Apolipoprotein C3 induces inflammasome activation only in its delipidated form

Abstract

Matters arising regarding the lipidation form of plasma APOC3 that induces an alternative NLRP3 activation pathway.

Extended Data Fig. 1. Characterization of small unilamellar vesicles (SUVs) and APOC3

a, IL-1β release from mouse bone marrow-derived monocytes incubated in indicated APOC3 concentrations. IL-1β measurements were conducted by ELISA and the data were normalized to total cellular protein determined by a BCA protein assay. Data show means ± SEM (n=5, 5, 5, 5, 5, 8 replicates/group, respectively). b, Endotoxin levels in the final concentrations of APOC3 used in panel a, measured by the amebocyte lysate assay (Pierce^™ Chromogenic Endotoxin Quant Kit). Data show means ± SEM (n=3 replicates/group). In a and b, statistical analyses were performed by Kruskal-Wallis tests and Dunn’s multiple comparisons tests. c, 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC) in PBS was sonicated on ice to allow the formation of small unilamellar vesicles (SUVs). The SUVs were purified on a Superose 6 column before the incorporation of APOC3. SUV-APOC3 was isolated with various centrifugation columns and was sterile filtered. The molar concentrations of APOC3 and SUVs were measured by ELISA, BCA protein assay, and phospholipid assay. d, FPLC profile of synthesized SUVs and fractions 16 and 17 (shown in red) were purified and used for the lipidation of APOC3. e, Ion mobility analysis on differential mobility analyzer (DMA) of SUVs from fractions 16 and 17, demonstrating a homogenized particle population around 27 nm. Extended Data Figure 1c was created with BioRender.com.

Extended Data Fig. 2. Distribution and density of serum triglyceride and high sensitivity C-reactive protein (hsCRP) values by APOC3 rs138326449 genotype in the UK Biobank.

Unadjusted serum levels of a, triglycerides (n=430,833) and b, hsCRP (n=426,774) among UK Biobank carriers of 0, 1 or 2 alternative (A) alleles of the APOC3 loss-of-function variant rs138326449 (IVS2 + 1G-A). Specifically, 0/0 indicates homozygosity of the reference (G) allele, 0/1 indicates heterozygosity and 1/1 indicates homozygosity of the alternative (A) allele. The data are shown in box-and-whisker plots (the bound of the box extends from the 25^th to 75^th percentiles and the whisker covers the minima and maxima) with the median marked by a center line, remaining distribution and kernel density estimations of each trait (outlier values ± 5 standard deviations from the mean were removed). Compared with rs138326449-G homozygotes, rs138326449-A heterozygotes demonstrate a 0.62 mmol/L decrease in median triglycerides (p = <2.00 × 10⁻¹⁶ by one-way ANOVA) and a 0.16 mg/L increase in median hsCRP (p = 1.54 × 10⁻⁵ by one-way ANOVA).

Figure 1.. De-lipidated APOC3, but not lipid-bound APOC3, induces IL-1β release from human and mouse monocytes.

a, IL-1β release from human blood monocytes incubated with or without ultrapure LPS (10 ng/mL), small unilamellar vesicles (SUVs) with an average diameter of 27 nm (70.5 mg/dL), de-lipidated human APOC3 (4 mg/dL), APOC3-SUVs complex (4 mg/dL APOC3 and 70.5 mg/dL SUVs), or human VLDL (4 mg/dL) for 16 hr. b, IL-1β release from mouse bone marrow-derived monocytes incubated as in a. IL-1β measurements were conducted by ELISA and the data were normalized to total cellular protein determined by a BCA protein assay. Data show means ± SEM (n=14, 13, 15, 13, 10, 15 replicates/group, respectively, of pooled samples isolated from 7 subjects in a and n=16, 18, 17, 8, 8, 4 individual mice/group in b). c-d, Serum samples from 10 individuals with T1D were used to investigate total serum levels and relative levels of APOC3 in different lipoprotein fractions and the lipoprotein-free (LP-free) fraction. Lipoprotein fractions were isolated by density ultracentrifugation and APOC3 levels were measured by targeted mass spectrometry (DALS peptide). c, APOC3 levels in whole serum versus the LP-free fraction (d>1.021 g/mL). d, APOC3 levels in HDL, LDL and VLDL+IDL. The data are shown in box-and-whisker plots (the bound of the box extends from the 25^th to 75^th percentiles and the whisker covers the minima and maxima) with the median marked by a center line. Statistical analyses were performed by Kruskal-Wallis test and Dunn’s multiple comparisons tests in a and b, two-sided, Mann-Whitney test in c, and one-way ANOVA followed by Holm-Šídák’s multiple comparisons test in d. Normality test was performed by D’Agostino & Pearson test. Outliers were removed based on robust regression and outlier removal method with Q=0.1%. (0, 0, 1, 2, 0, 0) datapoints were removed in a; no datapoint was removed in b, c, or d.

Figure 2.. Endogenous APOC3 does not alter plasma IL-18.

Female low-density lipoprotein receptor-deficient mice with a virus glycoprotein transgene (Ldlr^−/−;GP^Tg) were rendered diabetic (D) with lymphocytic choriomeningitis virus (LCMV). Saline was used as a control (non-diabetic, ND). At the onset of diabetes, the animals were switched to a low-fat, semipurified diet (LFD) and maintained for 4 weeks. Animals received weekly i.p. injections of 10 mg/kg control or APOC3 GalNAc ASOs. a, Schematic of study design for panel b-e. b, Blood glucose levels. c, Plasma triglyceride (TG) levels. d, Plasma APOC3. e, Plasma IL-18. Blood glucose was measured via a glucose meter. Plasma triglyceride levels were measured via colorimetric assay. Plasma APOC3 and IL-18 levels were measured via ELISA. f-h, Plasma samples collected from hAPOC3 transgenic mice treated with two different human APOC3 siRNAs were measured via ELISA. f, Plasma hAPOC3. g, Plasma TGs. h, Plasma IL-18. Control GalNAc ASO did not affect plasma TG, APOC3, or IL-18 levels. Data show means ± SEM (n=12, 12, 15, 15 individual mice in b and e; n=12, 12, 14, 15 in c; n=10, 15, 12, 12 in d) and (n=4 and n=7 individual mice for APOC3 siRNA 1 and siRNA 2 in f-h, except n=6 for t=14 for siRNA 2 in h); two-way ANOVA followed by Tukey multiple comparisons tests vs day 0. Outliers were removed based on robust regression and outlier removal method with Q=0.1%; (0, 0; 1, 0) datapoint was removed in c; (2, 0; 0, 3) datapoints in d; and (0,0,0; 0,0,1) datapoints in h. Figure 2a was created with BioRender.com.