Dysfunctional Extracellular Matrix Remodeling Supports Perianal Fistulizing Crohn's Disease by a Mechanoregulated Activation of the Epithelial-to-Mesenchymal Transition

Abstract

Background and aims: Perianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation.

Methods: Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed.

Results: A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli.

Conclusions: By mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.

Keywords: Crohn′s Disease; EMT; Extracellular Matrix; Perianal Fistula; TSG-6.

Figure 1

CD-associated fistula displays a distinct transcriptome profile compared with perifistula. (A) Histological representative images underline the differences from the same patient between perifistula and fistula (outlined by white dotted lines) regions by hematoxylin and eosin. Scale bar = 100 μm. (B) Relative transcript levels of IL-6,tumor necrosis factor α, and TGFβ1. The results were normalized to GAPDH and data presented as mean ± SEM.∗P < .05, ∗∗P < .01 by Mann-Whitney test. n = 22 patients. (C) t-Distributed stochastic neighbor embedding plot visualizing cluster assignments of samples, in which samples are projected in t-distributed stochastic neighbor embedding space. (D) Volcano plot representing upregulated and downregulated genes in fistula vs perifistula samples. (E) Heatmap of differentially expressed genes in fistula and perifistula samples. (F) Gene Ontology (GO) plot–enriched pathways in fistula vs perifistula biopsies with respective P values and enrichment score (n = 8, analysis between perifistula and fistula regions from the same patient).

Figure 2

ECM reorganization in CD-associated fistulae. (A) Heatmap highlighting the differences in genes related to ECM in fistula vs perifistula biopsies (n = 8, analysis between perifistula and fistula regions from the same patient). (B) HAS1, HAS2, and CD44 gene expression by qRT-PCR in fistula vs perifistula tract from the same patient by qRT-PCR (n = 14). (C) Sirius red staining showing collagen deposition in fistula tract from pooled samples of CD patients (n = 15) and in healthy tissue (HT) of anus from non-IBD patients (n = 7). Scale bar = 100 μm. In the right panel, the percentage of total collagen deposition is shown. (D) Representatives images of the polarized light microscopy showing the different type of collagen fibers (green-yellow and red) between fistula (n = 15) and HT (n = 7) areas and their enrichment in percentage in the right panel. (E) Heatmap representing the expression of collagen genes in fistula and perifistula areas from the same patient (n = 8). (F) Relative transcript levels by RNA-seq analysis (n = 8 patients) and (G) mRNA levels by qRT-PCR of MMP9, MMP13, MMP19, MMP25, and MMP28 in fistula and perifistula areas (n = 10 patients). Biological triplicates for each group. Data are presented as mean ± SEM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗ P < .0001 by Mann-Whitney test (B–D, G).

Figure 3

TSG-6 is upregulated in fistula compared with perifistula regions and mediates pathological HC•HA complex formation. (A) Heatmap representing the expression of matrisome core genes (n = 8 patients). (B) Relative transcript levels of TNFAIP6 in fistula and perifistula areas from the same patients (n = 20). (C) Representative Western blot analysis of TSG-6 and vinculin as control, in whole cell lysate from fistula and perifistula areas. Below, TSG-6 protein signal intensity quantification relative to vinculin (n = 5 patients for each group). (D) Immunofluorescence staining of TSG-6 (red) and HA (green) in fistula area (left panel) and in fistula tract (right panel) from the same patient. DAPI = nuclei in blue. Scale bar = 100 μm. Data are presented as mean ± SEM. ∗P < .05, ∗∗P < .01 by Mann-Whitney test.

Figure 4

Immunostaining for HA and Iαl expression in fistula and healthy anal tissue. Representative images of immunofluorescence staining of Iαl or TSG-6 (red) and HA (green) in perifistula (left panel) and fistula (middle panel) regions from the same CD patient and in healthy anal tissue of non-IBD patients (right panel). DAPI = nuclei in blue. Scale bar = 50 μm.

Figure 5

TSG-6 induces Snail and enhances the migratory capacity of Caco-2 cells. (A) Relative transcript levels of TNFAIP6 (which encodes the TSG-6 protein) in control (EV) and TSG-6 OE Caco-2 cells. Data are representative of 3 independent experiments, with biological triplicates for each group. (B) Western blot analysis of Caco-2 cells with EV and TSG-6 OE cells. (C) Western blot analysis of Caco-2 cell supernatants pretreated with 0.1 M NaOH or with 0.05 U chondroitinase ABC lyase. Shown are that 40 kDa and 150 kDa are the molecular weights expected for TSG-6 free molecules and the complexed TSG-6 forms, respectively. Data are representative of 3 independent experiments, with biological triplicates for each group. (D) Expression levels of CDH1 (which encodes E-cadherin) and SNAI1 (which encodes Snail) in fistula and perifistula regions from the same patients by RNA-seq analysis (left panel) (n = 8) and qRT-PCR (right panel) (n = 22). (E) Relative transcript levels of CDH1 and SNAI1 in control (EV) and TSG-6 OE Caco-2 cells. (F) [³H]-thymidine–based proliferation assay of control (EV) or TSG-6 OE Caco-2 cells. (G) Bright-field images of wound healing assay performed in control or TSG-6 OE Caco-2 cells for 24 and 48 hours, and quantification of the wound closure respective to time zero. Representative images of 3 independent experiments, with biological triplicates for each group. Data are presented as mean ± SEM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗ P < .0001 by Mann-Whitney test (A, C, E) and 1-way analysis of variance, with Tukey’s multiple comparisons (D, G).

