High intestinal and systemic levels of decoy receptor 3 (DcR3) and its ligand TL1A in active ulcerative colitis

Clinical Immunology (2010) 137, 242–249

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High intestinal and systemic levels of decoy receptor 3 (DcR3) and its ligand TL1A in active ulcerative colitis Giorgos Bamias a,⁎, Garyfallia Kaltsa a , Spyros I. Siakavellas a , Kostis Papaxoinis a , Evanthia Zampeli b , Spyros Michopoulos b , Irene Zouboulis-Vafiadis a , Spiros D. Ladas a Gastroenterology Division - First Department of Propaedeutic and Internal Medicine, “Laikon” General Hospital, Athens University Medical School, 17 Agiou Thoma st., 11527, Athens, Greece b GI Unit, “Alexandras” Hospital, 80 Vasilissis Sofias Ave., 11528, Athens, Greece a

Received 28 May 2010; accepted with revision 7 July 2010 Available online 2 August 2010 KEYWORDS Ulcerative colitis; DcR3; TL1A

Abstract Decoy receptor-3 (DcR3) is a member of the TNF receptor superfamily of proteins, which has been implicated in anti-apoptotic and anti-inflammatory pathways, via binding to TL1A, LIGHT and Fas-L. The role of the TL1A/DcR3 ligand/receptor pair in ulcerative colitis (UC) has not been studied. We investigated the systemic (peripheral blood) and local (large intestine) expression of DcR3 and TL1A in 64 patients with UC and 56 healthy controls. DcR3 serum concentrations were highly elevated in patients with active UC (P b 0.0001 vs. healthy controls). This elevation was clearly related to the presence of intestinal inflammation as it was less frequently observed in patients in remission (P = 0.003 vs. active UC) whereas effective treatment resulted in disappearance or significant decrease of serum DcR3 (P = 0.006 vs. pre-treatment). Furthermore, DcR3 mRNA transcripts were significantly elevated in inflamed areas of the colon (P = 0.002 vs. non-affected of the same patient). In addition to DcR3 elevation, we found increased circulating levels of TL1A in patients with either active or inactive UC in comparison to healthy controls (P b 0.001 for both). We conclude that elevated serum DcR3 may serve as an indicator of active colonic inflammation in patients with UC. TL1A/DcR3-mediated pathways may participate in the pathogenesis of UC. © 2010 Elsevier Inc. All rights reserved.

Introduction

⁎ Corresponding author. First Department of Propaedeutic and Internal Medicine, “Laikon” General Hospital, Athens University Medical School, 17 Agiou Thoma st., 11527, Athens, Greece. Fax: +30 210 7791839. E-mail address: [email protected] (G. Bamias).

The pathogenesis of Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as Inflammatory Bowel Diseases (IBD), remains largely unknown. Nonetheless, it is widely accepted that both conditions are mediated by aberrant immune responses towards constituents of commensal flora, which take place in the intestinal mucosa of genetically predisposed individuals [1]. In this context of immunological

