# Alirocumab Attenuated Plaque Inflammation and PCSK9-Induced Proinflammatory Signalling in M1 Macrophages Independently of Lipid Lowering

## TL;DR

Alirocumab reduces inflammation in arteries and macrophages by blocking PCSK9, without lowering cholesterol.

## Contribution

Alirocumab inhibits PCSK9-induced inflammation in macrophages and atherosclerosis independently of lipid lowering.

## Key findings

- Alirocumab reduced plaque lesions, lipid infiltration, and M1 macrophage activity in mice.
- PCSK9 activates TLR4-NFκB-NLRP3 signaling in macrophages, promoting inflammation.
- Alirocumab increased anti-inflammatory factors and M2 macrophages in mice.

## Abstract

Background: Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) has been implicated in vascular inflammation beyond its action on LDL-C degradation. We investigated whether PCSK9 may exacerbate proinflammatory signaling of M1 macrophages and if its neutralization with alirocumab could attenuate this effect and plaque progression by LDL-C independent mechanisms. Methods: ApoE−/− mice were treated with alirocumab for 13 weeks, and aortic arches were isolated for atherosclerotic plaque characterization based on lesion size and lipid and macrophage infiltration. Plasma and splenic monocytes/macrophages were also assessed by flow cytometry, and PCSK9, the lipid profile, and inflammatory cytokines were measured by qPCR or Western blot. Cultured THP-1-derived M1 macrophages were stimulated with PCSK9 and evaluated for TLR4-NFκB-NLRP3 activation and cytokine production. In addition, soluble PCSK9, LDL-C, and proinflammatory factors were analyzed in 1190 patients with acute coronary syndrome (ACS). Results: Alirocumab reduced plaque lesion (0.42-fold; p < 0.05) and lipid (0.63-fold; p < 0.01) and macrophage (0.61-fold; p < 0.05) infiltration, mainly the M1 subtype (0.37-fold; p < 0.01), as well as TLR4, NLRP3 and caspase-1 expressions (0.49-fold, 0.51-fold and 0.51-fold, respectively; p < 0.05), without altering LDL-C. Also, it decreased proinflammatory cytokines but enhanced anti-inflammatory factors and M2 markers at the descending aorta. Alirocumab enriched circulating Ly6Clow monocytes (1.51-fold; p < 0.05) and splenic M2 macrophages (1.32-fold; p < 0.01), while reducing M1 (0.62-fold; p < 0.05). In cultured M1 macrophages, PCSK9 overexpressed proinflammatory cytokines (i.e., CXCL9, CXCL10, TNF-α, IL-1β, and IL-6), downregulated anti-inflammatory mediators (i.e., CCL17, TGM2, TGF-β1, and IL-10), and promoted NFκB-p65 nuclear translocation and NLRP3 and gasdermin-D activation. However, TLR4 inhibition or silencing blunted these effects. In patients with AC, there was a positive association between PCSK9 and hsCRP and FGF-23 plasma levels, independently of LDL-C. Conclusions: PCSK9 may be released in parallel to proinflammatory factors such as hsCRP and FGF-23 in patients with ACS, independently of LDL-C levels. PCSK9 may directly promote macrophage-driven inflammatory responses through the TLR4-NFκB-NLRP3 signaling, but its neutralization with alirocumab attenuated this inflammatory axis and limited atherosclerotic progression, supporting an anti-inflammatory benefit secondary to PCSK9 inhibition.