Abstract
Nonsteroidal anti-inflammatory drugs have been reported to enhance oral sensitization to food allergens, potentially through disruption of the intestinal epithelial barrier. Epithelial-derived cytokines, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and IL-25, promote immunoglobulin E (IgE) production by inducing T helper type 2 immune responses and may contribute to allergen sensitization following epithelial injury. This study aimed to investigate oral sensitization to ovalbumin (OVA) in a rat model of intestinal epithelial injury induced by dextran sulfate sodium (DSS) or indomethacin (IND), focusing on the involvement of TSLP in relation to the anatomical site of epithelial injury. Rats were orally immunized with OVA by gavage every other day for 6 weeks. DSS was administered in drinking water from 2 weeks before immunization, whereas IND was administered orally at a dose of 6 mg/kg 30 min before each OVA administration. To explore the involvement of TSLP, anti-TSLP antibodies were administered intraperitoneally once a week throughout the immunization period. Histological analysis showed that DSS induced epithelial injury in both the jejunum and proximal colon, whereas IND caused milder epithelial injury limited to the jejunum. Plasma OVA-specific IgE (OVA-sIgE) levels were elevated in both models. Anti-TSLP antibody treatment attenuated DSS-enhanced OVA-sIgE production, but had limited effects in the IND model. In conclusion, intestinal epithelial injury may enhance oral sensitization through mechanisms that differ according to the anatomical site and extent of injury. TSLP appears to contribute predominantly to sensitization associated with epithelial damage involving both the jejunum and proximal colon.
INTRODUCTION
Production of allergen-specific immunoglobulin E (sIgE), a process known as sensitization, represents the initial step in the development of type-I food allergy. Recent studies have demonstrated that epithelial-derived cytokines, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and IL-25, promote IgE production through activation of innate lymphoid cells, antigen-presenting cells, and T helper type-2 (Th2) cells.1) These cytokines are expressed not only in bronchial epithelial cells and keratinocytes but are also detected in intestinal epithelial cells.2,3)
Nonsteroidal anti-inflammatory drugs, including aspirin, diclofenac, and indomethacin (IND), increase intestinal absorption of food allergens such as ovalbumin (OVA) through epithelial injury.4) This phenomenon is associated with enhanced OVA-sIgE production.4) However, enhanced sensitization was not observed in spermine-treated rats, in which OVA absorption was increased without epithelial injury. These findings suggest that epithelial injury, rather than increased allergen absorption alone, may be involved in the development of sensitization.
Based on these observations, we examined oral sensitization to OVA in a rat model of intestinal epithelial injury induced by dextran sulfate sodium (DSS), which is widely used to induce inflammatory bowel disease via epithelial injury,5) or IND, focusing on the involvement of TSLP in relation to the anatomical site of epithelial injury.
MATERIALS AND METHODS
Materials
OVA (grade V) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). DSS (molecular weight 5000–6000) and IND were purchased from Nacalai Tesque (Kyoto, Japan). Anti-TSLP antibody (Ab) and mouse anti-rat IgE (MARE-1) were obtained from Bio X Cell (Lebanon, NH, U.S.A.) and GeneTex (Irvine, CA, U.S.A.), respectively. All chemicals used were of analytical grade or higher purity.
Animals
Three-week-old female Brown Norway rats were obtained from Japan SLC (Hamamatsu, Japan). Prior to the initiation of the experimental procedures, the animals were provided with a standard laboratory diet and water ad libitum for one week. All animal experiments were performed in accordance with the Guidelines for Animal Experimentation established by the Committee of Research Facilities for Laboratory Animal Sciences, Hiroshima University (Approval No. A21-148-2, Approval date: April 15, 2024; Hiroshima, Japan).
Treatment with DSS, IND, or Anti-TSLP Abs
The experimental protocol is shown in Fig. 1. Rats were divided into control, DSS- or IND-treated, and anti-TSLP Ab-treated groups. All rats were orally administered OVA (50 mg) in phosphate-buffered saline (PBS; pH 7.4) three times per week for 6 weeks. In the DSS treatment, rats received 1% DSS in drinking water ad libitum from two weeks before OVA administration until the end of the experiment. In the IND treatment, IND (6 mg/kg) was orally administered 30 min before each OVA administration, while controls received saline. Anti-TSLP Abs (5.4 mg/kg) were administered intraperitoneally once weekly starting one day before sensitization. Blood was collected from the jugular vein at weeks 0, 4, and 6, and plasma was stored at −30°C for OVA-sIgE analysis after centrifugation. Body weight (BW) was monitored throughout the experiment, and the jejunum and proximal colon were excised at the end.
Histopathological Evaluation
The isolated intestine was incised and gently flushed with PBS. The tissue samples were then fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin–eosin (H&E). Histopathological evaluation was performed using a BZ-9000 microscope (Keyence, Osaka, Japan).
