Atherosclerotic plaques develop autophagy dysfunction

Although the link between deficient macrophage autophagy and increased atherosclerosis has been reported by several groups, including ours3,4,5,6, the exact nature of the autophagy dysfunction that takes place in atherosclerotic macrophages is unclear. Macrophages of the atherosclerotic plaque are known to be the predominant expressors of two commonly used autophagy markers, LC3 (an autophagosome coat protein and a direct marker of autophagy progression) and p62 (a selective autophagy chaperone that detects and delivers large biomolecules such as protein aggregates or organelles to autophagosomes)3. However, details about the expression of these markers with relation to plaque progression and their correlation with the observed autophagy dysfunction remain unknown. Since LC3 levels correlate with the progression of autophagy and the accumulation of p62 levels correlate with stalled or dysfunctional autophagy30, we first conducted detailed analysis of immunostained atherosclerotic plaques from atheroprone ApoE-null mice and atherosclerotic human carotid samples.

First, we compared the levels and co-localization of LC3 and p62 in aortic root plaques of ApoE-null mice at two stages of atherogenesis (‘early’ versus ‘advanced’ lesions as defined by the duration of western diet feeding). LC3 levels were significantly lower in the more advanced lesions, whereas p62 intensity remained elevated in both early and advanced lesions (Fig. 1a–c). In early lesions, LC3 levels were still high, and much of the p62 showed co-localization with LC3; this co-localization markedly waned in advanced lesions (Fig. 1d,e). Consistent with our previously reported observation, p62 (a chaperone for polyubiquitinated protein aggregates) showed high co-localization with polyubiquitin stains in both lesion types (Supplementary Fig. 1a)6. Overall, these data indicate that the observed dysfunction in plaque autophagy is manifested as a constellation of events (that is, p62 accumulation, decline in LC3/autophagosome formation and an inability to co-express autophagy markers).

Figure 1: Mouse and human atherosclerotic plaques develop features of a progressive autophagy dysfunction. (a–c) Representative immunofluorescence images of early-stage and more advanced atherosclerotic (ApoE-KO) aortic roots co-stained with antibodies against LC3 and p62. Early and advanced lesions were obtained from ApoE-KO mice fed a western diet for <2 months and 3–4 months, respectively (scale bar, 50 μm (a)). The mean intensity for LC3 and p62 stainings were analysed (n=5 mice for each group; b,c). (d,e) Co-localization of LC3 and p62 was also analysed in the same aortic roots. Representative co-localization images are shown from early and more advanced lesions (green indicates LC3/p62 co-localized, red indicates LC3-positive, and blue indicates p62-positive areas) (d). LC3/p62 co-localization is quantified as per cent of total signal (e). (f–k) Immunofluorescence analysis of human carotid endarterectomy specimens (n=8), which are separated as maximally- and adjacent minimally diseased regions (scale bar, 100 μm). Specimens were co-stained with LC3 and p62 (f), and co-localization (g) as well as correlation of staining intensity (h) between maximally and minimally diseased regions are quantified. Maximally diseased human atherosclerotic regions were co-stained for p62 and polyubiquitinated proteins (FK-1 antibody; i), and co-localization quantified (j). (k) Graph represents a comparison of the staining correlation between p62/LC3 versus p62/ubiquitin(FK-1) in maximally atherosclerotic regions. For all graphs, data are presented as mean±s.e.m. **P<0.01, ***P<0.001, two-tailed unpaired t-test. Max, maximum; Min, minimum; Ubiq., ubiquitination. Full size image

We were interested in determining whether our findings in mouse models are recapitulated in human atherosclerosis and repeated a similar analysis in human atherosclerotic plaques obtained from discarded carotid endarterectomy (CEA) specimens. CEA lesions were dissected immediately postoperatively into regions devoid of disease or regions with minimal or maximal disease, for LC3 and p62 staining. In areas lacking atherosclerosis, we only detected LC3 without any evidence for p62 or ubiquitin accumulation by immunofluorescence (IF), consistent with the presence of basal of autophagy without aberrant accumulation of autophagic cargo (Supplementary Fig. 1b). The accumulation of p62 was detected in minimally diseased regions and increased (that is, p62 staining increased in both size and number) in maximally diseased regions, although it did not reach statistical significance (Fig. 1f and Supplementary Fig. 1c). In contrast, the intensity of LC3 staining was both reduced (Fig. 1f and Supplementary Fig. 1d) and developed poor localization with p62 (Fig. 1g,h) in maximally diseased regions. Given the lower co-localization of p62 and LC3, we also evaluated the staining pattern of p62 and polyubiquitin in the same samples and found the large majority of p62 co-localized with polyubiquitin (Fig. 1i,j). Technical limitations precluded co-staining of plaques with LC3, p62 and polyubiquitin in unison and direct correlation between all markers. However, comparison of co-staining correlations between p62 and LC3 and p62 and polyubiquitin demonstrated significantly better correlation for p62 and polyubiquitin (Fig. 1k). Taken together, these data suggest that in advancing atherosclerotic plaques the formation and stagnation of p62-enriched polyubiquitinated aggregates is favoured over clearance and is a hallmark feature of the autophagy dysfunction in atherosclerosis.

