Investigation of immune regulation in pediatriceosinophilic esophagitis (EOE) patients

Abstract

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease of the esophagus, characterized by excessive eosinophil infiltration into the target tissue, ultimately causing inflammation and esophageal dysfunctions. EoE can affect both adults and children, atopic patients and adult males have a higher tendency to develop EoE. Once considered a rare disease, its incidence and prevalence has increased over the years. The most common risk factors are having food allergy or atopic conditions. Common symptoms in adults and children include; difficulty in eating/swallowing, abdominal pain, food impaction, heartburn and reflux. The first step in the diagnosis process is taking a careful patient history followed by an endoscopic evaluation by gastroenterologists and a histologic evaluation of the biopsy specimens by pathologists to detect EoE-compatible findings. The common approach for EoE treatment is elimination diets, coupled with the use of medications (topical steroids and acid suppressors) in order to to alleviate the symptoms and achieve remission. Some patients are responsive to the acid suppressor treatments, proton pump inhibitors (PPIs) and gain full disease remission while some patients are non-responsive to PPIs. There are no definite treatments to cure EoE and the disease significantly affects the life quality due to challenging symptoms. The pathogenesis of EoE is highly complex and involves epithelial barrier dysfunction, type 2 inflammatory response and tissue remodeling, in which each event contributes to the formation of one another and further enhances the disease outcome. The epithelial barrier integrity is impaired in EoE due to genetic and environmental factors and this issue allows facilitated penetration of food allergens into the deeper segments of the esophagus. EoE occurs mainly by the food allergens, triggering a type 2 inflammation mediated by Th2 cells leading to further epithelial barrier dysregulations and tissue remodeling. Inflammatory response begins when food allergens stimulate esophageal epithelial cells to secrete key cytokines called "alarmins" which leads to retention of immune cells, including Th2 cells. Th2 cells secrete type-2 cytokines (IL- 4, IL-5 and IL-13) that acts on: retention and degranulation of eosinophils and mast cells, alterations in gene expressions which leads to epithelial barrier impairments and fibrosis. The chronic inflammation causes constant damaging of the esophageal tissue and the body tries to repair this damage as long as the inflammation proceeds. Due to the intensity and the duration of this damage, tissue scaring and fibrosis is observed, which is critical for the patient and can even lead to organ malfunctions. Immune system is divided into innate and adaptive. Innate immune system is present from birth and provides rapid, first-line defense against pathogens. In contrast, adaptive immunity is the acquired over time and continuously develops. It involves T and B lymphocytes, which are responsible for cell-mediated and humoral immune responses, respectively. Both T and B cells have multiple subtypes, each with distinct functions. The ongoing clinical trials of monoclonal antibodies targeting type 2 cytokines still remains inadequate in treating EoE, indicating the presence of a complex but still not fully-known immune response specific to EoE. Studies focusing on the immune response seen in EoE revealed some differences between adults and children, the increased Treg levels in children being the prominent difference. Treg cells are important players of adaptive immunity, taking a part in regulation of excessive immune responses and sustaining the immune homeostasis. In pediatric patients, despite the high levels of Treg cells, the type 2 inflammation is not ceased, suggesting that the effector functions of Treg cells might be compromised however the exact explanation to this phenomenon is still unknown. Previous studies showed that TIGIT surface marker expressing Treg cells can suppress Th1 and Th17 responses while supporting the Th2 cells through secreting FGL2, an immunomodulatory protein. The presence of TIGIT+ Treg cells have been addressed in other pathologic conditions but not in EoE. Another recent study showed the presence of a novel B cell subset, characterized by CD73 and CD49b co-expression, that can promote angiogenesis in adult EoE patients. Angiogenesis is a key event during inflammatory response and these proangiogenic B cells are elevated in the blood of adult EoE patients, indicating that these cells might be playing an important role in the immune response however, their presence in pediatric patients is currently not addressed. Taken together, this information highlights gaps in the existing literature regarding the immune response and its regulatory mechanisms in pediatric EoE patients. The primary aim of this study was to examine various T and B cell subsets in the peripheral blood of pediatric EoE patients, offering deeper insight into the complex immune responses observed in this population. Considering the immune suppressive functions of Tregs and why these cells are elevated in pediatric EoE patients despite the continuing inflammation is a big question to answer and the response to this may involve various T and B cells and their cellular interactions. Since TIGIT-expressing Tregs cannot suppress Th2 responses and EoE is a Th2-mediated