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High intestinal and systemic levels of decoy receptor 3 and its ligand TL1A in ulcerative colitis over-reactivity, pivotal roles are reserved for members of the TNF and TNF receptor superfamilies of proteins (TNFSF and TNFRSF, respectively). TNFSF and TNFRSF consist of several molecules that are abundantly expressed in cells of the immune system [2]. These molecules are centrally involved in several aspects of immunological function, such as development of the immune system, activation, co-stimulation, and proliferation of immunocytes, as well as their removal through regulation of apoptosis [3]. The involvement of these molecules in the inflammatory pathways that take place during IBD has been established through several lines of evidence. First, mRNA and protein expression of several TNFSF and TNFRSF-related molecules is increased in affected areas of CD and UC patients [4,5]. Second, polymorphisms in the genes encoding for these proteins may modify clinical characteristics of IBD [6]. Third, animal studies have shown that overexpression of TNF in TNFΔARE mice, as well as of LIGHT (TNFSF14) or CD40L (TNFSF5) in transgenic mice leads to intestinal inflammation [7–9]. Finally, the most compelling evidence originates from the unequivocal efficacy of TNF neutralization to induce clinical remission and mucosal healing in patients with active CD or UC [10]. Recently, attention has been drawn to a novel TNF-like cytokine, TL1A (TNFSF15) and its two receptors, i.e. deathdomain receptor 3 (DR3, TNFRSF25) and decoy receptor 3 (DcR3, TNFRSF6B) and their functional importance during inflammatory conditions. Binding of TL1A to DR3 leads to activation, proliferation, and secretion of cytokines by lymphocytes and NK cells [11,12]. In addition, TL1A/DR3 association provides apoptotic signals to lymphocytes [11]. Accumulating evidence indicates that TL1A/DR3 mediated pathways may participate in the pathogenesis of clinical and experimental IBD. First, increased expression of TL1A and DR3 has been reported in both UC and CD [13]. Second, in animal models of IBD, TL1A/DR3 interactions have been shown to exacerbate intestinal inflammation via stimulation of TH1 and TH17 pathways [14] and by providing co-stimulatory signals to lymphocytes [15]. Finally, genetic studies have shown that polymorphisms of the tnfsf15 gene modify the individual risk for developing CD [16]. DcR3, on the other hand, acts as a decoy receptor which competes with DR3 for binding to TL1A and inhibits downstream signaling [11]. In addition to TL1A, DcR3 can also bind to CD95L (TNFSF6) and LIGHT (TNFSF14), inducing mainly anti-apoptotic signals [17,18]. It has been recently suggested that DcR3 may also be involved in the pathways of chronic inflammation acting either as an enhancer or a suppressor of immunological responses [19,20]. The functional importance of DcR3 in CD has been recently reported. In particular, Funke et al. proposed that DcR3 participates in the pathogenesis of chronic ileal inflammation by activating nuclear factor NF-κB, and protecting lamina propria T cells from undergoing apoptosis [21]. However, the role that DcR3 may play in UC has not been studied. In the present study we hypothesized that DcR3 and its ligand TL1A are elevated both locally and systemically in patients with active UC. We report that circulating levels of DcR3 are increased in patients with active UC. We also demonstrate that DcR3 expression is significantly upregulated in affected as compared to non-affected colonic segments. Additionally, we show a clear reduction of soluble DcR3 following effective treatment in patients with UC. Finally, we report that this

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augmentation of DcR3 is accompanied by a similar increase in the circulating levels of its ligand, TL1A, indicating a generalized upregulation of this novel TNF/TNFR system in IBD.

Materials and methods Patients Sixty-four patients with UC followed in our clinics were included in the present study. Demographic and clinical information was obtained from the medical records of the patients. Fifty-six age- and sex-matched healthy blood donors were studied in parallel and served as the control group. Diagnosis of IBD was confirmed by standard clinical, endoscopical, and histological criteria. The extent of colonic disease was determined by endoscopy and reported according to the Montreal classification [22]. Disease activity was estimated by a combination of clinical (number of bowel movements and blood in the stools) and serological parameters (ESR and CRP) and objectively determined by Truelove and Witts' severity criteria [23]. Evaluation of the disease activity was always performed at the same time as the blood collection. The study protocol was approved by the Hospital Ethics Committee and all subjects gave written informed consent.

Collection of samples Blood was collected from UC patients and controls and centrifuged at 3.000 rpm. Sera were separated, aliquoted, and stored at −80 °C until used. For mRNA studies, mucosal biopsies were obtained during colonoscopic investigation in 11 patients with active UC. Samples were taken from both healthy (proximal) and inflamed (distal) segments of the colon. The specimens were immersed directly into RNAlater® RNA stabilization solution (Ambion Inc, CA). After maintenance at 4 °C for 1–2 days, biopsies were then stored at −80 °C until use.

