Attenuation of the TGFB canonical pathway in UC
The profibrotic TGFB-SMAD pathway (illustrated in Figure 3) showed a significant dysregulated signaling pattern in acute UC and to a lesser extent in endoscopic remission (Figure 2b). Comparison of acute UC and healthy controls revealed an upregulation of TGFB2 and TGFB3, but not TGFB1. Furthermore, the corresponding receptors for these ligands, TGFB receptor 1 (TGFBR1) and TGFB receptor 2 (TGFB2), were downregulated along with intracellular SMAD signaling mediators SMAD2, SMAD3, SMAD4, SMAD6, and SMAD7. Inhibitors of the TGFB-SMAD pathway, THBS2 and BMP7, were increased in both the acute and endoscopically healed UC mucosa. Furthermore, TGFB3 normalized, whereas a persistent overexpression of TGFB2 was observed at the time of endoscopic remission.
Other relevant fibrotic mediators in UC
ECM remodeling enzymes MMP3 and MMP1 showed the most prominent log2 fold changes in acute and healed UC compared with controls. Proinflammatory cytokines and chemokines were strongly upregulated during acute disease, including IL 1 beta (IL1B), interferon gamma (IFNG), and TNF (Figure 2b). Comparison of the healed UC mucosa with the healthy control group revealed profibrotic genes IL 13 receptor subunit alpha 2 (IL13RA2) and Gremlin-1 (GREM 1) among the top overexpressed genes. Several upregulated inflammatory mediators normalized completely in the healed mucosa including MMP9, IFNG, IL 1alpha (IL1A), and C-C motif chemokine ligand 3 (CCL3). Immunostaining was consistent with the gene expression pattern of MMP9 (shown in Figure 4). In contrast, several fibrosis mediators showed little or no change in gene expression following anti-TNF therapy (BMP7, GREM1, CCL2, and CXCR4). In this cohort, TNF was still found to be upregulated in the mucosa following anti-TNF therapy. Comparison of endoscopic subscore 0 and 1 in disease remission revealed significant differences in 12 genes with log2 fold changes <1. Of these genes, IL10 and MMP14 were upregulated, whereas the remaining genes were downregulated (integrin subunit alpha 1 (ITGA1), integrin subunit beta 1 (ITGB1), integrin subunit beta 6 (ITGB6), SMAD4, signaling transducer and activator of transcription 6 (STAT6), epidermal growth factor, specificity protein 1 (SP1), and latent transforming growth factor–binding protein 1 (LTBP1). The remaining genes (n = 31) were not affected by endoscopic remission score 0 or 1. Correction for sex revealed only CCL3 to show a significant sex difference (log2 fold change male vs female: −0, 15).
We have clearly demonstrated that genes involved in ECM remodeling and fibrosis development do not all normalize in the endoscopically healed mucosa of UC (defined as Mayo endoscopic subscore of 0 or 1). Over half of the investigated fibrosis-associated genes were differentially expressed in the healed UC mucosa. This supports the notion that fibrosis is indeed an important component in the pathophysiology of UC. We found an increased TGFB2 and TGFB3 ratio in acute UC, with TGFB3 expression not normalizing in endoscopic remission. In contrast, the well-known profibrotic TGFB1 was present, but not significantly expressed in the UC mucosa. We believe that the balance of TGFB isoforms in different UC disease phases may be important for achieving mucosal healing and a potential factor to modulate. Indeed, others have reported differences in the TGFB ratio between patients with UC who respond or not to anti-TNF therapy (27). Furthermore, intestinal myofibroblasts isolated from healthy controls and patients with IBD have been shown to secrete different TGFB isoform ratios (28). All 3 TGFB isoforms are present in the intestine; however, their role in UC is unclear (19,21). Looking to other organ systems, TGFB1 and TGFB2 have been found to promote ECM deposition during skin wound healing, whereas TGFB3 reduces scarring (19,29). In sum, knowledge of the TFB isoforms role in UC is incomplete, warranting further research.
