Pro-efferocytic nanotherapies reduce vascular inflammation without inducing anemia in a large animal model of atherosclerosis

Abstract

Atherosclerosis is an inflammatory disorder responsible for cardiovascular disease. Reactivation of efferocytosis, the phagocytic removal of cells by macrophages, has emerged as a translational target for atherosclerosis. Systemic blockade of the key 'don't-eat-me' molecule, CD47, triggers the engulfment of apoptotic vascular tissue and potently reduces plaque burden. However, it also induces red blood cell clearance, leading to anemia. To overcome this, we previously developed a macrophage-specific nanotherapy loaded with a chemical inhibitor that promotes efferocytosis. Because it was found to be safe and effective in murine studies, we aimed to advance our nanoparticle into a porcine model of atherosclerosis. Here, we demonstrate that production can be scaled without impairing nanoparticle function. At an early stage of disease, we find our nanotherapy reduces apoptotic cell accumulation and inflammation in the atherosclerotic lesion. Notably, this therapy does not induce anemia, highlighting the translational potential of targeted macrophage checkpoint inhibitors.

Fig. 1. ‘Large batch’ SWNTs retain their expected physicochemical properties and induce phagocytosis in vitro.

Physicochemical characterization of large-batch SHP1i-loaded SWNTs displays successful SHP1i loading and release in acidic solutions. a DLS; b UV-Vis spectroscopy; and c FT-IR spectroscopy of SWNT and SWNT-Cy5.5-SHP1i; d SHP1i release from SWNTs at neutral and acidic pH. Data are presented as mean values ± SD. n = 3 over independent replicates; e In vitro Incucyte phagocytosis assay demonstrates large batch SWNT-SHP1i treatment of RAW264.7 macrophages significantly increases phagocytosis of apoptotic cells compared to SWNT treatment. ****P < 0.0001 by unpaired two-tailed t-test. Data are presented as mean values ± SEM. n = 16 per group over technical replicates. Source data are provided as a Source data file.

Fig. 2. SWNTs are preferentially taken up by monocytes in the circulation.

SWNTs are preferentially taken up and retained in the circulation by pig blood monocytes and not lymphocytes. a Representative dot plots and gating strategy for SWNT uptake and retention by different immune cell types 7 days after injection. CD3⁺ cells represent T cells, CD21⁺ cells represent B cells, and CD8a⁺ cells represent a subtype of Natural killer cells. CD163⁺CD14⁻, CD163⁺CD14⁺, CD163⁻CD14⁺ cells represent different monocyte subtypes, while CD163⁻CD14⁻ represent the remaining immune and non-immune cells. b Bar diagram of the percent SWNT⁺ cells in pig blood 7 days after SWNT or SWNT-SHP1i injection. **** indicates P < 0.0001 compared to CD3⁺, CD21⁺, CD8a⁺ NK subset and CD163⁻CD14⁻ cells, as analyzed by one-way ANOVA. Data are presented as mean values ± SEM, n = 5 [n = 2 (SWNT), n = 3 (SWNT-SHP1i)] from 5 biologically independent replicates. Source data are provided as a Source data file.

Fig. 3. SWNTs accumulate in atherosclerotic plaque and SHP1 inhibition reduces both vascular inflammation and apoptotic cell accumulation in transgenic atherosclerotic pigs, in vivo.

