Macrophage Tim-4 protects against deep vein thrombosis by binding CK2β to suppress inflammatory responses
Xiao Wang1,2, Zhen Zhang3, Weiwei Zheng4
1Department of Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China.
Background:
Deep vein thrombosis (DVT) is a venous reflux disorder caused by dysregulated coagulation, with macrophage inflammatory responses being critical for its progression. T-cell immunoglobulin and mucin domain containing 4 (Tim-4) is known as a key regulator of macrophage function and inflammation. However, its involvement in DVT remains completely unclear.
Methods:
Tim-4 expression was comprehensively assessed in peripheral blood mononuclear cells from DVT patients and inferior vena cava-associated macrophages in both clinical specimens and murine DVT models using integrated approaches including single-cell RNA sequencing, immunofluorescence, flow cytometry, quantitative PCR, and Western blot. Macrophage-specific Tim-4 knockout mice (LysM-Cre; Tim-4 fl/fl) and littermate controls (Tim-4 fl/fl) were constructed to investigate the role of macrophage Tim-4 in DVT. The interaction between Tim-4 and CK2β was verified by mass spectrometry and co-immunoprecipitation. The lncRNF219-3:1/miR-93-5p/Tim-4 regulatory axis was validated through RNA pull-down, RNA antisense purification, and luciferase reporter assays.
Results:
Tim-4 was significantly downregulated in DVT-associated macrophages, correlating with elevated proinflammatory cytokine levels. Macrophage-specific Tim-4 knockout aggravated DVT progression both in vitro and in vivo. Mechanistically, Tim-4 directly bound casein kinase 2β (CK2β) regulatory subunit, suppressing CK2 holoenzyme activity and subsequent NF-κB pathway activation (pP65 and pIκBα). Notably, pharmacologically blocking CK2 activation or the NF-κB pathway abolished the pro-thrombotic effects of Tim-4 deficiency. Furthermore, we identified a novel ceRNA network wherein lncRNF219-3:1 acted as a miR-93-5p sponge to indirectly upregulate Tim-4 expression, thereby enhancing anti-inflammatory macrophage responses and attenuating thrombus formation.
Conclusions:
Our findings demonstrate that macrophage Tim-4, regulated by lncRNF219-3:1/miR-93-5p axis, functions as a critical suppressor of DVT through hijacking and sequestering CK2β to dampen NF-κB mediated inflammation. The study unveils novel immunomodulatory mechanisms in DVT pathogenesis and highlights Tim-4 and its regulatory network as potential therapeutic targets for DVT in clinic.
