Expression Patterns of TNFα, MAdCAM1, and STAT3 in Intestinal and Skin Manifestations of Inflammatory Bowel Disease

Expression Patterns of TNFα, MAdCAM1, and STAT3 in Intestinal and Skin Manifestations of... Abstract Background Pathogenesis of cutaneous extraintestinal manifestations [EIM] in inflammatory bowel disease [IBD] remains elusive. Efficacy of anti-TNF agents suggests TNF-dependent mechanisms. The role of other biologics, such as anti-integrins or JAK-inhibitors, is not yet clear. Methods We performed immunohistochemistry for TNFα, NFκB, STAT1/STAT3, MAdCAM1, CD20/68, caspase 3/9, IFNγ, and Hsp-27/70 on 240 intestinal [55 controls, 185 IBD] and 64 skin biopsies [11 controls, 18 erythema nodosum [EN], 13 pyoderma gangenosum [PG], 22 psoriasis]. A semiquantitative score [0–100%] was used for evaluation. Results TNFα was upregulated in intestinal biopsies from active Crohn`s disease [CD] vs controls [36.2 vs 12.1, p < 0.001], but not ulcerative colitis [UC: 17.9]. NFκB, however, was upregulated in intestinal biopsies from both active CD and UC [43.2 and 34.5 vs 21.8, p < 0.001 and p = 0.017, respectively]. TNFα and NFκB were overexpressed in skin biopsies from EN, PG, and psoriasis. No MAdCAM1 overexpression was seen in skin tissues, whereas it was upregulated in active UC vs controls [57.5 vs 35.4, p = 0.003]. STAT3 was overexpressed in the intestinal mucosa of active and non-active IBD, and a similar upregulation was seen in skin biopsies from EN [84.7 vs 22.3, p < 0.001] and PG [60.5 vs 22.3, p = 0.011], but not in psoriasis. Caspase 3 and CD68 overexpression in skin biopsies distinguished EN/PG from psoriasis and controls. Conclusions Upregulation of TNFα/NFκB in EN and PG is compatible with the efficacy of anti-TNF in EIM management. Data on overexpressed STAT3, but not MAdCAM1, support a rationale for JAK-inhibitors in EN and PG, while questioning the role of vedolizumab. Extraintestinal manifestations, anti-TNF, anti-integrins, JAK-inhibitor, immunohistochemistry, microarray 1. Introduction Inflammatory bowel disease [IBD], with the two main subtypes Crohn`s disease [CD] and ulcerative colitis [UC], is a chronic inflammatory disorder of the gastrointestinal tract. The aetiopathogenesis of IBD is incompletely understood, although it is considered a multifactorial disease which arises from a complex interplay between genetic, environmental, and immunological factors, with an abnormal host immune response to environmental stimuli.1 Different cytokines and cell interaction proteins have been identified as key players in IBD pathogenesis, such as the TNFα-NFκB axis, the JAK-STAT pathway, and the integrin-vascular adhesion molecule interaction. Several already approved drugs or investigational agents take advantage of these pathomechanisms. Anti-TNF agents have been successfully used for more than a decade, whereas anti-integrins such as vedolizumab have been introduced into clinical practice only recently.2,3 The latter target the gut-specific interaction between integrin α4β7 on leukocytes and the adhesion molecule MAdCAM1 on endothelial cells in the intestine, thereby blocking leukocyte adhesion and migration to the site of inflammation. Few, if any, systemic side effects are observed.2,3 Tofacitinib represents a first-class oral agent inhibiting the janus kinase [JAK] family of proteins, which are important mediators in inflammation. Blocking JAK downregulates proinflammatory cytokines, such as interleukin [IL] 2, 4, 7, 9, 15, and 21, through the JAK-STAT pathway.4 Efficacy has been reported for UC but not for CD, but more specific JAK inhibitors such as filgotinib may also be efficacious in the latter indication.5–7 Approval process for tofacitinib`s use in UC patients has been recently initiated by the US Food and Drug Administration [FDA]. Extraintestinal manifestations [EIM] of IBD are common, with a frequency ranging from 6% to 47%.8–15 Besides arthritis and stomatitis, cutaneous manifestations are among the most prevalent EIM.15 IBD skin lesions mainly include erythema nodosum [EN] and pyoderma gangrenosum [PG]. Whereas EN usually parallels intestinal disease activity, PG may or may not be associated with intestinal disease. The pathomechanisms of EN and PG remain elusive, although efficacy of anti-TNF treatment suggests TNF-dependent mechanisms. Limited data from immunohistochemical evaluations and protein analyses from tissue lysates have demonstrated upregulation of several inflammatory cytokines, such as IL 8, 17, and 1β and TNFα in PG.16,17 Evidence for the efficacy of anti-TNF in EIM management is evolving. Recently, a thorough meta-analysis conducted by Peyrin-Biroulet and colleagues has shown response rates between 69% and 100% for PG and reduction of EN prevalence from 2.4% to 0.4%, with anti-TNF treatment.18,19 Whether other biologic agents are efficacious in the treatment of EN or PG has yet to be determined. Studies evaluating the role of vedolizumab are under way. It is yet unclear if the gut-selective mode of action limits its efficacy to the intestine or if indirect beneficial effects via decrease of intestinal inflammatory load might be relevant. No data are available so far regarding the influence of JAK inhibitors on EIM, although tofacitinib`s efficacy has been shown for other auto-inflammatory disorders such as rheumatoid arthritis and psoriasis.20,21 We herein investigated different proteins involved in intestinal and extraintestinal IBD manifestations, aiming at elucidating similarities and differences in IBD, cutaneous EIM, and psoriasis pathophysiology, with a special focus on possible therapeutic implications such as involvement of the TNFα-NFκB and JAK-STAT pathway as well as the integrin-MAdCAM interaction. 2. Methods 2.1. Study design In this observational single-centre study, we prospectively collected intestinal and cutaneous tissue samples from healthy individuals and patients with IBD, cutaneous EIM, and psoriasis. Sample collection was conducted between 2004 and 2011. All patients had previously given their written informed consent for tissue collection and review of patient charts. All data were anonymised. The study was supported by the Swiss National Science Foundation and was approved by the local ethics committee of the University Hospital Zurich [KEK-ZH-837]. 2.2. Patient and data collection All patients analysed in this study were treated for IBD and/or inflammatory skin disease such as EN, PG, or psoriasis, at the University Hospital Zurich, Switzerland. Diagnosis of UC and CD had been previously established according to the current ECCO guidelines.22,23 Inflammatory skin lesions had all been diagnosed by a dermatologist. Intestinal samples from healthy controls were collected at regular screening colonoscopies at the University Hospital Zurich. Patients had to be older than 18. Endoscopic disease activity was assessed using the Mayo Clinic endoscopy subscore for UC and the CD endoscopic index of severity [CDEIS] for CD.24–26 A score of ≥ 1 [Mayo Score] or ≥ 3 [CDEIS] was considered as active disease.25,27 Patients were excluded for: i] biologic treatment with anti-TNF within the past 8 weeks; ii] ongoing treatment for inflammatory skin disorder [topical or systemic]; iii] concomitant autoimmune disorders such as systemic lupus erythematodes or vasculitis; and iv] concomitant skin disorder such as eczema, atopic dermatitis, or paradoxical anti-TNF induced skin lesions. Clinical data were retrieved from paper-based and electronic patient records. The following data were collected: patient demographics [gender, age]; disease history; previous and current medications; and comorbidities. For this purpose, a standardised spreadsheet was used [see Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. 