USA) and 100 μg/mL DNase 1 (Sigma-Aldrich) for 30 minutes. The digested intestinal tissue and supernatant were passed through a 100 μm cell strainer (BD Biosciences). Cell suspensions were separated using 40% Percoll underlaid with 75% Percoll (GE Healthcare, Pittsburgh, PA, USA). Leukocytes were collected from the interface and subsequently washed and suspended in HBSS medium. The spleen was passed through a 40 μm cell strainer, and the obtained cells were centrifuged and washed with Roswell Park Memorial Institute (RPMI) medium containing 5% FBS. Ammonium-chloride-potassium lysis buffer was used to lyse red blood cells. Leukocytes were washed and suspended in RPMI medium containing 5% FBS.

Flow cytometry

Lamina propria mononuclear cell suspensions were obtained from small intestines as described previously herein. Cell suspensions were stained with the following monoclonal antibodies: anti-CD3 (PerCP-Cyanine5.5), anti-CD4 (allophycocyanin), anti-CD44 (phycoerythrin), anti-CD62 ligand (CD62L; fluorescein isothiocyanate), and anti-IL-18Rα (phycoerythrin-Cy7). Dead cells were excluded by staining with Fixable Viability Dye eFluor 780. All fluorochrome-labeled antibodies were purchased from eBioscience. Cells were analyzed using a BD LSR Fortessa™ X-20 (BD Biosciences) with FlowJo 10 software (Tree Star, Ashland, OR, USA).

Ex vivo cell culture and antibody treatment

WT and IL-18Rα−/− mice were sensitized intraperitoneally with 50 μg OVA plus 10 μg CT twice, with a 2-week interval in between. Leukocytes from the spleens of sensitized WT or IL-18Rα−/− mice were obtained as described previously herein. Splenocytes were stimulated with or without 10 mg/mL OVA, followed by treatment with 1 µg/mL anti-immunoglobulin G (Cell Signaling Technology) or anti-IL-18Rα antibody in a 96-well plate. After 5 days of culture, 1 × 106 cells per well plate were centrifuged, and supernatants were collected. In total, 2 × 105 cells per well plate were added to the Cell Counting Kit-8 solution (Dojindo Molecular Technologies, Rockville, MD, USA), and plates were incubated for 4 hours before absorbance was measured on a microplate reader.

CD4+ T cell sorting

The Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to remove dead cells among the lamina propria mononuclear cells using magnetic cell sorting (auto-MACS; Miltenyi Biotec). Thereafter, CD4+ T cells were isolated using a CD4+CD62L+ T Cell Isolation Kit (Miltenyi Biotec) with auto-MACS according to the manufacturer’s protocol.

Statistical analyses

All data were analyzed using Prism (GraphPad Software, San Diego, CA, USA). They are presented as the means ± standard error of the mean of at least 3 independent experiments. Comparisons of 2 groups were performed using a Student’s t-test. When more than 3 groups were compared, the one-way analysis of variance followed by Tukey’s test was used. P values < 0.05 were considered statistically significant.

RESULTS

IL-18Rα expression levels are increased in the food allergy mouse model

To investigate the pathophysiological relevance of IL-18Rα in food allergy, we established a mouse model of OVA-induced food allergy and analyzed IL-18Rα expression in the intestine. Mice were intraperitoneally sensitized and intragastrically challenged with OVA (Fig. 1A). OVA-challenged WT mice showed increased IL-18Rα mRNA expression compared to PBS-challenged WT mice (Fig. 1B). Additionally, the results of western blotting and ELISA showed elevated IL-18Rα protein levels in OVA-challenged WT mice (Fig. 1C-E), demonstrating the involvement of IL-18Rα in food allergy.

Fig. 1

Elevated IL-18Rα expression in murine models of food allergy. (A) Experimental scheme for the induction of food allergy in mice. Mice were sensitized via i.p. injection with 50 μg OVA plus 10 μg CT and challenged via i.g. administration with 100 mg OVA. The intestines were harvested 1 day after the last challenge. (B) IL-18Rα mRNA expression levels in the intestine were measured using real-time polymerase chain reaction. (C) IL-18Rα protein expression levels in the intestine were analyzed using western blotting, and (D) the signal intensity was quantified using ImageJ software. (E) Protein levels of IL-18Rα in intestinal lysates were assessed using an enzyme-linked immunosorbent assay. Data are representative of 3 independent experiments (n = 11 for each group) and presented as the mean ± standard error of the mean.