Macrophage TFEB overexpression rescues plaque autophagy

The progressive decrease in LC3 levels and p62/LC3 co-localization as well as the accumulation of polyubiquitinated proteins with advancing plaque formation led us to investigate methods by which the autophagic degradation machinery can be stimulated. TFEB is the predominant transcription factor capable of inducing coordinated expression of autophagy–lysosomal genes and the prodegradative response in several cell types including macrophages12,13,14. We utilized a previously described tissue-specific overexpression model of TFEB in mice and created macrophage-specific TFEB-transgenic mice by conducting crosses with mice expressing Cre under the control of Lysozyme-M promoter (hereafter referred to as mφTFEB-TG)12,13. Thioglycollate-elicited peritoneal macrophages (hereafter referred to as macrophages) from these mice showed increased TFEB expression, more TFEB nuclear localization and TFEB-induced target gene expression including p62 and LC3 (Fig. 2a–c, Supplementary Figs 2a and 12a). We also found enhanced autophagic flux in TFEB-TG (tissue-specific transgenic expression of TFEB) macrophages by observing baseline elevations in LC3-I levels with enhanced conversion to LC3-II upon bafilomycin incubation (a lysosomal inhibitor/blocker of autophagosome degradation; Fig. 2d and Supplementary Fig. 11a). In addition, p62 levels were persistently higher in agreement with our gene expression results (Fig. 2c,d). We crossed mφTFEB-TG mice with green fluorescent protein-tagged LC3 (GFP-LC3)-expressing mice to be able to further monitor autophagy in macrophages by live imaging. TFEB-TG macrophages showed significantly more GFP-LC3-positive area in 20 min of live imaging, suggesting induced autophagosome formation even under unstimulated conditions (Fig. 2e, Supplementary Fig. 2b,c and Supplementary Movies 1 and2). When these macrophages were incubated with bafilomycin, even higher GFP-LC3 fluorescence was observed consistent with TFEB-induced increases in autophagic flux (Fig. 2f, Supplementary Fig. 2d,e and Supplementary Movies 3 and 4). Increased flux was also corroborated by GFP-LC3 dots showing more co-localization with the lysosome marker Lysotracker in TFEB-TG macrophages (Fig. 2g, Supplementary Fig. 2f–h and Supplementary Movies 5 and 6). In agreement with our real-time live imaging experiments, we observed a significantly higher autophagic flux in TFEB-TG macrophages stained by LC3 immunocytochemistry at both baseline and after 3 h of treatment with another blocker of lysosomal degradation chloroquine (Fig. 2h). In keeping with TFEB’s ability to induce the autophagy–lysosome system as a whole, we also detected increases in LAMP1 expression (a commonly used marker of lysosomes and lysosomal mass) both in cultured peritoneal macrophages and splenic macrophages derived from mφTFEB-TG mice (Fig. 2i). In conclusion, these data suggest that overexpressing TFEB is a sufficient method to induce the autophagosome formation and autophagy–lysosomal biogenesis in macrophages.

Figure 2: TFEB overexpression induces autophagy and autophagy–lysosomal biogenesis in macrophages. (a–c) Control and TFEB-overexpressing (TFEB-TG) thioglycollate-elicited peritoneal macrophages (hereafter referred to as macrophages) were assessed as follows: transcript levels of (a) TFEB and (c) several autophagy–lysosome markers were evaluated by quantitative polymerase chain reaction (qPCR, n≥3 independent wells). (b) TFEB nuclear localization was assessed by immunofluorescence staining and quantified as percentage of TFEB-positive nuclei (n≥40 cells per group, scale bar, 20 μm). (d) Western blot analysis of p62 and LC3 in TFEB-TG macrophages after bafilomycin (200 nM) treatment for indicated times (C, control; T, TFEB-TG). (e–g) Control and TFEB-TG macrophages also co-expressing GFP-LC3 were evaluated by live imaging (every 30 s for the indicated times) while being incubated with either (e) DMEM, (f) bafilomycin (200 nM) or after staining with (g) Lysotracker-red. Graphs represent (e,f) GFP-LC3-positive areas or (g) per cent of GFP-LC3 co-localized with Lysotracker-red over the indicated times. For e,f each time point is compared with the control GFP-LC3 group (n≥10 cells for each treatment). (h) LC3 levels and the intracellular pattern were analysed by immunofluorescence staining of baseline (NoTX) or after 3 h of 10 μM chloroquine incubation. Graphs represent the mean LC3 intensity (n=16–46 cells, scale bar, 5 μm). (i) FACS analysis of peritoneal and splenic macrophages from control or macrophage-specific TFEB-TG mice for LAMP1 expression. For all graphs, three independent experiments were performed; data presented as mean±s.e.m. *P<0.05, ***P<0.001, two-tailed unpaired t-test. Baf,bafilomycin; CHQ, chloroquine; Ctrl, control; DAPI, 4,6-diamidino-2-phenylindole. Full size image

