IL1RAP Expression in Human Atherosclerosis: A Target of Novel Antibodies to Reduce Vascular Inflammation and Adhesion

Abstract

Background: Blockade of IL1RAP (interleukin 1 receptor associated protein) was recently shown to reduce atherosclerosis in mice, but the effect on human vascular cells is largely unknown. Targeting the IL1RAP coreceptor represents a novel strategy to block the IL1RAP-dependent cytokines IL (interleukin)-1, IL-33, and IL-36. In the present study, we aimed to evaluate the role of novel antibodies targeting IL1RAP to reduce the effects of IL-1β, IL-33, or IL-36γ in human vascular cells.

Methods: Expression of IL1RAP was observed in human atherosclerotic plaques by immunohistochemistry and microarray and in endothelial cells by flow cytometry. Endothelial cells were cultured with IL-1β, IL-33, or IL-36γ cytokines with or without IL1RAP antibodies and analyzed with Olink proteomics, ELISA, Western blot, and real-time quantitative polymerase chain reaction. The functional effect of IL1RAP antibodies on endothelial cells were analyzed with adhesion and permeability assays.

Results: Olink proteomics showed inhibition of the inflammatory proteins LIF (leukemia inhibitory factor), OPG (osteoprotegerin), CCL4 (C-C motif chemokine ligand 4), and MCP-3 (monocyte chemoattractant protein 3) by IL1RAP-blockade in endothelial cells after IL-1β stimulation. In addition, the IL1RAP antibodies inhibited IL-1β, and IL-33 induced IL-6 and IL-8 secretion. Secretion of MCP-1 (monocyte chemoattractant protein 1) was induced by IL-1β, IL-33, and IL-36γ, and subsequently was inhibited by IL1RAP antibodies. Similar effects were found on mRNA expression level. Endothelial expression of the adhesion markers ICAM1, VCAM1, and SELE were significantly reduced by IL1RAP antibodies, and neutrophil adhesion to endothelial cells induced by IL-1β and IL-33 was reduced by IL1RAP blockade. In human atherosclerotic lesions, IL1RAP expression correlated with markers of inflammation like IL6, IL8, and MCP1.

Conclusions: IL1RAP-targeting antibodies can reduce the expression of inflammatory cytokines and markers of adhesion in endothelial cells, which may be of importance for future putative targeted treatments against cardiovascular disease.

Keywords: HUVECs; IL‐1; IL‐33; IL‐36; endothelial cells.

Figure 1. Expression of IL‐1 receptor complex components in human atherosclerotic plaque and endothelial cells.

A, Immunofluorescent staining depicts IL1RAP (green) alongside VWF (red), SMA (red), and the macrophage marker CD68 (red) within different areas of a human atherosclerotic plaque (in gray). All scalebars=100 μm except for whole plaque images, where the scalebar=1 mm. B, Expression of IL1R1, IL1RAP, ST2, and IL36R in human atherosclerotic plaques compared with donor‐matched macroscopically intact carotid tissue (n=32 biological replicates in each group), from the Gene Expression Omnibus database of microarray data. C, Expression of IL1R1, IL1RAP, ST2, and IL36R in human umbilical vein endothelial cells detected by flow cytometry shown as mean fluorescence intensity of fluorophore (APC, PE, A647)‐conjugated antibodies (Ab) towards the receptor components (IL1R1, IL1RAP, ST2, IL36R). CD68 indicates cluster of differentiation 68; DAPI, 4′,6‐diamidino‐2‐phenylindole; FC, fold change; IL‐1, interleukin 1; IL1R1, IL‐1 receptor 1; IL1RAP, interleukin 1 receptor associated protein; IL36R, IL‐36 receptor; SMA, smooth muscle actin; ST2, supression of tumorigenicity 2; and VWF, von Willebrand factor

Figure 2. Inhibitory effect of IL1RAP antibodies on release of inflammatory proteins from human umbilical vein endothelial cells.

Protein content in cell culture media after 24‐hour treatment with IL‐1β (5 ng/mL) (A through D), IL‐33 (100 ng/mL) (E through G), or IL‐36γ (100 ng/mL) (H through J) in the presence of anti‐IL1RAP (20 μg/mL) or matching isotype control antibody. In (A), a proximity extension assay (Olink proteomics) using the inflammation panel was analyzed. Data are shown as FC of NPX units (log2) by anti‐IL1RAP compared with cytokine treatment alone and represents proteins with significant downregulation (P<0.05), FC >0.58, and Benjamini–Hochberg FDR <0.1 (n=3). ELISA was used to detect IL‐6 in (B), (E), and (H); IL‐8 in (C), (F), and (I); and MCP‐1 in (D), (G), and (J). Data are shown as mean±SD (n=3), ANOVA *P<0.05, **P<0.01, ***P<0.001. CCL4 indicates C‐C motif chemokine 4; expr, expression; FC, fold change; FDR, false discovery rate; IL, interleukin; IL1RAP, interleukin 1 receptor associated protein; LIF, leukemia inhibitory factor; MCP‐3, monocyte chemotactic protein 3; NPX, normalized protein expression; ns, not significant; and OPG, osteoprotegerin.

Figure 3. Effect of IL1RAP‐blocking antibodies on gene expression of inflammatory cytokines in endothelial cells.

HUVECs were treated for 4 hours with (A through C) IL‐1β (5 ng/mL), or (D through F) IL‐33 (100 ng/mL), or (G through I) IL‐36γ (100 ng/mL) in the presence of anti‐IL1RAP (20 μg/mL) or matching isotype control antibody. The mRNA expression was measured by RT‐qPCR of IL6 in (A), (D), and (G); IL8 in (B), (E), and (H); and MCP1 in (C), (F), and (I). Data are shown as mean±SD (n=3), ANOVA *P<0.05, **P<0.01, ***P<0.001. HUVECs indicates human umbilical vein endothelial cells; IL, interleukin; IL1RAP, interleukin 1 receptor associated protein; ns, not significant; and RT‐qPCR, real‐time quantitative polymerase chain reaction.

Figure 4. IL1RAP expression correlates to markers of inflammation in human atherosclerotic plaques.

Microarray data from the Gene Expression Omnibus database were used for correlation analysis of IL1RAP and (A) IL6, (B) IL8, (C) MCP‐1, (D) LIF, (E) OPG, (F) CCL4, and (G) MCP‐3 in human atherosclerotic plaque tissue (n=32 biological replicates).

Figure 5. Endothelial adhesion markers and neutrophil adhesion to endothelium are reduced by IL1RAP blocking antibodies.

HUVECs were treated with (A through D) IL‐1β (5 ng/mL), or (E through H) IL‐33 (100 ng/mL), or (I through L) IL‐36γ (100 ng/mL) in the presence of anti‐IL1RAP (20 μg/mL) or matching isotype control antibody. The mRNA expression was measured after 4 hours of treatment by RT‐qPCR of ICAM1 in (A), (E), and (I); VCAM1 in (B), (F), and (J); and SELE in (C), (G), and (K). HUVECs treated for 24 hours ([D], [H], and [L]) were coincubated with calcein‐AM‐labeled neutrophils obtained from human polymorphonucleated leukocytes isolation. Neutrophil adhesion was detected as number of fluorescent neutrophils attached to HUVECs after 30 minutes of coincubation at 37 °C in 5% CO₂ (n=3–5). Data are shown as mean±SD, ANOVA *P<0.05, **P<0.01, ***P<0.001. Correlation analysis of microarray data of IL1RAP and (M) ICAM1, (N) VCAM1, and (O) SELE from the Gene Expression Omnibus database of human atherosclerotic plaques (n=32 biological replicates). HUVECs indicates human umbilical vein endothelial cells; IL, interleukin; IL1RAP, interleukin 1 receptor associated protein; ns, not significant; and RT‐qPCR, real‐time quantitative polymerase chain reaction.

Figure 6. Reduction of IL‐1β‐induced endothelial permeability and NF‐κB signaling by IL1RAP blocking antibodies.

HUVECs were treated with (A through C) IL‐1β (5 ng/mL), or (D) IL‐33 (100 ng/mL, n=1), or (E) IL‐36γ (100 ng/mL, n=1) in the presence of anti‐IL1RAP (20 μg/mL) or a matching isotype control antibody. A, Permeability assay of FITC‐dextran on HUVECs. Permeability through the endothelial monolayer after 24 hours of treatment was measured as fluorescence intensity relative to control (n=3). B through E, IκBα protein expression was measured after 30 minutes of treatment by Western blot. GAPDH protein expression was analyzed as a loading control. B, Representative blots of (C) that show quantification and treatments (n=3). A and C, Data are shown as mean±SD, ANOVA *P<0.05, **P<0.01, ***P<0.001. FITC indicates fluorescein isothiocyanate; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells; IL, interleukin; IL1RAP, interleukin 1 receptor associated protein; IκBα, inhibitory κBα; and NF‐κB, nuclear factor κB.