Irritable Bowel Syndrome With Diarrhea in Pediatric Patients Is Associated With Type 2 and Type 9 T Cells in the Intestinal Mucosa

Irritable bowel syndrome (IBS) represents a complex and pervasive gastrointestinal disorder that is a highly prevalent disease in all ages and has a large impact on the quality of life for patients suffering from it; yet the pathogenesis of IBS is not understood. It is thought to involve structural, neurologic, and immune components. From the immune perspective, increased frequency and density of mast cells around the nerve fibers in the intestines have been identified consistently.¹ T cells and mast cells interact via various mechanisms: interleukin (IL)-4 produced by T cells induces intestinal mastocytosis² and induces expression of FcεRI, which enhances proliferation of mast cells³; IL-9 produced by T cells augments mast cell recruitment and effector function.⁴ Mast cells produce prostaglandin D2, IL-4, and IL-13, which enhance Th2 polarization. Few studies have examined the mucosa of patients with IBS for detailed changes in T cell populations and their interaction with mast cells, but there is evidence that immune activation plays a role in the pathogenesis of IBS. Sera from patients with IBS have increased IL-5, IL-13, IL-6, IL-8, IL-1β, and tumor necrosis factor-α, and decreased IL-10,⁵ suggesting a type 2 inflammatory response and a decrease in regulatory cytokines. In support of this, studies that examined the mucosa of the colon by histology found increased intraepithelial lymphocytes in the terminal ileum and in the cecum.⁶ Furthermore, flow cytometry evidence showed that the total numbers of lamina propria lymphocytes, as well as the amount of CD45RO⁺ CD4⁺ activated T cells and double positive CD4⁺CD8⁺ T cells, were all increased in the sigmoid colon of patients with IBS.⁷ Despite these findings, studies examining T cell populations in the mucosa of patients with IBS, in particular Type 2 and Type 9 T cells, are lacking. Importantly, most of the studies with human samples analyzed only material from adults. Because a more thorough understanding of aberrant T cell responses in the mucosa of patients with IBS can help to better explain the pathogenesis of the disease, we analyzed the production of cytokines by cells from colon and duodenum samples from children and adolescents diagnosed with IBS-diarrhea (IBS-D) as compared with control individuals. Control samples were obtained from patients undergoing diagnostic endoscopies that were not found to have an inflammatory bowel disease or fit the Rome IV criteria for IBS. Notably, some control patients had at least one atopic disease, but none of the patients had IgE-mediated food allergies (Table 1).

Table 1.

Patient Demographics

	IBS-D (n = 14)	Controls (n = 16)	Total (N = 30)	P value
Female gender, %	35.7	50.0	43.3	.676
Age, years	15.6 (8–18)	12.4 (2–17)	14 (2–18)	.055
Duodenum, n	8	8	16
Colon, n	7	8	16
Atopic disease (with at least 1)	1 (7.1)	5 (31.3)	6 (20.0)	.176
IgE mediated food allergy	0 (0.0)	0 (0.0)	0 (0.0)	> .999
Asthma	1 (7.1)	3 (18.8)	4 (13.3)	.602
Allergic rhinitis	0 (0.0)	2 (12.5)	2 (6.7)	.485
Eczema	0 (0.0)	2 (12.5)	2 (6.7)	.485

Note: Data are presented as number (%) or median (interquartile range) unless otherwise indicated.

IBS-D, irritable bowel syndrome-diarrhea.

Colon samples from subjects with IBS-D (n = 7) had significantly higher proportions of cells producing IL-5⁺ (Figure 1B), IL-13⁺ (Figure 1C), and IL-9⁺ cells (Figure 1D) within the memory CD4⁺ T cell population (CD45RA^-CD4⁺CD8^-) than controls (n = 8). Within the colon memory CD8⁺ T cell population, IL-4⁺ (Figure 1E), IL-5⁺ (Figure 1F), IL-13⁺ (Figure 1G), and IL-9⁺ (Figure 1H) cells were significantly increased in IBS-D compared with controls. We then examined the colonic resident memory CD4⁺ T cells (CD69⁺CD103⁺CD45RA^-CD4⁺CD8^-) in IBS-D as compared with controls and found no differences in cytokines (data not shown).

Figure 1.

Increased type 2 and type 9 memory T cells in the duodenum and colon of subjects with IBS-D. (A–D) Percentage of IL4⁺ (A), IL5⁺ (B), IL13⁺ (C), and IL9⁺ (D) in memory CD4⁺ T cells in colon. (E–H) Percentage of IL4⁺ (E), IL5⁺ (F), IL13⁺ (G), and IL9⁺ (H) in the colon memory CD8⁺ T cells. (I–K) Percentage of IL4⁺ (I), IL13⁺ (J), and IL9⁺ (K) in memory CD4⁺ T cells in duodenum. (L–N) Percentage of IL4⁺ (L), IL13⁺ (M), and IL9⁺ (N) in memory CD8⁺ T cells in duodenum samples (colon: IBS, n = 7; control, n = 8; duodenum: IBS, n = 8; control, n = 8; ∗P < .05; ∗∗P < .01).

In duodenum samples from subjects with IBS-D (n = 8), there was a significant rise in percentages of IL-13⁺ within the memory CD4⁺ T cell population (CD45RA^-CD4⁺CD8^-) as compared with controls (Figure 1I–K) (n = 8). In the duodenal memory CD8⁺ T cell population, IL-13⁺ and IL-9⁺ cells were significantly increased in IBS-D compared with controls (Figure 1L–N). We then examined the duodenal resident memory CD4⁺ T cells (CD69⁺CD103⁺CD45RA^-CD4⁺CD8^-) as compared with controls and found that IL-4⁺ and IL-9⁺ cells were increased in IBS-D (Supplementary Figure 1).

Collectively, these results suggest that IBS-D in pediatric patients is associated with a dysregulated immune response characterized by heightened Type 2 and Type 9 cytokine production in the mucosa of both duodenum and colon. This demonstrates a localized immune response in the mucosa of patients with IBS-D and is consistent with the increase in mast cells found within the mucosa in IBS.¹ The nature of the link between the mast cells and T cells has yet to be explored. This link may involve activation by antigens such as food antigens⁸ or microbial antigens. Type 9 T cell upregulation is a novel finding from our work, and studies have indicated a strong association between Type 9 T cells and atopic diseases. Although atopic disease is a recognized risk factor for IBS,⁹ only 1 of our patients with IBS-D had an atopic disease. It is important to note that most atopic diseases primarily involve a systemic IgE response, whereas IBS is likely associated with local IgE antibodies in the mucosa,¹⁰ given that studies examining the serum have only shown elevated food-specific IgG antibodies that have no correlation with symptom severity. Local pathogenic IgE in the mucosa is consistent with the increase in local mast cells and with our findings of Type 2 and Type 9 T cell activation. Alternatively, microbial antigens in the gut could be stimulating these immune responses.

IBS may be subclassified into different phenotypes based upon the presence of diarrhea, constipation, or both. Our study focused on IBS-D, but the pathophysiology of each IBS subtype likely varies. This variation could involve several factors that may differ between subtypes, including microbiota imbalances, low-grade inflammation, immune activation, and central nervous system involvement. Understanding the relationships between the immune response and the clinical subtypes of IBS is important as this may lead to better targeted treatments. Furthermore, the localization of the inflammatory response in different intestinal segments may vary among subjects with IBS and may be a factor in determining disease phenotype.

Understanding the underlying immune dysregulation in IBS can help us to better elucidate the pathophysiology of IBS and lead to the development of novel immunotherapies.