ORIGINAL PAPER ABSTRACT INTRODUCTION

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1 ORIGINAL PAPER Available from: URL: DOI: Mucosal Expression of Basic Fibroblastic Growth Factor, Syndecan 1 and Tumour Necrosis Factor-α in Crohn s Disease in Deep Remission under Treatment with Anti-TNFα Antibodies Antonio Tursi 1, Walter Elisei 2, Mariabeatrice Principi 3, Cosimo Damiano Inchingolo 4, Rosanna Nenna 4, Marcello Picchio 5, Floriana Giorgio 3, Enzo Ierardi 3, Giovanni Brandimarte 6 1) Gastroenterology Service, ASL BAT, Andria (BT) 2) Division of Gastroenterology, ASL Roma H, Albano Laziale (Roma) 3) Department of Medical Sciences and Organ Transplantation, Section of Gastroenterology, University of Bari, Bari 4) Division of Pathology, Lorenzo Bonomo Hospital, ASL BAT, Andria (BT) 5) Division of Surgery, Paolo Colombo Hospital, ASL Roma H, Velletri (Roma) 6) Division of Gastroenterology, Cristo Re Hospital, Roma Italy ABSTRACT Background & Aims: Both inflammation and fibrosis may be detected in Crohn s disease (CD). The molecular pattern of Basic Fibroblastic Growth Factor (bfgf) and Syndecan-1 (SD1) expression is altered in stenosing CD, but we do not know what the behaviour of this teamwork factor is in CD in deep remission under treatment with anti-tnfα antibodies. Our aim was to compare the expression of bfgf, SD1 and TNF-α in patients with CD in deep remission under treatment with Infliximab (IFX) or Adalimumab (ADA) and a control group of patients with active CD. Methods: We assessed the expression of bfgf, SD1 and TNF-α in 10 patients with active CD and in 28 patients with CD in sustained deep remission for at least 6 months. All patients underwent surveillance colonoscopy with biopsies, while receiving maintenance therapy with IFX or ADA. Analysis was conducted by real-time reverse transcriptase PCR (RT-PCR) in biopsy samples. Results: We found that bfgf, SD1 and TNF-α were significantly reduced under treatment with anti-tnfα versus controls (p=0.000). bfgf and SD1 expression were similar between IFX and ADA patients (p=0.335 and p=0.289, respectively), while TNF-α was significantly under-expressed in ADA patients (p=0.008). Conclusions: bfgf, SD1 and TNF-α are significantly reduced in CD patients in deep remission under treatment with anti-tnfα, likely as an expression of optimal control of inflammation. The significance of the TNF-α under-expression in patients under treatment with ADA with respect to those under treatment with IFX should be elucidated in further studies. Key words: adalimumab basic fibroblastic growth factor Crohn s disease infliximab syndecan 1 tumour necrosis factor α. Address for correspondence: Dr. Antonio Tursi, MD Servizio di Gastroenterologia Territoriale DSS n 4, ASL BAT Via Torino, Andria (BT) Italy antotursi@tiscali.it Received: Accepted: INTRODUCTION The goal of medical treatment in patients with Crohn s disease (CD) has shifted from symptom control alone to clinical remission in conjunction with mucosal healing [1]. This latter point seems to be particularly important, because reaching this goal would also produce a domino effect, resulting in fewer complications, hospitalization, and related surgical procedures [2, 3]. This is particularly true when considering the therapeutic use of biologic therapies. The pro-inflammatory cytokine Tumour Necrosis Factor-alpha (TNF-α) appears to play a pivotal role in the pathogenesis of mucosal inflammation, mediating the inflammatory cascade in CD [3]. TNF-α is mainly produced by monocytes and macrophages, although many other cells of the innate and adaptive immune system produce significant amounts of this cytokine [4]. Anti-TNFα therapy by using Infliximab (IFX) or Adalimumab (ADA) can lead to endoscopically assessed mucosal healing in patients with CD, avoiding surgery and maintaining a steroid-free remission up to 12 months [5]. Adhesion molecules containing heparan sulphate (syndecan family) are chemically proteo-glycans and play a significant role in tissue repair [6]. At the intestinal level, syndecan 1 (SD1) is located in the basolateral region of the columnar epithelium and is a relevant factor for damage reversal in inflammatory bowel disease [7]. In particular, it has been shown that SD1 is significantly decreased in mucosa and increased in serum of active CD, and seems to be able to differentiate between CD and