We next asked whether macrophage-specific TFEB overexpression can induce autophagy and autophagy–lysosomal biogenesis in atherosclerotic plaques in vivo. We crossed mφTFEB-TG mice with pro-atherogenic ApoE-KO mice and initiated plaque formation by western diet feeding. TFEB expression as gauged by IF staining of aortic roots was significantly elevated and coincided with plaque macrophages (Fig. 3a,b). More importantly, TFEB nuclear localization was particularly elevated in the atherosclerotic plaques of mφTFEB-TG mice using two independent TFEB-specific antibodies (Fig. 3c and Supplementary Fig. 3a). The expression of both LC3 and p62 was also increased in mφTFEB-TG aortic roots in agreement with our in vitro macrophage data (Fig. 3d,e). Interestingly, in direct contrast to what we observed in the progressive atherosclerosis of mice models and the maximally diseased regions of human plaques (Fig. 1), mφTFEB-TG atherosclerotic plaques showed remarkably higher p62-LC3 co-localization and co-staining correlation than control atherosclerotic lesions (Fig. 3f,g). Macrophages from mφTFEB-TG atherosclerotic aortas analysed by fluorescence activated cell sorting (FACS) analysis also displayed enhanced co-expression of autophagy and lysosomal markers as gauged by co-staining of LC3 and p62 as well as LC3 and Lamp2 (Fig. 3h,i). Overall, these data suggest that macrophage-specific TFEB overexpression is a viable approach to induce autophagy and autophagy–lysosomal biogenesis, reprogramme plaque macrophages to co-associate autophagy markers and their cargo, and reverse the autophagy dysfunction observed in advancing atherosclerosis.

Figure 3: TFEB overexpression in macrophages induces the autophagy markers LC3 and p62 and restores their co-localization in atherosclerotic aortic roots. (a,b) Representative immunofluorescence images of atherosclerotic aortic roots (2 months’ western diet) from control and mφTFEB-TG mice (ApoE-null background) stained with antibodies against TFEB (a), TFEB and MOMA-2 (b; scale bar, 50 μm). (c) Quantification of the average TFEB intensity and co-localization with nuclear marker DAPI (n=4-5 mice per group). (d) Representative immunofluorescence images of atherosclerotic aortic roots from control and mφTFEB-TG mice stained with p62 and LC3 (scale bar, 50 μm). (e) Quantification of the p62 and LC3 average intensity from control and mφTFEB-TG-stained roots (n=13–14 mice per group). (f) Representative pseudocolour image of these p62/LC3 images (green represents co-localization) and graph depicting the increased p62/LC3 correlation seen in a representative mφTFEB-TG as compared to a control lesion (scale bar, 50 μm). (g) Quantification of the p62/LC3 co-localization from control and mφTFEB-TG-stained roots shown (n=13–14 mice per group). (h,i) FACS analysis of aortic macrophages isolated from atherosclerotic aortas of Control or mφTFEB-TG mice (western diet-fed ApoE-KO background, n=3–4 pooled aortas) and stained for either (h) p62 and LC3, or (i) Lamp2 and LC3 antibodies (per cent of macrophages expressing each marker is shown below plots). For all graphs, data are presented as mean±s.e.m. *P<0.05, ***P<0.001, two-tailed unpaired t-test. Full size image

Macrophage TFEB overexpression reduces atherosclerosis

In order to assess the effect of macrophage TFEB overexpression on plaque progression, we fed cohorts of control and mφTFEB-TG (on a pro-atherogenic ApoE-null background) a Western diet for 8 weeks and quantified atherosclerotic lesion formation and parameters of plaque complexity (Fig. 4a details this study). mφTFEB-TG mice did not show any difference on serum cholesterol or other common serum metabolites compared to controls (Fig. 4b and Supplementary Fig. 4a,b). Lesion quantitation revealed mφTFEB-TG mice were significantly protected from atherosclerosis at both the level of the aortic root and whole aorta (Fig. 4c,d). Several features of plaque complexity were also concomitantly reduced: macrophage-positive and necrotic core areas were slightly reduced but the apoptotic-positive areas (as assessed by TUNEL staining) and the combined apoptotic/necrotic-positive areas were markedly reduced contributing to an approximately 50% reduction in lesion area (Fig. 4e,f and Supplementary Fig. 4c–e). The pro-inflammatory cytokine interleukin (IL)-1β, which has an inverse correlation with macrophage autophagy, was also substantially reduced in the serum of mφTFEB-TG mice (Fig. 4g). Given the ability of macrophage TFEB to induce autophagy and autophagy chaperones such as p62, we next evaluated the functional significance of some of these findings in cultured macrophages. In a recent detailed study of p62 in atherosclerosis, we have shown that atherogenic lipids such as cholesterol crystals render the macrophage autophagy–lysosome system dysfunctional and lead to accumulation of p62-enriched polyubiquitinated protein aggregates6. TFEB-TG macrophages reversed this effect and significantly reduced the accumulation of inclusion bodies (Fig. 4h,i). As we have previously shown, these inclusions are mostly composed of polyubiquitinated proteins, are largely devoid of significant amounts of lipids (Supplementary Fig. 4f) and disrupted autophagy of these p62-enriched aggregates is associated with increased apoptosis and inflammasome/IL-1β activation6. TFEB-TG macrophages also ameliorated these deleterious downstream effects by markedly reducing cholesterol crystal-induced macrophage apoptosis (Fig. 4j) and IL-1β secretion induced by various inflammasome-activating stimuli (Fig. 4k and Supplementary Fig. 4g). The anti-inflammatory phenotype of TFEB-TG macrophages appeared to be selective to IL-1β signalling because tumour necrosis factor secretion was not changed in those macrophages (Supplementary Fig. 4h). TFEB-TG macrophages also did not appear to show differences in lipid accumulation or foam cell formation upon challenge with modified lipids (Fig. 4l and Supplementary Fig. 4i–k) despite having a predicted rise in the activity of lysosomal acid lipase, a known transcriptional target of TFEB (Supplementary Fig. 4l)12.

