Genetic and pharmacologic disruption of interleukin-1β signaling inhibits experimental aortic aneurysm formation

Abstract

Objective: Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1β (IL-1β) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment.

Methods and results: IL-1β mRNA and protein levels were significantly elevated in abdominal aortas of 8- to 12-week-old male C57Bl/6 mice after elastase aortic perfusion (wild-type [WT]) compared with saline perfusion. Mice with genetic deletion of IL-1β (IL-1β knockout [KO]) or IL-1R (IL-1R KO) that underwent elastase perfusion demonstrated significant protection against AAA formation, with maximal aortic dilations of 38.0±5.5% for IL-1β KO and 52.5±4.6% for IL-1R KO, compared with 89.4±4.0% for WT mice (P<0.005). Correspondingly, IL-1β KO and IL-1R KO aortas had reduced macrophage and neutrophil staining with greater elastin preservation compared with WT. In WT mice pretreated with escalating doses of the IL-1R antagonist anakinra, there was a dose-dependent decrease in maximal aortic dilation (R=-0.676; P<0.0005). Increasing anakinra doses correlated with decreasing macrophage staining and elastin fragmentation. Lastly, WT mice treated with anakinra 3 or 7 days after AAA initiation with elastase demonstrated significant protection against AAA progression and had decreased aortic dilation compared with control mice.

Conclusions: IL-1β is critical for AAA initiation and progression, and IL-1β neutralization through genetic deletion or receptor antagonism attenuates experimental AAA formation. Disrupting IL-1β signaling offers a novel pathway for AAA treatment.

Figure 1

(A) RT-PCR for IL-1β gene expression of mRNA from C57Bl/6 mice perfused with elastase or saline over time. (B) ELISA for IL-1β protein from C57Bl/6 mice perfused with elastase or saline over the AAA model. * = p<0.05 for WT elastase vs. WT saline, † = p<0.005 for WT elastase vs. WT saline. (C) Aortic cross sectional histology with IL-1β antibody (brown, arrows) of elastase perfused mice at days 0, 3, 7, and 14. (D) Confocal immunohistochemistry performed on aortic tissue from a WT mouse 3 days following elastase perfusion. Macrophages (Mac-2) were shown in green, smooth muscle cells (smooth muscle actin, SMA) in red, IL-1β in purple, and cell nuclei (DAPI) in blue. Stacked image (below) demonstrated IL-1β co-localized with macrophages and aortic smooth muscle cells.

Figure 2

(A) Maximal % aortic dilation is shown for elastase perfused WT mice, saline perfused WT mice, elastase perfused IL-1β KO mice, and elastase perfused IL-1R KO mice with representative images displayed (B). * = p<0.0005 vs. WT Elastase. (C) Immunohistochemistry from WT elastase, IL-1β KO, and IL-1R KO aortas evaluating macrophages shown in brown, neutrophils shown in brown, and elastin fibers (arrows) shown in black with VVG. Scale bar = 200 μm. On the right, histograms are shown indicating the distribution of blinded histologic grading for each group (n=6/group). Significant differences existed for macrophage infiltration, neutrophil infiltration, and elastin degradation (p<0.0001 for all by χ² test).

Figure 3

(A) Gelatin zymography performed on protein isolated from WT Elastase and IL-1β KO aortas 3 and 7 days following elastase exposure demonstrating pro MMP9 (92kDa), active MMP9 (82 kDa, black boxes), pro MMP2 (72kDa), and active MMP2 (66kDa). Graphical display of the densitometry volume of active MMP9 is on the right. (B) Cytokine protein array for WT elastase, WT saline, IL-1β KO, and IL-1R KO at day 14 evaluating IL-1α, RANTES, IL-6, and complement component 5a. Significance from ANOVA with Tukey correction for multiple variables; * = p<0.005 compared to WT Elastase; † = p<0.005 compared to WT Saline.

Figure 4

(A) Study design to evaluate the effect of anakinra for prevention of AAA formation. Subcutaneous osmotic pumps were filled with escalating doses of anakinra solution 3 days prior to elastase infusion. Due to the amount of drug required, pumps were changed 9 days following insertion. Aortic harvest was performed on day 14. (B) Maximal aortic diameter with the four doses of anakinra (* = p 0.005 vs. vehicle, † = p<0.0005 vs. vehicle, ‡ = p<0.005 vs. 10mg/kg/day). (C) Aortic IL-1β protein levels by ELISA for aortas exposed to increasing doses of anakinra (* = p<0.05). (D) Representative histology sections for macrophages, neutrophils, and elastin fibers shown for anakinra at 0, 10, 30, and 100 mg/kg/day. Scale bar = 200μm. Histology slides were graded by a blinded reviewer with grading shown on the right with histograms (n=4/group, p<0.0001 for all by χ² test).

Figure 5

(A) Study design to evaluate anakinra for the treatment of experimental AAAs with anakinra started 3 or 7 days following elastase exposure. (B) Aortic dilation 14 days following elastase perfusion for WT mice treated with anakinra at day 3 compared to WT mice treated with vehicle only (*=p<0.0005). C) Aortic dilation of WT mice treated with anakinra 7 days following elastase exposure compared to control WT mice treated with vehicle alone (†=p<0.005). (D) Sample aortic cross sections of both day 3 and day 7 treatment groups compared to vehicle treated control groups evaluating IL-1β, macrophages, neutrophils, and elastin. Scale bar = 200μm. Histograms for blinded histology grading shown on the right (n=4/group, p<0.0001 for all by χ² test).

Figure 6

(A) Design for bone marrow transplantation of WT and IL-1R KO bone marrow cells delivered to irradiated WT and IL-1R KO hosts at 6 weeks of age with the elastase AAA model performed at 10 weeks. (B) Aortic dilation following elastase perfusion of the four bone marrow transplant groups (* = p<0.005 vs. WT→ WT, † = p<0.005 vs. IL-1R KO → WT, ‡ = p<0.05 vs. IL-1R KO → WT). IL-1R KO host mice had a high lethality following radiation and bone marrow transplantation with 47% lethality for WT bone marrow into IL-1R KO mice and 73% lethality for IL-1R KO bone marrow into IL-1R KO mice. (C) VVG stain for elastin fibers (white arrows) in representative aortic cross-sections. Scale bar = 200μm.