2 262 Tursi et al other granulomatous intestinal diseases, such as tuberculosis [8]. Moreover, basic fibroblast growth factor (bfgf) is a peptide able to repair ulcerative lesions, thanks to its capacity to bind epithelial and stromal cells [9]. It has been suggested that SD1 promotes bfgf morphology changes and modulates the structure of its receptors [10]. During the physiological restoration of mucosa in inflammatory bowel disease, SD1 migrates from basolateral epithelium to both epithelial apical surface and stroma and modulates bfgf activity. In detail, such a phenomenon favours the binding between bfgf and those cells dedicated to the repair process, which are located next to an ulcerative lesion. When it is not activated by SD1, bfgf may be destroyed by both luminal and circulating proteases, thus failing its role in the tissue restoration [11]. The induction of collagen secretion from fibroblasts by bfgf may be one of the mechanisms relevant to the stromal process disease in inflammatory bowel disease, in particular in CD, including transmural fibrosis and stricture formation, as well as tissue repair and healing [12]. TNF-α down-regulation presumably interacts with the adhesion molecules such as SD1 [3] and growth factors such as bfgf [4] in the process of mucosal repair. Indeed, SD1 is located in the basolateral region of the columnar epithelium and plays a relevant role in the course of inflammatory bowel disease damage reversal [5, 13]. It has been showed that CD, especially stenosing CD, shows impaired relation among this cytokine/adhesion molecule/ growth teamwork factor. This pro-fibrosis behavior could explain the tendency toward fibrosis in CD [14]. On the contrary, we do not know what the behaviour of this teamwork factor is in CD in deep remission under treatment with anti-tnfα antibodies. Moreover, we do not know whether a different relationship among the cytokine/adhesion molecule/ growth teamwork factor in patients taking IFX from those taking ADA is detectable. We aimed to assess the mucosal expression of this molecular framework in CD patients in deep remission with IFX or ADA, compared with a control group of patients with active CD. MATERIAL AND METHODS This was a retrospective, observational, multicentre study, performed on patients with CD in clinical, laboratory and endoscopic remission and undergoing surveillance colonoscopy while receiving maintenance therapy with IFX or ADA. Patients under treatment with IFX or ADA with a confirmed history of CD and under sustained deep remission were enrolled. All patients were under scheduled treatment with IFX 5 mg/kg/i.v. every 8 weeks or ADA 40 mg subcutaneously every other week in order to maintain remission. No patient received mesalamine enemas, corticosteroids, or immunosuppressants (tacrolimus, azathioprine, and 6-mercaptopurine) during the study period. According to our standard procedure, immunosuppressants were started at the beginning of the IFX and ADA treatment, in order to speed the response and to reduce the risk of anti-tnfα failure, and stopped six months after starting anti-tnfα therapy. Disease activity was assessed by the Harvey-Bradshaw Index (HBI) [15], endoscopic severity was assessed by Simple Endoscopic Score for CD (SES-CD) [16, 17]: to be considered in sustained deep remission for enrolment in this study, participants had to have a HBI score 5 points, a SES-CD score 0, and normal values of C-reactive protein (CRP) for at least 6 months. Moreover, patients should have had no changes in their CD medications (IFX or ADA) or any steroid use during the previous 6 months. Ten patients with active CD (HBI score 6 points and SES-CD score 2 points) and not taking anti-tnfα treatment served as control group. Biopsy samples were taken from the intestinal location in which the disease was active before and in remission thereafter (active group) or in which the disease was currently active (control group) (Table I). Molecular analysis Molecular analysis was independently performed by two expert gastrointestinal pathologists (E.I. and F.G.), blinded about the type and the degree of the disease. The method of detecting mucosal quantitative levels of bfgf, SD1 and TNF-α was a real-time reverse transcriptase PCR (RT-PCR) in biopsy samples from paraffin samples. We chose this technique because it is simpler and faster than classical immunohistochemistry, and it significantly correlates with semiquantitative evaluations obtained by immunohistochemistry [18]. RT-PCR has the ability to reflect the altered pattern of the expression of genes dedicated to the synthesis of a specific molecule and to quantify its transcription levels [18]. Therefore, in this study the technique allowed the assessment of the amount of the mrna codifying for the synthesis of these cytokines in intestinal biopsy samples. The amount was expressed by a numerical value (i.e. the fold change compared to controls, represented by patients undergoing colonic biopsy in absence of endoscopic and histological alteration, e.g., in the case of suspected microscopic or collagenous colitis) [19]. The relative