Measurement of soluble DcR3 and soluble TL1A For measurement of DcR3 we used the human DcR3 ELISA kit from Bender MedSystems GmbH, Austria, following the manufacturer's instructions. Absorbance was measured at 450 nm (primary wave length) and corrected at 620 nm (reference wave length). Absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human DcR3, run in duplicate (Bender MedSystems). The sensitivity of the ELISA is 7 pg/ml. Serum concentrations of TL1A were measured by ELISA, as previously reported [24]. Briefly, a rabbit anti-human anti-TL1A antibody (Peprotech, UK) was coated to flat-bottomed microplates (1 μg/ml) and incubated overnight. Non-specific binding was blocked for 2 h with 1% BSA in PBS. Wells were washed and 100 ml of diluted patient's sera was added and incubated for 2 h. After further washing, a biotinylated rabbit anti-human anti-TL1A antibody (Peprotech, UK) was added (0.5 μg/ml) and incubated for 2 h at room temperature. After further washing, avidin–HRP Conjugate (Peprotech, UK) was added for 30 min, the excess was washed off and 100 ml of ABTS Liquid Substrate Solution (Sigma, USA) was added. Absorbance was measured at

244 405 nm (primary wave length) and corrected at 655 nm (reference wave length). Absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human TL1A (Peprotech, UK), run in triplicate. The sensitivity of the ELISA is 62 pg/ml. For both TL1A and DcR3 measurements, concentrations were calculated via logarithmic analysis. All samples with an absorbance of less than the average of the zero standards + 2 SD were considered as non-detectable. In addition, to avoid any inter-assay variability, in each separate ELISA assay, we run in parallel similar numbers of samples from patients with UC (both active and remission) and non-IBD controls.

RNA extraction and real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) For measurements of DcR3 mRNA transcripts, intestinal mucosal samples obtained from endoscopical biopsies were homogenized by a Rotor stator Homogenizator. Total RNA was isolated from the homogenized tissue using a Purelink RNA Mini kit (Invitrogen, CA) following the manufacturer's protocol. The integrity and concentration of RNA was measured by the 2100 Bioanalyser (Agilent, CA). First-strand cDNA synthesis was conducted using the Roche Transcriptor High Fidelity cDNA Synthesis kit (Roche Diagnostics Corporation, IN). A total of 500 ng of RNA was used as a template. Specific primers for DcR3 (TNFRSF6B), and TNF-α were purchased from SABiosciences, MD. Real-time monitoring of PCR reactions was performed using the CFX96 Real-time system (Biorad, UK). Each reaction was conducted in a total of 20 μl as follows: 10 μl of Q™ SYBR Green Supermix (Biorad, UK), 5 μl of cDNA, 1 μl of Primers and 4 μl of H2O. Standard PCR conditions consisted of an initial denaturation step (95 °C for 10 min), followed by 40 amplification cycles (95 °C for 15 s, 60 °C for 60 s, and 75 °C for 5 s). The DcR3 or TNF mRNA was normalized to the ABL mRNA which was used as the internal control. ABL was selected because it demonstrated optimal stability of expression between different samples.

G. Bamias et al. Table 1

Clinical and demographic characteristics of patients.

Age, years [mean ± SD, (range)] Male sex (%) Disease duration, months, [mean ± SD, (range)] CRP, mg/l (mean ± SD) Disease location Proctitis Left colitis Pancolitis Extraintestinal manifestations a Myoskeletic Skin Eye a

UC active (n = 29)

UC remission (n = 35)

37.4 ± 16 (15–74) 15 (51.7%) 59.5 ± 78 (1–300) 25.6 ± 21.1

36.9 ± 14.7 (16–76) 18 (51.4%) 36.6 ± 45.4 (2–194) 4.5 ± 4.3

3 5 21 8 7 2 1

4 8 23 7 6 1 1

Some patients had more than one extraintestinal manifestation.

regard to age, gender or disease duration. In addition, healthy controls were matched for age and gender with the disease groups (data not shown). The majority of patients suffered from extensive colitis in both the UC-active (72.4%) and the UC-remission groups (65.7%). A personal history of extraintestinal manifestations was reported in 27.6% of patients with active disease and in 20% of patients in remission. At study enrollment patients with active IBD were receiving steroids (active: 45%, remission: 27.2%), 5-ASA preparations (active: 52%, remission: 57.6%), azathioprine or 6-MP (active: 11.5%, remission: 33.3%), antibiotics (active: 7.7%, remission: 0%) or infliximab (active: 3.8%, remission: 6.1%). In addition, 19.2% of patients in the active group (new diagnosis of UC) and 9.1% of patients in remission were not receiving any treatment.

