Gain-of-Function Variant in Spleen Tyrosine Kinase Regulates Macrophage Migration and Functions to Promote Intestinal Inflammation.

Abstract

Purpose: Spleen tyrosine kinase (Syk) is a widely-expressed cytoplasmic non-receptor tyrosine kinase involved in regulating various signaling pathways and plays an important role in chronic inflammation and autoimmune diseases. Gain-of-function SYK variants have been implicated in pediatric inflammatory bowel diseases. This study aimed to investigate the effects of gain-of-function SYK variants on the susceptibility to experimental colitis and macrophage function.

Methods: Colitis was induced using dextran sodium sulfate and dinitrobenzene sulfonic acid in mice harboring a gain-of-function variant in SYK (Syk^S544Y). Intestinal inflammation was assessed via disease activity index, histological analysis, and Western blotting. The frequencies of macrophages, phagocytosis, and reactive oxygen species (ROS) production in bone marrow-derived macrophages (BMDM) were measured via flow cytometry. Chemokines and BMDM chemotaxis were analyzed using real-time quantitative reverse transcription polymerase chain reaction and Transwell assays. The expression of nucleotide-binding domain leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome-related proteins were detected using immunohistochemistry, enzyme-linked immunoassay and Western blotting.

Results: Syk^S544Y mice exhibited increased susceptibility to experimental colitis, and macrophage infiltration in colon tissues significantly increased. We observed increased expression of macrophage chemokines in colon tissues and enhanced chemotaxis in Syk^S544Y BMDM. Additionally, we detected increased levels of fluorescent microspheres and 2.7-dichloride-hydro fluorescein diacetate-labeled ROS in Syk^S544Y BMDM. Moreover, enhanced levels of NLRP3 inflammasome-related proteins were observed in the colon tissues and BMDM from Syk^S544Y mice.

Conclusion: Gain-of-function variant in SYK may contribute to the pathogenesis of pediatric inflammatory bowel diseases by promoting macrophage migration, phagocytosis, ROS production and activation of NLRP3 inflammasomes.