Tu1629 Spleen Tyrosine Kinase Inhibition Reduces Intestinal Inflammation and Prevents Postoperative Ileus

Abstract

Background: Recent advances underline intestinal inflammation, induced by handling of the gut during surgery, as crucial mechanism in the pathophysiology of postoperative ileus (POI). Macrophages, in the muscularis layer, and mast cells are the key players in the induction of this inflammatory process. Spleen tyrosine kinase (Syk) is an important kinase involved in macrophage activation as well as in mast cell degranulation and therefore inhibition of Syk pathway may represent an interesting therapeutic approach for POI. In the current study, we have evaluated the effect of the Syk-inhibitor GSK143 as potential treatment to shorten POI. Methods: The effect of an oral single dose (3mg/kg, 1.5 hours before surgery) was evaluated in a mouse model of POI, by analyzing gastrointestinal transit and intestinal muscularis inflammation. The in vitro effect of GSK143 (1 and 3 μM) was evaluated on cultured peritoneal mast cells (pMCs) and bone marrow derived macrophages (BMDMs) stimulated with immune complexes with IgE-anti-TNP (40 ng/mL) and LPS (100 ng/ml) respectively. Results: Administration of GSK143 (3 mg/kg) resulted in a serum level of 0.38±0.24 μM 1.5 hours after oral delivery. Treatment with a single dose of GSK143 (3 mg/kg) significantly improved gastrointestinal transit after surgical intestinal manipulated mice compared to placebo (Geometric Center (GC): Placebo, 4.2 ±0.4 vs GSK143, GC; 6.9±0.6). In addition, GSK143 was able to reduce the number of recruited myeloperoxidase (MPO) positive cells (placebo, 187±30 vs GSK143, 44±8, per 0.5mm2), neutrophils (placebo, 4.6x10-5±0.2 x10-5 vs GSK143, 1.7x10-5 ±0.7 x10-5) and monocytes (placebo, 1.6x106±0.4 x10-6 vs GSK143, 0.8x10-6 ±0.25 x10-6) in the muscularis externa compared to placebo. To define the possible target cells affected, GSK143 was tested in vitro on pMCs and BMDMs. Thirty minutes after GSK143 (1 and 3 μM) or vehicle treatment, pMCs degranulation, quantified by assessment of β-hexosaminidase, was induced by immune complexes with IgE-anti-TNP. Interestingly, GSK143 significantly reduced pMCs degranulation compared to vehicle in a dose dependent manner (GSK143 1μM, 62±7.5% and GSK143 3μM, 76±3.3% inhibition vs vehicle). In line, pre-treatment of BMDMs with GSK143 (1 and 3 μM) prior to LPS challenge significantly lowered the expression of IL-6 (21±4% to 31± 3 % inhibition vs vehicle ), TNFα (39±0.8% to 54±0.7% inhibition vs vehicle ), IL1 β (31±1.3% to 39±1.06% inhibition vs vehicle) and CCL2 (33±5.3% to 43±7% inhibition vs vehicle). Conclusion: Pre-treatment with the Syk-inhibitor GSK143 attenuates intestinal inflammation and consequently restores gastrointestinal transit in a model of POI. In vitro data suggests that both macrophages and mast cells are targeted by GSK143. These findings strongly suggest that Syk inhibition may be a new therapeutical tool to shorten POI.