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Eosinophilic esophagitis (EE) involves marked accumulation of eosinophils in the esophageal mucosa that responds to swallowed fluticasone propionate (FP) in a subset of patients.
We aimed to uncover the mechanism of action of swallowed FP in patients with EE by providing evidence for a topical effect in the esophagus by identifying a molecular signature for FP exposure in vivo.
Global microarray expression profiles, immunofluorescence microscopy, and cell signaling in esophageal tissue and cell lines were analyzed.
Thirty-two transcripts exhibited altered expression in patients who responded to swallowed FP treatment. Esophageal FK506-binding protein 5 (FKBP51) mRNA levels were increased (P < .05) in FP responders compared with those seen in control subjects and patients with untreated active EE. After FP treatment of esophageal epithelial cells, FKBP51 mRNA and protein levels were increased in a dose- and time-dependent manner by FP treatment in vitro. FP-induced FKBP51 was steroid receptor dependent because RU486 completely inhibited gene and protein induction. The half-life of FKBP51 mRNA was 16 to 18 hours independent of FP treatment. FKBP51 overexpression reduced FP action as assessed by FP inhibition of IL-13–induced eotaxin-3 promoter activity.
Our results suggest that swallowed glucocorticoid treatment directly affects esophageal gene expression in patients with EE. In particular, increased FKBP51 transcript levels identify glucocorticoid exposure in vivo and distinguish FP responders from untreated patients with active EE and patients without EE. In addition, FKBP51 reduces glucocorticoid-mediated inhibition of IL-13 signaling in epithelial cells in vitro, suggesting that FKBP51 might influence FP responsiveness. We propose that esophageal FKBP51 levels have diagnostic and prognostic significance in patients with EE. (J Allergy Clin Immunol 2010;125:879-88.)
Eosinophilic esophagitis (EE) is characterized by an increased accumulation of eosinophils in the esophageal mucosa that is refractory to acid-suppressive therapy.1 Evidence suggests that the disease strongly associates with atopy and an antigen-driven, TH2-type immune response.2 EE predominantly affects male subjects in a ratio of approximately 3:1 (male/female).3,4 This chronic disease affects pediatric populations because age of onset can be as early as during the first year of life.3 Treatment strategies for EE include dietary restriction of food allergens or systemic glucocorticoid therapy. In addition, a subset of patients respond to swallowed glucocorticoid therapy delivered by means of asthma inhalation medications. The mechanism of action of swallowed glucocorticoids has not been examined, but the observation that patients with EE receiving inhaled corticosteroid therapy benefit from swallowing the same medication provides evidence for a local effect, although other reasons for the improvement exist (eg, increased systemic levels or delivery to the oral or gastric mucosa). Although swallowed or systemic glucocorticoids represent an effective therapy, their action is not sustained on discontinuation of the medicine.5 Additionally, a recent controlled clinical trial has reported that only 50% of patients with EE treated with swallowed fluticasone propionate (FP) respond to treatment compared with 9% in the placebo group.6 Determining the mechanism of action of swallowed corticosteroid therapy is likely to help improve efficacy, develop better drug formulation strategies, and identify mechanisms of steroid resistance, a common problem seen in patients with EE, as well as other allergic inflammatory disorders.7
Glucocorticoid treatment induces an anti-inflammatory effect by targeting various cell types involved in both the innate and adaptive immune responses. In the respiratory tract the action of glucocorticoids is mediated through effects on epithelial cells, at least in part.8,9 Several lines of evidence suggest that esophageal epithelial cells (keratinocytes) comprise a key cellular component involved in the pathogenesis of EE. Notably, on exposure to IL-13, a cytokine overproduced in patients with EE, esophageal epithelial cells express a transcriptome that markedly overlaps the EE transcriptome,10 a set of genes differentially expressed between biopsy specimens from control subjects and patients with EE.11 The gene exhibiting the highest increase in patients with EE and in IL-13–treated esophageal epithelial cells is eotaxin-3, an eosinophil chemoattractant and activator. It has therefore been proposed that IL-13 signaling in esophageal epithelial cells contributes to the pathogenesis of EE, particularly through induction of eotaxin-3. Global transcript profiling shows that 98% of the EE transcriptome is reversed to normal levels in patients who respond to FP.10 Given the importance of esophageal epithelial cells in patients with EE, these cells can serve as relevant targets of glucocorticoid treatment, particularly swallowed FP.
