Mucosal pathogenesis in gastro‐esophageal reflux disease

Despite gastro‐esophageal reflux disease affecting up to 20% of Western populations, relatively little is known about the molecular mechanisms underlying its most troublesome symptom: heartburn. Recent findings have unveiled the role of components of the esophageal mucosa in the pathogenesis of GERD including sensory nociceptive nerves and inflammatory mediators. Erosive esophagitis was long believed to develop as a result of acid injury at the esophageal lumen, but novel concepts suggest the generation of reflux‐induced esophageal injury as a result of cytokine‐mediated inflammation. Moreover, the localization and characterization of mucosal afferent nerves vary between GERD phenotypes and could explain the heterogeneity of symptom perception between patients who experience similar levels of acid reflux.

heartburn patients experience GERD symptoms despite having no association with reflux events, while many patients with Barrett's esophagus often do not present with heartburn despite having had years of pathological levels of acid reflux. Finally, although acid suppression with proton pump inhibitors (PPIs) is effective for many, over 30% of patients respond inadequately. 2,4,5 The components of the gastro-esophageal refluxate can vary, and systemic factors (e.g., psychological stress) will influence symptoms perception. However, a significant contribution to the pathogenesis of GERD and potentially the variability in disease expression may be explained at the mucosal level. This article discusses the current understanding of the mucosal pathogenesis of GERD.

| MUCOSAL PATHOG ENE S IS
Esophageal symptoms and/or injury develops when the contents of the gastro-esophageal refluxate interface with the mucosal epithelium, leading to a chain of events that can result in epithelial barrier disruption, activation of afferent nociceptive nerves, and inflammation. Although acid is believed to be the primary aggressor within the refluxate, other components including bile acids (from duodenogastro-esophageal reflux) and pepsin have also been implicated.
Our understanding of how these aggressive constituents lead to symptoms and injury in GERD has evolved over recent years, perhaps moving from a more simplistic view of barrier disruption and mucosal permeability to acid toward a more complex view, integrating this permeability change with epithelial cell and neuronal activation alongside cytokine-driven mucosal injury.
Here we look at the different aspects of esophageal mucosal pathogenesis of GERD and consider how the factors may interact according to our latest understanding.

| The epithelial barrier in reflux disease
Under normal circumstances, the threat imposed by noxious refluxate is met by mucosal defense mechanisms including local homeostatic repair induced by acid-sensing epithelial cells and neurons. 6 The stratified squamous epithelium of the esophagus itself provides a tight protective barrier against luminal contents. 7 Junctional complexes between esophageal epithelial cell membranes are composed of tight junctions, adherens junctions, and desmosomes which form a barrier against the diffusion of ions. 8 Where macroscopic erosions are visible (i.e., EE) there is clear evidence of mucosal barrier deficiency. However, macroscopic erosions occur in a minority of patients with GERD. Nevertheless, it is apparent that there is evidence of mucosal barrier deficiency even in NERD, and it is likely that this plays a role in symptom pathogenesis. 9 Seen under white light, but even more apparent under electron microscopy, epithelial dilated intercellular spaces (DIS) are seen in patients with NERD. 10 As the term suggests, increased distance between esophageal mucosal epithelial cells is seen in DIS.
Theoretically, DIS is a morphological representation of an impaired epithelial barrier whereby the increased space between neighboring epithelial cells enables noxious refluxate contents including H + to access nerve endings more readily and stimulate acid-sensitive nociceptors. 11 Experimental data demonstrate that DIS can be induced by acid exposure 12 and DIS in GORD is resolved by therapy with proton pump inhibitors. 13 The morphological findings of DIS are reinforced by functional studies of epithelial permeability. Measurements of transepithelial resistance (TER) allow dynamic monitoring of changes in epithelial permeability over time. When human esophageal biopsies are placed in Ussing chambers and exposed to acidic and weakly acidic solutions a reduction in TER occurs (suggesting that there is increased paracellular permeability within the biopsy specimen, as would be expected in the presence of DIS). Furthermore, these studies suggested that biopsies from patients with GERD had a greater reduction in TER upon acid exposure than biopsies from control subjects, perhaps suggesting an inherent vulnerability to barrier dysfunction in GERD. 14 However, the presence of transient DIS alone is unlikely sufficient for symptoms (perfusion-induced DIS in healthy volunteers does not induce symptoms. 12 Symptom generation is likely to require a long-lasting DIS, allowing prolonged epithelial exposure to noxious stimuli.
Functional changes in esophageal mucosal permeability can be demonstrated in vivo using intraluminal impedance techniques.
While impedance is used clinically in gastro-esophageal reflux measurements, in the collapsed esophagus the impedance electrodes lie in contact with the mucosa and are able to measure impedance to current flow within the mucosa (i.e., a low impedance suggests an easier ionic passage and thus increased mucosal permeability). 15 Indeed, baseline mucosal impedance is significantly lower in patients with GERD compared to controls, and the value of the impedance correlates inversely with the severity of 24-hour esophageal acid exposure. 16,17 Reinforcing the concept that increased epithelial permeability may contribute to symptom pathogenesis, low esophageal impedance is associated with increased

