In the present study we showed the gene expression of cytokines involved in the pathophysiology and immune response of GERD. This study analyzed gene expression profiling of inflammatory mediators (IL-1B, INF-γ,IL-6, TNF-α, IL-8 and IL-10) and two metalloproteases associated with damage of esophageal mucosa, according to literature, from patients with 3 different endoscopic phenotypes of the disease (BE, EE and NERD) and also compared those present in the non-erosive variety who were monitored for 24 hours of pH measures to differentiate cases of esophageal exposure to abnormal acid from normality.
Patients with BE showed an increased expression of IL-8, IL-10 and MMP-3; patients with EE had higher levels of IL-1B, IL-6 and IL-10. AAE stood out with higher levels of Il-1B, INF-γ, IL-6 , TNF-α and MMP3. AAE had a higher expression of IL-1B and TNF-α than NAE. A relevant difference in the groups with true pathological reflux and that do not present metaplasia (EE and AAE) is an increase expression in MMP3 and Il-10 in EE compared to AAE.
Even though, this is a descriptive study, the findings are of interest. As far as we know, is the first study to analyze all endoscopic phenotypes of patients with typical GERD symptoms and to analyze the non-erosive endoscopic phenotype with pH monitoring study and to characterize their genes according to this study.
The profile of mediators in BE showed an increased expression of IL-1B, IL-8 and IL-10. These results provide understanding of the pathogenesis of Barrett's esophagus and the possible immunoregulatory role of IL-10 associated with the permanent mucosal damage unable to counteract the aggression of other inflammation mediators.
This is in accordance with a previous reported in a mouse model of Barrett’s esophagus that showed an increased expression of IL -10, as compared to those in non-Barrett's esophagus while there were no differences in the levels of pro-inflammatory cytokines such as TNF-α or INF-γ [10] .
On the other hand, the expression of MMP3 and MMP9 were increased in BE compared to the control group and it is in the phenotype that these expressions predominates.
The primary function of matrix metalloproteinases (MMP) is in extracellular matrix (ECM) degradation and remodeling. They are secreted by T cells, neutrophils, keratinocytes, monocytes and macrophages.[11] It has been proposed that MMP-3 6A/5A polymorphism might also been involved in the presentation of patients with BE.[12]
Additionally, MMP-9 could be involved in the pathogenesis of BE and therefore of esophageal adenocarcinoma as well. We found this protease increased although our group of patients with BE did not show dysplasia. [13] Further studies are required to determine if metalloproteins play a role in mucosal damage in BE. Our study highlights the participation of MMP-9 and MMP-3 in patients with BE, possibly forming part of their pathological mechanism for this change of epithelium.
In relation to IL-8 measurements, we found an increased expression of this cytokine in patients with BE as compared to controls. Chronic inflammation in BE may play a critical role in the progression from benign to malignant esophageal disease[14]. The rate of progression from Barrett’s esophagus to esophageal adenocarcinoma is approximately 0.12– 0.4% per patient-year [15-17].
On the other hand, the erosive phenotype is a classic example of the balance that exists between the expression of Th1 and Th2 response, to the aggression presented (reflux).
In this study we showed an increased expression of pro inflammatory cytokines such as IL-1B and IL-6 in mucosal biopsies of patients with EE.
According to our results, Mönkemüller K, et al[19]; showed that IL-1B expression correlate with the histomorphological changes in esophageal mucosa of patients with Erosive and Non-Erosive Reflux Disease.
Previously, Rieder et al [20] demonstrated the increase production of IL-1B and IL-6 with EE compared with the controls and Fitzgerald et al[21] reported an inflammatory profile by increased production of IL-1B, IL-8 and INF-γ in esophageal epithelium and neutrophils of EE patients.
Blanchard et al[22] found an increased plasma cytokine levels of IL-8 in patients with EE and healthy subjects, differently to our results.
Interestingly, we found an increased gene expression of IL-10 in mucosa of patients with EE compared with controls, these results suggest the possible role of IL-10 as a critical cytokine for immunoregulatory mechanism in the inflammatory chronic response in the esophagus.
Similar to our results, there are reports in Asian populations that show association of IL-1B and IL-10 polymorphisms with an increased risk of erosive reflux esophagitis and gastritis[23].
Recent studies have provided greater insight into the pathophysiology and symptom generation in NERD[24].
In patients with non-erosive phenotype, this study allowed us to characterize patients with AAE and NAE. The AAE subgroup, being a real pathological reflux corroborated by the pH-monitorig study, allowed us to know that it has certain similarities with the EE group: increased expression of IL-1B and IL-6. These could be two markers in patients with real GERD who do not present metaplasia. AAE also presented unique differences within all groups, such as a significant increase in INF-γ and TNF-α compared with the control group. The expression of NAE cytokines were very similar to those of the control group. One finding to highlight the differences between AAE and NAE are the increased expression of IL-1B and TNF-α in AAE. In a subsequent study of diagnostic accuracy, it would be worth determining whether IL-1B / TNF-α could be biomarkers at the time of endoscopy by biopsies to define AAE / NAE for non-erosive endoscopic phenotypes.
In this study we were able to corroborate a similar expression profile between EE and AAE with increased expression of IL-1B and IL-6. But even these findings did not tell us why patients with pathological reflux can present erosions and others do not (despite the fact that the median of patients with % time with pH <4 was 13.65%, 3 times more than the point of cut). When comparing the expression of cytokines in these two groups, it was interesting that these differences lie in MMP3 and IL-10. It could indicate that these cytokines are involved in the mechanism of repair and damage to the mucosa. Further investigation is necessary to corroborate these findings.
As commented above, we found IL-8 mainly increased in BE. it is not part of the significant findings in EE or AAE. Previously, Kanazawa et al[25] in a Japanese study of patients with NERD with minimal mucosal involvement, determined by endoscopy , IL-8 mRNA levels were increased compared with NERD patients with no mucosal involvement and with controls, but in this study it is important to note the subgroup with NERD had not been studied by pH monitoring to truly corroborate this finding in patients who had normal acid exposure.
Interestingly, Kanasawa et al[25] detected an increase in the expression of IL-8 in NERD compared to asymptomatic subjects without pHmetry studies.[25] Yoshida[26] also found an increase the expression of this chemokine in patients with NERD.
It has also been demonstrated in patients with NERD, that exposure to GER in squamous cells increases the secretion of IL-8 and IL-1B causing an increase in the migration of T cells and neutrophils.[27]
Interestingly, we found a decreased expression of IL-10 in patients with AAE and NAE in compared with controls. IL-10 is a cytokine with potent anti-inflammatory properties that plays a central role in limiting host immune response, thereby preventing damage to the host and maintaining normal tissue homeostasis. Dysregulation of IL-10 is associated with enhanced immunopathology response to damage. Thus, a fundamental understanding of IL-10 gene expression is critical for our comprehension of disease progression and resolution of host inflammatory response.
This study showed the gene expression profiling of inflammatory mediators in the esophagus tissue from patients with different phenotypes of GERD.
Studying the molecular and genetic bases of a disease is of fundamental importance since it offers depth in its pathogenesis and opens new routes for diagnosis and specific treatment. Understanding of this pathway could lead us to the possibility of reversing the alteration of these genes and distinguish them from those that are involved in erosions or lesions from patients with GERD as compared to the NERD phenotype.