Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice

Abstract

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes ¹ . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL^2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr^-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr^-/- mice, Ldlr ^-/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

Extended Data Figure 1. E06-scFv expression and binding characteristics

a. Simply Blue staining of purified EO6-scFv from HEK293 cell lysates from two experiments. b. Western blot with anti-Myc of E06-scFv following purification on Ni-NTA agarose beads (representative of 4 independent experiments). c. Binding profile of purified E06-scFv using chemiluminescent ELISA (Binding data are mean ±SEM, using 3 independent samples, each determined in triplicate). d. Tissue distribution of E06-scFv gene transcript in Ldlr^−/−/E06-scFv mice determined by qPCR. (Data are mean ±SEM, determined from tissues of 3 Ldlr^−/−/E06-scFv mice. e. Competition immunoassays of Ldlr^−/−/E06-scFv plasma binding to plated OxLDL in the presence or absence of increasing amounts of indicated competitors. Results are ratio of binding of E06-scFv to OxLDL in the presence (B) or absence of competitor (B₀). AB1-2 is a T15 anti-idiotypic antibody; C16lysoPC: C16 lyso-phosphatidylcholine, DPPC: Dipalmitoyl phosphatidylcholine. Data shown are triplicates of each point from one competition experiment, representative of 4 separate studies of similar nature. f, Accumulation of desmosterol and other indicated sterols in TGEM from indicated mice fed a HC diet for 16 wks. TGEM were isolated from 3 mice in each group and each set of macrophages divided into 2 separate aliquots for analysis in triplicate. Data are mean ±SEM. There were no differences between respective sterol pairs, p>0.05 for all pairs.

Extended Data Figure 2. Expressed E06-scFv does not alter levels of total IgM or IgM E06 (detected by AB1-2) in transgenic mice

Comparison of plasma IgM titers to indicated antigens of Ldlr^−/−or Ldlr^−/−/E06-scFv mice at baseline or after 4 or 7 months of HC diets. Note significant increases in Total IgM and IgM to MDA-LDL and OxLDL at 4 and 7 months vs. respective baseline titers (all vales p< 0.001) except at 4 months, total IgM of Ldlr^−/−/E06-scFv mice and E06 (detected by AB1-2) in both mouse groups were not different than their respective baselines (p>0.05). Importantly, there were no significant differences in any antibody titers between Ldlr^−/− or Ldlr^−/−/E06-scFv mice at any time point, and in particular, note that endogenous IgM-E06 titers (detected by AB1-2 binding) were similar. As expected, the Rag1^−/− and Rag^−/−/E06-sc did not have any IgM.

Extended Data Figure 3. Plasma E06-scFv binds to atherosclerotic lesions and apoptotic thymocytes and is present in aorta of Ldlr^−/−/E06-scFv mice

a, Staining of atherosclerotic lesions of WHHL rabbit aorta with E06-scFv plasma (left panel), and Ldlr^−/− mice (right panel) (both at dilution of 1:20), visualized using biotinylated anti-Myc mAb and ABC-AP VectaStain kit. b, Deconvolution microscopy of E06-scFv plasma (1:20 dilution) binding to apoptotic but not normal cells. Blue, nuclei stained with Hoechst dye; Green, FITC-labeled anti-His tag mAb; Red, Annexin V-PE. c, Binding of E06-scFv plasma (1:20 dilution) to apoptotic thymocytes (7AAD+/Annexin V+) by FACS analysis. d, Expression of E06-scFv in aortic lesion of Ldlr^−/−/E06-scFv but not Ldlr^−/− mouse. Cross-sections at the AV were stained with biotinylated anti-Myc mAb to identify presence of E06-scFv in atherosclerotic lesion. Nuclei counterstained using Hematoxylin QS (Original ×200). Panels a-c are representative of similar studies with 5 other plasma samples. Panel d is representative of studies in 3 other aortic sections.

Extended Data Figure 4. Lipoprotein profiles of Ldlr^−/− and Ldlr^−/−/E06-scFv mice are similar in various studies

a, b, Plasma cholesterol (a), and triglycerides (b) distribution by FPLC in pools of equal aliquots of plasma from mice fed HC diet for 16 weeks (n=10, 11). c, Plasma cholesterol distribution in mice fed HC diet for 28 weeks (n=9,7). d, Plasma cholesterol and triglyceride distribution in BMT experiment: Lipoprotein profiles in Ldlr^−/− mice that received BM from wild type (control, n=9) or E06-scFv mice (n=13) and then fed a WD for 16 weeks.