Soluble DcR3 is increased in patients with active UC Statistical analysis Due to their highly skewed distributions DcR3 and TL1A concentrations were analysed with non-parametric tests. Comparison of values for serum DcR3 and TL1A between UC subgroups and control individuals was performed by Mann– Whitney (2 groups compared) and Kruskal–Wallis (more than 2 groups compared) tests. Serum values of DcR3 in the same patients before and after treatment were compared by Wilcoxon test. Spearman's r-test was used to assess correlations of DcR3 with other serum measurements. In all cases an alpha level of b 0.05 was considered significant.

Results Patient populations The characteristics of the patients included in the present study are depicted in Table 1. No significant differences existed between the groups with active or quiescent UC in

We, first, measured the concentration of DcR3 in sera from patients with active disease in comparison with those in remission and with healthy controls (Fig. 1). We observed that DcR3 levels were significantly higher in active UC in comparison with either the UC-remission (3.4× average increase, P =0.003) or the healthy control groups (9.5× average increase, P b 0.0001). In particular, high concentrations of DcR3 were detected in the majority of serum samples of patients with active UC (470.4, 0–5500 pg/ml, median, 95% CI). In contrast, the majority of patients with UC in remission or healthy controls did not have detectable values of serum (68% for UC-remission group and 75% for the control group, respectively). Overall, low concentrations of soluble DcR3 were measured in both UCremission (0, 0–2761.4 pg/ml) and healthy control groups (0, 0–726 pg/ml). We did not find any significant association between DcR3 and various clinical and epidemiological characteristics of the patients that we looked at (age, gender, duration of bowel disease, extent of colonic involvement, extraintestinal manifestations, and specific treatment).

High intestinal and systemic levels of decoy receptor 3 and its ligand TL1A in ulcerative colitis

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in all but one patient (10/11, 91%). More importantly, DcR3 was detected in all 11 patients pre-treatment, but it became undetectable in 7/11 patients (64%) after treatment. This decrease in DcR3 levels paralleled a significant drop of CRP levels which occurred in all patients (pre-treatment: 21.1 ± 22.5 mg/l vs. post-treatment: 3.6 ± 1.6 mg/l, mean ± SD, P = 0.009) (Fig. 2, right panel). Taken together, our findings indicate that during active UC there was a marked elevation of serum DcR3, which was the consequence of intestinal inflammation, as it was significantly reversed when effective treatment was administered. Figure 1 Serum concentration of DcR3 is elevated in patients with active UC. Sera were obtained from UC patients with active or inactive disease and from healthy controls. The concentration of soluble DcR3 was measured by ELISA. Levels of DcR3 were significantly higher in patients with active UC (median, 95% CI: 470.4, 0–5500 pg/ml) as compared with UC patients in remission (0, 0–2761.4 pg/ml, P b 0.005) or healthy control groups (0, 0– 726 pg/ml, P b 0.001). No significant difference was observed between UC-remission and healthy control groups. P values refer to Mann–Whitney test (for 2-group comparison). Horizontal lines represent median values for each group.

