Authors
M. STAKENBORG (1), B. VERSTOCKT (1), E. MERONI (1), W. WOLLANTS (1), M. FERRANTE (2), M. DI MATTEO (3), M. MAZZONE (3), G. BOECKXSTAENS (1), S. VERMEIRE (2), G. MATTEOLI (1) / [1] KU Leuven, , Belgium, TARGID, [2] University Hospitals Leuven, , Belgium, Gastro-enterology, [3] KU Leuven, , Belgium, Lab of Tumor Inflammation and Angiogenesis
Introduction
Neutrophils are crucial in the maintenance of intestinal homeostasis and inflammation. However, during chronic inflammatory conditions, like Inflammatory Bowel Disease (IBD), the intestinal immune system responds inaccurately resulting in excessive neutrophil infiltration and tissue damage.
Aim
Since MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF), we aim to identify the function of the HGF-MET axis in neutrophils during intestinal inflammation in a mouse model of colitis and in patients with ulcerative colitis (UC) and healthy controls (HC).
Methods
Acute colitis was induced in wild type mice (WT, C57BL/6) and mice with MET deficiency in neutrophils (MRP8-Cre MET-LoxP (KO)) by 2,5% dextran sodium sulfate (DSS). Disease progression was assessed via a standardized disease activity index (DAI) including body weight loss, stool consistency and blood in the feces. Immune cell infiltration in the colon was assessed by flow cytometry. Serum of HC (n=30) and inflamed UC patients (n=110) was collected, prior to the start of anti-TNF therapy, and at endoscopic reassessment (8-14 weeks after treatment initiation). Endoscopic remission was defined as a Mayo endoscopic sub-score ≤1. HGF was measured using electrochemiluminescence (MSD). Additionally, RNA sequencing (Illumina HiSeq4000) was performed on inflamed colonic biopsies in a subset of 24 UC patients and 11 HC.
Results
HGF was upregulated both during the acute phase of dextran sodium sulphate (DSS) colitis and in patients with active ulcerative colitis (UC). In addition, Met deletion in mouse neutrophils during acute DSS colitis improved disease severity together with reduced immune cell infiltration, in particular neutrophils, eosinophils and macrophages. Moreover, the percentage of FoxP3+ T regulatory cells was increased in KO mice compared to their WT counterparts, pointing towards a return to homeostasis in the KO colon. Strikingly, analysis of CD4+ T cells showed a predominant decrease of the percentage IL17A+ Th17 and IL17A+ IFNg+ Th1-like Th17 in KO mice compared to WT mice, while no differences were observed in the percentage of IFNg+ Th1 cells. Serum HGF was significantly upregulated in active UC patients compared to HC (p=0.001, fold change FC 1.5). Similarly, colonic HGF and MET expression were significantly upregulated compared to healthy individuals (p=3.2E10-6, FC 5.8; p=0.0007, FC 1.8 respectively). Serum HGF correlated significantly with tissue MET expression (r=0.47, p=0.03), but not with tissue HGF expression (r=0.23, p=0.30). Patients with a Mayo endoscopic sub-score of 3 had significantly higher serum HGF levels as compared to patients with a sub-score of 2 prior to therapy initiation (p=0.007, FC 1.2). Additionally, serum HGF levels correlated significantly with C-reactive protein (r=0.44, p=9.5E10-12) and absolute neutrophils counts (r=0.62, p=2.2E10-16). However, baseline HGF was not predictive for anti-TNF induced endoscopic remission later on (p=0.39). After anti-TNF administration, HGF levels overall decreased (p=1.2E10-7) and reached values similar to HC in case of endoscopic remission (p=0.35). At the time of endoscopic assessment, patients with endoscopic remission had significantly lower HGF levels than those without (p=0.0003, FC 0.72).
Conclusions
Colonic and serum HGF levels are significantly upregulated in active UC patients, with restoration towards physiological levels in patients with anti-TNF induced endoscopic remission. As murine findings suggest that absence of MET in neutrophils reduces intestinal inflammation, targeting MET could be considered as a novel therapeutic approach in UC therapy.