Extended Data Figure 5. E06-scFv reduces necrotic core formation and macrophage secretion of E06-scFv confers atheroprotection

a, E06-scFv reduces extent of necrosis within aortic root lesions after HCD for 7 months as shown in Fig 2c. Lesions of equal size were matched at each of the indicated sites in aortic root sections from 7 Ldlr^−/− and 9 Ldlr^−/−/E06-scFv mice and extent of necrosis measured as described in Methods. Necrosis was reduced by 43.9% in Ldlr^−/−/E06-scFv mice (AUC 113.4 vs. 63.6, p=0.015). b, Secretion of E06-scFv in cultured peritoneal macrophages in the absence or presence of LXR agonist T090137 from C57BL/6 (WT) and E06-scFv mice determined by PC-binding assay. Culture supernatants were concentrated 10-fold for ELISA (left panel). The E06-scFv expression, driven by the apoE promoter was stimulated by T0901317 as indicated by Western blots of cell lysates with anti-myc mAb (right panel). Representative of 4 separate experiments. c, Plasma E06-scFv titers following transplantation (baseline) in Ldlr^−/− mice transplanted with WT (n=7) or E06-scFv (n=7) bone marrow). E06-scFv titers (plasma from 7 WT and 7 E06-scFv) increased in mice transplanted with E06-scFv BM over 16 weeks of WD. d, Aortic root atherosclerosis in Ldlr^−/− mice transplanted with WT (n=9) or E06-scFv (n=13) bone marrow after 16 weeks of WD. As described in Methods, aortic root lesion areas were quantified from serial sections (9 sections per mouse) cut through the aorta at the origins of the AV leaflets and then stained with modified van Geison solution. Lesions at aortic root were reduced by 37% in mice that received BMT from E06-scFv mice (AUC 69.6 vs 110.6, p=0.02, two-sided-t-test).

Extended Data Figure 6. AV echocardiography and hepatic gene Expression

a Representative pulse wave Doppler derived aortic jet velocities in 12 month old Ldlr^−/−/E06-scFv (left) and Ldlr^−/− (right) mouse: ECG tracings are shown in green. Representative of studies in 9 Ldlr^−/− and 10 Ldlr^−/−/E06-scFv mice. b. Representative M-mode echocardiography images containing the AV in short axis through the right ventricular outflow tract (RVOT), aortic (Ao) root with AV, and left atrium (LA). The AV (arrows), best observed in diastole, is thinner in Ldlr^−/−/E06-scFv compared to Ldlr^−/− mice. ECG tracings shown in green. Representative of studies in 9 Ldlr^−/− and 10 Ldlr^−/−/E06-scFv mice. c. Decreased inflammatory gene expression in whole liver extracts of Ldlr^−/− and Ldlr^−/−/E06-scFv mice after 16 weeks of HC diet. Relative mRNA levels were determined by qPCR and normalized to GAPDH and expressed as means ± SEM. (n=4 mice each).

Figure 1. Transgenic mice expressing a single chain variant of E06 (E06-scFv)

a, The configuration of the E06 single-chain transgene, which encodes the E06 light and heavy chains with flexible linker (Gly4Ser)3 and epitope tags under the direction of ApoE promoter and LE6 enhancer. b, Binding properties of plasma from wild-type C57BL/6 and the E06-scFv mice to indicated antigens (mean±SEM, n=5 plasmas from each group, each point determined in triplicate). c, Plasma from E06-scFv mice reduced OxLDL binding by macrophages in culture. Shown is one experiment, each point in triplicate, and representative of 5 separate experiments. d, E06-scFv protected thioglycollate elicited peritoneal macrophages (TGEM) from OxPAPC induced inflammatory response. OxPAPC or vehicle (PBS) were injected ip into Ldlr^−/− or Ldlr^−/−/E06-scFv mice and TGEM macrophages were collected 4h after injection for qPCR analysis. Data are mean ± SEM normalized to control; (n=3,4 mice per group).

Figure 2. E06-scFv reduces atherosclerosis in HC-fed Ldlr_-/- mice

a, b, Examples of en face atherosclerosis in Ldlr^−/− and Ldlr^−/−/E06-scFv mice after 4 months (a) and 12 months (b) of 1% high cholesterol diet (HCD). c, d, Cumulative data for extent of en face lesion formation in the entire aorta (c) or at the aortic root (d) of Ldlr^−/− (n =8-10) and Ldlr^−/−/E06-scFv mice (n =7-11). Compared to Ldlr^−/− mice, atherosclerosis was significantly reduced in Ldlr^−/−/E06-scFv mice at 4, 7 and 12 months (en face by 57%, 34% and 28%, and aortic root by 55%, 41% and 27% respectively).