2.3. Tissue sample collection For intestinal samples, biopsies were taken from the terminal ileum [TI], caecum, and colon using a needle forceps. For skin samples, biopsies were taken from the affected lesion using a 4–6 mm surgical punch that collected epidermis, dermis and the upper subcutis. For healthy control skin, fresh, non-affected, and non-inflamed skin of unrelated surgical excisions was used. Biopsies were formalin-fixed, paraffin embedded and stored at -80°C for further use. 2.4. Tissue microarray and immunostaining Tissue microarrays [TMA] were constructed using formalin-fixed paraffin-embedded tissue blocks, which had been punched out from ileal, caecal, colonic, sigmoid, or skin tissue by a tissue cylinder [0.6 mm in diameter], as previously described.28,29 Immunohistochemical staining was performed using the automated system BOND RX [Leica Biosystems]. TMA sections were deparaffinised and rehydrated in dewax solution [Leica Biosystems]. Immunohistochemistry was carried out for the TNF-NFκB [TNFα, NFκB] and JAK-STAT pathway [STAT1, STAT3] as well as the MAdCAM-integrin interaction [MAdCAM1], which all represent targets of IBD treatment modalities. We further investigated markers for B lymphocytes [CD20], macrophages [CD68], Th1-cytokine mediated disease [IFNγ], apoptosis [caspase 3 and 9], and inflammation-induced anti-apoptotic factors [Hsp-27/70]. The following primary antibodies were used: TNFα [Abcam, ab6671, dilution 1:50], NFκB p65 [Abcam, ab32360, dilution 1:100], MAdCAM1 [clone CA102.2CI, Affimetrix BMS170, dilution 1:1000], STAT1 [R&D Systems, MAB1490, dilution 1:50], STAT3 [Abcam, ab50761-100, dilution 1:100], CD20 [Ventana Roche, prediluted], CD68 [Ventana Roche, prediluted], caspase 3 [Abcam, ab4051, dilution 1:200], caspase 9 [Invitrogen, PA5-17913, dilution 1:200], IFNγ [BD Biosciences, 560371, dilution 1:100], Hsp-27 [Leica Biosystems, NCL-HSP27, dilution 1:40], and Hsp-70 [Enzo Life Sciences, dilution 1:100]. Antibody was detected with the Bond Polymer Refine Detection kit [Leica Biosystems], using DAB [3-3’-diaminobenzidine] as chromogen and following the manufacturer’s instructions. A semiquantitative scoring system ranging from 0% to 100% [5% intervals] was used for evaluation of immunostaining.29,30 Scoring was performed by two reference pathologists in the field of tissue microarrays [AL, HD]. TMA review was blinded. All samples and controls were run in duplicate. Failure of analysis occurred in 15.1% [796/5040 spots] of all gastrointestinal samples and in 9.4% [72/765 spots] of skin samples. Reasons for such failure were missing samples [empty spots on microarray] or TMA technology. 2.5. Statistical analysis For all statistical analyses, IBM software SPSS [version 22.0.0, 2013 SPSS Science, Chicago, IL] was used. Each biopsy sample was analysed individually. Metric data are presented as means and standard deviation [SD]. Categorical data are depicted as percentage of the group total. For comparisons between continuous variables, two-sample t test and Mann-Whitney U test were used depending on whether data were normally distributed or not. A two-sided p-value of < 0.05 was regarded as statistically significant. 3. Results 3.1. Tissue samples A total of 304 intestinal and skin tissue samples were collected; 240 intestinal biopsies were taken from a total of 106 patients. These samples showed active endoscopic IBD in 108 cases and endoscopic inactivity in 77 cases; 55 intestinal biopsies were taken from 29 healthy controls; 64 skin samples were collected from 64 patients: 53 patients had an inflammatory skin disorder [18 EN, 13 PG, 22 psoriasis] whereas 11 patients [controls] had normal skin. Two of the 53 patients with an inflammatory skin disorder [1 EN and 1 PG] had corresponding intestinal biopsies showing active ileocaecal CD [for EN] and active UC pancolitis [for PG]. In detail, the following samples were analysed: 74 from TI [19 HC, 19 CD inflamed, 13 CD not inflamed, 23 UC not inflamed], 79 from the caecum [21 HC, 17 CD inflamed, 13 CD not inflamed, 28 UC not inflamed], 57 from the sigmoid colon [15 HC, 42 UC inflamed], 30 from the colon [30 UC inflamed], and 64 from the skin [11 HC, 18 EN, 13 PG, 22 psoriasis]. 3.2. Patient demographics Of the samples, 156 [51.3%] were taken from male patients, whose mean age was 43.7 years [SD 9.7]. Patients with PG were significantly older, and there were significantly less males in the EN group. The other groups were comparable regarding gender and age. Of the 185 IBD intestinal samples, 36 showed endoscopically active CD, 26 non-active CD, 72 endoscopically active UC, and 51 non-active UC. Of the 72 samples with endoscopic UC activity, 31 were collected from patients with pancolitis and 41 from patients with left-sided UC. All of the 36 samples with active CD were taken from ileal [n = 19] or caecal [n = 17] disease, and the samples for inactive CD were collected from non-inflamed TI [n = 13] or non-affected caecum [n = 13]. Two IBD patients reported a previous anti-TNF treatment which was, however, stopped more than 8 weeks before biopsy. At the time of skin biopsy, none of the patients with skin disorders were treated with systemic or topical anti-inflammatory drugs. None of them had a history of a previous use of biologic treatment. None of the patients with psoriasis had underlying IBD. Table 1 summarises demographic data and previous medication of all patients and the respective subgroups. Table 1. Demographic data and previous medication.   All samples n = 304  Intestinal samples from IBD n = 185  Skin samples from EN n = 18  Skin samples from PG n = 13  Skin samples from psoriasis n = 22  Skin and intestinal samples from controls n = 66  Age  Mean in years [SD]  43.7 [9.7]  41.9 [14.5]  43.8 [20.7]  61.0 [17.4]  48.7 [16.3]  43.1 [10.6]  Males  156 [51.3%]  101 [54.6%]  5 [27.8%]  6 [46.2%]  12 [54.5%]  32 [48.5]  Concurrent medications   -none  164 [53.9%]  50 [27.0%]  18 [100%]  13 [100%]  22 [100%]  61 [92.4%]   -5-ASA  107 [35.2%]  106 [57.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  1 [1.5%]   -topical steroids  15 [4.9%]  15 [8.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -systemic steroids  21 [6.9%]  21 [11.4%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antibiotics  10 [3.3%]  8 [4.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]   -AZA  9 [3.0%]  9 [4.9%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antihistamines  4 [1.3%]  4 [2.2%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -biologics  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -NSAR  3 [1.0%]  1 [0.5%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]  Previous biologic treatment   -anti-TNF  2 [0.7%]a  2 [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -vedolizumab  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -JAK inhibitor  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  Surgical history  2 [0.7%]b  2b [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]    All samples n = 304  Intestinal samples from IBD n = 185  Skin samples from EN n = 18  Skin samples from PG n = 13  Skin samples from psoriasis n = 22  Skin and intestinal samples from controls n = 66  Age  Mean in years [SD]  43.7 [9.7]  41.9 [14.5]  43.8 [20.7]  61.0 [17.4]  48.7 [16.3]  43.1 [10.6]  Males  156 [51.3%]  101 [54.6%]  5 [27.8%]  6 [46.2%]  12 [54.5%]  32 [48.5]  Concurrent medications   -none  164 [53.9%]  50 [27.0%]  18 [100%]  13 [100%]  22 [100%]  61 [92.4%]   -5-ASA  107 [35.2%]  106 [57.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  1 [1.5%]   -topical steroids  15 [4.9%]  15 [8.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -systemic steroids  21 [6.9%]  21 [11.4%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antibiotics  10 [3.3%]  8 [4.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]   -AZA  9 [3.0%]  9 [4.9%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antihistamines  4 [1.3%]  4 [2.2%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -biologics  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -NSAR  3 [1.0%]  1 [0.5%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]  Previous biologic treatment   -anti-TNF  2 [0.7%]a  2 [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -vedolizumab  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -JAK inhibitor  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  Surgical history  2 [0.7%]b  2b [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  5-ASA, 5-aminosalicylates; AZA, azathioprine; EN, erythema nodosum; IBD, inflammatory bowel disease; NSAR, non-steroidal antirheumatic agent; PG, pyoderma gangrenosum; SD, standard deviation. a Treatment was stopped 8 weeks before biopsies. b Ileocecal resection (1) and partial colectomy (1). View Large 3.3. Expression of the TNFα/NFκB pathway in intestinal disease and cutaneous manifestations Subepithelial TNFα expression was significantly higher in active CD compared with controls [36.2 vs 12.1, p < 0.001], but was not elevated in inactive CD (20.9 vs 12.1, not significant [n.s.]). Difference between active and inactive CD was also significant [p = 0.019]. No difference was seen regarding TNFα expression in UC patients [regardless of disease activity] vs controls [17.9 and 13.1 vs 12.1, respectively, n.s.]. TNFα expression was significantly higher in CD than in UC [36.2 vs 17.9, p = 0.001]. However, NFκB, a downstream signal of TNF, was upregulated in both active UC [34.5] and active CD [43.2] vs controls [21.8, p = 0.017, p < 0.001, respectively] with no significant differences between the two diseases. In inactive disease, NFκB was not significantly elevated in either CD or UC. Similarly to what was seen in intestinal samples, subepithelial TNFα was overexpressed in all inflammatory skin disorders [EN 25.6, PG 37.7, psoriasis 14.7] compared with controls [3.2, p < 0.001, p = 0.001, and p = 0.001, respectively]. PG demonstrated even higher TNFα expression levels than psoriasis [37.7 vs 14.7, p = 0.022], and for EN vs psoriasis at least a clear trend was seen [25.6 vs 14.7, p = 0.052]. Similar results were observed for NFκB expression with, however, no difference between PG and psoriasis, whereas upregulation of NFκB was significant in EN vs psoriasis [p = 0.002]. NFkB expression was 41.8 in EN, 25.0 in PG, 14.3 in psoriasis, and 5.9 in healthy controls. For details regarding TNFα and NFκB expression, see Figures 1 and 2. Figure 1. View largeDownload slide Mean TNF expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 1. View largeDownload slide Mean TNF expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 2. View largeDownload slide Mean NFkB expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 2. View largeDownload slide Mean NFkB expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. 