Céline Mamie

Anti-MMP-9 Antibody: A Promising Therapeutic Strategy for Treatment of Inflammatory Bowel Disease Complications with Fibrosis.

Background:Despite medical treatments or surgical options, more than one-third of patients with Crohns disease suffer from recurring fistulae. Matrix metalloprotease 9 (MMP-9), a type IV collagenase that cleaves components of the extracellular matrix leading to tissue remodeling, is upregulated in crypt abscesses and around fistulae suggesting an important role for this enzyme in fistula formation. Our aims were (1) to correlate serum levels of MMP-9 degradation products in patients with CD with the presence of fistulae and (2) to investigate the impact of selective MMP-9 inhibition in a mouse model of intestinal fibrosis. Methods:Serum MMP-9 degradation products were quantified in subjects affected with nonstricturing and nonpenetrating CD (n = 50), stricturing CD (n = 41), penetrating CD (n = 22), CD with perianal fistula (n = 29), and healthy controls (n = 10). Therapeutic efficacy of anti-MMP-9 monoclonal antibodies was assessed in a heterotopic xenograft model of intestinal fibrosis. Results:C3M, an MMP-9 degradation product of collagen III, demonstrated the highest serum levels in patients with penetrating CD and differentiated penetrating CD from other CD subgroups and healthy controls, P = 0.0005. Anti-MMP-9 treatments reduced collagen deposition and hydroxyproline content in day-14 intestinal grafts indicating reduced fibrosis. Conclusions:The serologic biomarker C3M can discriminate penetrating CD from other CD subgroups and could serve as marker for the development of penetrating CD. Anti-MMP-9 antibody has therapeutic potential to prevent intestinal fibrosis in CD complications.

Decreased Fibrogenesis After Treatment with Pirfenidone in a Newly Developed Mouse Model of Intestinal Fibrosis.

Background:Fibrosis as a common problem in patients with Crohns disease is a result of an imbalance toward excessive tissue repair. At present, there is no specific treatment option. Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis with both antifibrotic and anti-inflammatory effects. We subsequently investigated the impact of pirfenidone treatment on development of fibrosis in a new mouse model of intestinal fibrosis. Methods:Small bowel resections from donor mice were transplanted subcutaneously into the neck of recipients. Animals received either pirfenidone (100 mg/kg, three times daily, orally) or vehicle. Results:After administration of pirfenidone, a significantly decreased collagen layer thickness was revealed as compared to vehicle (9.7 ± 1.0 versus 13.5 ± 1.5 µm, respectively, **P < 0.001). Transforming growth factor–&bgr; and matrix metalloproteinase–9 were significantly decreased after treatment with pirfenidone as confirmed by real-time PCR (0.42 ± 0.13 versus 1.00 ± 0.21 and 0.46 ± 0.24 versus 1.00 ± 0.62 mRNA expression level relative to GAPDH, respectively, *P < 0.05). Significantly decreased transforming growth factor–&bgr; after administration of pirfenidone was confirmed by Western blotting. Conclusion:In our mouse model, intestinal fibrosis can be reliably induced and is developed within 7 days. Pirfenidone partially prevented the development of fibrosis, making it a potential treatment option against Crohns disease–associated fibrosis.

The oxysterol synthesizing enzyme CH25H contributes to the development of intestinal fibrosis

Intestinal fibrosis and stenosis are common complications of Crohn’s disease (CD), frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidized cholesterol derivatives, with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase (CH25H) converts cholesterol to 25-hydroxycholesterol (25-HC), which modulates immune responses and oxidative stress. In human intestinal samples from CD patients we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme using the sodium dextran sulfate (DSS)-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.

Severity of local inflammation does not impact development of fibrosis in mouse models of intestinal fibrosis

Intestinal fibrosis is thought to be a consequence of excessive tissue repair, and constitutes a common problem in patients with Crohn’s disease (CD). While fibrosis seems to require inflammation as a prerequisite it is unclear whether the severity or persistence of inflammation influences the degree of fibrosis. Our aim was to investigate the role of sustained inflammation in fibrogenesis. For the initiation of fibrosis in vivo the models of Il10−/− spontaneous colitis, dextran sodium sulfate (DSS)-induced chronic colitis and heterotopic transplantation were used. In Il10−/− mice, we determined a positive correlation between expression of pro-inflammatory factors (Il1β, Tnf, Ifnγ, Mcp1 and Il6). We also found a positive correlation between the expression of pro-fibrotic factors (Col3a1 Col1a1, Tgfβ and αSma). In contrast, no significant correlation was determined between the expression of pro-inflammatory Tnf and pro-fibrotic αSma, Col1a1, Col3a1, collagen layer thickness and the hydroxyproline (HYP) content. Results from the DSS-induced chronic colitis model confirmed this finding. In the transplantation model for intestinal fibrosis a pronounced increase in Mcp1, inos and Il6 in Il10−/− as compared to WT grafts was observed, indicating more severe inflammation in Il10−/− grafts. However, the increase of collagen over time was virtually identical in both Il10−/− and WT grafts. Severity of inflammation during onset of fibrogenesis did not correlate with collagen deposition. Although inflammation might be a pre-requisite for the initiation of fibrosis our data suggest that it has a minor impact on the progression of fibrosis. Our results suggest that development of fibrosis and inflammation may be disconnected. This may be important for explaining the inefficacy of anti-inflammatory treatments agents in most cases of fibrotic inflammatory bowel diseases (IBD).