Key Points
• As the first line of defence against noxious gastric contents, the esophageal mucosa has a key role in disease and symptom pathogenesis in gastroesophageal reflux disease.
• Even in non-erosive reflux disease, the esophageal mucosal barrier is defective when compared to healthy controls.
• Symptoms occur after stimulation of afferent nerves, which can often be found in the mucosa.
• Mucosal inflammation is important in disease and symptom generation, and occurs due to a "cytokine sizzle" rather than due to direct caustic injury. sensitivity to perception of infused acid. 17 These results collectively suggest that excessive acid reflux leads to an increase in epithelial permeability as evidenced by DIS, TER, and low mucosal impedance, and this may allow noxious luminal contents an easier passage to activate submucosal nerve endings and lead to heartburn symptom generation. 11,18,19 Animal experiments have suggested that it is not just reflux exposure that may drive changes of epithelial permeability in GERD.
Exposure of rats to acute stress was found to induce DIS and increase esophageal mucosal permeability to small molecules. 20 In the mouse colon, exposure to acute stress altered expression of tight junction proteins, ZO-2, and occludin, while in the skin, acute stress stimulated mast cell degranulation and altered barrier function. 21,22 The role of mast cells in stress-induced permeability has also been suggested in the esophagus, where the stress response mediator corticotrophin-releasing hormone receptor subtype 2 expression was identified in the rat esophageal mucosa. 23 Moreover, many GERD patients report a higher symptom burden with increased stress, 24,25 and these findings suggest that the mechanism may involve not only increased central sensitization, but peripheral sensitization driven by permeability changes at the level of the esophageal mucosa. 26,27 The molecular events that account for defects in barrier function involve changes in the structure and function of adhesion its subsequent cleavage of the adherens junction protein e-cadherin have been identified as major contributors of a disturbed junctional barrier in the esophageal epithelium. 28 In line with the association between enhanced mucosal permeability and GERD pathophysiology, in vitro and animal studies have also described the dysregulation of tight junction molecules. [29][30][31] A rat model of EE identified interleukin-6 as a mediator for the downregulation of desmosomes, the onset of DIS, and subsequently defective cell-cell contacts. 31 Moreover, distinct patterns of localization and expression of tight junction proteins (Occludin, Claudin-1-4) have been described. While Claudin-3 expression decreases, Claudin-1 and Claudin-2 become upregulated in EE only. 29,32 In addition, acid exposure reduced the expression of tight junction protein 1 (ZO-1) by human esophageal epithelial cells, whereas incubation with epithelial growth factor reversed this effect and inhibited barrier function impairment. 33

| MUCOSAL RE S P ON S E IN G ERD
For years, the pathogenesis of esophageal mucosal inflammation in GERD was thought to proceed in a "top-down" fashion, with acidpeptic injury starting at the luminal surface. 34 According to this traditional acid burn model of EE, the process begins with refluxed acid and pepsin damaging proteins in the tight and adherens junctional complexes between esophageal squamous cells, thus enabling hydrogen ions (i.e., acid) to penetrate the epithelium and kill the cells at the luminal surface. The death of surface epithelial cells is assumed to trigger an acute inflammatory response with the infiltration of granulocytes (neutrophils and eosinophils) into the epithelium and to induce a proliferative response in esophageal basal cells attempting to replace the damaged surface cells. The characteristic GERD histologic abnormalities of basal cell hyperplasia and elongation of the squamous papillae (projections of lamina propria with capillary vessels) were believed to be manifestations of this reflux injury-induced proliferative response. 11,35 In addition to enabling the entry of hydrogen ions, the increased epithelial permeability caused by acid-peptic damage to tight and adherens junctional complexes also allows chloride ions to diffuse into the intercellular spaces. These This finding suggests that reflux-induced esophageal inflammation increases vascular permeability, and that fluid leakage from inflammation-damaged blood vessels contributes to DIS formation. 48 Furthermore, blood vessels are located in the papillae and significant papillary hyperplasia was observed by 2 weeks off PPI therapy. Thus, vascular fluid leakage could explain why DIS are most frequently observed in the basal and suprabasal epithelial layers adjacent to hyperplastic papillae during mucosal inflammation.

