Macrophage-derived galectin-3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF-κB/NLRP3 during atherosclerosis

Abstract

Background: Pyroptosis, apoptosis and necroptosis (PANoptosis) simultaneously occur and are extensively cross-linked in infectious and inflammatory diseases. However, the co-existence and regulation of macrophage pyroptosis, apoptosis and necroptosis in atherosclerosis have not yet been investigated.

Methods: Atherosclerotic specimens from human lower extremity amputation and carotid endarterectomy were analysed. Ox-LDL-induced macrophages and high-fat diet (HFD)-fed ApoE^−/− mice were employed as in vitro and in vivo models, respectively. Galectin-3 was recognised as a key differentially expressed protein and gene related to PANoptosis by integrative proteomic and transcriptomic analysis of atherosclerotic murine aortas. Single-cell transcriptomic analysis of human carotid endarterectomy specimens investigated the cellular distribution of galectin-3. Galectin-3 is a potent driver of macrophage activation and elicits inflammation through NLRP3 inflammasome activation. To elucidate the role of galectin-3/NLRP3 in atherosclerosis, galectin-3 siRNA transfection in macrophages was conducted, galectin-3 and ApoE dual-deficiency mice were produced, AAV-F4/80-shGalectin-3 was injected, and NLRP3 agonist nigericin was administered.

Results: A substantial content of inflammatory factors, the activation of NLRP3/GSDMD/CASP3/CASP8/RIPK3/pMLKL, and the upregulation of galectin-3 were detected in advanced human and mouse atherosclerotic lesions. Galectin-3 was predominantly expressed in atherosclerotic macrophages, and Galectin-3-positive macrophages were mainly distributed in the atherosclerotic core in comparison with the proximal adjacent artery. Ox-LDL induced apoptosis, pyroptosis and necroptosis in macrophages, as evidenced by the activation of NLRP3/GSDMD/CASP3/CASP8/RIPK3/pMLKL and the secretion of proinflammatory cytokines. Galectin-3 interacted with NLRP3. Genetic knockdown of galectin-3 alleviated ox-LDL-induced activation of inflammatory cell death, which was pronouncedly abrogated by NLRP3 agonist nigericin. Genetic galectin-3 deficiency attenuated, and conversely nigericin exacerbated macrophage death, vascular inflammation and atherosclerosis in HFD-fed ApoE^−/− mice. Mechanistically, galectin-3 activated the TLR4/MyD88/NF-κB/NLRP3 axis and induced pyroptosis, apoptosis and necroptosis in macrophages.

Conclusions: Macrophage-derived galectin-3 contributed to pyroptosis, apoptosis and necroptosis in concert, promoted vascular inflammation and atherosclerosis through the upregulation of TLR4/MyD88/NF-κB/NLRP3 pathway.

Key points:

Keywords

apoptosis / atherosclerosis / galectin-3 / necroptosis / NLRP3 / pyroptosis

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