Figure 4: Macrophage-specific TFEB overexpression is atheroprotective. (a) Experimental protocol and mouse cohorts used for assessment of atherosclerosis. (b–g) Control and mφTFEB-TG mice (all on ApoE-KO background) were fed a western diet for 2 months for lesion development. (b) Serum cholesterol levels at 2 months (n≥14 mice per group). (c) Quantification of atherosclerotic plaque burden using Oil Red O-stained aortic root sections with representative roots shown on right (scale bar, 0.4 mm) and (d) en face analysis of whole aorta (statistical significance of differences calculated using Mann–Whitney U-test). (e) Macrophage content in aortic root sections was analysed by MOMA-2-positive area (n≥12 mice per group). (f) Apoptotic, necrotic core and combined apoptotic–necrotic core areas of aortic root sections were determined by quantifying TUNEL immunofluorescence staining, acellular areas or the combined area, respectively (n≥12 mice per group). (g) Serum IL-1β concentration was measured from n≥6 independent samples derived by pooling serum from two to three mice per sample (>12 mice per group). (h) Control and TFEB-TG macrophages treated with or without cholesterol crystals for 24 h and stained using DAPI (nuclei) and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (i) Quantification of average p62/ubiquitin-positive dots and average p62 intensity per cell from immunofluorescence experiment described in h (numbers of cells under each bar). (j) Control and TFEB-TG macrophages were incubated with cholesterol crystals and per cent of caspase 3/7-positive cells quantified in three independent experiments. Representative immunofluorescence images are shown on left and numbers of cells shown under each bar (scale bar, 50 μm). (k) Control and TFEB-TG macrophages were treated with LPS (lipopolysaccharide)+cholesterol crystals (hereafter referred to as LPS+CC) for 24 h or with LPS followed by ATP for 3 h. Culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). (l) Control and TFEB-TG macrophages were treated with DiI-acetylated LDL for 12 h and intracellular lipid accumulation quantified by immunofluorescence microscopy (n≥187 cell per group, scale bar, 5 μm). For all graphs, data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired t-test, except c,d. Full size image

TFEB-driven atheroprotection is autophagy and p62 dependent

Our characterization of mφTFEB-TG mice in vivo and cultured macrophages in vitro demonstrates TFEB’s ability to increase macrophage autophagy and autophagy–lysosomal biogenesis, increase aggrephagy and the clearance of p62-enriched protein aggregates, decrease macrophage apoptosis and the pro-inflammatory cytokine IL-1β, and decrease atherosclerosis and plaque complexity. In order to determine whether the TFEB-autophagy-p62-mediated effects are causally linked to the observed reduction in atherosclerosis, we developed two new lines of mice by crossing mφTFEB-TG mice with either macrophage-specific autophagy-deficient (mφATG5-KO) or p62-deficient (p62-KO) mice (on a pro-atherogenic ApoE-null background). Autophagy remained fully inactive in ATG5-KO macrophages even with concomitant TFEB overexpression as gauged by absence of the autophagosome marker LC3-II (Supplementary Figs 5a and 12b).

Consistent with TFEB’s dependence on autophagy, the dual mφTFEB-TG/ATG5-KO mice were no longer protected from atherosclerosis (Fig. 5a) with similar serum cholesterol and other common serum metabolites to controls (Supplementary Fig. 5b–d). We also observed no differences in parameters of plaque complexity (Fig. 5b,c). In addition, dual TFEB-TG/ATG5-KO macrophages subjected to atherogenic lipids were no longer able to (1) reduce the number of p62-enriched protein aggregates or (2) blunt the degree of apoptosis (Fig. 5d–f). We were surprised to find out, however, that TFEB retained its ability to specifically diminish the inflammasome/IL-1β levels even in the absence of autophagy, suggesting an independent mechanism that does not appear to be relevant to the mechanism of plaque reduction (Fig. 5g and Supplementary Fig. 5e,f).

Figure 5: Macrophage-specific TFEB overexpression requires an intact autophagy pathway for atheroprotection including efficient clearance of polyubiquitinated protein aggregates and reductions in macrophage apoptosis. (a) Cohorts of control and mφTFEB-TG mice (all on mφATG5-KO and ApoE-KO background) were fed a western diet for 2 months to develop lesions—exact genotypes are provided at the top of the graph. Atherosclerotic plaque burden was quantified by computer image analysis of Oil Red O-stained aortic root sections with representative Oil Red O-stained aortic roots shown on right (scale bar, 0.4 mm; statistical significance of differences was calculated using Mann–Whitney U-test). (b) Macrophage content in aortic root sections was analysed by immunofluorescence staining using an antibody against MOMA-2 (n≥10 mice per group). (c) Apoptotic, necrotic core and combined apoptotic–necrotic core areas of aortic root sections were determined by quantification of TUNEL immunofluorescence staining, acellular areas or a combination of the two, respectively (n≥10 mice per group). (d) Immunofluorescence images of ATG5-KO and ATG5-KO/TFEB-TG macrophages treated with or without cholesterol crystals for 24 h and stained using DAPI (nuclei) and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (e) Quantification of average p62/ubiquitin-positive dots and average p62 intensity per cell from immunofluorescence experiment described in d (numbers of quantified cells are shown under each bar). (f) ATG5-KO and ATG5-KO/TFEB-TG macrophages were incubated with cholesterol crystals and the per cent of caspase 3/7-positive cells was quantified in three independent experiments (numbers of quantified cells are shown under each bar). (g) ATG5-KO and ATG5-KO/TFEB-TG macrophages were treated with LPS+CC for 24 h or with LPS, followed by ATP for 3 h. Culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). For all graphs, data are presented as mean±s.e.m. *P<0.05, **P<0.01, NS: not significant, two-tailed unpaired t-test, except a. Full size image

Our findings with dual mφTFEB-TG/ATG5-KO macrophages were mirrored with dual mφTFEB-TG/p62-KO mice where again TFEB’s protective effects on atherosclerosis and lesion complexity were abrogated in the absence of p62 (Fig. 6a–c and Supplementary Fig. 6a–c). As we have described before, the absence of p62 in macrophages leads to a disruption and further accumulation of polyubiquitinated proteins upon atherogenic lipid treatment6. TFEB overexpression was unable to reverse this effect, resulting in a similar degree of polyubiquitinated proteins in dual TFEB-TG/p62-KO macrophages (Fig. 6d,e). Consistent with the involvement of cell death pathways, TFEB overexpression was unable to prevent atherogenic lipid-induced apoptosis (Fig. 6f). With regard to the inflammasome and IL-1β, we were again surprised to discover that TFEB’s ability to dampen IL-1β secretion was completely independent of p62 and reaffirmed the notion that TFEB mediates these IL-1β-suppressive effects via an alternative mechanism that appears to have no physiological significance for atherosclerotic plaque formation (Fig. 6g and Supplementary Fig. 6d,e).

Figure 6: The atheroprotective effect of macrophage-specific TFEB overexpression is also p62-dependent. (a) Cohorts of control and mφTFEB-TG mice (all on p62-KO and ApoE-KO background) were fed a western diet for 2 months to develop lesions—exact genotypes are provided at the top of the graph. Atherosclerotic plaque burden was quantified by computer image analysis of Oil Red O-stained aortic root sections with representative Oil Red O-stained aortic roots shown on right (scale bar, 0.4 mm; statistical significance of differences was calculated using Mann–Whitney U-test). (b) Macrophage content in aortic root sections was analysed by immunofluorescence staining using an antibody against MOMA-2 (n≥11 mice per group). (c) Apoptotic, necrotic core and combined apoptotic–necrotic core areas of aortic root sections were determined by quantification of TUNEL immunofluorescence staining, acellular areas or a combination of the two, respectively (n≥11 mice per group). (d) Immunofluorescence images of p62-KO and p62-KO/TFEB-TG macrophages treated with or without cholesterol crystals for 24 h and stained using DAPI (nuclei), and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (e) Quantification of average ubiquitin intensity per cell from immunofluorescence experiment described in d (numbers of quantified cells are shown under each bar). (f) p62-KO and p62-KO/TFEB-TG macrophages were incubated with cholesterol crystals and the per cent of caspase 3/7-positive cells was quantified in three independent experiments (numbers of quantified cells are shown under each bar). (g) p62-KO and p62-KO/TFEB-TG macrophages were treated with LPS+CC for 24 h or with LPS followed by ATP for 3 h. Culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). For all graphs, data are presented as mean±s.e.m. *P<0.05, ***P<0.001, NS: not significant, two-tailed unpaired t-test, except a. Full size image

Trehalose induces macrophage autophagy–lysosomal biogenesis

In order to leverage the therapeutic benefit of TFEB activation in macrophages, we were interested in evaluating compounds capable of stimulating a similar response. The natural disaccharide trehalose has been reported to have autophagy-inducing and protein aggregate-reducing effects that parallel those observed for TFEB, which led us to assess trehalose’s ability to stimulate macrophage autophagy, autophagy–lysosomal biogenesis and downstream effects on atherosclerosis.