3.4. Expression of MAdCAM1 MAdCAM1 was upregulated in inflamed intestinal tissue. Compared with healthy controls, active UC showed a significant MadCAM1 overexpression [57.5 vs 35.4, p = 0.003]. MadCAM1 was also upregulated in active UC when compared with inactive UC [57.5 vs 39.3, p = 0.027]. Expression from intestinal samples with active CD did not show significant differences when compared with healthy controls [30.8 vs 35.4]. In contrast to the upregulation of MAdCAM1 in UC patients, no overexpression compared with healthy controls was seen for EN, PG, or psoriasis. In fact, expression levels of MAdCAM1 were below the threshold of 10% in all samples with inflammatory skin disorders. Figure 3 shows representative tissue samples from the intestine [IBD and healthy controls, Figure 3a] and inflammatory skin diseases [Figure 3b], stained for TNFα and MAdCAM1. Representative tissue samples for inactive intestinal disease stained for TNFα and MAdCAM1 are depicted in Supplementary Figure 1, available as Supplementary data at ECCO-JCC online. For expression levels of intestinal samples see Supplementary Figure 2, available as Supplementary data at ECCO-JCC online. Figure 3. View largeDownload slide Immunohistochemical analysis of intestinal and skin tissue tissue microarrays [TMA] for TNF and MAdCAM1 protein expression: a] intestinal tissue; A-C: MAdCAM1 immunostaining, D-F: TNF immunostaining, A/D: normal colon, B/E active CD, C/F active UC; b] skin tissue: A-D: MAdCAM1 immunostaining, E-H: TNF immunostaining, A/E: normal skin, B/F: erythema nodosum, C/G: pyoderma gangrenosum, D/H: psoriasis. Figure 3. View largeDownload slide Immunohistochemical analysis of intestinal and skin tissue tissue microarrays [TMA] for TNF and MAdCAM1 protein expression: a] intestinal tissue; A-C: MAdCAM1 immunostaining, D-F: TNF immunostaining, A/D: normal colon, B/E active CD, C/F active UC; b] skin tissue: A-D: MAdCAM1 immunostaining, E-H: TNF immunostaining, A/E: normal skin, B/F: erythema nodosum, C/G: pyoderma gangrenosum, D/H: psoriasis. 3.5. Expression of STAT proteins in intestinal disease and cutaneous manifestations STAT3, which is involved in the JAK-STAT pathway and which can be blocked by JAK inhibitors such as tofacitinib, was elevated in both active and inactive CD [73.4 and 81.5 vs 60.5, p = 0.028 and p = 0.002, respectively]. The same results were seen for active and inactive UC [79.2 and 72.7 vs 60.5, p < 0.001 and p = 0.012, respectivle]. There was no difference between active and inactive disease, nor between UC and CD [n.s.]. Of note, the same significant upregulation of STAT3 was observed in EN [84.7] and PG [60.5] compared with controls [22.3] and psoriasis [26.8]. STAT3 was even more highly expressed in EN compared with PG [p = 0.033]; for details see Figure 4. Although there was also some significant overexpression of STAT1 in EN, absolute numbers [EN 17.1, PG 1.4, psoriasis 2.9, controls 0.0] were considerably lower than those seen for STAT3. Figure 4. View largeDownload slide Mean STAT3 expression in intestinal and skin tissue. Standard error of the mean bars are shown; p -value calculated by Mann-Whitney U test. Figure 4. View largeDownload slide Mean STAT3 expression in intestinal and skin tissue. Standard error of the mean bars are shown; p -value calculated by Mann-Whitney U test. 3.6. Expression of other proteins We assessed the expression of several other proteins involved in inflammatory processes and cell injury. Caspase 3 upregulation significantly distinguished PG and EN from healthy controls and psoriasis: caspase 3 was higher in both EN [20.0] and PG [17.7] vs in psoriasis [2.8, p < 0.001 and p = 0.002, respectively] and in controls [0.9, p < 0.001 and p < 0.001, respectively]. Caspase 9 was overexpressed in EN [13.1], PG [10.9], and psoriasis [6.0] compared with controls [2.3], although upregulation in PG was not significant due to a high variation. Nonetheless, these results highlight caspases as potential targets in EIM management. No upregulation of IFNγ in intestinal disease nor in skin lesions was seen, but there was a significant overexpression of CD68 in inflammatory skin disorders [EN 54.7, PG 32.3, psoriasis 10.2 vs controls 3.9, p < 0.001, p < 0.001, and p = 0.003, respectively]. Upregulation was significantly higher in EN compared with PG [p = 0.042] and psoriasis [p < 0.001]. Difference between PG and psoriasis was also significant, highlighting the possible pathogenic role of CD68 and therefore of macrophages in PG and EN pathophysiology, but not in psoriasis. However, absence of CD68 overexpression in intestinal IBD, as well as low absolute numbers of CD20 [controls 0.0, EN 2.2, PG 2.1, psoriasis 1.4] and Hsp-27 expression [controls 5.9, EN 11.8, PG 6.8, psoriasis 4.5] do not allow clear conclusions. Hsp-70 did not show any overexpression in the skin samples [controls 8.2, EN 9.4, PG 8.2, psoriasis 5.6]. Expression levels of caspase 3, caspase 9, Hsp-27, Hsp-70, CD20, CD68, and IFNγ are depicted in Supplementary Figure 3, available as Supplementary data at ECCO-JCC online. 4. Discussion This single-centre observational study, with assessment of protein expression patterns in IBD, cutaneous EIM, and psoriasis compared with healthy controls, shows similar upregulation of TNFα and STAT3 in both intestinal and cutaneous disease, but absence of MAdCAM1 overexpression in inflammatory skin disorders. TNFα and STAT3 are both considerably upregulated in EN and PG, with even higher expression levels compared with psoriasis. Protein expression patterns of EN and PG show many similarities, with only few differences revealing important therapeutic implications. The TNFα-NFκB axis is overexpressed in both intestinal disease and inflammatory skin disorders, with an even more pronounced upregulation in PG and EN compared with psoriasis. TNFα was recognised as an important inflammatory parameter in different autoimmune diseases, including IBD and psoriasis, many years ago. Anti-TNF agents take advantage of this canonical inflammatory pathway. They have been successfully introduced in the past decade and have changed IBD management dramatically. Their potential beneficial role in EIM management had been first proposed based on small case series and case reports.31 Very recently, Peyrin-Biroulet and colleagues nicely summarised their efficacy in a meta-analysis demonstrating high response rates for PG [69–100%] and EN [prevalence reduction from 2.4% to 0.4%]. Complete remission rates for PG were however lower [25–100%].18,19 PG actually is the only cutaneous IBD manifestation where a randomised controlled trial is available for showing clinical improvement in 69% and a complete remission in 25% of the patients after an induction treatment with infliximab.32 Further data from the Swiss IBD cohort study support these data, demonstrating improvement rates of 60% for PG and 80% for EN.33 This has led to the current understanding of a TNF-dependent pathogenesis in cutaneous EIM. In PG samples, TNFα upregulation has been reported previously, but no such data are available for EN. Our data showing overexpression in both PG and EN, even higher levels when compared with psoriasis, are compatible with the known efficacy of anti-TNF efficacy in cutaneous EIM.16 Although the TNFα-NFκB axis was upregulated in both CD and UC with a particular overexpression of intracellular NFκB, TNFα was actually only significantly increased in intestinal samples from active CD, but not from UC. There are three possible explanations: 1] the finding that TNFα levels in the lamina propria are indeed higher in CD than UC has been already described more than two decades ago;34 2] for detection of the small difference between healthy controls and UC, our study was probably underpowered; and 3] other factors such as bacterial products [e.g. lipopolysaccharides, LPS] can lead to NFκB upregulation independent of TNFα, so [over]expression of the two proteins do not necessarily go in parallel. The anti-integrin vedolizumab has been introduced into clinical practice very recently. Data from randomised controlled trials demonstrated vedolizumab`s efficacy in both CD and UC.2,3 Vedolizumab`s role in EIM management has not yet been defined. Its gut-selective mechanism advocates against, whereas some preliminary data