Glucocorticoids exert their action through a variety of mechanisms, including transcriptional inhibition of specific promoter response elements, destabilization of cytokine mRNA, and direct induction of cellular apoptosis. The action of glucocorticoids varies among cell types, and few studies exist12–15 that have examined the action of glucocorticoids on the esophagus in vivo or on esophageal cell lines. Accordingly, in this study we aimed to identify targets of swallowed FP treatment in the esophagus of patients with EE to further understand the molecular mechanisms underlying patient response to this drug. Using global expression profile analysis of esophageal tissue, we identified a panel of 32 transcripts altered by FP treatment in vivo in responders compared with untreated control patients and patients with active EE. Of these transcripts, only 1 gene exhibited levels that were differentially regulated in both responders and nonresponders; the remainder exhibited altered regulation only in responders and not in nonresponders. This suggested that nonresponders might exhibit a fundamental dysfunction in the glucocorticoid signaling pathway in their esophagus. Notably, levels of mRNA for FKBP51, a known steroid-induced gene in respiratory epithelial cells and lymphocytes,16–19 were increased in FP responders. FKBP51 was directly induced by glucocorticoids in esophageal epithelial cells, and we developed evidence that it acts as a negative regulator of FP action. We propose that swallowed FP therapy acts topically and mediates its effects by directly regulating gene expression in esophageal epithelial cells. Furthermore, we suggest that levels of esophageal transcripts, in particular FKBP51, serve as in vivo signatures for steroid exposure.
Patients included in the study include those recruited from either Cincinnati Children’s Hospital Medical Center (50 patients) or Children’s Hospital, San Diego (1 patient). Patients ranged in age from 1.5 to 22.4 years (mean age, 8.9 ± 4.8 years). The population included 38 male and 13 female subjects. Samples were divided into groups according to the following characteristics: control patients (n = 14)—no history of EE, 0 eosinophils per high-powered field (hpf) in the esophagus at the time of biopsy, and no concomitant swallowed glucocorticoid treatment; patients with EE (n = 14)—24 or more eosinophils/hpf at the time of biopsy and no concomitant swallowed glucocorticoid treatment); patients with EE who responded to FP treatment (n = 13)—history of EE, 0 to 1 eosinophils/hpf at the time of biopsy, and concomitant swallowed fluticasone treatment; and patients with EE who did not respond fully to FP treatment (n = 8)—history of EE, 6 or more eosinophils/hpf at the time of biopsy, and concomitant swallowed FP treatment (see Table E1 in this article’s Online Repository at www.jacionline.org). It was noted that no significant difference existed in the average eosinophil count of untreated biopsy samples between FP responders and nonresponders (see Figure E1 and Table E2 in this article’s Online Repository at www.jacionline.org).
Patients taking swallowed FP were generally instructed to use an inhaler without a spacer, spray the medication into the pharynx, and not eat, drink, or rinse for 30 minutes after administration. Doses of swallowed FP are indicated for individual patients; when available, the time of the last dose of FP before endoscopy is listed (see Table E3 in this article’s Online Repository at www.jacionline.org). Biopsy specimens were collected from the distal esophagus on routine endoscopy and submerged in RNAlater (for RNA isolation), modified F-media (for culture), or 4% paraformaldehyde (for immunofluorescence studies). This study was approved by the Institutional Review Board of the Cincinnati Children’s Hospital Medical Center.
RNA extraction and microarray analysis were performed as previously described.11 To identify glucocorticoid-regulated genes, those transcripts differentially regulated between control subjects (n = 14) and patients with EE (n = 14) were identified by using the t test (P < .01). Genes differentially regulated between control subjects and FP responders (n = 13) were then identified by using a 2-fold change filter. Subsequently, the genes differentially regulated between control subjects and patients with EE were subtracted from those differentially regulated between control subjects and FP responders. These transcripts were then subjected to ANOVA (P < .01). Although not involved in the initial analysis, transcript values were also included for FP nonresponders (n = 8).