We first aimed to determine a therapeutically relevant trehalose dose that could be tested both in macrophages in vitro and then in our atherosclerosis mouse models in vivo. This was an important initial evaluation due to the discrepancy in prior literature on what constitutes a physiologically effective dose of trehalose for autophagy induction; many studies use concentrations as high as 100 mM in cultured cells that appear to differ greatly from doses likely achievable in vivo22,23,24,25,26,27,28. We thus administered trehalose to a cohort of mice intraperitoneally (i.p. 3 g kg−1 body weight), a dose similar to that used in prior studies involving mouse models22,26,28,29, and determined simple pharmacokinetics of serum trehalose (Fig. 7a). Over a 2 h time course after bolus injection, trehalose clearly peaks between 30 and 60 min (up to a concentration of ∼10 mM) and then largely redistributes or is cleared by 2 h (Fig. 7a). We further determined the kinetics of trehalose in relevant target tissues (aorta, heart and spleen) by mass spectrometry, and found more sustained tissue levels with trehalose being detectable for up to 4 h (Fig. 7b). Trehalose levels in the aorta were also corroborated by a colorimetric assay (Supplementary Fig. 7a). On the cellular level, we also found that trehalose is indeed capable of being taken by macrophages in a concentration-dependent manner (Fig. 7c).

Figure 7: Trehalose induces autophagy and the transcription of autophagy–lysosomal genes in macrophages. (a,b) Time course of serum and tissue trehalose levels from wild-type mice (n≥4) after trehalose administration (3 g kg−1 i.p.) by colorimetric method and mass spectrometry, respectively. (c) Macrophages were treated with vehicle or trehalose at indicated concentrations for 3 h and intracellular trehalose levels measured by mass spectrometry (n=2 independent wells). (d) GFP-LC3-expressing macrophages were imaged live every 30 s while being incubated in DMEM (control)±bafilomycin (200 nM), PBS (starvation) or trehalose (1 mM, 100 μM) for 20 min. GFP-LC3-positive area was quantified (n≥10 cells for each treatment) and plotted relative to 0 min. No significant difference seen for DMEM and 100 μM trehalose treatments. Trehalose (1 mM), bafilomycin and PBS significance is demarcated by *, # and ‡, respectively (P<0.05 for all cases). (e) Protocol as in d but macrophages were imaged live for 1 h (DMEM and 1 mM trehalose) or 2 h (100 μM trehalose). No significant difference seen for DMEM and 100 μM trehalose treatments. *P<0.05 for 1 mM trehalose treatment time points. (f) Wild-type macrophages were incubated with trehalose (1 mM; 3 and 6 h), bafilomycin (200 nM; 6 h) or both (6 h trehalose pretreatment and 6 h co-incubation) and stained with LC3 antibody and DAPI. Representative images are shown at left and quantification of average LC3 intensity with each condition at right (number of cells under each bar). #shows significant difference compared to vehicle (using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test; scale bar, 5 μm). (g) Wild-type macrophages were treated with 1 mM trehalose for indicated time points and transcripts of autophagy–lysosomal genes detected by qPCR (n≥3 independent wells for each gene). (h) Western blot analysis of Cathepsin D, Lamp1, p62 and LC3 in macrophages after 1 mM trehalose treatment for indicated times. Ponceau S staining used as loading control. For graphs in c,f,g, data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, NS: not significant, two-tailed unpaired t-test compared to zero time point or vehicle, except f. Full size image

On the basis of this analysis, we chose reasonable doses of trehalose at 1/10th and 1/100th (that is, 1 mM and 100 μM) of the observed peak serum dose to conduct all of our culture analyses. Using macrophages from GFP-LC3 mice, we first conducted live-cell imaging to quantify autophagosome formation upon short-term trehalose exposure. Trehalose (1 mM but not 100 μM) was able to minimally elevate autophagy within minutes that persisted at low levels for up to 1 h but was clearly not as potent as nutrient starvation (PBS) or the autophagosome buildup seen with the lysosomal inhibitor bafilomycin (Fig. 7d,e, Supplementary Fig. 7b–g and Supplementary Movies 7–10). In contrast, macrophages treated with trehalose for longer periods (3, 6 and 12 h) displayed more potent autophagy-inducing effects (Fig. 7f and Supplementary Fig. 7h). The need for longer incubation times raised the possibility that trehalose’s effects might also relate to transcriptional activation of autophagy and induction of autophagy–lysosomal biogenesis. Indeed, beginning at 3 h, trehalose significantly induced transcripts for a variety of autophagy and lysosomal genes (Fig. 7g) and concomitantly induced their protein expression (Fig. 7h and Supplementary Figs 7i and 11b). Moreover, trehalose was still able to induce some transcriptional activation of autophagy–lysosomal genes even at 100 μM, a dose at which we observed no autophagy induction during the short time course experiments (Supplementary Fig. 7j), suggesting a more potent role for trehalose in autophagy–lysosomal biogenesis. In this regard, we also tested whether relatively short exposure to trehalose could potentially sustain autophagy for longer durations. Interestingly, macrophages transiently incubated with trehalose for 3 h still maintained elevations in the autophagy markers LC3 and p62 for an additional 9 h even in the absence of additional trehalose exposure (Supplementary Figs 7k and 12c). These sets of experiments suggested that trehalose can promote a sustained autophagy–lysosomal biogenesis programme in macrophages with peak effect on the order of 6–12 h.

Trehalose drives and functionally mimics TFEB activation

Supporting a role for trehalose in autophagy–lysosomal biogenesis and TFEB activation, trehalose was able to increase both TFEB expression at both 1 mM and 100 μM doses as well as TFEB nuclear translocation, a surrogate marker for TFEB activation, and its transcriptional effects (Fig. 8a,b and Supplementary Fig. 11c). Time course experiments conducted over 24 h suggested maximal trehalose-induced TFEB expression to occur at approximately the 6-h time point coinciding with the transcriptional activation of the autophagy–lysosomal genes (Supplementary Figs 8a and 12d). Since TFEB is a member of the MiTF transcription factor family (which includes MiTF and TFE3) and those transcription factors have also been previously implicated as inducers of autophagy–lysosomal biogenesis in different tissues31,32, we also tested the effects of trehalose on MiTF and TFE3. Interestingly, trehalose had similar stimulatory effects on TFE3 (as gauged by increased TFE3 transcripts and nuclear translocation) but not MiTF (Fig. 8c–e and Supplementary Figs 8b–e and 11d).

Figure 8: Trehalose induces TFEB nuclear localization and protects from atherogenic lipid-induced protein aggregation and related sequelae of apoptosis and inflammasome activation. (a) Western blot analysis of TFEB in macrophages after trehalose treatment for indicated times. Ponceau S staining is shown as loading control and densitometric quantification from three separate experiments is shown below. (b) TFEB nuclear localization is analysed by immunofluorescence staining after trehalose treatment and graphed as nuclear TFEB intensity (n≥40 cells per group, scale bar, 10 μm). (c) Western blot analysis of other MiTF transcriptional family members (TFE3 and MiTF) after trehalose or chloroquine (CHQ, 10 μM) treatments for indicated doses and times. Ponceau S staining is shown as loading control. (d,e) TFE3 and MiTF nuclear localization was analysed by immunofluorescence staining after trehalose treatment for indicated times and quantified by the intensity of nuclear staining (n≥500 cells per group). (f) Western blot analysis of polyubiquitinated proteins (FK-1 antibody) in detergent-soluble and detergent-insoluble lysate fractions of vehicle (V) or trehalose (T) treated wild-type macrophages. Lanes 3 and 4 were either vehicle or trehalose pretreated for 3 h, and then co-treatment with cholesterol crystals is performed for 12 h. Lanes 5 and 6 were cholesterol crystal-treated for 6 h and then either treated with vehicle or trehalose alone for another 6 h. (g) Densitometric quantification of f from three similar separate experiments. (h) Immunofluorescence images of wild-type macrophages after indicated treatments using DAPI and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (i) Graphs represent average p62/ubiquitin+ dot numbers and average p62 intensity per cell for immunofluorescence images in h (numbers of quantified cells are shown under each bar). (j) Wild-type macrophages were co-incubated with cholesterol crystals and trehalose (or vehicle); the per cent of caspase 3/7-positive cells was quantified in three independent experiments (numbers of quantified cells are shown under each bar). (k) Wild-type macrophages were treated as indicated and cell culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). For all graphs data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired t-test compared to zero time point or vehicle treatment group. Full size image

Because trehalose has the capacity to induce transcriptional activation of autophagy and autophagy–lysosomal biogenesis in macrophages, we sought to assess its potential to recapitulate the functional benefits of macrophage TFEB activation we had observed in the mφTFEB-TG system. Indeed, trehalose reduced atherogenic lipid-induced p62-enriched polyubiquitinated protein accumulation as assessed by both immunoblot and IF (Fig. 8f–i and Supplementary Figs 8f and 11e). Trehalose also concomitantly blunted macrophage apoptosis and IL-1β secretion in assays similar to those we conducted for the TFEB-overexpressing macrophages above (Fig. 8j,k).

Trehalose induces autophagy in vivo and is atheroprotective

To evaluate whether trehalose has autophagy- and TFEB-inducing properties directly in the plaque, we fed a cohort of GFP-LC3 mice (on a pro-atherogenic ApoE-null background) a western diet for 6 weeks to develop atherosclerotic lesions followed by an additional 2 weeks of trehalose administration. A combined strategy of i.p. (2 g kg−1 body weight, daily) and oral (3% w/v, ad libitum) trehalose administration was pursued since similar dosing strategies have been used in prior trehalose studies of neurodegenerative/protein aggregation disorders22,26,28,29. IF quantification of aortic root atherosclerotic lesions revealed both increased GFP-LC3 fluorescence (Fig. 9a,b), together with enhanced TFEB staining and TFEB nuclear localization (Fig. 9c–e and Supplementary Fig. 9a,b). Splenic macrophages derived from trehalose-treated mice also showed increased transcripts for a variety of autophagy and lysosomal genes (Supplementary Fig. 9c). These data indicate trehalose’s ability to induce autophagy as well as TFEB activation in vivo in agreement with our cultured macrophage data and provided the impetus to conduct a longer-term atherosclerosis study.

Figure 9: Trehalose administration in mice is atheroprotective. (a,b) GFP-LC3 mice (ApoE-KO background) were fed western diet for 2 months and administered vehicle or trehalose (2 g kg−1 given five times per week i.p.) in the final 2 weeks of diet (n=4 mice per group). Representative GFP fluorescence (scale bar, 100 μm; a) and quantification of GFP intensity in aortic roots by confocal microscopy (n=4 mice per group; b) is shown. (c–e) TFEB intensity and nuclear localization in aortic roots of the same cohort used in a was detected by immunofluorescence. Shown are (c) representative aortic root TFEB staining (scale bar, 100 μm), (d) average aortic root TFEB intensity; and (e) TFEB-DAPI co-localization. (f) Diagram summarizing experimental protocol and mice cohorts used for in vivo assessment of trehalose in atherosclerosis. Mice were fed a western diet for 2 months while being administered either vehicle, trehalose or sucrose (disaccharides given both i.p. 2 g kg−1 for three times per week and orally 3% ad libitum in drinking water). (g) Serum cholesterol levels at 2 months of western diet (n=7 mice per group). (h) Quantification of atherosclerotic plaque burden by computer image analysis of Oil Red O-stained aortic root sections in the experiment summarized in f (statistical significance of differences was calculated using Mann–Whitney U-test). (i) Serum trehalose levels in wild-type mice (n=4 per group) after administration of trehalose (3 g kg−1) either by i.p. injection or oral gavage at indicated time points. (j) Quantification of atherosclerotic plaque burden by computer image analysis of Oil Red O-stained aortic root sections in mice fed 2 months of western diet while being administered only oral trehalose (statistical significance of differences was calculated using Mann–Whitney U-test). All data are presented as mean±s.e.m. *P<0.05, two-tailed unpaired t-test except h,j. Full size image

A large cohort of pro-atherogenic ApoE-null mice were placed on a western diet, treated with vehicle, trehalose or sucrose (a similar non-reducing disaccharide), and atherosclerosis was quantified at the level of the aortic root (Fig. 9f, details are this study). Trehalose administration had no effects on serum cholesterol levels or other common metabolic parameters (Fig. 9g and Supplementary Fig. 9d,e). Lesion quantitation revealed that trehalose-treated mice were significantly protected from atherosclerosis compared to vehicle (Fig. 9h). Furthermore, the administration of sucrose had no effect and was, in fact, slightly atherogenic (Fig. 9h). Although the non-reducing nature of sucrose makes it the ideal control disaccharide in comparison to trehalose, sucrose is α,β-1,2-linkage of glucose with fructose rather than trehalose’s α,α-1,1-linkage of glucose with glucose. Thus, we also administered the reducing disaccharide maltose (α,α-1,4-linkage of glucose and glucose) to ApoE-null mice in order to ascertain the uniqueness of trehalose as an atheroprotective disaccharide. Maltose administration had no detectable effect on atherosclerotic plaque burden or on cholesterol levels (Supplementary Fig. 9f,g).

Finally, our dual i.p. and oral dosing strategy raised the question of which route of administration contributes most significantly to serum trehalose levels and the observed protection from atherosclerosis. We were surprised to discover that i.p. administration of trehalose leads to far greater elevations in serum trehalose than oral dosing (Fig. 9i). Serum trehalose levels rose minimally after oral dosing despite the far greater excess (by weight) provided than i.p. dosing. Thus, to rule-out off-target effects of oral trehalose in affecting atherosclerotic lesion formation (for example, effects of trehalose on the gastrointestinal system or gut microbiota), we conducted an oral-only trehalose experiment in a cohort of ApoE-null mice over 8 weeks of concomitant western diet feeding. Oral trehalose had no observable effects on either atherosclerosis or serum cholesterol (Fig. 9j and Supplementary Fig. 9h).

Trehalose reduces atherosclerosis through autophagy and p62

Similar to mφTFEB-TG mice, the effects of trehalose in vivo and on cultured macrophages in vitro demonstrate this unique disaccharide’s ability to increase macrophage autophagy and autophagy–lysosomal biogenesis, to increase aggrephagy and the clearance of p62-enrich protein aggregates, to decrease macrophage apoptosis and the pro-inflammatory cytokine IL-1β and to an overall reduction in atherosclerosis. We set out to determine whether trehalose’s autophagy-p62-mediated effects are mechanistically linked to the observed reduction in atherosclerosis by administering trehalose to mφATG5-KO and p62-KO mice (both on pro-atherogenic ApoE-null backgrounds).

Consistent with its specificity towards macrophage autophagy, trehalose was no longer able to reduce atherosclerosis in mφATG5-KO mice (Fig. 10a and Supplementary Fig. 10a–c). In addition, trehalose was no longer able to reduce the number of p62-enriched protein aggregates or blunt the degree of apoptosis when subjected to atherogenic lipids (Fig. 10b,c). Interestingly, trehalose’s blunting effect on IL-1β secretion was independent of autophagy, again suggesting an independent mechanism that appears to have no physiological significance for atherosclerotic plaque formation (Fig. 10d). Finally, abrogation of the atheroprotective effects of trehalose, its ability to reduce polyubiquitinated protein aggregates and macrophage apoptosis, and apparent irrelevance of IL-1β reduction to these phenotypes were entirely recapitulated in p62-KO mice (Fig. 10e–h and Supplementary Fig. 10d–f).