presented at recent congresses advocate for, an at least partial efficacy in the treatment of EIM.35 Consistently with the literature, we herein report on isolated MAdCAM1 expression in the inflamed intestine, but not in inflammatory skin disorders, which makes a direct effect on cutaneous EIM extremely unlikely. However, vedolizumab might have a role in EIM management by treating intestinal disease activity in EIM that parallels IBD. The third biologic, which is currently being reviewed by the US FDA for treatment in UC patients, is the JAK inhibitor tofacitinib, which has been successfully used in other diseases such as rheumatoid arthritis and psoriasis.20,21 Tofacitinib inhibits the JAK-STAT pathway which incites pro-inflammatory signals. Biopsies from active intestinal disease and inflammatory skin disorders demonstrated similar results regarding STAT3 overexpression in our patient cohort. Difference between healthy controls and inflamed tissue was even more prominent in skin biopsies. Of note, STAT3 was significantly more upregulated in PG/EN than in psoriasis, shedding light on the potential benefit from JAK inhibition in those patients. Clinical data are not available so far, but our findings make a therapeutic response to JAK inhibitors very likely. Given tofacitinib`s current blackbox warning for serious infections, possible systemic side effects from pan-JAK inhibitors should be kept in mind; these, however, occur less frequently with more specific JAK inhibitors such as filgotinib. With the microarray approach, several other interesting targets for EIM management have been identified by screening markers for B lymphocytes [CD20], macrophages [CD68], apoptosis [caspase 3 and 9], and inflammation-induced anti-apoptotic factors [Hsp-27/70]. Both caspase 3 and CD68 were overexpressed in EN and PG compared with healthy controls and psoriasis, highlighting the overlap between cutaneous EIM with a protein expression profile distinct from that of other inflammatory skin diseases. Anti-caspases are currently tested in liver diseases [emricasan in non-alcoholic steatohepatitis, NCT02686762], but no data are available so far for EIM or IBD. Our study has several strengths, but also some limitations. To the best of our knowledge, it is so far the only study investigating protein expression patterns in IBD and cutaneous EIM in IBD and comparing them with healthy controls and psoriasis. Microarray is a widely used and reliable tool to study expression of various proteins. Variation due to TMA technology has been limited using only duplicate samples. None of the patients with inflammatory skin disorders was treated with either topical or systemic anti-inflammatory drugs, which makes the TMA results more reliable. In addition, previous anti-TNF treatment was reported in two IBD patients only, and treatment was stopped more than 8 weeks before study evaluation, making interference with biopsy results less likely. All TMA were analysed by experts in TMA evaluation, and involvement of only two pathologists has limited inter-observer variability. We were able to include 13 patients with PG despite its rarity [prevalence 2% of Swiss IBD population, incidence 0.63/100000] due to the close collaboration with our department of dermatology and a study enrolment over several years.15,36 A limitation of the TMA technology is that tissue sample heterogeneity is not completely taken into account. The study was cross-sectional without any follow-up, and therefore changes in expression levels in response to any biologic treatment could not be assessed. The study relied on protein expression levels only, and did not include analysis of mRNA levels. A further drawback of this study is that we only identified MAdCAM1 overexpression in active UC, not in active CD samples. This might be due to: 1] the low number of samples from active CD [n = 36] compared with the relatively high number of active UC [n = 72]; and 2] the inclusion of non-severe CD cases given the very low cut-off of CDEIS ≥ 3.37 Whereas upregulation of TNFα in cutaneous EIM compared with healthy controls is compatible with the known efficacy of anti-TNF in EIM management, the lack of MAdCAM1 overexpression in the human skin, even in the context of inflammation, questions the role of vedolizumab in EIM treatment. Its only effect might be indirect, through inducing and maintaining intestinal disease remission in those EIM parallelling IBD activity. Data on upregulated STAT give a rationale for JAK inhibitors in both EN and PG management, particularly in the current context of missing clinical studies. Data from randomised controlled trials evaluating the effect of anti-integrins and JAK inhibitors on EIM are, however, urgently needed. Funding This work was supported by a research grant from the Swiss National Science Foundation (Swiss IBD Cohort Study [Grant No. 3347CO-108792]). Conflict of Interest None declared. Author Contributions SRV and TG take responsibility for the integrity of the work as a whole, from inception to published article. Author contributions: Study concept [AL, IZ, SRV and TG], data collection [AMS, GR, LB, MBP, MS, SRV, TG], tissue microarray immunohistochemistry and analysis [AL, AS, HD, IZ, JAG, LT], data analysis [AMS, SRV, TG], drafting of manuscript [SRV, TG], critical review of manuscript [AL, AMS, AN, AS, GR, IZ, JAG, LB, LT, MBP, MS]. All authors approved the final version of the manuscript. Supplementary Data Supplementary data are available at ECCO-JCC online. References 1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol  2010; 28: 573– 621. Google Scholar CrossRef Search ADS PubMed  2. Feagan BG, Rutgeerts P, Sands BEet al.  ; GEMINI 1 Study Group. Vedolizumab as induction and maintenance therapy for ulcerative colitis. N Engl J Med  2013; 369: 699– 710. Google Scholar CrossRef Search ADS PubMed  3. Sandborn WJ, Feagan BG, Rutgeerts Pet al.  ; GEMINI 2 Study Group. Vedolizumab as induction and maintenance therapy for Crohn’s disease. 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Med Clin [Barc]  2005; 125: 297– 300. Google Scholar CrossRef Search ADS PubMed  11. Ricart E, Panaccione R, Loftus EVJret al.   Autoimmune disorders and extraintestinal manifestations in first-degree familial and sporadic inflammatory bowel disease: a case-control study. Inflamm Bowel Dis  2004; 10: 207– 14. Google Scholar CrossRef Search ADS PubMed  12. Rankin GB, Watts HD, Melnyk CS, Kelley MLJr. National Cooperative Crohn’s Disease Study: extraintestinal manifestations and perianal complications. Gastroenterology  1979; 77: 914– 20. Google Scholar PubMed  13. Su CG, Judge TA, Lichtenstein GR. Extraintestinal manifestations of inflammatory bowel disease. Gastroenterol Clin North Am  2002; 31: 307– 27. Google Scholar CrossRef Search ADS PubMed  14. Veloso FT, Carvalho J, Magro F. Immune-related systemic manifestations of inflammatory bowel disease. A prospective study of 792 patients. J Clin Gastroenterol  1996; 23: 29– 34. Google Scholar CrossRef Search ADS PubMed  15. Vavricka SR, Brun L, Ballabeni Pet al.   Frequency and risk factors for extraintestinal manifestations in the Swiss inflammatory bowel disease cohort. Am J Gastroenterol  2011; 106: 110– 9. Google Scholar CrossRef Search ADS PubMed  16. Marzano AV, Cugno M, Trevisan Vet al.   Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases. Clin Exp Immunol  2010; 162: 100– 7. Google Scholar CrossRef Search ADS PubMed  17. Marzano AV, Fanoni D, Antiga Eet al.   Expression of cytokines, chemokines and other effector molecules in two prototypic autoinflammatory skin diseases, pyoderma gangrenosum and Sweet’s syndrome. Clin Exp Immunol  2014; 178: 48– 56. Google Scholar CrossRef Search ADS PubMed  18. Peyrin-Biroulet L, Van Assche G, Gómez-Ulloa Det al.   Systematic review of tumor necrosis factor antagonists in extraintestinal manifestations in inflammatory bowel disease. Clin Gastroenterol Hepatol  2017; 15: 25– 36.e27. Google Scholar CrossRef Search ADS PubMed  19. Löfberg R, Louis EV, Reinisch Wet al.   Adalimumab produces clinical remission and reduces extraintestinal manifestations in Crohn’s disease: results from CARE. Inflamm Bowel Dis  2012; 18: 1– 9. Google Scholar CrossRef Search ADS PubMed  20. Fleischmann R, Kremer J, Cush Jet al.  ; ORAL Solo Investigators. Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N Engl J Med  2012; 367: 495– 507. Google Scholar CrossRef Search ADS PubMed  21. Bachelez H, van de Kerkhof PC, Strohal Ret al.  ; OPT Compare Investigators. Tofacitinib vs etanercept or placebo in moderate-to-severe chronic plaque psoriasis: a phase 3 randomised non-inferiority trial. Lancet  2015; 386: 552– 61. Google Scholar CrossRef Search ADS PubMed  22. Gomollón F, Dignass A, Annese Vet al.  ; ECCO. Third European evidence-based consensus on the diagnosis and management of Crohn’s disease 2016. Part 1: Diagnosis and medical management. J Crohns Colitis  2017; 11: 3– 25. Google Scholar CrossRef Search ADS PubMed  23. Magro F, Gionchetti P, Eliakim Ret al.  ; European Crohn’s and Colitis Organisation [ECCO]. Third European evidence-based consensus on diagnosis and management of ulcerative colitis. Part 1: Definitions, diagnosis, extra-intestinal manifestations, pregnancy, cancer surveillance, surgery, and ileo-anal pouch disorders. J Crohns Colitis  2017; 11: 649– 70. Google Scholar CrossRef Search ADS PubMed  24. Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study. N Engl J Med  1987; 317: 1625– 9. Google Scholar CrossRef Search ADS PubMed  25. Rutgeerts P, Sandborn WJ, Feagan BGet al.   Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med  2005; 353: 2462– 76. Google Scholar CrossRef Search ADS PubMed  26. Mary JY, Modigliani R. Development and validation of an endoscopic index of the severity for Crohn’s disease: a prospective multicentre study. Groupe d’Etudes Thérapeutiques des Affections Inflammatoires du Tube Digestif [GETAID]. Gut  1989; 30: 983– 9. Google Scholar CrossRef Search ADS PubMed  27. Sipponen T, Savilahti E, Kolho KL, Nuutinen H, Turunen U, Färkkilä M. Crohn’s disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn’s disease activity index and endoscopic findings. Inflamm Bowel Dis  2008; 14: 40– 6. Google Scholar CrossRef Search ADS PubMed  28. Sauter G, Simon R, Hillan K. Tissue microarrays in drug discovery. Nat Rev Drug Discov  2003; 2: 962– 72. Google Scholar CrossRef Search ADS PubMed  29. Eppenberger M, Zlobec I, Baumhoer D, Terracciano L, Lugli A. Role of the VEGF ligand to receptor ratio in the progression of mismatch repair-proficient colorectal cancer. BMC Cancer  2010; 10: 93. Google Scholar CrossRef Search ADS PubMed  30. Fischer CA, Zlobec I, Green Eet al.   Is the improved prognosis of p16 positive oropharyngeal squamous cell carcinoma dependent of the treatment modality? Int J Cancer  2010; 126: 1256– 62. Google Scholar PubMed  31. Vavricka SR, Scharl M, Gubler M, Rogler G. Biologics for extraintestinal manifestations of IBD. Curr Drug Targets  2014; 15: 1064– 73. Google Scholar CrossRef Search ADS PubMed  32. Brooklyn TN, Dunnill MG, Shetty Aet al.   Infliximab for the treatment of pyoderma gangrenosum: a randomised, double blind, placebo controlled trial. Gut  2006; 55: 505– 9. Google Scholar CrossRef Search ADS PubMed  33. Vavricka SR, Gubler M, Gantenbein Cet al.  ; Swiss IBD Cohort Study Group. Anti-TNF treatment for extraintestinal manifestations of inflammatory bowel disease in the Swiss IBD Cohort Study. Inflamm Bowel Dis  2017; 23: 1174– 81. Google Scholar CrossRef Search ADS PubMed  34. Murch SH, Braegger CP, Walker-Smith JA, MacDonald TT. Location of tumour necrosis factor alpha by immunohistochemistry in chronic inflammatory bowel disease. Gut  1993; 34: 1705– 9. Google Scholar CrossRef Search ADS PubMed  35. Tadbiri S, Grimaud JC, Peyrin-Biroulet Let al.   DOP025 Efficacy of vedolizumab on extraintestinal manifestation in patients with inflammatory bowel disease: a post-hoc analysis of the OBSERV-IBD cohort from the GETAID. Gastroenterology  2017; 152[ Suppl 1]: S396. Google Scholar CrossRef Search ADS   36. Langan SM, Groves RW, Card TR, Gulliford MC. Incidence, mortality, and disease associations of pyoderma gangrenosum in the United Kingdom: a retrospective cohort study. J Invest Dermatol  2012; 132: 2166– 70. Google Scholar CrossRef Search ADS PubMed  37. Peyrin-Biroulet L, Panés J, Sandborn WJet al.   Defining disease severity in inflammatory bowel diseases: current and future directions. Clin Gastroenterol Hepatol  2016; 14: 348– 54.e17. Google Scholar CrossRef Search ADS PubMed  Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Crohn's and Colitis Oxford University Press

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Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Background Pathogenesis of cutaneous extraintestinal manifestations [EIM] in inflammatory bowel disease [IBD] remains elusive. Efficacy of anti-TNF agents suggests TNF-dependent mechanisms. The role of other biologics, such as anti-integrins or JAK-inhibitors, is not yet clear. Methods We performed immunohistochemistry for TNFα, NFκB, STAT1/STAT3, MAdCAM1, CD20/68, caspase 3/9, IFNγ, and Hsp-27/70 on 240 intestinal [55 controls, 185 IBD] and 64 skin biopsies [11 controls, 18 erythema nodosum [EN], 13 pyoderma gangenosum [PG], 22 psoriasis]. A semiquantitative score [0–100%] was used for evaluation. Results TNFα was upregulated in intestinal biopsies from active Crohn`s disease [CD] vs controls [36.2 vs 12.1, p < 0.001], but not ulcerative colitis [UC: 17.9]. NFκB, however, was upregulated in intestinal biopsies from both active CD and UC [43.2 and 34.5 vs 21.8, p < 0.001 and p = 0.017, respectively]. TNFα and NFκB were overexpressed in skin biopsies from EN, PG, and psoriasis. No MAdCAM1 overexpression was seen in skin tissues, whereas it was upregulated in active UC vs controls [57.5 vs 35.4, p = 0.003]. STAT3 was overexpressed in the intestinal mucosa of active and non-active IBD, and a similar upregulation was seen in skin biopsies from EN [84.7 vs 22.3, p < 0.001] and PG [60.5 vs 22.3, p = 0.011], but not in psoriasis. Caspase 3 and CD68 overexpression in skin biopsies distinguished EN/PG from psoriasis and controls. Conclusions Upregulation of TNFα/NFκB in EN and PG is compatible with the efficacy of anti-TNF in EIM management. Data on overexpressed STAT3, but not MAdCAM1, support a rationale for JAK-inhibitors in EN and PG, while questioning the role of vedolizumab. Extraintestinal manifestations, anti-TNF, anti-integrins, JAK-inhibitor, immunohistochemistry, microarray 1. Introduction Inflammatory bowel disease [IBD], with the two main subtypes Crohn`s disease [CD] and ulcerative colitis [UC], is a chronic inflammatory disorder of the gastrointestinal tract. The aetiopathogenesis of IBD is incompletely understood, although it is considered a multifactorial disease which arises from a complex interplay between genetic, environmental, and immunological factors, with an abnormal host immune response to environmental stimuli.1 Different cytokines and cell interaction proteins have been identified as key players in IBD pathogenesis, such as the TNFα-NFκB axis, the JAK-STAT pathway, and the integrin-vascular adhesion molecule interaction. Several already approved drugs or investigational agents take advantage of these pathomechanisms. Anti-TNF agents have been successfully used for more than a decade, whereas anti-integrins such as vedolizumab have been introduced into clinical practice only recently.2,3 The latter target the gut-specific interaction between integrin α4β7 on leukocytes and the adhesion molecule MAdCAM1 on endothelial cells in the intestine, thereby blocking leukocyte adhesion and migration to the site of inflammation. Few, if any, systemic side effects are observed.2,3 Tofacitinib represents a first-class oral agent inhibiting the janus kinase [JAK] family of proteins, which are important mediators in inflammation. Blocking JAK downregulates proinflammatory cytokines, such as interleukin [IL] 2, 4, 7, 9, 15, and 21, through the JAK-STAT pathway.4 Efficacy has been reported for UC but not for CD, but more specific JAK inhibitors such as filgotinib may also be efficacious in the latter indication.5–7 Approval process for tofacitinib`s use in UC patients has been recently initiated by the US Food and Drug Administration [FDA]. Extraintestinal manifestations [EIM] of IBD are common, with a frequency ranging from 6% to 47%.8–15 Besides arthritis and stomatitis, cutaneous manifestations are among the most prevalent EIM.15 IBD skin lesions mainly include erythema nodosum [EN] and pyoderma gangrenosum [PG]. Whereas EN usually parallels intestinal disease activity, PG may or may not be associated with intestinal disease. The pathomechanisms of EN and PG remain elusive, although efficacy of anti-TNF treatment suggests TNF-dependent mechanisms. Limited data from immunohistochemical evaluations and protein analyses from tissue lysates have demonstrated upregulation of several inflammatory cytokines, such as IL 8, 17, and 1β and TNFα in PG.16,17 Evidence for the efficacy of anti-TNF in EIM management is evolving. Recently, a thorough meta-analysis conducted by Peyrin-Biroulet and colleagues has shown response rates between 69% and 100% for PG and reduction of EN prevalence from 2.4% to 0.4%, with anti-TNF treatment.18,19 Whether other biologic agents are efficacious in the treatment of EN or PG has yet to be determined. Studies evaluating the role of vedolizumab are under way. It is yet unclear if the gut-selective mode of action limits its efficacy to the intestine or if indirect beneficial effects via decrease of intestinal inflammatory load might be relevant. No data are available so far regarding the influence of JAK inhibitors on EIM, although tofacitinib`s efficacy has been shown for other auto-inflammatory disorders such as rheumatoid arthritis and psoriasis.20,21 We herein investigated different proteins involved in intestinal and extraintestinal IBD manifestations, aiming at elucidating similarities and differences in IBD, cutaneous EIM, and psoriasis pathophysiology, with a special focus on possible therapeutic implications such as involvement of the TNFα-NFκB and JAK-STAT pathway as well as the integrin-MAdCAM interaction. 