Figure 3. Mechanisms by which E06-scFv decreases atherosclerosis

a, AlexaFluor 488 labeled OxLDL was injected i.p. and macrophage uptake of OxLDL was determined by FACS and expressed as % macrophages taking up OxLDL. Macrophage uptake was reduced in Ldlr^−/−/Rag1^−/−/E06-scFv compared to Ldlr^−//⁻Rag1^−/− mice (% uptake 91±1.03 vs. 62±5.01, 4 mice each). b, AlexFluor labeled OxLDL was pre-incubated with plasma from Ldlr^−//⁻Rag1^−/− or Ldlr^−/− or Ldlr^−/−/Rag1^−/−/E06-scFv mice, and injected into corresponding mice (n =4 each) and macrophage uptake determined as in a. The % of macrophages that took up OxLDL was 98.83±0.44, 66.06±5.87, and 26.48±3.90 respectively. The ~ 25% decrease observed following incubation with Ldlr^−/− plasma possibly reflects the presence of endogenous anti-OxLDL antibodies. c, TGEM from 16 week HCD mice (n=4 mice each) were isolated and cellular cholesterol and desmosterol and other oxysterol levels determined and normalized to cellular protein. Total cholesterol accumulation shown here was reduced 48% in Ldlr^−/−/E06-scFv mice (Data are mean ± SD; n = 4). Desmosterol and oxysterol levels were similar (see Extended Data Fig 1d). d, Heat map of RNAseq data from Ldlr^−/− and Ldlr^−/−/E06-scFv TGEM collected after 16 weeks of HCD (n = 4 each group). e, Selected list of differentially expressed transcripts in TGEM (Mϕ) based on clustering analysis on log ratio RPKM data from d. All values represent FDR-adjusted p values < 0.05 as described in Methods. f, Aortic wall tissue-resident macrophage phenotypes were determined by FACS analysis of CD45⁺ viable cells. Macrophages were defined as M1-like (CD11b⁺CD11c⁺Arg1⁻) or M2-like (CD11b⁺CD11c⁻Arg1⁺) and expressed as % Frequency of parent (CD11b⁺CD11C^+/−) (Ldlr^−/−chow and Ldlr^−/−/E06-scFv HCD (n=4), Ldlr^−/−HCD (n=3).

Figure 4. E06-scFv decreases early aortic valve stenosis, hepatic steatosis, and systemic inflammation

a, b, Ldlr^−/− (n=11) and Ldlr^−/−/E06-scFv (n=10) mice were fed HCD for 15 months and prospectively examined at 3 time points for aortic valve hemodynamics. a, Mean pressure gradients across the aortic valve, determined by Doppler echocardiography. At 12 months there was a 49% lower mean gradient in the Ldlr^−/−/E06-scFv mice (2.4±1.9mmHg vs. 4.8±2.4mmHg, mean±SD, p = 0.026, Ldlr^−/−/E06-scFv (n=10) and Ldlr^−/− (n=9). c, d, Calcification in aortic valve leaflets was determined by von Kossa staining of serial aortic valve sections and AUC compared. AV calcium was reduced in Ldlr^−/−/E06-scFv mice by 41.5% (p=0.045, one-tailed-t-test, Ldlr^−/−/E06-scFv (n=9) and Ldlr^−/− (n=8)). e, Survival of mice used in AV hemodynamic study over 15 months. f, Hepatic cholesterol and triglyceride (TG) levels were reduced by 42% and 47% respectively in Ldlr^−/−/E06-scFv mice, Ldlr^−/− (n=10) and Ldlr^−/−/E06-scFv (n=12) mice g, Livers of mice fed HCD for 16-wks were immunostained with biotinylated E06 IgM (brown) and compared to chow-fed C57BL/6 mice. Shown are representative photomicrographs, representative of 7 Ldlr^−/−, 7 Ldlr^−/−/E06-scFv and 3 WT (C57BL/6) mice. h, Plasma serum amyloid A (SAA) was decreased 32% in HC fed Ldlr^−/−/E06-scFv mice (Ldlr^−/− (n=10) and Ldlr^−/−/E06-scFv (n=12) mice).