a Experimental workflow including downstream analyses. b Bar diagram of SWNT uptake in monocytes/macrophages versus non-monocytes/macrophages in multiple vascular beds of the pooled SWNT and SWNT-SHP1i treated pigs. MO stands for monocytes and MΦ stands for macrophages. SWNT/SWNT-SHP1i uptake by MO/MΦ was significantly higher than non-MO/MΦ in vascular beds including the carotid artery (***p = 0.0001), iliac artery (**p = 0.0066), and renal artery bifurcation (**p = 0.0049) as analyzed by an unpaired two-tailed t-test with Welch’s correction. No significant difference was observed in SWNT/SWNT-SHP1i uptake by MO/MΦ compared to non-MO/MΦ in non-vascular tissue such as the bone marrow (p = 0.6314). Data are presented as mean values ± SEM. Carotid artery n = 2 (SWNT), n = 2 (SWNT-SHP1i); iliac artery n = 2 (SWNT), n = 3 (SWNT-SHP1i); renal artery bifurcation n = 2 (SWNT), n = 3 (SWNT-SHP1i); bone marrow n = 2 (SWNT), n = 3 (SWNT-SHP1i) over biologically independent samples. c Pigs treated with SWNT-SHP1i had similar atherosclerotic lesion sizes as SWNT controls in the RCA ostium (p = 0.5763). Data are presented as mean values ± SEM. n = 6 (SWNT) and n = 5 (SWNT-SHP1i) over 11 independent biological samples. d ¹⁸F-FDG PET/CT imaging and analysis of the carotid arteries demonstrate that SWNT-SHP1i significantly reduces vascular inflammation. *p = 0.0438 by an unpaired two-tailed t-test with Welch’s correction. Data are presented as mean values ± SEM. n = 5 (SWNT) and n = 5 (SWNT-SHP1i) over 10 independent biological samples. e In SWNT-SHP1i treated pigs, macrophage infiltration into the atherosclerotic lesion was not significantly lower than in SWNT controls (p = 0.4339). Data are presented as mean values ± SEM. n = 6 (SWNT) and n = 5 (SWNT-SHP1i) over 11 independent biological samples. f The RCA ostium of SWNT-SHP1i treated pigs contained significantly fewer TUNEL-positive apoptotic bodies compared to SWNT controls. *p = 0.0280 by unpaired two-tailed t-test with Welch’s correction. Data are presented as mean values ± SEM. n = 6 (SWNT) and n = 5 (SWNT-SHP1i) over 11 independent biological samples. Source data are provided as a Source data file. Figure 3/panel a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 4. Pro-efferocytic therapies induce favorable immunological gene expression changes across species.

a Bulk porcine RNA-sequencing experimental workflow. b Volcano plot of differentially expressed porcine genes from bulk RNA-sequencing in SWNT-SHP1i (n = 3) versus SWNT control (n = 3) common carotid arteries. Red and blue points represent up- and downregulated transcripts (log2 fold change >1 or <−1), respectively. Orange points indicate significantly differentially expressed genes, selected by log2 fold change (>2 or <−2) and adjusted p-value (<0.05, Benjamin–Hochberg correction). Gray points represent nonsignificant genes. Statistical significance was assessed using a likelihood ratio test (LRT) with two-sided testing. c Venn Diagram representing overlap of 35 significant differentially expressed genes between pig bulk RNA-sequencing results and published mouse scRNA-sequencing data from monocytes and macrophages. d Biological Process GO enrichment and pathway analysis of the 35 DEGs shared across species reveal pathways governing immune system response, response to an external biotic stimulus, endocytosis, and phagocytosis in the SWNT-SHP1i group. Adjusted p-value derived from the Benjamini & Hochberg test. Figure 4/panel a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 5. Pro-efferocytic SWNTs do not induce the hematologic abnormalities associated with anti-CD47 Ab therapy.

a Pigs treated with SWNT-SHP1i do not develop anemia or thrombocytopenia, with no significant difference in CBC lab values (WBC, neutrophils, lymphocyte, monocyte) between the SWNT and SWNT-SHP1i treated cohorts at each timepoint. Data are presented as mean values ± SEM. SWNT and SWNT-SHP1i cohort per group n = 7 (Baseline), n = 7 (Month 1), n = 6 (Month 2), n = 5 (Month 3) from independent biological samples. b SWNT-SHP1i was not significantly associated with derangements in lipid levels compared to SWNT controls. Data are presented as mean values ± SEM. SWNT cohort n = 8 (Baseline), n = 7 (Month 1), n = 7 (Month 2), n = 6 (Month 3); SWNT-SHP1i cohort n = 7 (Baseline), n = 8 (Month 1), n = 7 (Month 2), n = 7 (Month 3) from independent biological samples. c, d SWNT-SHP1i was not associated with derangements in c renal function, or d hepatic function compared to SWNT treated controls. Data are presented as mean values ± SEM. SWNT cohort n = 8 (Baseline), n = 7 (Month 1), n = 7 (Month 2), n = 6 (Month 3); SWNT-SHP1i cohort n = 7 (Baseline), n = 7 (Month 1), n = 6 (Month 2), n = 6 (Month 3) from independent biological samples. e There was no significant difference in body weight between SWNT and SWNT-SHP1i treated pigs. Data are presented as mean values ± SEM. SWNT cohort n = 8 (Weeks 1–3), n = 7 (Weeks 4–8), n = 6 (Weeks 9–11), n = 3 (Week 12); SWNT-SHP1i cohort n = 7 (Weeks 1–4), n = 6 (Weeks 5–11), n = 4 (Week 12) from independent biological samples. Source data are provided as a Source data file.