Serum DcR3 is decreased in response to effective treatment in patients with UC Based on our previous results, we, next, hypothesized that when patients with active UC and detectable DcR3 enter remission there should be a significant decrease in serum DcR3. We, therefore, compared serum DcR3 concentrations in 11 individuals with active UC before and after treatment, which led to clinical remission (Fig. 2). The latter was induced medically in 10 patients and surgically in the remaining one patient. The average interval between the two measurements was 5.1 ± 4.3 months. Our results clearly showed that post-treatment DcR3 values were significantly reduced when compared with the respective pre-treatment ones (0, 0–1840.9 pg/ml vs. 822.5, 333.8–4951.1, median, 95% CI, P = 0.006) (Fig. 2, left panel). Serum concentration of DcR3 was decreased post-treatment

DcR3 mRNA expression is upregulated in the inflamed intestinal mucosa of patients with UC Next, we hypothesized that if the increase in serum DcR3 levels was the result of intestinal inflammation, DcR3 expression should be elevated locally, i.e. in the colon of patients with UC. To test our hypothesis, we compared the relative DcR3 mRNA expression between pairs of samples obtained at endoscopy from affected and non-affected colonic segments from the same UC patient. Results from these experiments are shown in Figure 3. There was a highly significant difference in the relative number of DcR3 transcripts between non-affected and inflamed colonic mucosa (mean ± SD: 12.5 ± 10.4 vs. 144.3 ± 97.6, respectively, P = 0.002). Overall, the relative average DcR3 mRNA elevation was 14.7 ± 11.2 (range 1.5–38.3). Since TNF-α is a molecule with accepted involvement in active UC, we also compared the relative expression of TNF-α (Fig. 3) between the two groups of samples. In comparison to DcR3, we observed a clear difference between affected and nonaffected areas regarding TNF-α expression. The relative average TNF-α mRNA elevation was 4.8 ± 7.3 (range 0.6–25.3), the difference being highly significant (relative expression of TNF-α mRNA; non-affected: 74.1 ± 46.4, affected: 293.5 ± 316.6, P = 0.006). Interestingly, upregulation of DcR3 was more prominent than that of TNF-α. Since comparisons were performed between paired samples, which only differed in the presence or absence of inflammation, these data confirm that upregulation of DcR3 related only to the presence of intestinal inflammation.

Figure 2 Serum concentration of DcR3 is decreased following effective treatment in patients with UC. Concentration of DcR3 was measured by ELISA in sera obtained from 11 patients with active UC, before and after treatment that resulted in disease remission (left graph). Serum DcR3 became undetectable in 7/11 patients (64%) after treatment and was overall significantly decreased (P b 0.01 vs. pre-treatment). Serum CRP level, which served as an indicator of decrease in inflammation was also measured before and after treatment and values are shown in the right graph. Serum values of DcR3 or CRP in the same patients before and after treatment were compared by Wilcoxon test (paired comparison). Horizontal lines represent the mean expression for each group.

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Figure 3 DcR3 mRNA transcripts are significantly increased in affected colonic segments of patients with UC. Mucosal biopsies were obtained during colonoscopy from normal-looking and inflamed segments of the colon. Ten patients with UC were studied. Total RNA was extracted from intestinal tissue and cDNA generated as described in Materials and methods. Relative DcR3 (left) and TNF-α (right) mRNA expression were determined by real-time RT-PCR. Expression was normalized against abl expression (internal control). There was a highly significant increase in the expression of DcR3 in areas with inflammation as compared to normal-looking ones (P b 0.005). P values refer to Wilcoxon test (paired comparison). Horizontal lines represent the mean expression for each group.

Soluble TL1A, a ligand for DcR3 is elevated in the serum of patients with active UC Since our tissue and serum data clearly showed local and systemic elevation of DcR3, we, next, examined whether the concentration of circulating TL1A, a TNF-like cytokine with the ability to bind to DcR3, is also altered during active UC. Serum TL1A concentrations were significantly elevated in patients with active UC (432.4, 174–7691.5 pg/ml, median, 95% CI) and also in patients with UC in remission (389.3, 0–6654.8 pg/ml) as compared to healthy controls (187.4, 0–2166.5, P b 0.001 for both comparisons) (Fig. 4). The average concentrations for DcR3 were comparable between the UC-active and UC-remission groups. This indicates that

