ABCC7/CFTR Expression is Down-regulated in Patients with Active Ulcerative Colitis

Background Inammatory Bowel Disease includes Ulcerative Colitis (UC) and Crohn´s disease (CD) of unknown etiology. The expression of ATP Binding Cassette Family proteins (ABC) has been associated with drug resistance and development of UC. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or also known as ABCC7 is involved in the inammatory chronic response. The aim of the study was to evaluate the role of ABCC7/CFTR in UC patients and normal controls without inammation. Results A total of 62 patients with UC and normal controls were included. We found a signicant downregulation of CFTR gene expression in patients with active UC compared to remission UC and normal controls without inammation (P<0.004 and P<0.0001 respectively) even the gene expression of CFTR was decreased in remission UC patients compared to normal controls (P=0.047). The CFTR gene expression was associated with persistent activity of UC and young age at diagnosis before 40 years. The protein expression of CFTR was decreased in severe active UC patients compared to normal controls without inammation. Conclusion

The protein expression of CFTR was decreased in severe active UC patients compared to normal controls without in ammation.
Conclusion The CFTR gene and protein expression were signi cantly decreased in active UC patients and it was also associated with clinical outcomes.

Background
The Ulcerative Colitis (UC) is a chronic in ammatory condition of the colon that affects only the colonic mucosa of unknown etiology. The clinical symptoms includes rectal bleeding, chronic diarrhea and abdominal pain 1,2 . It has been proposed that innate and adaptive immunity are involved in the in ammatory response, speci cally UC had increase the production of pro-in ammatory interleukins such as IL1, IL6 and IL8 as a result of activation of the transcriptional nuclear factor Nuclear ĸB (NF-ĸB) 3-5 .
Recently, genes that encodes for a family of ABC transmembranal multidrug resistant proteins (ABC, ATP Binding Cassette family) have been associated with medical response and clinical course of disease 6 . The ABC family is composed by 48 transmembranal proteins grouped in 7 subfamilies (ABCCA-ABCCG) 7,8 . The ABCC member 7 (ABCC7), also known Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) is an ionic channel that actively participates in the regulation of elimination and absorption of chloride ion in different tissues including the gastrointestinal tract 9,10 .
The presence of mutation in the ABCC7/CFTR gene has been associated with cystic brosis (CF) which is a prevalent disease in white populations, dysfunction and changes in gene expression has been implicated in other diseases development, including chronic in ammation 11,12 . In patients with CF has been observed an increase of fecal calprotectin as same as happens in the in ammatory bowel disease (IBD) [13][14][15] . The ABCC7/CFTR knock-out mice model developed intestinal in ammation suggesting a role of this gene in the gut in ammation 12,16 . The levels of ABCC7/CFTR expression have been involved in modulating the NF-ĸB activity and the production of IL8 and IL6 production in lung cell lines [17][18][19] . The aim of this study was determinate the ABCC7/CFTR gene and protein expression in UC patients and its association with clinical outcomes.

Demographic and clinical characteristics of UC patients
A total of 41 patients with UC were included in the study, 52% were male, mean age of 43.5 ± 15.1 years, age at diagnosis < 40 years in 83%. All demographic and clinical characteristics are shown in Table 1. We found a down-regulation of the gene expression of CFTR in active UC group compared to control without in ammation and UC remission patients (P < 0.001, P < 0.004 respectively). We also identify that the expression of CFTR was down-regulated in UC remission group compared with control without in ammation (P < 0.04) as shown in Fig. 1. In protein expression we observed low expression of CFTR/ABCC7 in biopsies of the active UC compared with remission UC and control group (P = 0.046), no difference was observed in remission UC group compared with control (Fig. 2).
The IL-6 gene expression was measured in colonic biopsies from UC patients in order to determine the presence of in ammation and it was also correlated with histological activity. The IL-6 gene expression was low in controls without in ammation and UC remission patients compared to active UC group (P < 0.001). The gene expression of IL-6 in UC remission group and control without in ammation was similar in both groups as shown in Fig. 3.

