Alirocumab attenuated plaque inflammation and PCSK9-induced proinflammatory signalling in M1 macrophages independently of lipid lowering

Abstract

BackgroundProprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) may directly promote vascular inflammation beyond its action on LDL-C degradation. We investigated whether PCSK9 may exacerbate proinflammatory signalling of M1 macrophages and if alirocumab could attenuate this effect and plaque progression by LDL-C independent mechanisms. MethodsApoE-/- mice were treated with alirocumab for 13 weeks and aortic arches were isolated for atherosclerotic plaques characterization based on lesion size and lipid and macrophage infiltration. Also, plasma and splenic monocytes/macrophages were assessed by flow cytometry, and PCSK9, the lipid profile, inflammatory cytokines were measured by qPCR or western blot. Cultured THP-1-derived M1 macrophages were stimulated with PCSK9 and evaluated for TLR4-NF{kappa}B-NLRP3 activation and cytokine production. PCSK9 and proinflammatory factors were analysed in 1190 patients with acute coronary syndrome (ACS). ResultsAlirocumab 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 expression (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-, IL-1{beta}, IL-6) and downregulated anti-inflammatory mediators (i.e., CCL17, TGM2, TGF-{beta}1, IL-10), promoted NF{kappa}B-p65 nuclear translocation, and NLRP3 and gasdermin-D activation. However, TLR4 inhibition or silencing blunted these effects. In addition, there was a positive association between PCSK9 with hsCRP and FGF-23 plasma levels in ACS patients. ConclusionsPCSK9 may be released in parallel to proinflammatory factors such as hsCRP and FGF-23 in ACS patients. However, alirocumab could exert lipid-independent anti-inflammatory effects through activation of the TLR4-NF{kappa}B-NLRP3 signaling in M1 macrophages, promotion of M2, and reducing plaque inflammation.