Figure 4 Serum TL1A is elevated during both active and quiescent UC. Sera were obtained from UC patients with active (n = 29) or inactive (n = 35) disease and healthy controls (n = 56). Concentration of TL1A was measured by ELISA. Levels of TL1A were significantly higher in patients with active UC (median, 95% CI: 432.4, 174–7691.5 pg/ml) or with UC in remission (389.3, 0– 6654.8 pg/ml) as compared to healthy controls (187.4, 0– 2166.5, P b 0.001 for both comparisons). No significant difference was observed between UC-active and UC-remission groups. P values refer to Mann–Whitney test (for 2-group comparison). Horizontal lines represent median values for each group.

the increase in TL1A is not solely related to the presence of intestinal inflammation. Taken together, our results indicate a clear and generalized upregulation of the TL1A/DcR3 ligand/receptor pair in active UC.

Discussion In the present study we report that patients with active UC have increased intestinal and systemic levels of DcR3, a member of the TNF receptor superfamily. We provide evidence that intestinal inflammation is the source of this elevation as DcR3 transcripts are significantly increased in affected areas of the colon and remission of colitis leads to significant reduction or disappearance of circulating DcR3. Finally, we demonstrate a parallel increase in the serum concentration of TL1A, a novel cytokine that acts as ligand for DcR3, in both active and quiescent UC. We observed a remarkable increase in the number of DcR3 transcripts in mucosal biopsies obtained from areas with colonic inflammation. To avoid interindividual variability, mRNA comparisons were done between pairs of affected and non-affected areas from the same individual. Therefore, interference of genetic variability was omitted and upregulation of DcR3 in afflicted areas was solely associated with the presence of mucosal inflammation. This is important in light of a recent study which showed that polymorphisms in the tnfsf6b gene (DcR3) are carried by a subgroup of UC patients and may, actually, affect the expression level of DcR3 [25]. Although the authors reported a correlation between these polymorphisms and pediatric onset UC, in our study we did not observe an association between age at disease onset or duration of UC and DcR3 levels. Nevertheless, similarly to our results, increased expression of DcR3 mRNA in affected areas of IBD patients was also noted in the aforementioned genetic study. In an earlier study, Fayad et al. reported augmented DcR3 protein expression by immunohistochemistry, in the intestinal lamina propria of patients with UC [26]. In addition to enhanced mucosal expression, we observed a significant elevation of circulating DcR3 in patients with active UC. DcR3 presumably exists only in soluble form as it

High intestinal and systemic levels of decoy receptor 3 and its ligand TL1A in ulcerative colitis lacks a transmembrane domain [18]. Under healthy conditions, DcR3 usually is not detected in the serum, as has been constantly shown in previous studies [24,27]. In sharp contrast, as our results show, during flares of UC, DcR3 was present in the serum in substantial concentrations. Our findings support the hypothesis that elevation of soluble DcR3 is driven by mucosal inflammation. First, similar to healthy controls, the majority of patients with UC did not have detectable serum DcR3. Second, non-inflamed parts of the colon had significantly lower expression of DcR3. Third, when inflammation was abrogated with treatment, DcR3 became undetectable in the sera of most patients. Therefore, it appears that the presence of intestinal inflammation is the critical factor that induces elevation of DcR3 both locally and systemically. In that sense it was interesting to note that it was not the extent of the disease (proctitis vs. left-sided vs. extensive colitis) but the inflammatory activity that induced DcR3 increase. In support of our findings, published studies have also shown elevated local or systemic expression of DcR3 in other inflammatory conditions, including appendicitis [28], systemic lupus erythematosus [29], rheumatoid arthritis [24,30], and bacterial infections [31]. We speculate that, in UC, detectable DcR3 in the serum may serve as a marker of ongoing intestinal inflammation. Interestingly, on several cases we observed elevated values of serum DcR3 whereas CRP was within normal limits. This lack of correlation between DcR3 and CRP, which was the case in other inflammatory conditions as well, may indicate that DcR3 could represent a more specific serum marker of tissue inflammation. Further studies are needed to examine whether the presence of mildly elevated DcR3 in a minority of patients in remission indicates the presence of residual subclinical intestinal inflammation or, alternatively, is the result of the aforementioned genetic variability. Given the immunosuppressive role that is attributed to decoy receptors [32], the local and systemic upregulation of DcR3 during a pro-inflammatory state such as active UC is, at first, surprising. One possible explanation is that this increase is a compensatory mechanism, aiming to downregulate an excessive pro-inflammatory response driven by the DcR3 ligand, TL1A. The latter has been shown to be directly involved in clinical and experimental intestinal inflammation [14,15,21,33]. This is supported not only by our findings of increased circulating TL1A in patients with UC, but also of elevated expression of TL1A mRNA and protein at the mucosal level by other groups [13,34]. Differently from DcR3, its ligand TL1A was elevated in the sera from patients with either active or quiescent UC. This may indicate that TL1A may be an IBD specific molecule. In contrast, DcR3 upregulation appears to represent a secondary response to the presence of inflammation. This may explain why we observed no association between DcR3 and TL1A levels (data not shown). It follows that the predominance of proinflammatory pathways during UC implies a relative insufficiency of DcR3 to suppress intestinal inflammation. A second explanation for the increased expression of DcR3 in UC is offered by emerging recent data which points to the possibility that DcR3 may be involved in the induction and maintenance of chronic pro-inflammatory pathways. First, DcR3 skews the immune response towards a Th2, i.e. IL-4, IL-5, and IL-13 dominated, phenotype, possibly through modulation of dendritic cell function and in response to