2. Methods 2.1. Study design In this observational single-centre study, we prospectively collected intestinal and cutaneous tissue samples from healthy individuals and patients with IBD, cutaneous EIM, and psoriasis. Sample collection was conducted between 2004 and 2011. All patients had previously given their written informed consent for tissue collection and review of patient charts. All data were anonymised. The study was supported by the Swiss National Science Foundation and was approved by the local ethics committee of the University Hospital Zurich [KEK-ZH-837]. 2.2. Patient and data collection All patients analysed in this study were treated for IBD and/or inflammatory skin disease such as EN, PG, or psoriasis, at the University Hospital Zurich, Switzerland. Diagnosis of UC and CD had been previously established according to the current ECCO guidelines.22,23 Inflammatory skin lesions had all been diagnosed by a dermatologist. Intestinal samples from healthy controls were collected at regular screening colonoscopies at the University Hospital Zurich. Patients had to be older than 18. Endoscopic disease activity was assessed using the Mayo Clinic endoscopy subscore for UC and the CD endoscopic index of severity [CDEIS] for CD.24–26 A score of ≥ 1 [Mayo Score] or ≥ 3 [CDEIS] was considered as active disease.25,27 Patients were excluded for: i] biologic treatment with anti-TNF within the past 8 weeks; ii] ongoing treatment for inflammatory skin disorder [topical or systemic]; iii] concomitant autoimmune disorders such as systemic lupus erythematodes or vasculitis; and iv] concomitant skin disorder such as eczema, atopic dermatitis, or paradoxical anti-TNF induced skin lesions. Clinical data were retrieved from paper-based and electronic patient records. The following data were collected: patient demographics [gender, age]; disease history; previous and current medications; and comorbidities. For this purpose, a standardised spreadsheet was used [see Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. 2.3. Tissue sample collection For intestinal samples, biopsies were taken from the terminal ileum [TI], caecum, and colon using a needle forceps. For skin samples, biopsies were taken from the affected lesion using a 4–6 mm surgical punch that collected epidermis, dermis and the upper subcutis. For healthy control skin, fresh, non-affected, and non-inflamed skin of unrelated surgical excisions was used. Biopsies were formalin-fixed, paraffin embedded and stored at -80°C for further use. 2.4. Tissue microarray and immunostaining Tissue microarrays [TMA] were constructed using formalin-fixed paraffin-embedded tissue blocks, which had been punched out from ileal, caecal, colonic, sigmoid, or skin tissue by a tissue cylinder [0.6 mm in diameter], as previously described.28,29 Immunohistochemical staining was performed using the automated system BOND RX [Leica Biosystems]. TMA sections were deparaffinised and rehydrated in dewax solution [Leica Biosystems]. Immunohistochemistry was carried out for the TNF-NFκB [TNFα, NFκB] and JAK-STAT pathway [STAT1, STAT3] as well as the MAdCAM-integrin interaction [MAdCAM1], which all represent targets of IBD treatment modalities. We further investigated markers for B lymphocytes [CD20], macrophages [CD68], Th1-cytokine mediated disease [IFNγ], apoptosis [caspase 3 and 9], and inflammation-induced anti-apoptotic factors [Hsp-27/70]. The following primary antibodies were used: TNFα [Abcam, ab6671, dilution 1:50], NFκB p65 [Abcam, ab32360, dilution 1:100], MAdCAM1 [clone CA102.2CI, Affimetrix BMS170, dilution 1:1000], STAT1 [R&D Systems, MAB1490, dilution 1:50], STAT3 [Abcam, ab50761-100, dilution 1:100], CD20 [Ventana Roche, prediluted], CD68 [Ventana Roche, prediluted], caspase 3 [Abcam, ab4051, dilution 1:200], caspase 9 [Invitrogen, PA5-17913, dilution 1:200], IFNγ [BD Biosciences, 560371, dilution 1:100], Hsp-27 [Leica Biosystems, NCL-HSP27, dilution 1:40], and Hsp-70 [Enzo Life Sciences, dilution 1:100]. Antibody was detected with the Bond Polymer Refine Detection kit [Leica Biosystems], using DAB [3-3’-diaminobenzidine] as chromogen and following the manufacturer’s instructions. A semiquantitative scoring system ranging from 0% to 100% [5% intervals] was used for evaluation of immunostaining.29,30 Scoring was performed by two reference pathologists in the field of tissue microarrays [AL, HD]. TMA review was blinded. All samples and controls were run in duplicate. Failure of analysis occurred in 15.1% [796/5040 spots] of all gastrointestinal samples and in 9.4% [72/765 spots] of skin samples. Reasons for such failure were missing samples [empty spots on microarray] or TMA technology. 2.5. Statistical analysis For all statistical analyses, IBM software SPSS [version 22.0.0, 2013 SPSS Science, Chicago, IL] was used. Each biopsy sample was analysed individually. Metric data are presented as means and standard deviation [SD]. Categorical data are depicted as percentage of the group total. For comparisons between continuous variables, two-sample t test and Mann-Whitney U test were used depending on whether data were normally distributed or not. A two-sided p-value of < 0.05 was regarded as statistically significant. 3. Results 3.1. Tissue samples A total of 304 intestinal and skin tissue samples were collected; 240 intestinal biopsies were taken from a total of 106 patients. These samples showed active endoscopic IBD in 108 cases and endoscopic inactivity in 77 cases; 55 intestinal biopsies were taken from 29 healthy controls; 64 skin samples were collected from 64 patients: 53 patients had an inflammatory skin disorder [18 EN, 13 PG, 22 psoriasis] whereas 11 patients [controls] had normal skin. Two of the 53 patients with an inflammatory skin disorder [1 EN and 1 PG] had corresponding intestinal biopsies showing active ileocaecal CD [for EN] and active UC pancolitis [for PG]. In detail, the following samples were analysed: 74 from TI [19 HC, 19 CD inflamed, 13 CD not inflamed, 23 UC not inflamed], 79 from the caecum [21 HC, 17 CD inflamed, 13 CD not inflamed, 28 UC not inflamed], 57 from the sigmoid colon [15 HC, 42 UC inflamed], 30 from the colon [30 UC inflamed], and 64 from the skin [11 HC, 18 EN, 13 PG, 22 psoriasis]. 3.2. Patient demographics Of the samples, 156 [51.3%] were taken from male patients, whose mean age was 43.7 years [SD 9.7]. Patients with PG were significantly older, and there were significantly less males in the EN group. The other groups were comparable regarding gender and age. Of the 185 IBD intestinal samples, 36 showed endoscopically active CD, 26 non-active CD, 72 endoscopically active UC, and 51 non-active UC. Of the 72 samples with endoscopic UC activity, 31 were collected from patients with pancolitis and 41 from patients with left-sided UC. All of the 36 samples with active CD were taken from ileal [n = 19] or caecal [n = 17] disease, and the samples for inactive CD were collected from non-inflamed TI [n = 13] or non-affected caecum [n = 13]. Two IBD patients reported a previous anti-TNF treatment which was, however, stopped more than 8 weeks before biopsy. At the time of skin biopsy, none of the patients with skin disorders were treated with systemic or topical anti-inflammatory drugs. None of them had a history of a previous use of biologic treatment. None of the patients with psoriasis had underlying IBD. Table 1 summarises demographic data and previous medication of all patients and the respective subgroups. Table 1. Demographic data and previous medication.   All samples n = 304  Intestinal samples from IBD n = 185  Skin samples from EN n = 18  Skin samples from PG n = 13  Skin samples from psoriasis n = 22  Skin and intestinal samples from controls n = 66  Age  Mean in years [SD]  43.7 [9.7]  41.9 [14.5]  43.8 [20.7]  61.0 [17.4]  48.7 [16.3]  43.1 [10.6]  Males  156 [51.3%]  101 [54.6%]  5 [27.8%]  6 [46.2%]  12 [54.5%]  32 [48.5]  Concurrent medications   -none  164 [53.9%]  50 [27.0%]  18 [100%]  13 [100%]  22 [100%]  61 [92.4%]   -5-ASA  107 [35.2%]  106 [57.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  1 [1.5%]   -topical steroids  15 [4.9%]  15 [8.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -systemic steroids  21 [6.9%]  21 [11.4%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antibiotics  10 [3.3%]  8 [4.