Although we found an upregulation of the TGFB2 and TGFB3, the canonical TGFB pathway showed an attenuated response with downregulated TGFB receptors (TGFBR1 and TGFBR2), together with intercellular SMAD signaling mediators. This might be an appropriate response in the acute phase of inflammation, as TGFB is recognized to be released from its latent bound form in the ECM by MMPs and other ECM–degrading factors (19). Indirectly, our findings support this with overexpression of MMPs and extracellular remodeling enzymes in acute UC, suggesting an abundance of released and activated TGFB. However, the canonical TGF pathway does not normalize in the endoscopically healed UC mucosa, suggestive of a persistent dysregulation.
As expected, proinflammatory genes showed a pattern of reduction, and even normalization following anti-TNF therapy in agreement with other studies (27,30–33). In contrast, several fibrosis mediators showed little or no change in gene expression following anti-TNF therapy including BMP7, GREM1, CCL2, and CXCR4. Specific to the endoscopically healed mucosa, a set of genes associated with collagen synthesis and degradation (ACTA2, COL3A1, LOX, and TIMP3) were upregulated compared with controls. In summary, our findings show that fibrosis-associated mediators are still dysregulated in the endoscopically healed UC mucosa compared with healthy controls. This is in keeping with other reports that the healed UC mucosa remains dysregulated (27,32). A recent study by Arijs et al. found a large overlap of mucosal genes that were persistently dysregulated in patients with UC responding to infliximab (anti-TNF) or vedolizumab (anti-α4βb7 integrin), suggesting that unidentified pathways are yet to be targeted (34). Furthermore, vedolizumab was found to influence a unique set of mucosal genes independent of anti-TNF therapy. It is exceedingly interesting whether the new biological therapies, including vedolizumab and ustekinumab (inhibitor of p40 subunit of IL12 and IL23), will target additional pathways and influence fibrosis. Our findings highlight the current knowledge gap of fibrosis-associated pathways in UC, also evident in the clinics as no effective antifibrotic therapies exist.
Our finding of an upregulated CAV-1 in the healed UC mucosa may reflect a fibroprotective mechanism, as reported in murine studies (35). The protein Cav-1 acts as the scaffolding in caveolae of the cell membrane, important for endocytosis and cell signaling (36). However, to our knowledge, the role of CAV-1 in IBD is little explored. One study supports the location of CAV-1 and -2 expression in the colonic mucosa (37). Interestingly, the literature reports CAV-1 to exhibit antifibrotic properties by binding and internalizing TGFB receptors on the cell membrane, effectively attenuating TGFB signaling and reducing collagen production (38). A low gene expression of CAV-1 has been reported in other fibrotic disorders including idiopathic pulmonary fibrosis and scleroderma (38). In addition, we found an interesting gene expression pattern for AGT, being upregulated in acute disease and downregulated in the healed UC mucosa. Mediators of the renin-angiotensin-aldosterone system are found in many tissues including the colon (39). Inhibitors of the renin-angiotensin-aldosterone system are well-established therapies for prevention of end-organ damage in cardiac and renal disease, and also of interest in IBD as therapeutic targets for inflammation and fibrosis.
IL13RA2 was among the top DEGs in our study. Interestingly, high mucosal expression of this receptor in IBD has been linked to anti-TNF unresponsiveness and impaired restoration of the intestinal barrier in a murine Dextran sulfate sodium colitis model (40). Both profibrotic and antifibrotic properties have been described in mouse models of colitis and pulmonary fibrosis, respectively; thus, tissue and cellular context is important (41,42). Taken together, IL13RA2 is emerging as an interesting future target both for refractory UC and in modulating fibrosis.