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microbial stimuli [35,36]. Second, DcR3 impairs the phagocytic activity of macrophages and their response to LPS [37]. Third, DcR3 upregulates the expression of adhesion molecules, in particular α4 integrin on macrophages [38]. These functions may be of particular importance during active UC, as defective microbial clearance by cells of the innate immunity at the mucosa, enhanced inflammatory cell trafficking, as well as excessive Th2 pathways dominated by IL-13 production are considered central to the pathogenesis of UC [39]. Finally, induction of anti-apoptotic pathways may explain the involvement of DcR3 in mucosal inflammation. DcR3 inhibits apoptosis by blocking TL1A/DR3, LIGHT/ HVEM, and FAS-L/FAS interaction [11,17,18]. Abrogation of apoptosis in effector immunocytes at the intestinal mucosa would offer them a survival advantage and help them remain activated, thus perpetuating inflammation, and mediating tissue injury. In fact, defective apoptosis of activated lymphocytes that infiltrate the lamina propria of patients with UC has been previously shown [40]. Interestingly, it was reported that effector T cells at the lamina propria of patients with UC highly express DcR3, along with other decoy receptors, and are rendered resistant to apoptosis [26]. Similarly, in systemic lupus erythematosus DcR3 was shown to enhance T-cell proliferation while concomitantly abrogating activation-induced apoptosis, having an overall proinflammatory effect [29]. A recent study has shed light on the possible role that DcR3 may play during chronic intestinal inflammation, and in particular CD [21]. Similarly to our results in UC, Funke et al. reported increased mucosal expression as well as elevated circulating levels of DcR3 in patients with CD. The authors proposed that DcR3 may have a dual function by inducing both pro-inflammatory, NF-κB-mediated, and anti-apoptotic signals. An interesting finding from the previous study was that DcR3 protein localized primarily to the epithelial layer of the intestine and inhibited CD95L-induced apoptosis in epithelial cells [21]. A similar anti-apoptotic function of DcR3 may also take place in UC. This combined with the increased mucosal expression of DcR3 shown in our study, even in the presence of low-grade inflammation, may point to a possible role of DcR3 in the increased colonic carcinogenesis that is observed in UC. In conclusion, we provide evidence for increased intestinal and systemic expression of DcR3 and TL1A during active UC. Our results further expand recent data on the significance of DcR3 in CD and of TL1A and its functional receptor DR3 in both forms of IBD. Overall, these data support a central role of the TL1A/DR3/DcR3 system in the pathogenesis of acute and chronic intestinal inflammation. These molecules may represent markers of active inflammation in this patient population. More importantly, modification of such pathways may be of therapeutic significance for IBD.

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