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]   -AZA  9 [3.0%]  9 [4.9%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antihistamines  4 [1.3%]  4 [2.2%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -biologics  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -NSAR  3 [1.0%]  1 [0.5%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]  Previous biologic treatment   -anti-TNF  2 [0.7%]a  2 [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -vedolizumab  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -JAK inhibitor  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  Surgical history  2 [0.7%]b  2b [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]    All samples n = 304  Intestinal samples from IBD n = 185  Skin samples from EN n = 18  Skin samples from PG n = 13  Skin samples from psoriasis n = 22  Skin and intestinal samples from controls n = 66  Age  Mean in years [SD]  43.7 [9.7]  41.9 [14.5]  43.8 [20.7]  61.0 [17.4]  48.7 [16.3]  43.1 [10.6]  Males  156 [51.3%]  101 [54.6%]  5 [27.8%]  6 [46.2%]  12 [54.5%]  32 [48.5]  Concurrent medications   -none  164 [53.9%]  50 [27.0%]  18 [100%]  13 [100%]  22 [100%]  61 [92.4%]   -5-ASA  107 [35.2%]  106 [57.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  1 [1.5%]   -topical steroids  15 [4.9%]  15 [8.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -systemic steroids  21 [6.9%]  21 [11.4%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antibiotics  10 [3.3%]  8 [4.3%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]   -AZA  9 [3.0%]  9 [4.9%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -antihistamines  4 [1.3%]  4 [2.2%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -biologics  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -NSAR  3 [1.0%]  1 [0.5%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  2 [3.0%]  Previous biologic treatment   -anti-TNF  2 [0.7%]a  2 [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -vedolizumab  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]   -JAK inhibitor  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  Surgical history  2 [0.7%]b  2b [1.1%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  0 [0.0%]  5-ASA, 5-aminosalicylates; AZA, azathioprine; EN, erythema nodosum; IBD, inflammatory bowel disease; NSAR, non-steroidal antirheumatic agent; PG, pyoderma gangrenosum; SD, standard deviation. a Treatment was stopped 8 weeks before biopsies. b Ileocecal resection (1) and partial colectomy (1). View Large 3.3. Expression of the TNFα/NFκB pathway in intestinal disease and cutaneous manifestations Subepithelial TNFα expression was significantly higher in active CD compared with controls [36.2 vs 12.1, p < 0.001], but was not elevated in inactive CD (20.9 vs 12.1, not significant [n.s.]). Difference between active and inactive CD was also significant [p = 0.019]. No difference was seen regarding TNFα expression in UC patients [regardless of disease activity] vs controls [17.9 and 13.1 vs 12.1, respectively, n.s.]. TNFα expression was significantly higher in CD than in UC [36.2 vs 17.9, p = 0.001]. However, NFκB, a downstream signal of TNF, was upregulated in both active UC [34.5] and active CD [43.2] vs controls [21.8, p = 0.017, p < 0.001, respectively] with no significant differences between the two diseases. In inactive disease, NFκB was not significantly elevated in either CD or UC. Similarly to what was seen in intestinal samples, subepithelial TNFα was overexpressed in all inflammatory skin disorders [EN 25.6, PG 37.7, psoriasis 14.7] compared with controls [3.2, p < 0.001, p = 0.001, and p = 0.001, respectively]. PG demonstrated even higher TNFα expression levels than psoriasis [37.7 vs 14.7, p = 0.022], and for EN vs psoriasis at least a clear trend was seen [25.6 vs 14.7, p = 0.052]. Similar results were observed for NFκB expression with, however, no difference between PG and psoriasis, whereas upregulation of NFκB was significant in EN vs psoriasis [p = 0.002]. NFkB expression was 41.8 in EN, 25.0 in PG, 14.3 in psoriasis, and 5.9 in healthy controls. For details regarding TNFα and NFκB expression, see Figures 1 and 2. Figure 1. View largeDownload slide Mean TNF expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 1. View largeDownload slide Mean TNF expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 2. View largeDownload slide Mean NFkB expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. Figure 2. View largeDownload slide Mean NFkB expression in intestinal and skin tissue. Standard error of the mean bars are shown; p-value calculated by Mann-Whitney U test. 3.4. Expression of MAdCAM1 MAdCAM1 was upregulated in inflamed intestinal tissue. Compared with healthy controls, active UC showed a significant MadCAM1 overexpression [57.5 vs 35.4, p = 0.003]. MadCAM1 was also upregulated in active UC when compared with inactive UC [57.5 vs 39.3, p = 0.027]. Expression from intestinal samples with active CD did not show significant differences when compared with healthy controls [30.8 vs 35.4]. In contrast to the upregulation of MAdCAM1 in UC patients, no overexpression compared with healthy controls was seen for EN, PG, or psoriasis. In fact, expression levels of MAdCAM1 were below the threshold of 10% in all samples with inflammatory skin disorders. Figure 3 shows representative tissue samples from the intestine [IBD and healthy controls, Figure 3a] and inflammatory skin diseases [Figure 3b], stained for TNFα and MAdCAM1. Representative tissue samples for inactive intestinal disease stained for TNFα and MAdCAM1 are depicted in Supplementary Figure 1, available as Supplementary data at ECCO-JCC online. For expression levels of intestinal samples see Supplementary Figure 2, available as Supplementary data at ECCO-JCC online. Figure 3. View largeDownload slide Immunohistochemical analysis of intestinal and skin tissue tissue microarrays [TMA] for TNF and MAdCAM1 protein expression: a] intestinal tissue; A-C: MAdCAM1 immunostaining, D-F: TNF immunostaining, A/D: normal colon, B/E active CD, C/F active UC; b] skin tissue: A-D: MAdCAM1 immunostaining, E-H: TNF immunostaining, A/E: normal skin, B/F: erythema nodosum, C/G: pyoderma gangrenosum, D/H: psoriasis. Figure 3. View largeDownload slide Immunohistochemical analysis of intestinal and skin tissue tissue microarrays [TMA] for TNF and MAdCAM1 protein expression: a] intestinal tissue; A-C: MAdCAM1 immunostaining, D-F: TNF immunostaining, A/D: normal colon, B/E active CD, C/F active UC; b] skin tissue: A-D: MAdCAM1 immunostaining, E-H: TNF immunostaining, A/E: normal skin, B/F: erythema nodosum, C/G: pyoderma gangrenosum, D/H: psoriasis. 3.5. Expression of STAT proteins in intestinal disease and cutaneous manifestations STAT3, which is involved in the JAK-STAT pathway and which can be blocked by JAK inhibitors such as tofacitinib, was elevated in both active and inactive CD [73.4 and 81.5 vs 60.5, p = 0.028 and p = 0.002, respectively]. The same results were seen for active and inactive UC [79.2 and 72.7 vs 60.5, p < 0.001 and p = 0.012, respectivle]. There was no difference between active and inactive disease, nor between UC and CD [n.s.]. Of note, the same significant upregulation of STAT3 was observed in EN [84.7] and PG [60.5] compared with controls [22.3] and psoriasis [26.8]. STAT3 was even more highly expressed in EN compared with PG [p = 0.033]; for details see Figure 4. Although there was also some significant overexpression of STAT1 in EN, absolute numbers [EN 17.1, PG 1.4, psoriasis 2.9, controls 0.0] were considerably lower than those seen for STAT3. Figure 4. View largeDownload slide Mean STAT3 expression in intestinal and skin tissue. Standard error of the mean bars are shown; p -value calculated by Mann-Whitney U test. Figure 4. View largeDownload slide Mean STAT3 expression in intestinal and skin tissue. Standard error of the mean bars are shown; p -value calculated by Mann-Whitney U test. 3.6. Expression of other proteins We assessed the expression of several other proteins involved in inflammatory processes and cell injury. Caspase 3 upregulation significantly distinguished PG and EN from healthy controls and psoriasis: caspase 3 was higher in both EN [20.0] and PG [17.7] vs in psoriasis [2.8, p < 0.001 and p = 0.002, respectively] and in controls [0.9, p < 0.001 and p < 0.001, respectively]. Caspase 9 was overexpressed in EN [13.1], PG [10.9], and psoriasis [6.0] compared with controls [2.3], although upregulation in PG was not significant due to a high variation. Nonetheless, these results highlight caspases as potential targets in EIM management. No upregulation of IFNγ in intestinal disease nor in skin lesions was seen, but there was a significant overexpression of CD68 in inflammatory skin disorders [EN 54.7, PG 32.3, psoriasis 10.2 vs controls 3.9, p < 0.001, p < 0.001, and p = 0.003, respectively]. Upregulation was significantly higher in EN compared with PG [p = 0.042] and psoriasis [p < 0.001]. Difference between PG and psoriasis was also significant, highlighting the possible pathogenic role of CD68 and therefore of macrophages in PG and EN pathophysiology, but not in psoriasis. However, absence of CD68 overexpression in intestinal IBD, as well as low absolute numbers of CD20 [controls 0.0, EN 2.2, PG 2.1, psoriasis 1.4] and Hsp-27 expression [controls 5.9, EN 11.8, PG 6.8, psoriasis 4.5] do not allow clear conclusions. Hsp-70 did not show any overexpression in the skin samples [controls 8.2, EN 9.4, PG 8.2, psoriasis 5.6]. Expression levels of caspase 3, caspase 9, Hsp-27, Hsp-70, CD20, CD68, and IFNγ are depicted in Supplementary Figure 3, available as Supplementary data at ECCO-JCC online. 