Real-time PCR was performed with the IQ5 system (Bio-Rad Laboratories, Hercules, Calif). Reactions were carried out with SYBR green mix (Bio-Rad Laboratories). The value obtained for each primer set was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) value for the corresponding sample. Primer sequences are listed in Table E4 (available in this article’s Online Repository at www.jacionline.org).
Primary esophageal epithelial cells were generally cultured as previously described.10 Detailed methods of culture and treatment can be found in the Methods section in this article’s Online Repository at www.jacionline.org. This study was approved by the Institutional Review Board of the Cincinnati Children’s Hospital Medical Center.
The human esophageal cell line TE-7 was kindly provided by Dr Hainault (IARC, Lyon, France). These cells were maintained in RPMI medium (Invitrogen, Carlsbad, Calif) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, Ga) and 1% penicillin/streptomycin (Invitrogen). Details regarding cell treatment and transfection can be found in the Methods section in this article’s Online Repository.
RNA was isolated from biopsy specimens as previously described11 by using the RNeasy kit (Qiagen, Hilden, Germany) per the manufacturer’s instructions. RNA was isolated from cells with Trizol (Invitrogen) according to the manufacturer’s instructions, except in actinomycin experiments, in which RNA was isolated with the RNeasy kit (Qiagen). RNA samples (100–1000 ng) were subjected to reverse transcription with Superscript II Reverse Transcriptase (Invitrogen) per the manufacturer’s protocol.
TE-7 or primary esophageal epithelial cells were washed with PBS and incubated with MPER lysis buffer (Thermo Fisher Scientific, Rockford, Ill) supplemented with aprotinin (10 µg/mL), leupeptin (10 µg/mL), pepstatin (10 µg/mL), and sodium orthovanadate (1 mmol/L). Total protein (5–10 mg) was loaded onto 4–12% NuPage Tris-bis gels (Invitrogen), electrophoresed for 1.5 hours at 150 V, and transferred to nitrocellulose membranes, followed by Western blot analysis. Primary antibodies were diluted in TBS/0.1% Tween 20 containing 5% milk: goat anti-FKBP51 (1:1,000; R&D Systems, Minneapolis, Minn) and murine anti–β-actin (1:5,000; Sigma-Aldrich, St Louis, Mo). Secondary antibodies were incubated with the membranes: anti-goat horseradish peroxidase (1:10,000; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) and anti-mouse horseradish peroxidase (1:10,000; Cell Signaling Technology, Inc, Danvers, Mass). Blots were developed with ECL Plus reagent (GE Healthcare, Piscataway, NJ). Densitometric measurements were performed with Multi Gauge V3.0 (Fujifilm, Tokyo, Japan).
Biopsy samples were collected as described above. After incubation in 4% paraformaldehyde, biopsy specimens were submerged in 30% sucrose and then embedded in OCT compound. Frozen sections cut from OCT compound–embedded esophageal biopsy samples were mounted on slides. Sections were fixed in ice-cold acetone for 10 minutes, incubated in blocking buffer (PBS, 1% saponin, and 3% FBS), and then incubated with either primary antibody or an equal concentration of control antibody in blocking buffer: anti-FKBP51, 10 µg/mL (R&D Systems, Inc); control, normal goat IgG (R&D Systems, Inc). Sections were incubated with Alexa 594–conjugated secondary antibody (1:250, Invitrogen). Sections were washed 3 times with PBS after each antibody incubation. Fluromount G containing 4′-6-diamidino-2-phenylindole, dihydrochloride was used for mounting. Sections were visualized with the BX51 microscope and MagnaFire imaging software (Olympus America, Inc, Center Valley, Pa).
pHRL-TK, pGL3-Basic, and pCDNA3.1 were obtained from Promega (Madison, Wis). Construction of pEotaxin-3 has been described.20 The pEotaxin-3 3′ untranslated region (UTR) consists of coordinates 394 to 562 of NM_006072.4 (GenBank) in the XbaI/SalI sites of the pGL3-Promoter. pFKBP51 was constructed by inserting the FKBP51 open reading frame (coordinates 154 to 1535 ofNM_004117.2,GenBank) into the pCDNA3.1 polylinker.
