Interleukin-13 Mediates Non-Steroidal Anti-Inflammatory-Drug-Induced Small Intestinal Mucosal Injury with Ulceration

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs), which are antipyretics and analgesics, cause gastrointestinal disorders, such as inflammation and ulcers. To prescribe NSAIDs more safely, it is important to clarify the mechanism of NSAID-induced gastrointestinal mucosal injury. However, there is a paucity of studies on small intestinal mucosal damage by NSAIDs, and it is currently unknown whether inflammation and ulceration also occur in the small intestine, and whether mediators are involved in the mechanism of injury. Therefore, in this study, we created an animal model in which small intestinal mucosal injury was induced using NSAIDs (indomethacin; IDM). Focusing on the dynamics of immune regulatory factors related to the injury, we aimed to elucidate the pathophysiological mechanism involved. We analyzed the pathological changes in the small intestine, the expression of immunoregulatory factors (cytokines), and identified cytokine secretion and expression cells from isolated lamina propria mononuclear cells (LPMCs). Ulcers were formed in the small intestine by administering IDM. Although the mRNA expression levels of IL-1β, IL-6, and TNFα were decreased on day 7 after IDM administration, IL-13 mRNA levels increased from day 3 after IDM administration and remained high even on day 7. The IL-13 mRNA expression and the secretion of IL-13 were increased in small intestinal LPMCs isolated from the IDM-treated group. In addition, we confirmed that IL-13 was expressed in CD4-positive T cells. These results provided new evidence that IL-13 production from CD4-positive T cells in the lamina propria of the small intestine contributes to NSAID-induced mucosal injury.

Keywords: IL-13 (Interleukin 13); LPMCs (lamina propria mononuclear cells); NSAIDs (non-steroidal anti-inflammatory drugs); small intestine; ulceration.

Figure 1

Biological and histological changes in the indomethacin (IDM)-induced intestinal mucosal injury model. Seventy-two mice (36 mice each for the control and IDM groups) were used. The IDM group was administered a single dose of IDM by subcutaneous injection. From day 1 of IDM administration (which was defined as day 0), body weight (a), the length of the small intestine (b), and the villus height (c,d) were measured. Statistical analyses were performed using two-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Ulceration in the small intestine of the indomethacin (IDM)-induced intestinal mucosal injury model. Forty-eight mice (12 mice for each point) were used. The control and indomethacin (IDM) groups were tail-injected with Evans Blue and left for 30 min. After perfusion with PBS, the small intestine was harvested and observed. The blue spots were inflammatory sites with increased vascular permeability (a). The ulcer tissues observed in (a) -1 or -2 were stained with H&E (b). The ulcers (c) were counted and the extravasation of Evans blue in the tissue (d) was measured via absorbance. Statistical analyses were performed using the two-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

mRNA expression analysis of cytokines in small intestinal tissues of the indomethacin (IDM)-induced intestinal mucosal injury model. Seventy mice (seven mice for each point) were used. mRNA expressions of several cytokines in ileal tissues were sequentially analyzed via real-time PCR (a). From the analysis results of Figure 3a, the expression differences were confirmed for each small intestine site on days when mRNA expressions were significantly increased (b). Statistical analyses were performed using the two-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

mRNA expression analysis of IL-13 in small intestinal tissues divided by parts of the indomethacin (IDM)-induced intestinal mucosal injury model. Sixteen mice (eight mice for each point) were used on day 7 after IDM administration. The small intestinal tissue was separated into each site, namely, the ulcer (U), near ulcer (NU), and the morphologically preserved part (MPP), via the laser microdissection method (a). mRNA expressions of IL-13 were analyzed using real-time PCR (b). Statistical analyses were performed using the two-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Figure 5

IL-13 detection in the small intestine of the indomethacin (IDM)-induced intestinal mucosal injury model. Twenty-eight mice (14 mice each for the control and IDM groups) were used. Jejunal and ileal tissues were determined using IL-13 immunohistochemistry (a). Percentages of IL-13-positive areas per villi were calculated (b). Statistical analyses were performed using the t test. ** p < 0.01, *** p < 0.001.

Figure 6

IL-13 mRNA expression and secretion in the LPMCs of the small intestine in the indomethacin (IDM)-induced intestinal mucosal injury model. Twenty-four mice (12 mice each for the control and IDM groups) were used. The separated lamina propria mononuclear cells (LPMCs) were stimulated with PMA and ionomycin for 24 h. The LPMCs were counted (a). mRNA expressions of IL-13 in LPMCs were analyzed via real-time PCR, while IL-13 secretions in the culture supernatant of LPMCs were analyzed using ELISA (b,c). Statistical analyses were performed using the t test (a) and two-way analysis of variance (ANOVA) with Tukey’s post hoc test (b,c). ** p < 0.01, *** p < 0.001.

Figure 7

Identification and characterization of the cell subpopulations of LPMCs of small intestine in indomethacin (IDM)-induced intestinal mucosal injury model. Twenty-four mice (12 mice each for the control and IDM groups) were used. Cell type identification was performed on stimulated LPMCs via flow cytometry (FACS).

Figure 8

Identification of cells expressing IL-13 in the small intestine of the indomethacin (IDM)-induced intestinal mucosal injury model. Twenty-four mice (12 mice each for the control and IDM groups) were used. CD4-positive and IL-13-positive cells were detected (a). The CD4-positive and IL-13-positive cells in jejunal tissues were determined via CD4 and IL-13 double staining (b).

Figure 9

Cytotoxicity of IL-13 in intestinal epithelial cells. The cell viability of the human colonic carcinoma cell line (Caco-2 cells) added to recombinant IL-13 proteins in the presence of IDM was evaluated. Statistical analyses were performed using the two-way analysis of variance (ANOVA) with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.