IL-18Rα, interleukin-18 receptor α; i.p., intraperitoneal; OVA, ovalbumin; CT, cholera toxin; i.g., intragastric; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

*P < 0.05; †P < 0.001.

IL-18Rα regulates systemic immune reactions

To investigate the effect of IL-18Rα in a mouse model of food allergy, WT and IL-18Rα−/− mice were sensitized and challenged with OVA, and their immune responses were compared. Following the last challenge with OVA, WT mice showed a significant decrease in rectal temperature (−1.34°C ± 0.21°C), whereas IL-18Rα−/− mice had a relatively lower drop (−0.63°C ± 0.12°C, Fig. 2A). Furthermore, OVA-challenged WT mice exhibited profuse diarrhea compared with PBS-challenged WT mice. However, OVA-challenged IL-18Rα−/− mice showed a relatively low incidence of allergic diarrhea compared to OVA-challenged WT mice (Fig. 2B). In serum, antigen-specific IgE level was increased in food allergy-induced WT mice. The expression of MCPT-1, which is released from mucosal mast cells upon allergen-dependent crosslinking of IgE, was also increased upon allergen challenge. However, OVA-challenged IL-18Rα−/− mice showed reduced levels of antigen-specific IgE and MCPT-1 compared to OVA-challenged WT mice (Fig. 2C and D). We also observed histopathological changes in the intestine using toluidine blue and H&E staining. The number of migrated mast cells was increased in OVA-challenged WT mice; however, these were relatively less abundant in OVA-challenged IL-18Rα−/− mice (Fig. 2E and G). H&E staining illustrated histological changes such as a damaged villus and the infiltration of immune cells. Intestinal damage and eosinophilic infiltration were milder in OVA-challenged IL-18Rα−/− mice than in OVA-challenged WT mice (Fig. 2F and H). These findings collectively demonstrated that IL-18Rα mediates the systemic immune responses of food allergy.

Fig. 2

Attenuated inflammatory responses in OVA-challenged IL-18Rα−/− mice. (A) Rectal temperature was measured in WT and IL-18Rα−/− mice. (B) The percentage of diarrhea-positive mice was calculated in WT and IL-18Rα−/− mice. (C) OVA-specific IgE and (D) MCPT-1 levels in serum were measured using an enzyme-linked immunosorbent assay. (E) Mast cell number per HPF in the intestine; (G) black arrowheads indicate toluidine blue positive mast cells (scale bars = 50 μm). (F) Eosinophil cell number per HPF in the intestine; (H) black arrowheads indicate hematoxylin and eosin-stained eosinophil cells (scale bars = 50 μm). Data are representative of at least 3 independent experiments (n = 9–15 for each group) and are presented as the mean ± standard error of the mean.

OVA, ovalbumin; IL-18Rα−/−, interleukin-18 receptor α-null mutant; WT, wild-type; IgE, immunoglobulin E; MCPT-1, mouse mast cell protease-1; HPF, high-power field; O.D., optical density; PBS, phosphate-buffered saline.

*P < 0.05; †P < 0.01; ‡P < 0.0001.

IL-18Rα activates CD4+ T cells

A diverse range of effector and regulatory CD4+ T cells are distributed in the lamina propria, and CD4+ T cells constitutively express IL-18Rα.2430 To determine whether IL-18Rα is expressed on CD4+ T cells of the intestinal lamina propria during the development of food allergy, we first assessed the population of IL-18Rα+ T cells in WT mice using flow cytometry. The percentage of CD3+CD4+ T cells and IL-18Rα+ T cells were higher in OVA-challenged WT mice than in PBS-challenged WT mice (Fig. 3A and B). As T cells are the important cellular source of IL-18Rα, we investigated whether IL-18Rα deficiency affected T cell activation in the intestinal lamina propria. OVA-challenged IL-18Rα−/− mice had a reduced rate of effector T cell populations (CD3+CD4+CD44highCD62Llow) in the intestinal lamina propria compared to their OVA-challenged WT counterparts (Fig. 3C and D). In addition, real-time PCR revealed decreased expression of Th2 cytokines such as IL-4, IL-5, and IL-13 in the intestinal tissue of OVA-challenged IL-18Rα−/− mice compared to that in OVA-challenged WT mice (Fig. 3E-G). Additionally, the expression of Th1 cytokines such as IFN-γ and tumor necrosis factor-α was also decreased in the intestine of OVA-challenged IL-18Rα−/− mice compared to that in OVA-challenged WT mice (Supplementary Fig. S1). These data demonstrated that IL-18Rα+ T cells were enriched in the lamina propria by food allergy induction and suggested that IL-18Rα plays an important regulatory role in T cell activation and intestinal allergic inflammation.