A limitation to the study is the lack of a general consensus on what constitutes a “truly healed” mucosa. The widely used Mayo score includes endoscopic subgrades 0 and 1 in disease remission, with a score of 1 allowing for the presence of mild erythema, decreased vascular pattern, and mild friability. The definitions of mucosal healing are currently being debated, with endoscopic subscore 0 and histological remission being associated with improved clinical outcomes (43). However, histological remission does not necessary imply endoscopic remission and vice versa. In a recent study, Magro et al. (44) showed that histological indexes could be associated with endoscopic outcomes with a high sensitivity when the Mayo endoscopic subscore was set at 1. Of the 7 patients in our study with available histological assessment, 1 patient met the criteria for histological remission with an endoscopic subscore of 1. This raises the questions of how do we distinguish between transcription patterns of inflammation, tissue restitution, and those representing the underlying disease? Some overlap is likely. Is the UC mucosa ever truly inactive? Arguably, we could have used stricter remission criteria; on the other hand, most genes did not show any significant difference when corrected for endoscopic score of 0 and 1 in disease remission. Although the paired samples design is a strength reducing interpatient variation, our sample size was limited by available biopsies in the IBD Biobank and at risk of type II error. Another limitation is the use of a preselected gene panels associated with general human fibrosis, thus not comprehensive. The heterogeneity of cell populations across biopsies is also important to take into account when interpreting results (45).
Apart from CCL3, we did not uncover any sex-dimorphic patterns for our gene set. In our study, most patients with UC had well-established UC; however, the role of disease duration on fibrosis development is not clear. A large histopathology study of fibrosis in UC found that the degree of fibrosis was linked to the severity ad chronicity of disease and, interestingly, not to disease duration (46). Thus, intestinal fibrosis is also a complication occurring in short-standing UC. Although different disease entities, we and others have previously found that the mucosal gene expression of inflammatory cytokines between UC and Crohn's disease is remarkably similar (47,48). In view of this, our findings could be relevant for future research in Crohn's disease; however, this remains to be explored. Comparison of mucosal fibrosis mediators in patients with UC with established fibrotic disease was not within the scope of the present study, but of interest for future work. Combining knowledge of mucosal fibrosis mediators between anti-TNF responders and nonresponders may be useful as therapeutic biomarkers, providing clinicians with tools to personalize therapy.
In conclusion, we have seen a significant modulation of genes associated with both inflammation and fibrosis in patients treated to a clinical and endoscopically verified healed mucosa. The mucosa of UC in disease remission showed a persistent dysregulation of fibrosis-associated genes, with an attenuated pattern of TGFB signaling mediators in UC. We identified mucosal TGFB isoforms, CAV-1 and AGT as potential antifibrotic targets in UC, of interest for future research.
CONFLICTS OF INTEREST
Guarantor of the article: Mona Dixon Gundersen, MD.
Specific author contributions: M.D.G: planning, conducting the study, analysis, interpretation, drafting the manuscript, and approved the final submitted draft. R.G.: planning the study, manuscript review and writing, and approved the final submitted draft. C.G.F: statistical analysis and data interpretation, review of the manuscript, and approved the final submitted draft. E.A: statistical analysis and data interpretation, visualization and editing/writing of the manuscript, and approved the final submitted draft. S.W.S.: analysis of the study material, interpretation, review of the manuscript, and approved the final submitted draft. J.R.F.: planning the study, interpretation of data, manuscript review and editing, and approved final submitted draft. R.H.P: planning and conducting the study, interpretation and data analysis, manuscript review and editing, and approved the final submitted draft.
Financial support: This work was supported by the Northern Norway Regional Health Authority (SFP-1134-13).
Potential competing interests: None to report.
WHAT IS KNOWN
- ✓ Intestinal fibrosis is a severe complication in IBD.
- ✓ No effective antifibrotic therapies exist for intestinal fibrosis.
- ✓ Recent studies report that intestinal fibrosis is underestimated in UC.
WHAT IS NEW HERE
- ✓ Profibrotic mediators are dysregulated in the endoscopically healed mucosa of UC, including transforming growth factor beta 2 (TGFB2).
- ✓ Genes involved in ECM remodeling were uniquely upregulated in the endoscopically healed mucosa of UC compared with healthy controls.
- ✓ Identification of mucosal markers warranting further exploration as potential antifibrotic targets in UC therapy.
We thank colleagues at the Gastroenterology Department, University Hospital North Norway, for inclusion and follow-up of study participants; Hagar Taman and Kristine Osther for excellent assistance with qPCR array; and the Imaging Core Facility at UiT—The Arctic University of Norway for assistance with image acquisition.
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© 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology