Figure 10. Atheroprotective effects of trehalose are dependent on macrophage autophagy and p62.

(a,e) mφATG5-KO (a) or p62-KO mice (e; all on ApoE-null background) were fed a western diet for 2 months with concurrent vehicle or trehalose administration (2 g kg⁻¹ given three times per week i.p. and 3% ad libitum in drinking water). Graphs represent quantification of Oil Red O-stained atherosclerotic plaques at the level of aortic root (statistical significance of differences was calculated using Mann–Whitney U-test). (b,f) Immunofluorescence images of ATG5-KO (b) or p62-KO (f) macrophages using DAPI and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). Average p62 intensity (b) or ubiquitin intensity (f) per cell and number of cells is shown. (c,g) ATG5-KO (c) or p62-KO (g) macrophages were co-incubated with cholesterol crystals and trehalose (or vehicle). Per cent of caspase 3/7+ cells were quantified in three independent experiments (number of cells shown under each bar). (d,h) ATG5-KO (d) or p62-KO (h) macrophages were treated as indicated and cell culture media assayed for IL-1β by ELISA (n=3 independent wells for each treatment). Data are presented as mean±s.e.m. *P<0.05, NS: not significant, two-tailed unpaired t-test except a,e. (i) Graphical summary of the benefits of harnessing macrophage autophagy–lysosomal biogenesis in atherosclerosis: TFEB overexpression in macrophages initiates its nuclear localization and autophagy–lysosomal biogenesis (1 and 2). This activation modulates several downstream mechanisms in macrophages that contribute to a reduction in atherosclerosis (3 and 4), such as decreased IL-1β secretion (3), p62-dependent polyubiquitinated protein sequestration and autophagic clearance and decreased apoptosis (4). Our data largely implicate the atheroprotective effects of TFEB to be dependent on the aggrephagy of p62-enriched inclusion bodies and associated reductions in apoptotic cell death (4). The disaccharide trehalose is an inducer of macrophage autophagy and autophagy–lysosomal biogenesis and reduces atherosclerosis by recapitulating these TFEB-induced pathways (5).