Fig. 3

Diminished T cell responses in food allergy-induced IL-18Rα−/− mice. (A) IL-18Rα expression in intestinal CD3+CD4+ T cells from WT mice was quantified using flow cytometry. (B) The graph represents the percentage of CD3+CD4+IL-18Rα+ cells. (C) Effector T cell populations (CD3+CD4+CD44highCD62Llow) in the intestinal lamina propria of WT and IL-18Rα−/− mice were quantified using flow cytometry. (D) The graph represents the percentage of CD3+CD4+CD44highCD62Llow cells. mRNA expression levels of (E) IL-4, (F) IL-5, and (G) IL-13 in the intestines of WT and IL-18Rα−/− mice were analyzed using a real-time polymerase chain reaction. Data are representative of at least 3 independent experiments (n = 7–15 for each group) and are presented as the mean ± standard error of the mean.

IL-18Rα−/−, interleukin-18 receptor α-null mutant; WT, wild-type; CD62L, CD62 ligand; IL, interleukin; PBS, phosphate-buffered saline; OVA, ovalbumin.

*P < 0.05; †P < 0.01; ‡P < 0.001; §P < 0.0001.

IL-18Rα affects T cell proliferation and differentiation

Given that IL-18Rα might play an important role in T cell activation, we further evaluated T cell responses in an ex vivo experiment using splenocytes from OVA-sensitized WT and IL-18Rα−/− mice. The results revealed higher cell proliferation rates in OVA-treated WT splenocytes than in media-treated WT splenocytes and lower cell proliferation rates in OVA-treated IL-18Rα−/− splenocytes than in OVA-treated WT splenocytes (Fig. 4A). Moreover, Th2 cytokines such as IL-5 and IL-13 showed decreased levels in the supernatant of cultured splenocytes from OVA-treated IL-18Rα−/− mice compared to those from OVA-treated WT mice (Fig. 4B and C). Additionally, anti-IL-18Rα antibody treatment reduced cell proliferation and Th2 cytokine secretion in WT splenocytes upon OVA treatment, similar to that observed in OVA-treated IL-18Rα−/− splenocytes (Fig. 4). These results suggested that IL-18Rα could mediate Th2 inflammation in secondary lymphoid tissue via the proliferation and differentiation of T cells and IL-18Rα neutralization could be a potential therapeutic target for Th2 inflammatory disease.

Fig. 4

Decreased T cell proliferation and differentiation in IL-18Rα−/− cells and anti-IL-18Rα antibody-treated WT cells after OVA treatment. WT and IL-18Rα−/− mice were sensitized for a 2-week interval. Two weeks after secondary sensitization, splenic single cells were cultured for 5 days in the presence or absence of OVA with anti-IgG or anti-IL-18Rα antibody treatment. (A) Cell proliferation was measured using the Cell Counting Kit-8 assay. Levels of T-helper type 2 cytokines, (B) IL-5 and (C) IL-13, in the supernatant from splenocyte cultures were investigated using an enzyme-linked immunosorbent assay. Data are representative of 3 independent experiments (n = 9–13 for each group) and are presented as the mean ± standard error of the mean.

IL-18Rα−/−, interleukin-18 receptor α-null mutant; WT, wild-type; OVA, ovalbumin; IgG, immunoglobulin G; IL, interleukin; n.s., not significant.

*P < 0.05; †P < 0.01; ‡P < 0.001; §P < 0.0001.

IL-18Rα is involved in STAT3 activation and SOCS3 and SOCS1 expression

IL-18R is known to induce two major intracellular pathways. One involves the adaptor molecule of MyD88, and the other induces STAT3 phosphorylation. STAT3 plays an important role in allergy, and SOCS protein, a direct target gene of STAT, contributes to the pathogenesis of inflammatory diseases caused by Th cell differentiation.3132 To evaluate the involvement of IL-18Rα signaling in the CD4+ T cell-mediated induction of intestinal inflammation, we investigated the interaction between IL-18Rα and the STAT/SOCS signaling pathway in food allergy. The ratio of STAT3 phosphorylation in the intestine was increased in OVA-challenged WT mice compared to that in PBS-challenged WT mice. OVA-challenged IL-18Rα−/− mice demonstrated inhibited STAT3 phosphorylation compared to OVA-challenged WT mice (Fig. 5A). SOCS, the target gene for STAT, is predominantly expressed in T cells and plays an important role in regulating the onset and maintenance of allergic immune disease.13 Therefore, we hypothesized that dysregulation of SOCS expression in T cells might play a role in food allergy. Further experiments on sorting intestinal CD4+ T cells showed that SOCS3 and SOCS1 mRNA expression was significantly upregulated in OVA-challenged IL-18Rα+/+ intestinal T cells compared with PBS-challenged IL-18Rα+/+ intestinal T cells. However, the expression of SOCS3 and SOCS1 in IL-18Rα−/− intestinal T cells was significantly lower than that in IL-18Rα+/+ intestinal T cells after food allergy induction (Fig. 5B and C). Thus, our findings demonstrated that IL-18Rα activated STAT3 signaling pathways by targeting SOCS3 and SOCS1 in T cells.

Fig. 5

Suppressed phosphorylation of STAT3 and expression of SOCS3 and SOCS1 in food allergy-induced IL-18Rα−/− mice. (A) STAT3 phosphorylation at Tyr705 (P-STAT3) and total STAT3 (T-STAT3) levels were examined in the intestine of WT and IL-18Rα−/− mice using an enzyme-linked immunosorbent assay. mRNA expression levels of (B) SOCS3 and (C) SOCS1 in intestinal CD4+ T cells were measured using a real-time polymerase chain reaction. Data are representative of 3 independent experiments (n = 8–15 for each group) and are presented as the mean ± standard error of the mean.

STAT, signal transducer and activator of transcription; SOCS, suppressors of cytokine signaling; IL-18Rα−/−, interleukin-18 receptor α-null mutant; WT, wild-type; OVA, ovalbumin.

*P < 0.05; †P < 0.001.

DISCUSSION

In this study, we used a food allergy mouse model and showed that IL-18Rα expression is significantly increased in the intestine, especially in intestinal CD4+ T cells. IL-18Rα deficiency attenuated systemic food allergic reactions and decreased T cell activation, proliferation, and differentiation. Moreover, IL-18Rα affected STAT3 phosphorylation in the intestine by targeting SOCS3 and SOCS1 in T cells (Fig. 6).

Fig. 6

IL-18Rα modulates allergic reactions and immune responses by regulating the T cell responses in food allergy. IL-18Rα expression was significantly elevated in the intestine. IL-18Rα activated STAT3 phosphorylation, which induced SOCS3 and SOCS1 expression. Furthermore, IL-18Rα regulated T cell responses and systemic food allergic reactions.

IL-18Rα, interleukin-18 receptor α; STAT, signal transducer and activator of transcription; SOCS, suppressors of cytokine signaling; Th2, T-helper type 2 cell; IL, interleukin; EOS, eosinophil; MC, mast cell; IgE, immunoglobulin E; MCPT-1, mouse mast cell protease-1.

Several studies have demonstrated that IL-18 is an important mediator in the pathogenesis of inflammatory diseases such as asthma, rheumatoid arthritis, and colitis.202123 IL-18 and IL-18Rα proteins were strongly expressed in an allergic asthmatic patient.33 Additionally, the IL-18R chromosome (2q12) was observed as a candidate gene associated with elevated susceptibility to asthma in pediatric patients, and polymorphisms of this gene are associated with airway hyperresponsiveness.223435 Moreover, IL-18Rα+ cells and IL-18Rα mRNA levels are increased in patients with eosinophilic esophagitis, and serum IL-18 levels correlate with esophageal eosinophilia.36 However, little is known about the function of IL-18Rα in food allergy pathogenesis. In the present study, we demonstrated that IL-18Rα expression was increased in the intestines of food allergy-induced mice. The elevated levels of IL-18Rα are strongly associated with various inflammatory diseases, thereby making IL-18Rα a potentially useful prognostic or diagnostic marker.

A better understanding of the underlying mechanisms is needed to develop more accurate diagnostic methods and prevent and treat food allergy.37 The present study showed that the decrease in body temperature and occurrence of diarrhea was reduced in the food allergy-induced IL-18Rα−/− group compared to those in the WT group. In addition, IL-18Rα might play a pivotal role in food allergic reactions by regulating IgE production, degranulation of mast cells, and inflammatory cell infiltration. These findings indicated that IL-18Rα promotes immune responses in food allergy. Our observation correlates well with previous studies in which IL-18 was found to be a cofactor for IgE production and Th2 cell development.2538 IL-18 is also involved in the pathogenesis of eosinophilic esophagitis, a food allergen-induced inflammatory disease.36 Additionally, IL-18Rα regulates intestinal inflammation by regulating Foxp3+ regulatory T cells in colitis.39 Thus, these studies suggested that IL-18/IL-18Rα is involved in food allergy and intestinal inflammatory responses.

IL-18/IL-18R signaling is primarily involved in Th1 cell polarization and acts as a cofactor in Th2 cell development and IgE production by promoting Th2 cytokine production. It also contributes to Th17 cell differentiation; thus, IL-18/IL-18R signaling plays an important role in the T cell immune response.253940 In this study, the development of OVA-induced food allergy in WT mice was dependent on CD4+ T cell infiltration into the intestine, and IL-18Rα was predominantly expressed in intestinal CD4+ T cells. IL-18Rα deficiency reduced the intestinal effector CD4+ T cell populations and Th2 cytokine expression compared to those in WT mice after food allergy induction. Similarly, an ex vivo study demonstrated that IL-18Rα deficiency attenuated the cell proliferation and Th2 cytokine production in OVA-stimulated leukocytes. Consistent with our findings, another study showed IL-18Rα involvement in rheumatoid arthritis, mediated by reducing proinflammatory cytokine expression and suppressing T cell accumulation in IL-18Rα−/− mice.21 Furthermore, IL-18Rα expression is enhanced on both effector and regulatory CD4+ T cells in the intestinal lamina propria, and the neutralization of IL-18 or IL-18 binding protein ameliorates colitis.394142 Moreover, anti-IL-18 antibody administration to mice protects against eosinophil-mediated allergic airway inflammation.43 Similarly, in our ex vivo studies, IL-18Rα blockade attenuated cell proliferation and Th2 cytokine expression. Collectively, IL-18Rα neutralization could be a potential therapeutic strategy for the treatment of patients with food allergy.

Th2 cytokines play an important role in allergic diseases and exert their biological functions through JAK and STAT transcription factors. STAT3 is a well-known critical transcription factor for cytokine signaling and allergic immune responses.32 A previous study showed that the inhibition of STAT3 phosphorylation prevents Th2 cell differentiation and lung inflammation in an asthmatic mouse model.44 In addition, STAT protein activation is regulated by SOCS proteins, which contribute to Th cell differentiation during immune responses.12 SOCS3 and SOCS1 expression levels are also associated with allergic and inflammatory diseases, such as asthma and atopic dermatitis.1345 SOCS3 is predominantly expressed in Th2 cells and participates in intestinal inflammation.46 SOCS3 silencing in primary CD4+ T cells attenuates Th2 responses in vitro.47 Moreover, the STAT/SOCS signaling pathway is a well-known mediator of several biological processes, and STAT3 is associated with IL-18R-related intracellular pathways. However, the role of the STAT/SOCS signaling pathway in food allergy is poorly studied. Our results revealed that IL-18Rα increased STAT3 phosphorylation in the intestine and the expression levels of SOCS3 and SOCS1 in intestinal CD4+ T cells. Therefore, we conclude that in IL-18Rα-mediated food allergy, dysregulation of the STAT/SOCS signaling pathway contributes to intestinal inflammation. IL-18/IL-18Rα is also involved in other signaling pathways such as mitogen-activated protein, phosphoinositide-3, and AMP-activated protein kinases.4849 Therefore, further studies of various pathways are needed to confirm the role of IL-18Rα in the pathogenesis of food allergy.

In conclusion, our current findings define a novel role for IL-18Rα in the pathogenesis of food allergy via T cell immune responses and provide evidence that IL-18Rα might play a pivotal role in food allergy by activating the STAT/SOCS signaling pathway. Collectively, our results suggest that IL-18Rα is a potential biomarker and therapeutic target to prevent and treat food allergy.