Our data indicate that fibroblast growth factor is among a set of factors that are differentially regulated in the periphery in EoE, suggesting that eosinophilic esophagitis is not only a local condition but also a systemic disorder that may be detected through analysis of plasma samples. Although symptoms of disease of may be localized at just the esophagus, our results show that EoE subjects have a set of unique peripheral immune indicators that could be used as diagnostic indicators. In addition, through analysis of PBMCs (which consist mainly of lymphocytes, monocytes, macrophages, and dendritic cells rather than granulocytes), we were able to detect an activation state present in other immune cells in EoE that could lead to subsequent activation of eosinophils. This would prove especially useful since current diagnosis involves an esophageal tissue biopsy, which carries potentially unnecessary risks.
The plasma fluid assay data provided here show that bFGF was upregulated in EoE subjects. Along with bFGF upregulation, an increase in eotaxin was also seen in EoE subjects. Interestingly, IL-5 was increased in the plasma of EoE subjects compared to that of GERD or HC. The subject with the highest IL-5 expression level did have the highest percent eosinophils in the blood (10.2%). Further work on the role of individual immune indicators vs. a composite biomarker tool using bFGF, eotaxin, and IL-5 simultaneously in blood samples from EoE vs. GERD vs. HC could be studied. Recent studies have also shown that IL-13 is overexpressed in the esophagus of EoE patients [20–22].
Recently, Mulder et al. found increased FGF-9 and FGF-Receptor expression levels in tissue from EoE subjects (n = 7) compared to HC (n = 7) and GERD (n = 7). However, there was no analysis of FGF expression in plasma or peripheral blood mononuclear cells. There was no evaluation of other FGF isoforms . Moreover, a possible link to the mechanism of EoE pathology was still to be identified.
The fibroblast growth factor (FGF) family consists of 22 members that are essential to normal cell development, cell differentiation, and tissue repair after injury. The receptors for FGFs (FGFR) are tyrosine kinase receptors that are specific for particular FGF subsets and can be found in a variety of tissues, including epithelial and mesenchymal tissue . bFGF increases the half-life of cells and could possibly increase the overall lifetime of the eosinophil in the esophagus . Our data showing that the antiapoptotic pathways are associated with EoE confirm this possibility. Eosinophils are also known to secrete bFGF  and FGF-9  upon the presence of necrotic epithelial cells. bFGF is also known to be secreted in case of cellular damage, such as the esophageal epithelial tissue damage experienced by EoE subjects . Both eotaxin and bFGF mediate their biological effects through the ERK pathway [18, 19]. Our data showed an increased expression of ERK in the PBMC of EoE subjects, and it is possible that, together with eotaxin, FGF enhances activation of eosinophils in EoE specifically.
The increased global expression of bFGF led to the investigation of the local expression bFGF of the esophageal biopsies of the subjects. Our data show that FGF, as assessed by IHC of local tissue and bFGF assessed by QT-PCR of PBMC, are upregulated in EoE. Initial immunohistochemistry antibody stains of FGF demonstrate an increase in local esophageal FGF expression levels in the lamina propria. FGF is diffusely spread over the lamina propria, close to the surface mucinous epithelium and covers mucosal glands as well. While healthy control and GERD subjects have very low expression of FGF, EoE subjects show higher FGF expression. It is important to note that bFGF and FGF-9 are highly homologous, sharing over 54% identical or similar amino acids (Protein BLAST). Since the FGF-9 antibody was made against the entire length of the amino acid, it is likely that homologous isoforms of FGF such as bFGF were also recognized. These data further suggested that FGF expression levels may help differentiate EoE from GERD, since EoE subjects have a significantly higher FGF expression level than GERD.
bFGF may be involved in the esophageal tissue fibrosis that is common to many EoE subjects. Past research has demonstrated that bFGF is a pro-fibrotic cytokine that may promote both fibrosis and angiogenesis by binding to the extracellular matrix [25, 26]. The release of FGF by a wide variety of cells, especially in conditions of cellular damage, may explain the high levels of systemic FGF in EoE subjects. Additionally, FGF is irreversibly bound to the extracellular matrix after its release, further amplifying its fibrotic capabilities . FGF may be further upregulated in the repair response after injury to the esophageal endothelium, leading to proliferation of fibroblasts and resulting fibrosis.
It has been shown that recurrent stricture formation and dysphagia can be associated with esophageal subepithelial fibrosis . Studies on animal models have shown that stricture can form in the epithelium repair process, leading to an overly dense extracellular matrix, an overproduction of fibroblasts, and the development of scar tissue. In comparison to a previous study showing that EoE subjects have an increase in local FGF production , our study demonstrates that there is increase in FGF production locally and this increase is also reflected systemically. In addition, the extent of increase in FGF and ERK pathways was closely associated with the severity of the disease and endoscopic findings in the EoE patients; further patients will help determine if FGF and other immune indicators are correlated closely with EoE disease. Interestingly, we did not detect a difference in FGF and eotaxin expression in atopic (allergic) vs. non atopic EoE patients. However, further studies are needed to seek to identify possible pathological differences associated with atopic vs. non atopic EoE.
bFGF expression is essential to the transcription of ERK  and ERK feeds into the eotaxin-3 pathway, potentially further activating the eosinophil and improving its sensitivity and migration towards eotaxin in the esophageal tissue. Activation of ERK may induce both degranulation and chemotaxis of eosinophils . It does appear that eosinophils also express bFGF . The eosinophils may be operating in a positive feedback loop in which the expression bFGF is encouraging the increased activation of the eosinophil, which then leads to more bFGF expression and better chemotaxis. Additionally, like bFGF, FGF-9 can activate both ERK1 and ERK2 .
Further analysis of more subjects is necessary to identify additional immune indicators that constitute a unique EoE plasma protein composite or individual marker profile. Lower esophageal eosinophilia is common in GERD, and further determination of numbers of eosinophils in the tissues of GERD subjects is needed; we believe our current results reflect a relative difference in GERD and EoE subjects. We have focused on EoE to determine the involvement of specific blood immune indicators in the disease; we will continue to study other eosinophilic gastroenterological disorders such as eosinophilic gastroenteritis and eosinophilic colitis to determine the role of FGF in those disease entities.
We have demonstrated that fibroblast growth factors may play an important role in the pathophysiology of EoE and may be part of a set of immune indicators that could, without biopsy, differentiate EoE subjects from subjects with other clinically similar symptoms such as GERD. In this effort to determine possible pathological mechanisms, we found that FGF increase was associated with activation of ERK and of anti-apoptotic, pathways which could enhance eotaxin signaling and increase eosinophil lifespan, respectively.