| Molecular basis of GORD-induced cytokine production
The molecular pathways linking reflux esophagitis to pro-inflammatory cytokine production involve the generation of reactive oxygen species (ROS), which stabilize and activate hypoxia-inducible factor (HIF)-2α. HIFs are heterodimeric transcription factors comprised of oxygen-regulated HIF-α subunits and a constitutively-expressed HIF-1β subunit. 51 Hypoxia and ROS can stabilize HIFs, enabling them to accumulate in the cytoplasm and translocate to the nucleus where they induce transcription of their target genes, which include a number of pro-inflammatory cytokines such as IL-8 and IL-1β. 52 Cultures of human esophageal squamous cells exposed to acidic bile salts in vitro produce ROS, stabilize HIF-2α, and secrete pro-inflammatory cytokines that trigger T-lymphocyte migration. 53,54 Moreover, all of these acidic bile salt-induced effects can be blocked by inhibition of HIF-2α either through genetic knockdown or by administration of a highly selective small molecule pharmacologic inhibitor. 54 Acidic (an NFκ-B subunit), as well as increases in mRNA for a number of pro-inflammatory cytokines including IL-8, IL-1β, TNF-α, and cyclooxygenase-2. 54 These biopsy specimens also demonstrated associations between levels of HIF-2α and levels of mRNA for these pro-inflammatory mediators, and between HIF-2α and phospho-p65, observations supporting the hypothesis that reflux esophagitis is initiated through a reflux-induced, cytokine-mediated process in which HIF-2α plays a central role.