Measurement of Plasma Levels of OVA-sIgE
Enzyme-linked immunosorbent assay (ELISA) was performed to determine plasma OVA-sIgE levels. Briefly, 100 ng of MARE-1 was added to each well of a 96-well plate and incubated overnight at 4°C. The wells were blocked with 1% Block Ace® for 1 h at 37°C. Plasma samples diluted 1 : 10 with 1% Block Ace® were added and incubated for 1 h at 37°C, followed by incubation with biotinylated OVA for 1 h at the same temperature. Horseradish peroxidase-conjugated streptavidin was then added and incubated for 1 h at 37°C. After washing, 3,3′,5,5′-tetramethylbenzidine was added for color development and incubated for 15 min at 37°C. The reaction was stopped with 1 M phosphoric acid, and absorbance was measured at 450 nm with 630 nm as a reference wavelength using a microplate reader.
Statistical Analysis
Data are presented as means ± standard error. Differences between groups were analyzed using ANOVA followed by the Tukey–Kramer post hoc test. A p-value of <0.05 was considered statistically significant.
RESULTS
DSS Induces Histopathological Damage in the Small Intestine and Proximal Colon
To confirm DSS-induced intestinal damage, changes in BW as a supportive indicator of intestinal damage and intestinal histopathological damage were evaluated. DSS treatment slightly suppressed BW gain compared with the controls (Supplementary Fig. 1A). H&E staining of the jejunum revealed well-preserved mucosal architecture, including intact villi and a normal epithelial structure, in control rats at Week 6 (Fig. 2A). DSS-treated rats showed shortened villi and severe mucosal erosion, which were not affected by anti-TSLP Ab treatment (Figs. 2B, 2C). In the proximal colon, control rats exhibited an intact epithelial lining and clearly defined crypt structures (Fig. 2D). By contrast, DSS-treated rats showed marked mucosal injury, including epithelial disruption, shortened mucosal folds, and infiltration of inflammatory cells into the submucosa (Fig. 2E).
Anti-TSLP Ab Decreases DSS-Enhanced Oral Sensitization to OVA
To evaluate the effects of DSS and anti-TSLP Ab treatment on oral sensitization, plasma levels of OVA-sIgE were determined by ELISA (Fig. 3). Plasma OVA-sIgE levels were higher in DSS-treated rats than in control rats at Weeks 4 and 6, and this increase was reduced by anti-TSLP Ab treatment, almost to control levels.
IND Induces Histopathological Damage in the Small Intestine but Not in the Proximal Colon
To assess intestinal damage induced by IND treatment, BW changes and intestinal histopathological damage were evaluated. IND treatment slightly suppressed BW gain compared with control rats (Supplementary Fig. 1B). IND induced mucosal erosion and inflammatory cell infiltration in the jejunum (Figs. 4A, 4B); however, these changes were less severe than those observed in DSS-treated rats. Anti-TSLP Ab treatment did not affect IND-induced intestinal damage (Fig. 4C). In contrast to DSS-treated rats, no apparent epithelial injury was observed in the proximal colon of either control or IND-treated rats (Figs. 4D, 4E).
Anti-TSLP Ab Fails to Decrease IND-Enhanced Oral Sensitization to OVA
To evaluate the effects of IND and anti-TSLP Ab treatment on oral sensitization, plasma levels of OVA-sIgE were determined by ELISA (Fig. 5). Plasma OVA-sIgE levels were slightly higher in IND-treated rats than in control rats at both time points examined. Anti-TSLP Ab treatment slightly reduced IND-enhanced OVA-sIgE levels at Week 4 but had no effect at Week 6.
DISCUSSION
In this study, we found that TSLP may contribute to the enhancement of oral sensitization under conditions of DSS-induced epithelial injury affecting both the jejunum and proximal colon. In contrast, oral sensitization associated with IND-induced mild epithelial injury localized to the jejunum appeared to be less dependent on TSLP. These findings suggest that the immunological pathways leading to oral sensitization may differ according to the anatomical site and extent of intestinal epithelial injury.
Anti-TSLP Ab treatment attenuated the DSS-induced increase in plasma OVA-sIgE levels, suggesting that TSLP may contribute to the enhancement of oral sensitization associated with epithelial injury in both the jejunum and proximal colon. In preliminary studies, plasma TSLP levels were not elevated following DSS treatment. TSLP released from injured intestinal epithelial cells has been reported to act in a paracrine manner on immune cells in the underlying lamina propria, including dendritic cells and T cells.6) Therefore, locally released TSLP may regulate Th2 immune responses and sIgE production, even when systemic levels are undetectable.