expression of the studied genes levels was calculated using the 2CT method. Statistics The collection and analysis of data were performed using MedCalc Release Chi-square test for categorical variables and Mann-Whitney U test for continuous variables were used. All tests were two-tailed, and the level of significance was p=0.05. Ethics approval This study was approved by the Institutional Review Board and each subject gave written informed consent. RESULTS A total of 28 patients received maintenance therapy and were in deep remission with IFX (11 patients) or ADA (17 patients). The characteristics of the treated patients and controls are summarized in Table I. The characteristics of the CD treated groups are summarized in Table II. When evaluating the cytokine expression, we found that bfgf, SD1 and TNF-α were significantly reduced under

3 TNF-α, SD1 and bfgf expression in Crohn s disease 263 Table I. Characteristics of the study groups Treated patients (n=28) Controls (n=10) P* Gender Male n (%) 17 (60.7) 6 (60.0) Age median (range), years 34 (20-63) 35 (21-64) Age > 40 years n (%) 12 (42.9) 4 (40.0) CD duration median (range), months 19.5 (9-60) 18 (10-56) Previous surgery n (%) 3 (10.7) Smoking n (%) 4 (14.3) 1 (10.0) Family history n (%) 4 (14.3) 2 (20.0) Location n (%) Terminal ileum 6 (21.4) 2 (20.0) Ileo-colon 18 (64.3) 6 (60.0) Colon 4 (14.3) 2 (20.0) Extraintestinal diseases n (%) 7 (25.0) 2 (20.0) Crohn s disease type n (%) Inflammatory 21 (75.0) 7 (70.0) Stenosing 3 (10.7) 2 (20.0) Fistulizing 4 (14.3) 1 (10.0) Medications n (%) Mesalazine 28 (100.0) 10 (100.0) Immunosuppressors 0 (0) 0 (0.0) Steroids 0(0) 2 (10) *Chi-square test for categorical variables and Mann-Whitney U test for continuous variables treatment with anti-tnfα versus controls (p=0.000; Fig.1). Cytokine expression under therapy with IFX and ADA is reported in Fig. 2; bfgf and SD1 expression were similar between IFX and ADA patients (p=0.335 and p=0.289, Table II. Characteristics of the CD treated population IFX-treated patients (n=13) ADA-treated patients (n=15) P* Gender Male n (%) 8 (61.5) 9 (60.0) Age median (range), years 34 (20-63) 32 (19-66) Age > 40 years n (%) 5 (38.5) 7 (46.6) CD duration median (range), months 19.5 (9-44) 18 (9-60) Previous surgery n (%) 1 (7.7) 1 (6.6) CRP values (in mg, n.v: <5 mg) 3 (2-5) 3 (1-5) SES-CD score (median) HBI score (median) 3 (2-5) 3 (2-5) Median TNFα treatment duration, months 20 (12-44) 24 (12-60) Smoking n (%) 1 (7.7) 3 (20.0) Family history n (%) 2 (14.3) 2 (20.0) Location n (%) Terminal ileum 3 (23.) 3 (20.0) Ileo-colon 8 (61.5) 10 (66.6) Colon 2 (15.3) 2 (13.3) Extraintestinal diseases n (%) 4 (30.7) 3 (20.0) Crohn s disease type n (%) Inflammatory 9 (69.2) 12 (80.0) Stenosing 2 (14.3) 1 (6.6) Fistulizing 2 (15.3) 2 (13.3) *Chi-square test for categorical variables and Mann-Whitney U test for continuous variables

4 264 Tursi et al Fig. 1. Expression of bfgf, SD1, and TNF-alpha, and SD1 in controls and treated patients. Median values and minimum to maximum values are denoted by bars and error bars. Fig. 2. Expression of bfgf, SD1, and TNF-alpha, and SD1 in the Infliximab and Adalimumab group. Median values and minimum to maximum values are denoted by bars and error bars. respectively), while TNF-α was significantly under-expressed in ADA patients (p=0.008). DISCUSSION The achievement of mucosal healing is a critical endpoint in the treatment of CD patients. Achievement of mucosal healing in patients with inflammatory bowel disease carries the prospect of influencing the natural history of this disease by the prevention of complications, such as the need for surgery or hospitalization. The understanding of basic mechanisms of wound generation and healing is crucial for the improvement of existing and the development of future therapies. The mechanism by which it occurs has been recently hypothesized. Ierardi et al demonstrated that a decrease of TNF-α, induced by IFX treatment is accompanied by a decrease in both SD1 and bfgf when mucosal healing occurs [20]. A possible explanation is that IFX may down-regulate, via a marked reduction of TNF-α mucosal levels, the bfgf/ SD1 link. This molecular profile might represent a pathway of mucosal healing. However, the parallel trend of TNF-α, SD1 and bfgf might be just a simultaneous consequence of the control of inflammation. To clarify this last point, a further study on cultured biopsy samples, taken from patients with both CD and ulcerative colitis and incubated in a medium containing comparable amounts of IFX similar to those reached in the serum of treated patients was performed. After 24 hours, TNF-α, SD1 and bfgf were assayed in tissue homogenates. TNF-α was decreased, while SD1 and bfgf levels were