inflammatory condition, investigating the potential contribution of TIGIT+ Tregs to EoE would be important. To address this question, we analyzed the presence of TIGIT-expressing T cells in the peripheral blood and compared their levels between patients in remission and non-EoE controls. In addition, we examined the levels of proangiogenic B cells in pediatric patients, along with PD-1- and TIGIT-expressing B cells, which have been associated with regulatory and suppressive functions in other diseases. Above mentioned T and B cell subtypes were never been addressed in pediatric EoE patients in the literature, therefore this study holds an importance in terms of contributing to the literature and filling some of the missing gaps. Some of the analyzed cells were previously addressed in the literature and were included in this study to further support the novel findings of this research. To investigate this aim, blood samples were collected from a total of 24 pediatric patients: 9 EoE, 6 remission and 9 non-EoE controls, between the ages 2 to 17. PBMC cells were isolated and serum samples were collected from each blood sample. To investigate T and B cell subtypes in the PBMCs, antibody panels were generated for flow cytometry analysis. FOXP3+ Tregs were identified as FOXP3+CD4+ Th cells, Th2 cells as IL-4+CD4+ Th cells and proangiogenic B cells as CD73+CD49b+ B cells in this study. Our findings revealed several results consistent with the literature, such as; elevated CD3+ T cells in EoE and decreased IL-4+ Th (indicating the Th2 cells) cells in the remission patients. Conversely, some of our findings were different from those reported in the previous studies. While other studies reported elevated levels of peripheral CD4+ Th cells and FOXP3+ Treg cells in pediatric EoE patients, our study found no significant difference between EoE and control groups. However, we observed a decrease in peripheral FOXP3+ Treg cell levels in patients during remission. IL-10 is an anti-inflammatory cytokine, often attributed to FOXP3+ Treg effector functions and in this study IL-10+ Th cell levels did not show a significant difference among the groups yet their frequency was slightly higher in EoE group. CD39+CD73+ B cells, attributed to regulatory functions due to converting pathogenic ATP to free adenosine thereby supporting angiogenesis, were significantly decreased in the remission patients compared to patient and control groups. Proangiogenic B cells were shown to be elevated in adult patients but according to our study, their levels are significantly lower in pediatric patients compared to non-EoE controls, suggesting a different immune regulatory mechanism in children. PD-1 and TIGIT expressing B cells, attributed to regulatory functions, were decreased in the patient group compared to controls, indicating a link between the absence of B cell regulatory functions and active inflammation. Surprisingly, TIGIT expressing T cells (both CD3+ and CD4+) were elevated in the peripheral blood of pediatric EoE patients compared to remission and control groups. In addition to that, TIGIT+ T cells were significantly reduced in remission patients, overall supporting the idea that TIGIT+ T cells might be involved in the complex immune response seen in EoE as their levels dropped once the patients were in remission and using medications. Once it was established that TIGIT+ Th cells were elevated in EoE patients, the next step was to investigate the FGL2 levels. The second aim of this study was to evaluate the levels of FGL2, a multifunctional protein secreted by TIGIT+ Treg that suppresses Th1 and Th17 responses while promoting Th2 responses. Given that EoE is a Th2-mediated disease, we hypothesized that FGL2 may play a role in the Th2-mediated inflammatory response, potentially explaining why Treg levels are elevated in pediatric patients despite their immune- suppressive functions. To investigate this aim, esophageal tissue samples collected by the clinicians during endoscopy were used to assess the relative mRNA expression of FGL2 in the local tissue. Additionally, GATA3, the master transcription factor of Th2 cells, was analyzed to evaluate the Th2 levels in the esophageal tissue of pediatric patients. Our findings showed that tissue FGL2 mRNA expression was significantly higher in pediatric EoE patients compared to non-EoE controls, while no change was observed in the relative mRNA expression GATA3. FGL2 production may not be confined to the local inflamed tissue; it could also be secreted by cells and circulate in the bloodstream. To address this question, serum samples from pediatric EoE patients were analyzed for secreted FGL2 levels using an ELISA assay. Our results showed no significant difference between the groups. Altogether, these findings demonstrate that TIGIT+ T cells are elevated in the peripheral blood of pediatric EoE patients, with levels decreasing during remission, suggesting a potential role of these cells in the EoE immune response. Although tissue FGL2 mRNA expression was higher in pediatric EoE patients, soluble FGL2 levels in in the serum showed no significant differences between groups indicating that FGL2 may act as a key mediator at the local site of inflammation but may be less involved in the systemic response. In conclusion, this study contributes valuable insights and helps fill some gaps in the literature regarding the complex immune response in EoE, while also raising new questions for further investigation.