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

## TL;DR

Macrophage-produced galectin-3 promotes multiple forms of cell death in atherosclerosis by activating a key inflammatory pathway.

## Contribution

Galectin-3 is identified as a novel driver of PANoptosis in atherosclerosis via the TLR4/MyD88/NF-κB/NLRP3 axis.

## Key findings

- Galectin-3 is highly expressed in macrophages within atherosclerotic plaques in both humans and mice.
- Galectin-3 activates the TLR4/MyD88/NF-κB/NLRP3 pathway, inducing pyroptosis, apoptosis, and necroptosis.
- Genetic galectin-3 deficiency reduces atherosclerosis, while NLRP3 activation worsens it.

## Abstract

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.

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.

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.

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.

Pyroptosis, apoptosis, and necroptosis of macrophages occur concurrently in atherosclerosis.Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.Galectin‐3 is predominantly expressed in macrophages within atherosclerotic plaques.Galectin‐3 interacts with NLRP3, activates TLR4/MyD88/NF‐kB/NLRP3 signal axis, and induces PANoptosis‐like cell death.Galectin‐3 deficiency attenuates, whereas the NLRP3 agonist nigericin exacerbates, atherosclerotic lesion development.

Pyroptosis, apoptosis, and necroptosis of macrophages occur concurrently in atherosclerosis.

Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Galectin‐3 is predominantly expressed in macrophages within atherosclerotic plaques.

Galectin‐3 interacts with NLRP3, activates TLR4/MyD88/NF‐kB/NLRP3 signal axis, and induces PANoptosis‐like cell death.

Galectin‐3 deficiency attenuates, whereas the NLRP3 agonist nigericin exacerbates, atherosclerotic lesion development.

Pyroptosis, apoptosis and necroptosis of macrophages occur concurrently during atherosclerosis. Galetein‐3 and NLRP3 are highly expressed in atherosclerotic lesions. Galectin‐3 is principally expressed in macrophages within atherosclerotic plaques. Galectin‐3 interacts with NLRP3, activates TLR4/MyD88/NF‐κB/NLRP3 signal axis, and promotes PANoptosis‐like cell death. Galectin‐3−/− relieves, and NLRP3 agonist nigericin enlarges atherosclerotic lesions.