still high when evaluated by both a molecular method and immunohistochemistry [21]. This last finding supports the hypothesis that a mucosal TNF-α reset, induced by biological drugs, is followed by a mucosal restoration in which SD1 modulates the strong reparative bfgf aptitudes. This mechanism of mucosal restoration has been recently confirmed by De Bruyn et al [22]. The authors found that gene expression of many matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and metalloprotease with thrombospondin motifs [ADAM(TS)s] and growth factor was upregulated in patients with active IBD. After controlling inflammation with IFX, most gene dysregulations observed at baseline were restored in responders. This confirms that suppression of inflammation results in the arrest of epithelial damage and subsequent mucosal healing [22]. However, two issues arise from these preliminary analyses: first, what happens when patients are in deep remission (namely under clinical, laboratory and endoscopic remission); second, what happens when we use ADA instead of IFX to treat CD? With regard to the first question, we found that the framework TNF-α, SD1 and bfgf is significantly decreased in CD in deep remission when compared with active disease. In particular, all molecules are simultaneously decreased. These results differ from what was previously described in ulcerative colitis, in which TNF-α rapidly decreases under IFX, followed by SD1 and bfgf [21]. The first and simple hypothesis explaining this result is that the mechanisms involved in the reparative process may be different in the two diseases. In fact, we cannot forget that ulcerative colitis is a mucosal disease, while CD is a wall disease, often causing fibrosis. Therefore, in CD, but not in ulcerative colitis, the framework TNF-α, SD1 and bfgf may decrease simultaneously under effect of anti-tnfα drugs. However, a more attractive hypothesis is that the parallel trend of TNF-α, SD1 and bfgf in CD in deep remission might be just a consequence of the control of inflammation. A mucosal TNF-α reset, induced by biological drugs, is followed by a mucosal restoration in which SD1 modulates the strong reparative bfgf aptitudes. Finally, in healed mucosa as in our population, cytokines, adhesion molecules and growth factors resume their normal pattern. Thus, the parallel trend of TNF-α, SD1 and bfgf in CD in deep remission might be just a consequence of the optimal control of inflammation. Regarding the second question, we did not find any difference in SD1 and bfgf expression between patients under treatment with IFX or ADA, but TNF-α was significantly under-expressed in ADA than in IFX patients. It is not easy to explain these surprising results. We know that IFX heals ulcers by the down-regulation of metallo-proteinase [23] and

5 TNF-α, SD1 and bfgf expression in Crohn s disease 265 enhancement of tissue inhibitor of metalloproteinase [24], and that it has distribution and elimination half-lives of 4.3 and 18.5 days, respectively [25]. On the contrary, mean concentrations for patients receiving ADA quickly reach the steady-state, and remain relatively constant during therapy, ranging from 15 to 19 days in relation to the dose administered [26, 27]. Indeed, it is hypothesized that ADA inhibits TNF-α in a deeper way thanks to its stable half-life, due to a 2-week administration. However, there are many variables which could have biased the results, i.e. site of biopsy sampling, time point of biopsy sampling, TNFα antibody trough levels at the time point of biopsy sampling, etc. Hence, this hypothesis needs to be supported by further studies assessing the possible link between ADA levels in blood/colonic mucosa and TNF-α expression in intestinal mucosa. CONCLUSION Our results are limited by the small sample number of CD patients enrolled to draw reliable conclusions. However, the present study shows for the first time that bfgf, SD1 and TNF-α are significantly reduced in CD patients in deep remission under treatment with anti-tnfα, likely to be an expression of optimal control of inflammation. The significance of the TNF-α under-expression in patients under treatment with ADA with respect to those under treatment with IFX should be elucidated in further studies. Conflicts of interest: None to declare. REFERENCES 1. Travis S, Van Assche G, Dignass A, Cabré E, Gassull MA. On the second ECCO Consensus on Crohn s disease. J Crohns Colitis 2010;4: Peyrin-Biroulet L, Ferrante M, Magro F, et al; Scientific Committee of the European Crohn s and Colitis Organization. Results from the 2nd scientific workshop of the ECCO. I: Impact of mucosal healing on the course of inflammatory bowel disease. 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