Data are expressed as means ± SEMs. Statistical significance was determined by using the Student t test (normal distribution, equal variance), 1-way ANOVA followed by the Tukey post test (>2 groups), or the Kruskal-Wallis test followed by a Dunn multiple comparison test (nonparametric test, >2 groups) with Prism 5.0 Software.
We performed genome-wide expression analysis of esophageal biopsy samples derived from 4 distinct patient populations, including control patients (n = 14), untreated patients with EE (n = 14), patients with EE who responded to FP treatment (n = 13), and patients with EE who did not respond fully to FP treatment (n = 8; see Table E1). We aimed to identify the subset of genes regulated exclusively by FP treatment and not by EE disease. More specifically, we sought to identify genes differentially regulated between untreated control subjects and FP responders, excluding genes that were dysregulated in untreated patients with EE. Accordingly, genes exhibiting altered expression between control subjects and untreated patients with EE were subtracted from the subset of genes that were differentially expressed between control subjects and FP responders (Fig 1, A). Thirty-two transcripts exhibited differential expression in FP responders compared with control subjects but were not included in the genes differentially expressed in untreated patients with EE at baseline (Fig 1, B, and see Table E5).
Eight transcripts represented 6 genes that exhibited increased expression in responders but not in nonresponders (Fig 1, C). Of the 17 transcripts (representing 15 genes) with decreased levels in responders, all showed levels in nonresponders that were similar to those seen in control subjects (Fig 1, D). Notably, 4 MHC class II genes were identified, as well as 3 genes of the collagen family. Six transcripts identified in the original analysis (Fig 1, B) were not included in either Fig 1, C or D, because they exhibited a change in expression in control subjects versus that seen in untreated patients with EE despite the analysis scheme devised to eliminate such transcripts. Of the subset of transcripts with increased levels in responders, 1 gene (F3, encoding for coagulation factor III or tissue factor) was identified that exhibited increased expression in both responders and nonresponders (Fig 1, C). Taken together, these results indicate that nonresponders have markedly blunted steroid-induced signaling responses in the esophagus because only 1 of 32 of the genes exhibited differential regulation in both responders and nonresponders.
We next validated the expression pattern of 3 of the identified candidate genes using real-time PCR analysis. Genes were selected based on their known biology. FK506-binding protein 5 (FKBP51), which has a known role in glucocorticoid receptor biology; keratin 7 (KRT7), which promotes epithelial cell integrity and protects against physical damage; and H19, which is processed to a microRNA, showed increased transcript levels in FP responders compared with untreated patients by means of both microarray (Fig 2, A) and real-time PCR (Fig 2, B) analysis. On average, FKBP51 mRNA levels were 2-fold higher in FP responders compared with those seen in control subjects, whereas nonresponders exhibited levels of this transcript that were not significantly higher than those seen in control subjects (Fig 2, A).
We delineated the cell type or types responsible for FKBP51 expression in patients’ biopsy samples. Esophageal biopsy specimens were immunostained with antibody specific for FKBP51, and nuclei were stained with 4′-6-diamidino-2-phenylindole, dihydrochloride. In biopsy specimens obtained from untreated patients with EE, the basal layer of the esophageal tissue exhibited the strongest signal (Fig 3, A). The signal appeared most intense in the nuclei, although some cytoplasmic staining was apparent. Weaker cytoplasmic staining was also observed in the cells near the luminal side of the biopsy specimen (Fig 3, A). When compared with the hematoxylin and eosin–stained biopsy specimen (data not shown), the expression pattern of FKBP51 in patients’ biopsy specimens was consistent with its being present in epithelial cells. Staining with control antibody confirmed that the observed signal was specific for the FKBP51 antibody (Fig 3, B).