| ACID -S EN S ING RECEP TOR S AND MUCOSAL INNERVATI ON
After esophageal epithelial exposure to acid, induction of inflammation and symptoms is likely, at least in part, to be transduced via acidsensitive receptors expressed within the mucosa. Transient receptor potential vanilloid type-1 (TRPV1) is a non-selective Ca 2+ -permeant channel that has been widely proposed to be a candidate sensory transductor of reflux-induced symptoms since it can be activated by acid and is often involved in pain pathways. 26,[55][56][57] Neutrophils and other immune cells can also release protons from their exocytic granules and lysosomes into the microenvironment 58-60 and this inflammation-induced microenvironment acidification conceivably could activate TRPV1 even in the absence of reflux episodes. [61][62][63] TRPV1 expression has been demonstrated to be increased in biopsies taken from GERD patients compared to controls. 64 Acidactivation of TRPV1 in cat esophageal mucosa has been shown to result in release of substance P and CGRP, strongly implicating a nociceptive role. 65 Similarly, TRPV1 activation in cultured human esophageal epithelial cells has been shown to result in the release of adenosine triphosphate (ATP), a neurotransmitter involved in pain signaling and inflammation. 66 Moreover, a recent in vivo study demonstrated reduced acid-induced damage to the mucosal integrity of murine esophageal epithelium upon pharmacological blockade of TRPV1, suggesting a potential role for TRPV1 in mucosal barrier impairment in NERD. 67 The protease-activated receptor 2 (PAR2): a receptor for trypsin, is reportedly upregulated in the esophageal mucosa of GORD patients, coupled with the expression of inflammatory mediators such as IL-8. 68,69 The acid-induced activation of PAR2 has been shown to enhance ATP release from cultured esophageal epithelial cells, to increase TRPV1 phosphorylation, and to lead to heightened sensitivity to acid. 66 Other TRP channels may also be candidates for sensory trans- Acid-sensing ion channels (ASICs) are members of the voltage-insensitive, amiloride-sensitive degenerin family of cation channels 72 .
As their name suggests they can be activated by protons, and thus are candidates for a role in GERD pathogenesis. While ASIC1, ASIC2, and ASIC3 have been shown to be expressed in the rat esophageal mucosa, ASIC3 alone is expressed in the human esophageal epithelium. 73,74 The importance of ASICs in nociception and inflammation is highlighted in transgenic animal studies which show changes in response to mucosal acid exposure, and their upregulation during gastrointestinal inflammation regulated by nerve growth factor and serotonin which directly interact with the ASIC3 gene promoter region and induce its transcription in peripheral sensory neurons. [75][76][77] In cultured human esophageal epithelial cells, the potentiating effect of trypsin on PAR2 activation was found to enhance weak acid-induced ATP release from esophageal epithelial cells through both TRPV1 and ASIC3. While the sensitization mechanisms of ASIC3 in esophageal epithelial cells are not well known, current studies suggest that the generation of heartburn perception and esophageal hypersensitivity is likely to involve the interaction of PAR2, TRPV1, and ASICs. 66 After gastric contents have injured or inflamed the esophageal mucosa, the ultimate pathway by which heartburn will be felt is via activation of nociceptive nerves by the receptors discussed above.
The distribution and characterization of mucosal sensory nerves may play an important role in reflux hypersensitivity in NERD, and hyposensitivity in Barrett's esophagus. In asymptomatic human subjects, a network of intra-mucosal nerves expressing calcitonin gene-related protein (CGRP) can be seen throughout the esophagus. In usual circumstances, these nerves lie deep in the esophageal mucosa in the distal esophagus, but lie very close to the luminal surface in the proximal esophagus (likely explaining the relative hypersensitivity of the proximal esophagus, in turn likely a protective feature to defend against aspiration). 78,79 Interestingly, it has recently been shown that in NERD, but not erosive esophagitis, Barrett's esophagus, or functional heartburn, the mucosal nerves lie close to the lumen in the distal esophagus of patients with non-erosive reflux. 80 Recently these superficial mucosal nerves in NERD have been shown to strongly express TRPV1, suggesting that an acid-transduced nociceptive role of these nerves in heartburn perception is highly likely. 81 Intriguingly these findings also suggest that DIS may play less of a causative role than was previously thought, since deep penetration of acid would not be required to activate these nerves. Deep nerves in EE, however, do not express TRPV1 and may express inflammatory receptors such as bradykinin receptors as in the colon and become activated by inflammatory mediators. 82 (Figure 1). 85 Release of inflammatory mediators such as IL-8, IL-1β, peripheral prostaglandin E2, and TNF-α is likely to directly activate or heighten the sensitization of peripheral afferent nerves 85,86 as seen in animal studies of colonic tissue where previously silent visceral afferent nerves are activated by chemical and inflammatory mediators, and lead to continuous neuronal firing. 87,88 Growth and location of nerves may also be regulated by inflammation, such as in the colon where tryptase-positive mast cells release nerve growth factor during mucosal inflammation. 89 Acid may upregulate both the expression and activation of PAR-2 in esophageal squamous cells. 68 Activation of PAR-2 might increase permeability of the esophageal epithelium by causing the redistribution of tight junction proteins. 68 Generation of inflammation within the mucosa is likely to cause in- This paper demonstrates that much progress has been made in the understanding of the mucosal changes that develop after the occurrence of excessive gastro-esophageal reflux. It also illustrates that more research is needed to fully understand the role and nature of the interaction between the gastro-esophageal refluxate and the mucosal epithelium, afferent nerves, and inflammatory pathways. Such research includes studies to understand the receptors by which the epithelial cells and nerves interact with the refluxate, to understand the mechanisms for afferent nerve migration and location, and to understand how inflammation interacts with nerve activation. As we further study the mucosal pathogenesis, we can hope to identify targets for treatment (likely topical therapies) to help a significant number of GORD patients including those with refractory symptoms.

D I SCLOS U R E
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
AU and AN developed and wrote the scope of the review, DS and SS contributed to the revision of the manuscript, and RS and PW performed extensive manuscript revision. All authors reviewed the final version of the manuscript.

Daniel Sifrim
https://orcid.org/0000-0002-4894-0523 Philip Woodland https://orcid.org/0000-0003-2445-9724 F I G U R E 1 A proposed integrated mucosal pathogenesis of GERD: Esophageal epithelial cells secrete pro-inflammatory cytokines such as IL-8 upon exposure to noxious substances in the luminal refluxate, triggering immune cell infiltration into the esophageal tissue beginning in the submucosa and progressing into the esophageal epithelium. Sensory nerve endings innervating the esophageal mucosa express acid-sensing ion channels can be activated by the acidic microenvironment created by cytokine-induced inflammation, while deep nerves innervating the papillae can become directly activated by pro-inflammatory cytokines. Upon activation, nociceptive nerves can release CGRP and cause pain even in the absence of macroscopic injury