MET deletion in MRP8+ neutrophils is protective during DSS-induced colitis via Th17 pathway

Background: Neutrophils play an essential role in the maintenance of intestinal homeostasis as they provide a first line of defense against invading pathogens. During inflammation, neutrophils also aid in the recruitment of other immune cells and facilitate mucosal healing by the release of factors necessary for the resolution of inflammation. However, during chronic inflammatory conditions, such as Inflammatory Bowel Disease (IBD), excessive neutrophil accumulation can lead to loss of homeostasis, tissue damage and delayed tissue repair. In the current study, we aim to identify the role and function of neutrophils recruited during colitis. Methods: To block neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor, we used the neutrophil-specific Mrp8-Cre line backcrossed with Metfl/fl. Acute colitis was induced in MRP8Cre/WT METfl/fl(KO) mice and MRP8WT/WT METfl/fl littermate controls (WT) by 2,5% dextran sodium sulfate (DSS) in drinking water for 5 days. Disease progression was assessed via a standardized disease activity index (DAI) including body weight loss, stool consistency and blood in the faeces. During chronic colitis, mice were subjected to 3 cycles of 2.5% DSS for 5 days followed by 2 weeks of drinking water. Colonic immune cells were assessed by flow cytometry. Data are expressed as mean ± SEM; t-test was performed; p<0.05 is considered significant. Results: During the third cycle of chronic DSS colitis, KO mice displayed a decreased DAI (p<0.01) and body weight loss (p<0.05) compared to WT mice. Moreover, flow cytometric analysis revealed a reduced amount of ROS+ neutrophils (WT; 3.17 ± 0.84 x 105, KO; 0.69 ± 0.26 x 105, p<0,05), eosinophils (WT; 3.00 ± 0.62 x 105, KO; 0.80± 0.20 x 105, p<0,05), and macrophages (WT; 9.98 ± 0.59 x 105, KO; 6.46 ± 0.73 x 105, p<0,05), implying a protective effect of MET deletion in MRP8+ neutrophils during chronic intestinal inflammation. To further elucidate the observed phenotype, we performed acute DSS colitis. In line, KO mice subjected to acute DSS colitis showed an improved disease course with reduced body weight loss and DAI and a comparable decrease in the amount of neutrophils, eosinophils and macrophages. Moreover, the percentage of FoxP3+ T regulatory cells was increased in KO mice compared to their WT counterparts (WT; 29,27 ± 2,14%, KO; 41,05 ± 2,11%, p<0,05), pointing towards a return to homeostasis in the KO colon. Strikingly, analysis of CD3+ CD4+ T cells showed a predominant decrease of the percentage IL17A+ Th17 (WT; 26,87 ± 1,85%, KO; 14,19 ± 2,11%, p<0,01) and IL17A+ IFNg+ Th1-like Th17 (WT; 10,62 ± 0,50%, KO; 4,70 ± 1,51%, p