4. Discussion This single-centre observational study, with assessment of protein expression patterns in IBD, cutaneous EIM, and psoriasis compared with healthy controls, shows similar upregulation of TNFα and STAT3 in both intestinal and cutaneous disease, but absence of MAdCAM1 overexpression in inflammatory skin disorders. TNFα and STAT3 are both considerably upregulated in EN and PG, with even higher expression levels compared with psoriasis. Protein expression patterns of EN and PG show many similarities, with only few differences revealing important therapeutic implications. The TNFα-NFκB axis is overexpressed in both intestinal disease and inflammatory skin disorders, with an even more pronounced upregulation in PG and EN compared with psoriasis. TNFα was recognised as an important inflammatory parameter in different autoimmune diseases, including IBD and psoriasis, many years ago. Anti-TNF agents take advantage of this canonical inflammatory pathway. They have been successfully introduced in the past decade and have changed IBD management dramatically. Their potential beneficial role in EIM management had been first proposed based on small case series and case reports.31 Very recently, Peyrin-Biroulet and colleagues nicely summarised their efficacy in a meta-analysis demonstrating high response rates for PG [69–100%] and EN [prevalence reduction from 2.4% to 0.4%]. Complete remission rates for PG were however lower [25–100%].18,19 PG actually is the only cutaneous IBD manifestation where a randomised controlled trial is available for showing clinical improvement in 69% and a complete remission in 25% of the patients after an induction treatment with infliximab.32 Further data from the Swiss IBD cohort study support these data, demonstrating improvement rates of 60% for PG and 80% for EN.33 This has led to the current understanding of a TNF-dependent pathogenesis in cutaneous EIM. In PG samples, TNFα upregulation has been reported previously, but no such data are available for EN. Our data showing overexpression in both PG and EN, even higher levels when compared with psoriasis, are compatible with the known efficacy of anti-TNF efficacy in cutaneous EIM.16 Although the TNFα-NFκB axis was upregulated in both CD and UC with a particular overexpression of intracellular NFκB, TNFα was actually only significantly increased in intestinal samples from active CD, but not from UC. There are three possible explanations: 1] the finding that TNFα levels in the lamina propria are indeed higher in CD than UC has been already described more than two decades ago;34 2] for detection of the small difference between healthy controls and UC, our study was probably underpowered; and 3] other factors such as bacterial products [e.g. lipopolysaccharides, LPS] can lead to NFκB upregulation independent of TNFα, so [over]expression of the two proteins do not necessarily go in parallel. The anti-integrin vedolizumab has been introduced into clinical practice very recently. Data from randomised controlled trials demonstrated vedolizumab`s efficacy in both CD and UC.2,3 Vedolizumab`s role in EIM management has not yet been defined. Its gut-selective mechanism advocates against, whereas some preliminary data presented at recent congresses advocate for, an at least partial efficacy in the treatment of EIM.35 Consistently with the literature, we herein report on isolated MAdCAM1 expression in the inflamed intestine, but not in inflammatory skin disorders, which makes a direct effect on cutaneous EIM extremely unlikely. However, vedolizumab might have a role in EIM management by treating intestinal disease activity in EIM that parallels IBD. The third biologic, which is currently being reviewed by the US FDA for treatment in UC patients, is the JAK inhibitor tofacitinib, which has been successfully used in other diseases such as rheumatoid arthritis and psoriasis.20,21 Tofacitinib inhibits the JAK-STAT pathway which incites pro-inflammatory signals. Biopsies from active intestinal disease and inflammatory skin disorders demonstrated similar results regarding STAT3 overexpression in our patient cohort. Difference between healthy controls and inflamed tissue was even more prominent in skin biopsies. Of note, STAT3 was significantly more upregulated in PG/EN than in psoriasis, shedding light on the potential benefit from JAK inhibition in those patients. Clinical data are not available so far, but our findings make a therapeutic response to JAK inhibitors very likely. Given tofacitinib`s current blackbox warning for serious infections, possible systemic side effects from pan-JAK inhibitors should be kept in mind; these, however, occur less frequently with more specific JAK inhibitors such as filgotinib. With the microarray approach, several other interesting targets for EIM management have been identified by screening markers for B lymphocytes [CD20], macrophages [CD68], apoptosis [caspase 3 and 9], and inflammation-induced anti-apoptotic factors [Hsp-27/70]. Both caspase 3 and CD68 were overexpressed in EN and PG compared with healthy controls and psoriasis, highlighting the overlap between cutaneous EIM with a protein expression profile distinct from that of other inflammatory skin diseases. Anti-caspases are currently tested in liver diseases [emricasan in non-alcoholic steatohepatitis, NCT02686762], but no data are available so far for EIM or IBD. Our study has several strengths, but also some limitations. To the best of our knowledge, it is so far the only study investigating protein expression patterns in IBD and cutaneous EIM in IBD and comparing them with healthy controls and psoriasis. Microarray is a widely used and reliable tool to study expression of various proteins. Variation due to TMA technology has been limited using only duplicate samples. None of the patients with inflammatory skin disorders was treated with either topical or systemic anti-inflammatory drugs, which makes the TMA results more reliable. In addition, previous anti-TNF treatment was reported in two IBD patients only, and treatment was stopped more than 8 weeks before study evaluation, making interference with biopsy results less likely. All TMA were analysed by experts in TMA evaluation, and involvement of only two pathologists has limited inter-observer variability. We were able to include 13 patients with PG despite its rarity [prevalence 2% of Swiss IBD population, incidence 0.63/100000] due to the close collaboration with our department of dermatology and a study enrolment over several years.15,36 A limitation of the TMA technology is that tissue sample heterogeneity is not completely taken into account. The study was cross-sectional without any follow-up, and therefore changes in expression levels in response to any biologic treatment could not be assessed. The study relied on protein expression levels only, and did not include analysis of mRNA levels. A further drawback of this study is that we only identified MAdCAM1 overexpression in active UC, not in active CD samples. This might be due to: 1] the low number of samples from active CD [n = 36] compared with the relatively high number of active UC [n = 72]; and 2] the inclusion of non-severe CD cases given the very low cut-off of CDEIS ≥ 3.37 Whereas upregulation of TNFα in cutaneous EIM compared with healthy controls is compatible with the known efficacy of anti-TNF in EIM management, the lack of MAdCAM1 overexpression in the human skin, even in the context of inflammation, questions the role of vedolizumab in EIM treatment. Its only effect might be indirect, through inducing and maintaining intestinal disease remission in those EIM parallelling IBD activity. Data on upregulated STAT give a rationale for JAK inhibitors in both EN and PG management, particularly in the current context of missing clinical studies. Data from randomised controlled trials evaluating the effect of anti-integrins and JAK inhibitors on EIM are, however, urgently needed. Funding This work was supported by a research grant from the Swiss National Science Foundation (Swiss IBD Cohort Study [Grant No. 3347CO-108792]). Conflict of Interest None declared. Author Contributions SRV and TG take responsibility for the integrity of the work as a whole, from inception to published article. 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Journal

Journal of Crohn's and ColitisOxford University Press

Published: Mar 1, 2018

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