To determine whether FP directly induced FKBP51 in esophageal epithelial cells, we treated primary esophageal epithelial cells with FP and observed an increase (2.15 ± 0.4–fold) in FKBP51 protein expression after FP treatment (Fig 4, A). In addition, FP also induced FKBP51 mRNA and protein in the esophageal epithelial cell line TE-7 (Fig 4, B–D). Transcript levels of FKBP51 were increased by 4 hours after treatment and were further increased after 8 hours, after which a plateau was observed (Fig 4, C). Similarly, FKBP51 protein levels in TE-7 cells increased in a time-dependent manner, with maximum levels of protein observed by 24 hours after treatment (Fig 4, D). The induction of FKBP51 by FP was not specific for this steroid because FKBP51 mRNA (Fig 5, A) and protein (Fig 5, B and C) levels increased after treatment with dexamethasone. FKBP51 protein levels were similarly increased in primary esophageal epithelial cells after dexamethasone treatment (Fig 5, D).
Cells were pretreated with the glucocorticoid receptor antagonist RU486 followed by FP or dexamethasone treatment. Western blot analysis showed that RU486 treatment abolished the FP- and dexamethasone-mediated increase in FKBP51 protein levels. Dexamethasone required an equimolar amount of RU486 for the inhibition, whereas FP-mediated induction of FKBP51 protein levels was inhibited by a 100-fold molar excess of RU486 (Fig 4, E). As such, FKBP51 induction is mediated through a glucocorticoid receptor ligand binding–dependent process.
TE-7 cells were treated with FP for 24 hours, followed by addition of actinomycin D to inhibit de novo transcription, to determine whether glucocorticoid treatment increased the stability of FKBP51 mRNA. At the indicated time points after initiation of actinomycin D treatment, FKBP51 mRNA levels were monitored. The half-life of FKBP51 mRNA in the absence and presence of FP treatment was similar (17.84 ± 2.03 hours and 16.44 ± 1.11 hours, respectively; P = .35; Fig 4, F). We next tested whether de novo protein synthesis was required for the increased transcript levels of FKBP51. TE-7 cells were pretreated with cycloheximide (CHX) at a dose (10 µg/mL) that inhibited protein synthesis in these cells more than 95%, as measured based on [35S]-methionine incorporation into protein (data not shown) followed by addition of vehicle or FP (10−6 mol/L) for 24 hours. Although an increase of FKBP51 transcripts was observed at baseline after CHX treatment, FKBP51 mRNA levels were even further increased in FP-treated cells in which protein synthesis was inhibited (Fig 4, G), suggesting that de novo protein synthesis was not absolutely required for subsequent FP-mediated induction of FKBP51 gene expression.
IL-13 signaling in esophageal epithelial cells has been shown to have a critical role in EE pathogenesis by inducing the EE transcriptome, at least in part.10 When TE-7 cells were treated with IL-13, an increase in eotaxin-3 transcript levels was observed (Fig 6, A). When the cells were treated concomitantly with FP, the IL-13–mediated increase in eotaxin-3 mRNA and protein decreased significantly at all doses tested (Fig 6, A and B).
To understand the contribution of the promoter and 3′ UTR sequences of eotaxin-3 to the control of its gene expression in esophageal epithelial cells after IL-13 and FP treatment, we performed luciferase assays with TE-7 cells transiently transfected with constructs containing the eotaxin-3 5′ UTR upstream or the 3′ UTR downstream of a luciferase expression cassette. The cells were transfected with a luciferase reporter construct containing either 800 bp of the eotaxin-3 promoter 5′ of the firefly luciferase gene in the pGL3-Basic vector or a plasmid containing 164 bp of the eotaxin-3 3′ UTR downstream of the firefly luciferase gene in the pGL3-Promoter vector (Fig 7, A).10,11 Experiments with the promoter construct demonstrated a decrease in IL-13–induced eotaxin-3 promoter activity on concomitant FP treatment (Fig 7, B). Experiments with the 3′ UTR construct did not reveal significant modulation of mRNA stability on IL-13 or FP treatment (Fig 7, C). Taken together, these findings indicate that at least part of the observed decrease in IL-13–induced eotaxin-3 mRNA levels after FP treatment is mediated through modulation of the promoter activity through cis-acting sequences located within the immediate 5′ UTR of eotaxin-3.
