Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation

Abstract

Aims: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis.

Methods and results: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade.

Conclusion: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

Trial registration: ClinicalTrials.gov NCT06143371.

Keywords: Atherosclerosis; IL-1; IL1RAP; Immunomodulation; Inflammation.

Graphical Abstract

Figure 1

Expression of IL1RAP family receptors and cytokines in human atherosclerotic plaques. Single-cell RNA sequencing data of human carotid plaques (n = 18) from patients that underwent carotid endarterectomy. (A) tSNE depicting 14 distinct cell clusters consisting of T cells, myeloid cells, endothelial cells, smooth muscle cells, B cells, and mast cells (image from Depuydt et al.). (B) Feature plot of IL1RAP gene expression. Dot plots of gene expression of (C) IL1RAP-related receptors and (D) IL1RAP-related cytokines. (E) Human carotid plaques (n = 4) were digested, stained with anti-IL1RAP-Alexa Fluor 647 or isotype control IgG, and analysed for IL1RAP MFI by flow cytometry. ΔMFI defined as the difference in MFI in each cell population between anti-IL1RAP and isotype antibody staining. (F) Representative histogram of IL1RAP expression on carotid plaque CD14⁺ myeloid cells.

Figure 2

IL1RAP is present in atherosclerotic plaques of Apoe^−/− mice. Immunohistochemical staining of IL1RAP and quantification of IL1RAP-expressing cells (per cent of total nucleated cells) in the aortic root of (A and B) wild-type (C57Bl/6, n = 7) and (C and D) hyperlipidaemic Apoe^−/− mice (n = 6). The media layer is outlined by the dashed line. Flow cytometric analysis was performed to identify IL1RAP expression on leucocytes in the blood and digested atherosclerotic aortas of hyperlipidaemic Apoe^−/− mice (n = 7). (E) Representative histograms of IL1RAP expression on monocytes (CD115⁺Ly6G⁻CD11b⁺), neutrophils (Ly6G⁺CD11b⁺), and T cells (CD11b⁻TCRβ⁺) in the blood and (F) quantification of IL1RAP MFI (ΔMFI) on leucocytes in the blood. (G) Representative histograms of IL1RAP expression on macrophages, neutrophils, and T cells in digested aorta and (H) quantification of IL1RAP (ΔMFI) on leucocytes in digested aorta. ΔMFI defined as the difference in MFI in each cell population between anti-IL1RAP and isotype antibody staining. Analysed with (B) Student’s unpaired t-test and (D) Kruskal–Wallis test with Dunn’s multiple comparisons test.

Figure 3

IL1RAP blockade reduces plaque burden. (A) Female Apoe^−/− mice were treated biweekly with i.p. injections of either anti-IL1RAP antibody or control isotype IgG (Ctrl IgG) (n = 14/group) for 6 weeks, for a total of 10 weeks HCD. (B) Representative lipid staining (Oil Red O) of HCD-fed Apoe^−/− mice treated with anti-IL1RAP antibodies or isotype. (C) Quantification of average subvalvular plaque area progressing through the aortic valve. Dots denote average of all mice in each treatment group at indicated distance within the aortic valve. Quantification of (D) average plaque area (P = 0.0016) and (E) plaque volume (P = 0.0016). (F) Representative collagen staining (Masson’s trichrome) and (G) quantification of relative collagen area in aortic root plaques. (H) Total plasma cholesterol levels. Analysed with Student’s unpaired t-test.