Figure 6

TSG-6 induces migration in Caco-2 cells in a dose-dependent manner. Bright-field images of migration assay performed in Caco-2 cells for 24 and 48 hours in presence of low (5 ng) and high (10 ng) concentrations of rhTSG-6 and quantification of the wound closure respective to time zero. Representative images of 3 independent experiments, with biological triplicates for each group. Data are presented as mean ± SEM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗ P < .0001 by 1-way analysis of variance, with Tukey’s multiple comparisons.

Figure 7

Characterization of HD-iPSCs. (A) Representative images of iPSC-like colony morphology of iPSC cells cultured under feeder-free conditions. Scale bar = 50 μm. (B) HD-iPSC exhibited a normal diploid karyotype (46,XX). (C) Immunostaining for stemness markers (green): OCT4, NANOG, and SOX-2. Scale bar = 15 μm. (D) Immunostaining for markers of the 3 germ layers (green): endoderm (GATA4), mesoderm (smooth muscle actin SMA), and ectoderm (NESTIN). Nuclei are counterstained with DAPI (blue). Scale bar = 15 μm.

Figure 8

TSG-6 expression in iHOs derived from human dermal iPSCs. (A) Schematic reprogramming process of MET, when mesenchymal genes are repressed through the generation of human iPSC-derived iHOs from adult fibroblasts. (B) Representative image of cryosection iPSC-derived iHO (C, D) immunostained for the presence of cytokeratin 19 and villin indicating the presence of a brush border and columnar epithelial cells; (E–H) zona occludens-1 and epithelial cellular adhesion molecule indicating the presence of the tight junction facing the lumen of the organoid; villin for enterocytes; anti-mucin-2 indicating the presence of goblet cells; and chromogranin A indicating the presence of enteroendocrine cells; and (I) TSG-6. DAPI = nuclei in blue. Scale bars = 100 and 20 μm. (J) Relative transcript levels of TNFAIP6 encoding for TSG-6, SNAI1 encoding for Snail, Vimentin, CDH1 encoding E-cadherin, and LGR5 in fibroblasts and iHOs by qRT-PCR. Data are presented as mean ± SEM. ∗P < .05, ∗∗∗P < .001 by Mann-Whitney test (n = 3).

Figure 9

TSG-6 overexpression in PFFs triggers the acquisition of fistula-like features. (A) Relative transcript levels of TNFAIP6 (which encodes for TSG-6 protein) and SNAI1 (which encodes for Snail) in PFFs or FFs isolated from the same patient (n = 5). (B) Relative transcript levels of TNFAIP6 and SNAI1 in PFF control (EV) and overexpressing TSG-6. Data are representative of 2 independent experiments, with biological triplicates for each group (n = 3). (C) Relative transcript levels of HAS2 (encoding for HA synthase 2) and ACTA2 (encoding for α-smooth muscle actin) in control (EV) or TSG-6 OE perifistula-derived human fibroblasts. (D) Bright-field images (left panel) and F-actin immunolabeling (right panel) of human fibroblasts derived from fistula and perifistula areas of the same patient and of perifistula fibroblasts overexpressing TSG-6. Data are representative of 2 independent experiments, with biological triplicates for each group (n = 3). DAPI = nuclei in blue. Scale bar = 30 μm. (E) Nuclei surface (cm²) of human fibroblasts derived was quantified by ImageJ. Data are presented as mean ± SEM. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 by Mann-Whitney test (A, D, E) and 1-way analysis of variance (F, H).

Figure 10

TSG-6 expression in the fistula positively correlates with mediators of mechanotransduction. (A) Heatmap representing the expression of genes related to mechanotransduction in fistula and perifistula biopsies. (B) Paired analysis of mechanosensitive proteins in fistula and perifistula areas. Gene expression of ITGA2, ITGA2B, ITGA4, PTK2B, FUT8, ITGAL, ITGB7, ITGA3, and NEDD9. For all qRT-PCR results, data reflect mean ± SEM from 3 biological replicates; the results were normalized to GAPDH and P values were measured by paired t test (n = 10 patients for each group). (C) Correlation matrix displaying gene-gene Pearson correlation coefficients. Ellipses show gene expression value distributions with red and blue ellipses representing positive and negative correlations, respectively. Cells marked with X represent not significant correlations. n = 8 patients for each group.

Figure 11

The ECM reorganization mediated by HA•TSG-6 complex promotes the activation of mechanosensors in PFFs. (A) Schematic representation of experimental procedure, in which PFFs were plated on HA scaffold ± rhTSG-6. (B) Quantification of collagen deposition in PFFs plated on scaffold ± TSG-6. (C) IL-6 (pg/mL) levels in PFFs plated on scaffold ± TSG-6. (D–G) Relative transcript level of SNAI1 (encoding for Snail), ITGA2B (encoding for integrin alpha 2B), ITGA4 (encoding for integrin alpha-4), and PTK2 (encoding for protein tyrosine kinase 2) in FFs vs PFFs plated or not on scaffold ± rhTSG-6. Data are presented as mean ± SEM from 3 independent experiments, with biological triplicates for each group (n = 3). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗ P < .0001 by 1-way analysis of variance with Tukey’s multiple comparison.

Figure 12

Relative transcript levels of IL-13 by qRT-PCR. Data are presented as mean ± SEM from 3 biological replicates; the results were normalized to GAPDH, and P values were measured by paired t test (n = 10 patients for each group).