IND treatment induced mild epithelial injury characterized by mucosal erosion and inflammatory cell infiltration in the jejunum, whereas no apparent histopathological injury was observed in the proximal colon. Unlike DSS treatment, the increase in OVA-sIgE levels induced by IND was not markedly affected by TSLP inhibition, suggesting a limited contribution of TSLP under conditions of localized jejunal injury. These findings suggest that the immunological pathways underlying oral sensitization are determined not only by the type of epithelial insult but also by the anatomical site and extent of intestinal epithelial injury. Previous studies have reported higher TSLP expression in the proximal colon than in the small intestine.7) Accordingly, site-dependent differences in epithelial cytokine expression may underlie the distinct sensitization patterns observed. Clinically, these results suggest that anti-TSLP–based interventions may show variable efficacy depending on the anatomical distribution of intestinal injury. Pathophysiologically, sensitization associated with localized small intestinal damage may involve epithelial-derived mediators other than TSLP.
In addition to TSLP, other epithelial-derived cytokines may also contribute to oral sensitization. Sensitization to egg-white proteins enhanced by medium-chain triglycerides has been reported to involve multiple epithelial-derived cytokines, including TSLP, IL-25, and IL-33.8) Consistent with this finding, inhibition of TSLP alone did not completely suppress IND-enhanced oral sensitization, suggesting that additional mechanisms may contribute to sensitization under these conditions. However, the contribution of these cytokines remains uncertain because the local expression of TSLP, IL-25, and IL-33 was not evaluated in the present study. Further studies are required to determine whether cytokines other than TSLP participate in sensitization associated with localized jejunal injury.
This study has several limitations. First, we did not use oral adjuvants, and considerable inter-individual variation in OVA-sIgE levels was observed, which may have reduced statistical sensitivity. Second, local expression of epithelial-derived cytokines was not examined in the jejunum or proximal colon. Although plasma levels of IL-25 and IL-33 were below the detection limit under the present assay conditions, these cytokines are known to act predominantly in a local manner. Therefore, the present study could not directly determine whether differences in local cytokine expression contributed to the distinct sensitization pathways observed following DSS- and IND-induced epithelial injury. Third, the present findings support an association between the anatomical site of epithelial injury and oral sensitization pathways but do not establish a causal relationship. Finally, the effects of anti-TSLP Ab treatment on intestinal histopathology were not specifically evaluated. Therefore, it remains unclear whether TSLP inhibition directly influenced epithelial injury in the jejunum or proximal colon. Future studies examining local cytokine expression and functional blockade of candidate cytokines will be necessary to clarify the mechanisms linking epithelial injury at specific intestinal sites to oral sensitization.
In conclusion, intestinal epithelial injury enhances oral sensitization through mechanisms that differ according to the anatomical site and extent of injury. TSLP may be involved in sensitization associated with epithelial damage involving both the jejunum and proximal colon. These findings suggest that the anatomical distribution of intestinal epithelial injury may be a key determinant of the cytokine pathways involved in oral sensitization.
Acknowledgements
This work was supported by JSPS KAKENHI (Grant Number: 22K06741).
DECLARATIONS
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
This article contains supplementary materials.
REFERENCES
- 1) Stanbery AG, Smita S, von Moltke J, Tait Wojno ED, Ziegler SF. TSLP, IL-33, and IL-25: Not just for allergy and helminth infection. J. Allergy Clin. Immunol., 150, 1302–1313 (2022).
- 2) Tahaghoghi-Hajghorbani S, Ajami A, Ghorbanalipoor S, Hosseini-Khah Z, Taghiloo S, Khaje-Enayati P, Hosseini V. Protective effect of TSLP and IL-33 cytokines in ulcerative colitis. Auto Immun. Highlights, 10, 1 (2019).
- 3) Latiano A, Palmieri O, Pastorelli L, Vecchi M, Pizarro TT, Bossa F, Merla G, Augello B, Latiano T, Corritore G, Settesoldi A, Valvano MR, D’Incà R, Stronati L, Annese V, Andriulli A. Associations between genetic polymorphisms in IL-33, IL1R1 and risk for inflammatory bowel disease. PLoS One, 8, e62144 (2013).
- 4) Fukushima T, Yokooji T, Hirano T, Kataoka Y, Taogoshi T, Matsuo H. Aspirin enhances sensitization to the egg-white allergen ovalbumin in rats. PLoS One, 14, e0226165 (2019).
- 5) Wirtz S, Popp V, Kindermann M, Gerlach K, Weigmann B, Fichtner-Feigl S, Neurath MF. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat. Protoc., 12, 1295–1309 (2017).
- 6) Ito T, Wang YH, Duramad O, Hori T, Delespesse GJ, Watanabe N, Qin FX, Yao Z, Cao W, Liu YJ. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J. Exp. Med., 202, 1213–1223 (2005).
- 7) Taylor BC, Zaph C, Troy AE, Du Y, Guild KJ, Comeau MR, Artis D. TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis. J. Exp. Med., 206, 655–667 (2009).
- 8) Khodoun MV, Tomar S, Tocker JE, Wang YH, Finkelman FD. Prevention of food allergy development and suppression of established food allergy by neutralization of thymic stromal lymphopoietin, IL-25, and IL-33. J. Allergy Clin. Immunol., 141, 171–179.e1 (2018).