Figure 4

IL1RAP blockade limits leucocyte accumulation in the atherosclerotic plaque and adventitia. Representative immunohistochemical images of subvalvular aortic plaques of HCD-fed Apoe^−/− mice treated with anti-IL1RAP antibodies or isotype control (Ctrl IgG) (n = 14/group) stained for (A) neutrophils (Ly6G), (B) macrophages (CD68), (C) CD4⁺ T cells (CD4), and (D) CD8⁺ T cells (CD8). Quantification of (E) neutrophil area within the plaque and (F) number of CD68⁺ cells per plaque section (P = 0.0014). Quantification of adventitial (G) CD4⁺ T cells (P = 0.0248) and (H) CD8⁺ T cells (P = 0.0047). Full aortas from the same mice were enzymatically digested and single-cell suspensions were analysed via flow cytometry. (I–J) Representative flow cytometry plots and quantification of numbers of (K) neutrophils (CD11b⁺Ly6G⁺), (L) total T cells (CD11b⁺TCRβ⁺), (M) CD4⁺ T cells, and (N) CD8⁺ T cells (P = 0.0427) in digested aortas, as determined by flow cytometry. Analysed with Student’s unpaired t-test or Mann–Whitney U test.

Figure 5

Systemic effects of IL1RAP blockade in Apoe^−/− mice. Immune cell composition was analysed in spleen, blood, and bone marrow from HCD-fed Apoe^−/− mice treated with anti-IL1RAP antibodies or isotype control (Ctrl IgG) (n = 14/group). (A) Quantification of counts of regulatory T cells (FoxP3⁺CD4⁺) per spleen. Splenocytes were stimulated with PMA/ionomycin and Brefeldin A for 4 h to analyse cytokine production and T cell subsets. Quantification of counts of IFN-γ-producing (B) CD4⁺ T cells and (C) CD8⁺ T cells per spleen. Quantification of (D) counts per spleen and (E) proportion of IL-17-producing CD4⁺ T cells. (F) Representative flow cytometry plot of circulating neutrophils. Quantification of neutrophils (CD11b⁺Ly6G⁺), Ly6C^low monocytes (CD11b⁺Ly6G⁻CD115⁺), and Ly6C^high monocytes, both as (G) counts per 100 μL of blood and (H) per cent of CD11b⁺ myeloid cells. (I) Representative flow cytometry plots of LT-HSC, ST-HSC, and MPP populations in bone marrow. Quantification of (J) numbers of live lineage-negative cells (P = 0.0283). Percentages and counts of (K and L) LT-HSCs (P = 0.0323), (M and N) ST-HSCs, (O and P) MPP3 cells (P = 0.024), and (Q and R) MPP4 cells (P = 0.0156). Bone marrow population percentages given as per cent of LSK cells, counts given as per leg (one tibia and one femur). Analysed with Student’s unpaired t-test or Mann–Whitney U test.

Figure 6

Reduced expression of chemokines in anti-IL1RAP-treated mice. RNA was isolated from BCAs from HCD-fed Apoe^−/− mice treated with anti-IL1RAP antibodies or isotype control (Ctrl IgG) (pooled samples, n = 2/pool; n = 7/group). Real-time PCR quantification of relative expression (2^−ΔΔCt) of (A) adhesion molecules, (B) chemokines, and (C) inflammatory cytokines. (D) Dot plot of gene expression of chemokines and adhesion molecules in human carotid artery plaques. (E) BMDMs from wild-type (C57Bl/6) mice and a mouse fibroblast cell line (NIH3T3) were stimulated with either IL-1α, IL-1β, IL-33, or IL-36 in the presence of anti-IL1RAP antibody or isotype control (Ctrl IgG). (F and G) Expression of IL1RAP on BMDMs and NIH3T3 fibroblasts determined by flow cytometry. (H) Quantification of release of CXCL1 by NIH3T3 fibroblasts under each treatment condition (n = 6). Quantification of release of CXCL1 by (I) BMDMs (n = 3) stimulated with IL-1α, IL-1β, or IL-33 or (J) BMDMs (pre-treated with GM-CSF/TGF-β; n = 5) stimulated with IL-36 (IL-36α, IL36-β, and IL-36γ). (A–C) Bars denote median, analysed with Mann–Whitney U test (*P = 0.0262). (H–J) Bars denote mean.