Discussion
Our best knowledge, this is the rst study that showed a down-regulation of the gene and protein expression of CFTR or ABCC7 in patients with active UC compared to UC in remission and normal controls without in ammation. It is important to note that low CFTR gene expression was signi cant associated with young age at diagnosis (< 40 years old) and a clinical course characterized by persistent UC activity.
It is well known that CFTR has been widely studied in cystic brosis (CF) and recently, it has grown interest in to study its role in other diseases or in ammatory conditions such as in the biliary epithelium and intestine 12 25 . In other cell lines from colorectal carcinoma, Crites K et al reported that a down-regulation of ABCC7/CFTR increased the production of IL-6, IL-1β and IL-8 in CACO2 and HT29 cells due to the activation of ERK1/2, MAPK, IΚBα and NF-ĸB pathways 26 . In a knock-out animal model of ABCC7/CFTR, they found that the down regulation of CFTR produced in ammation through the increased of NF-ĸB, TNFα and IL-6 in the mouse intestine, CACO2 cells and colangiocytes stimulated by lipopolysaccharides from Escherichia coli 2326, 27 . In other study performed in rats and 16HBE140 cells found that down-regulation of CFTR increased the production of IL-8 compared to those that overexpressed CFTR with lack of MAPK/NF-ĸB activation 19 and the same nding was also observed in mutant CFTR (DF508) mice and HaCaT cells that the down-regulation of the CFTR expression exhibited in ammation and delayed cutaneous wound healing 28 .
Our ndings are similar to above mentioned studies con rming the role of down-regulation of CFTR in the development of in ammatory process in patients with active UC.
Nevertheless, it already known that CFTR interacts with a variety of proteins and regulates its function 10 .
Recent studies have demonstrated in Cftr knock-out mice model higher levels of IL-17 compared to wild type mouse during infection with Pseudomonas aeruginosa and resulting in the perpetuation of in ammatory process by neutrophil recruitment suggesting that the absence of CFTR contributes to the in ammation by IL17 production 29 and have been also observed in patients with active UC 30 .
The CFTR is a channel expressed in the intestine that regulates the e ux of chloride and bicarbonate ions and also participates actively in the pH regulation 9,10 . A proposed hypothesis explaining the role of CFTR in UC patients is that down-regulation of CFTR affects the pH balance in the colon producing a decreased expression of MUC11 and MUC12 contributing to the adherence of bacteria and promoting the colonic in ammation 10,11,31 and it was reported by our group a decreased gene expression of MUC20 in patients with active UC compared to UC remission and normal controls 32 .
On the other hand, changes in the intestinal pH and decreased MUC11 and MUC12 expression promotes the colonization of phatobiontic microbiota in the intestinal lumen as it has been reported in IBD patients 11,23,33 . Phatobiontic microbiota increased the expression, recognition and activation of TLR4 with activation of NF-ĸB and subsequent production of pro-in ammatory cytokines such as IL-6 and IL-8. 23,26,27,33,34 Microbiota dysbiosis is associated with reduction of short-chain fatty acids, an antiin ammatory molecules that regulates the immune response in the intestine, this fatty acids ameliorates the intestinal in ammation in IBD in animal model. 35 But, is possible that down-regulation of ABCC7/CFTR contribute to IL17 up-regulation in patients with UC allowing the persistent activity as we observed in the active group in our study. 29,30 Conclusions In conclusion, a down-regulation of gene and protein expression of CFTR was found in patients with active UC suggesting the role of CFTR in the development of colonic in ammation. The CFTR downregulation was associated with young age at diagnosis and persistent activity clinical course.

Material And Methods
We included 62 individuals who were divided in 3 groups: active UC (n = 20), remission UC (n = 21) and normal controls without in ammation (n = 21). All patients belonged to the In ammatory Bowel Disease Clinic at the Instituto Nacional de Ciencias Medicas y Nutrición Salvador Zubirán. All patients had a de nitive diagnosis of UC con rmed by histopathology. All demographical and clinical variables were collected from interview and medical records such as: age at diagnosis, gender, type of medical treatment (5-aminosalycilates, steroids, thiopurines, biologic therapy), disease extension or location according to Montreal Classi cation, the presence of extra-intestinal manifestations such as articular affection, ankylosing spondylitis, sacroiliitis, sclerosing cholangitis, pyoderma gangrenosum, erythema nodosum or uveitis and clinical course classi ed as: initially active and then prolonged remission ( rst episode with activity and then long-term remission for more than 5 years), intermittent activity (> 2 relapses per year) and chronic continual activity (persistent activity despite medical conventional therapy). The clinical and endoscopic activity was determined by Mayo and histological activity by Riley score. 20,21 Tissue samples and Gene Expression Analysis All colonic biopsies were taken by colonoscopy with informed consensus of all the patients that participate in the study, immediately placed in RNA Later (Ambion, Austin, Tx, USA) and stored at -70 °C until processing. Total RNA extraction from colonic biopsies was made using RNA extraction kit (High Pure RNA Tissue Kit, Roche). The biological sample was mixed by homogenizer Polytron→ LD1300 for 1 minute, the RNA was extracted by commercial kit for RNA extraction High Pure RNA Tisseu kit (Roche ) after the incubation with DNAase for 20 minutes and was diluted in 50 µl of elution buffer and stored at -70°C. The integrity of RNAm was evaluated by electrophoresis in an agarose gel at 1% with ethidium bromide, it was then visualized using an UVP dual-intensity transluminator.
The cDNA was synthetized through reverse transcription with the cDNA Transcription Synthesis kit

Protein Expression Analysis by Western Blot Analysis
The total protein of colon tissues samples 4 patients per group (active and remission UC and control group) was extracted with solution of RIPA buffer (Siagma-Aldrich) and cocktail of Proteases inhibitor (Roche), and quanti ed by Bradford assay (Bio-Rad, Hercules, CA, USA), and stored at -70C. The protein detection was performed by electrophoresis in SDS-PAGE and then transferred to polyvinylidene di uoride membranes. All blots were blocked with 3% BSA for 60 min at room temperature and incubated overnight at 4 with primary antibodies. The primary antibodies for CFTR/ABCC7 were used in a concentration of 1:1000 and β-actin was used to normalize the data in a concentration of 1:10000. The blots were incubated with anti-rabbit secondary antibodies conjugated with horseradish peroxidase (1:3000).
Images were analyzed with a ChemiDocTM XRS + System Image LabTM Software (Bio-Rad, Hercules, CA, USA) and the western blot analysis was performed using independent blots.

Statistical Analysis
The statistical analysis was performed using SPSS version 17 Figure 1 Relative gene expression of ABCC7/CFTR in biopsies of UC patients. The relative gene expressions of ABC7/CFTR were compared in patients with active, remission UC and control group. The medians of relative expression in the tree groups were compared with H-Kruskall Wallis and individual groups were analyzed by U-Mann-Withney test.

Figure 2
Relative gene protein expression of ABCC7/CFTR in biopsies of UC patients. The relative protein expressions of ABC7/CFTR were compared in biopsies of patients with active, remission UC and control group (Figure 2A). The qualitative protein expression is showed in gure 2B and was performed by Western blot analysis. The medians of relative expression in the tree groups were compared with by Dunn's method for all pairwise multiple comparison procedure.