To test whether increased FKBP51 levels affected the FP-mediated decrease in IL-13–induced eotaxin-3 promoter activity, TE-7 cells were transfected with either empty vector (pCDNA3.1) or an expression construct that contained FKBP51 under the control of the CMV promoter (pFKBP51; Fig 7, A) to simulate high baseline levels of FKBP51. Cells transfected with the empty vector showed a 2-fold increase in eotaxin-3 promoter activity on IL-13 treatment, and this was reduced by 50% when the cells were concomitantly treated with FP (Fig 7, D). However, when cells were transfected with the FKBP51 expression vector, the FP-mediated decrease in IL-13–induced eotaxin-3 promoter activity was reduced (Fig 7, D).
In this study we identified a set of genes that exhibited differential expression in the esophagus of treated, FP-responsive patients with EE compared with untreated subjects. Our analysis uncovered several genes previously shown to be glucocorticoid responsive, including those encoding FKBP51, MHC class II, and collagen molecules. Glucocorticoids upregulate FKBP51, whereas they decrease expression of MHC class II and collagen genes in several cell types.16–19,21,22 The identification of such genes validates the analysis performed. Of the transcripts identified, all but 1 exhibited expression in nonresponders similar to that observed in untreated patients. This indicates that nonresponders might have a fundamental dysfunction in glucocorticoid signaling, at least in the esophagus. Tissue-specific glucocorticoid resistance has been reported in other inflammatory diseases, including rheumatoid arthritis, osteoarthritis, Crohn disease, ulcerative colitis, and asthma.23 Multiple mechanisms have been shown to account for glucocorticoid resistance, including aberrant interactions between transcription factors, coactivators, and corepressors; posttranslational modification of inflammatory mediators; and expression of glucocorticoid receptor isoforms.23 Interestingly, only 1 gene (F3) showed differential expression in both FP responder and FP non-responders compared with untreated subjects. This implies that F3 gene expression could be influenced by a nongenomic mechanism of glucocorticoid signaling. This gene might be especially useful to monitor patient compliance because it is expressed in all patients who take the drug, regardless of responsiveness.
The observed change in patients’ biopsy specimen gene expression in addition to previous literature reporting a low systemic bioavailability of oral FP24 supports the interpretation that swallowed FP treatment exerts a topical effect in the esophagus. The oral bioavailability of FP has been reported to be less than 1% at a dose greater than 20-fold higher than the dose for patients with EE in this current study24; this, coupled with the fact that FP undergoes extensive first-pass metabolism in the liver,25 makes it less likely that swallowed FP acts through a systemic route to alter gene expression in the esophagus. To our knowledge, this represents the first report suggesting direct evidence of a topical effect of glucocorticoids in the esophagus.
Here we report that FKBP51 was upregulated at the mRNA and protein levels in esophageal epithelial cells, including primary esophageal epithelial cells that were cultured from esophageal biopsy specimens. Based on the ability of RU486 to inhibit FKBP51 protein induction, the increased FKBP51 levels are likely glucocorticoid receptor dependent. We observed that increased transcript stability did not account for the upregulation of FKBP51 mRNA. Instead, increased FKBP51 mRNA levels are likely caused by increased transcription of the gene. Numerous putative glucocorticoid response elements (GREs) exist within the FKBP51 promoter and introns, and such sequences might mediate the increased transcription in cell types, including A549 cells.26 We observed that FKBP51 levels were increased at baseline on CHX treatment, suggesting that inhibition of protein synthesis up-regulates FKBP51. This could occur if a repressor of FKBP51 transcription required ongoing protein synthesis; alternatively, FKBP51 levels might be upregulated as part of a stress response. Despite this increased baseline expression, FKBP51 transcription still increased on glucocorticoid treatment in the presence of CHX, suggesting that at least a portion of the increased FKBP51 transcripts do not require de novo protein synthesis. These data collectively suggest that glucocorticoid signaling directly affects FKBP51 transcription in esophageal epithelial cells.
We observed that FP treatment inhibited IL-13–mediated eotaxin-3 promoter activity. This appears to be mediated through the 800 bp of promoter that contains 1 canonical GRE sequence. Previous reports have suggested that repression of IL-4–induced eotaxin-3 expression by FP occurs independently of this GRE sequence in lung epithelial cells.27 It remains to be tested whether this is the case in esophageal epithelial cells.
FKBP51 has been shown to act as a negative regulator of glucocorticoid signaling.28,29 In fact, baseline FKBP51 levels in airway epithelial cells negatively correlate with response to glucocorticoid treatment in asthmatic patients.30 Additionally, new world primates exhibit high levels of FKBP51, and this correlates with general glucocorticoid resistance in these animals. 17,31,32 Herein, we show that increasing baseline FKBP51 levels in the esophageal cell line TE-7 correlate with a decreased ability of glucocorticoid to repress IL-13–mediated eotaxin-3 promoter activity (Fig 8).
Biopsy specimens of patients who respond to swallowed FP treatment exhibit high levels of FKBP51, a negative regulator of glucocorticoid signaling.28,29,31,32 However, we note that the observed high FKBP51 transcript levels occurred after treatment. In contrast, we showed that high baseline FKBP51 levels before treatment negatively affect glucocorticoid signaling in vitro. Therefore we propose that although increased FKBP51 levels after FP treatment serve as a readout of functional glucocorticoid signaling, the actual biological function of FKBP51 in part involves negative regulation of glucocorticoid signaling to dampen the anti-inflammatory response so that homeostatic conditions can be restored after the glucocorticoid-mediated resolution of inflammation.
In summary, we have shown that (1) FP responders express a unique set of esophageal genes (including FKBP51), providing evidence that swallowed glucocorticoids mediate their role through a topical effect in the esophagus in vivo, and (2) FP nonresponders have a blunted expression of steroid-induced esophageal transcripts, suggesting impaired glucocorticoid signaling in these patients. Further proof for the ability of glucocorticoids to induce esophageal transcripts is derived by in vitro studies with esophageal epithelial cells, which have demonstrated that (1) glucocorticoids directly induce FKBP51 through a RU486-inhibited mechanism, indicating the direct involvement of the glucocorticoid receptor; (2) FKBP51 mRNA half-life is approximately 16 to 18 hours and importantly not regulated by glucocorticoids; and (3) FKPB51 overexpression inhibits glucocorticoid-mediated transcriptional repression of eotaxin-3. Taken together, our data provide molecular evidence that swallowed glucocorticoids mediate their action through a topical effect in the esophagus and that posttreatment FKBP51 esophageal levels serve as a measure of functional glucocorticoid signaling.
Clinical implications: Swallowed glucocorticoids induce disease remission by regulating local gene expression in the esophagus. Understanding the mechanism by which topical glucocorticoids induce remission from EE will provide insight into disease pathogenesis, steroid resistance, and additional molecular pathways to serve as targets for therapeutic intervention.
Supported in part by National Institutes of Health grant RO1 DK76893 and U19 AI070235, American Heart Association grant 09POST2180041, the Food Allergy Project, the Campaign Urging Research for Eosinophilic Disorders (CURED), and the Buckeye Foundation.
We thank Drs Michael Farrell, Michael Bates, Kathleen Campbell, and Mitchell Cohen for their referral of patients. We are grateful to Bridget Buckmeier, Emily Stucke, and Tommie Grotjan for research assistance, as well as to the Food Allergy Project, the Campaign Urging Research for Eosinophilic Disorders (CURED), and the Buckeye Foundation for their generous support.
Disclosure of potential conflict of interest: J. M. Caldwell has received a postdoctoral grant from the American Heart Association. C. Blanchard has received research support from the National Institutes of Health, the Digestive Health Center CCHMC, and the American Partnership for Eosinophilic Disorders. M. H. Collins was a subcontractor as a clinical study central review pathologist for GlaxoSmithKline and Ception Therapeutics; was a consultant as a clinical study central review pathologist for Meritage Pharma; and is a Member of the Medical Advisory Panel for the American Partnership for Eosinophilic Diseases. S. S. Aceves has intellectual property patent royalties in Meritage Pharma and is on the Medical Advisory Board for the American Partnership for Eosinophilic Disorders. M. E. Rothenberg is a speaker and consultant for Merck; is a consultant for Centocor, Ception Therapeutics, Nycomed, and Array Biopharmra; has received research support from the National Institutes of Health, the Food Allergy and Anaphylaxis Network, and the Dana Foundation; is on the Medical Advisory Board for the American Partnership for Eosinophilic Disorders; and is on the Executive Council for the International Eosinophil Society. The rest of the authors have declared that they have no conflict of interest.