Intestinal Activation of pH-Sensing Receptor OGR1 [GPR68] Contributes to Fibrogenesis

Background and Aims pH-sensing ovarian cancer G-protein coupled receptor-1 [OGR1/GPR68] is regulated by key inflammatory cytokines. Patients suffering from inflammatory bowel diseases [IBDs] express increased mucosal levels of OGR1 compared with non-IBD controls. pH-sensing may be relevant for progression of fibrosis, as extracellular acidification leads to fibroblast activation and extracellular matrix remodelling. We aimed to determine OGR1 expression in fibrotic lesions in the intestine of Crohns disease [CD] patients, and the effect of Ogr1 deficiency in fibrogenesis. Methods Human fibrotic and non-fibrotic terminal ileum was obtained from CD patients undergoing ileocaecal resection due to stenosis. Gene expression of fibrosis markers and pH-sensing receptors was analysed. For the initiation of fibrosis in vivo, spontaneous colitis by Il10-/-, dextran sodium sulfate [DSS]-induced chronic colitis and the heterotopic intestinal transplantation model were used. Results Increased expression of fibrosis markers was accompanied by an increase in OGR1 [2.71 ± 0.69 vs 1.18 ± 0.03, p = 0.016] in fibrosis-affected human terminal ileum, compared with the non-fibrotic resection margin. Positive correlation between OGR1 expression and pro-fibrotic cytokines [TGFB1 and CTGF] and pro-collagens was observed. The heterotopic animal model for intestinal fibrosis transplanted with terminal ileum from Ogr1-/- mice showed a decrease in mRNA expression of fibrosis markers as well as a decrease in collagen layer thickness and hydroxyproline compared with grafts from wild-type mice. Conclusions OGR1 expression was correlated with increased expression levels of pro-fibrotic genes and collagen deposition. Ogr1 deficiency was associated with a decrease in fibrosis formation. Targeting OGR1 may be a potential new treatment option for IBD-associated fibrosis.

BCL2 Regulates Differentiation of Intestinal Fibroblasts

Background Fibrosis in patients with Crohns disease (CD) results from an imbalance toward excessive fibrous tissue formation driven by fibroblasts. Activation of fibroblasts is linked to the B-cell lymphoma 2 (BCL2) family, which is involved in the induction of apoptosis. We investigated the impact of BCL2 repression on fibrogenesis. Methods The model of dextran sodium sulfate (DSS)-induced chronic colitis and the heterotopic transplantation model of fibrosis were used. Following the administration of the BCL2 antagonist (ABT-737, 50 mg/kg/d), collagen layer thickness and hydroxyproline (HYP) content were determined. Fibroblasts were stimulated with the BCL2 antagonist (0.01-100 µM). BCL2, alpha smooth muscle actin (αSMA), and collagen I (COL1A1) were determined by quantitative polymerase chain reaction (qPCR), immunofluorescence microscopy (IF), and western blot (WB). mRNA expression pattern was determined by next-generation sequencing (NGS). Results Collagen layer thickness was significantly decreased in both DSS-induced chronic colitis and the transplantation model of fibrosis upon BCL2 antagonist administration compared with vehicle. Decreased HYP content confirmed the preventive effects of the BCL2 antagonist on fibrosis. In vitro, a significant increase in PI+/annexin V+ human colonic fibroblasts was determined by fluorescence-activated cell sorting upon treatment with high-dose BCL2 antagonist; at a lower dose, αSMA, COL1A1, and TGF were decreased. NGS, IF, and qPCR revealed decreased expression and nuclear translocation of GATA6 and SOX9, known for reprogramming fibroblasts. Conclusion BCL2 antagonist administration partially prevented fibrogenesis in both fibrosis models. The BCL2 antagonist reduced the expression of TGFβ-induced factors involved in differentiation of myofibroblasts, and therefore might represent a potential treatment option against CD-associated fibrosis.

BCL-2 levels do not predict azathioprine treatment response in inflammatory bowel disease, but inhibition induces lymphocyte apoptosis and ameliorates colitis in mice: BCL-2 selective inhibition by a-1211212 ameliorates colitis

In inflammatory bowel disease (IBD), inflammation is sustained by an exaggerated response of lymphocytes. This results from enhanced expression of anti‐apoptotic B cell lymphoma (BCL‐2) and BCL‐XL associated with a diminished turnover. Azathioprine (AZA) directly targets BCL‐2 family‐mediated apoptosis. We investigated whether the BCL‐2 family expression pattern could be used to predict treatment response to AZA and determined whether BCL‐2 inhibitor A‐1211212 effectively diminishes lymphocytes and ameliorates inflammation in a model of colitis. BCL‐2 family expression pattern was determined by next‐generation sequencing (NGS). BCL‐2 inhibitor was administered orally to Il10‐/‐ mice. Haematological analyses were performed with an ADVIA 2120 and changes in immune cells were investigated using quantitative polymerase chain reaction (qPCR) and fluorescence activated cell sorter (FACS). We determined similar expression levels of BCL‐2 family members in patients with remission and patients refractory to treatment, showing that BCL‐2 family expression can not predict AZA treatment response. Expression was not correlated with the modified Truelove and Witts activity index (MTWAI). BCL‐2 inhibitor initiated cell death in T cells from patients refractory to AZA and reduced lymphocyte count in Il10‐/‐ mice. FACS revealed diminished CD8+ T cells upon BCL‐2 inhibitor in Il10‐/‐ mice without influencing platelets. Tnf, Il1β, IfnƔ and Mcp‐1 were decreased upon BCL‐2 inhibitor. A‐1211212 positively altered the colonic mucosa and ameliorated inflammation in mice. Pro‐apoptotic BCL‐2 inhibitor A‐1211212 diminishes lymphocytes and ameliorates colitis in Il10‐/‐ mice without inducing thrombocytopenia. BCL‐2 inhibition could be a new therapy option for patients refractory to AZA.