Figure 4.

IL-1β antagonism decreases neutrophil content and NETosis in atherosclerotic lesions of Ldlr^−/− mice with myeloid Abca1/g1 deficiency. Ldlr^−/− mice were transplanted with BM from LysmCreAbca1^fl/flAbcg1^fl/fl (Myl-Abc^dko) mice and fed WTD. The anti-mouse monoclonal antibodies (IgG2a) that selectively neutralize IL-1β or isotype-matched control IgG2a were administered subcutaneously at 6, 7, and 8 weeks of WTD feeding (three doses of antibodies in total). At 8.5 weeks of WTD, mice were sacrificed and neutrophils were stained in atherosclerotic lesions using Ly6G (A) or MPO (B), and Ly6G⁺ (A) or MPO⁺ (B) percentages of lesion size were quantified. Concomitantly, lesions were stained for 3HCit. To assess NETs, the overlap of Ly6G and 3HCit (A) or MPO and 3HCit (B) was quantified as a percentage of the total lesion area. Representative pictures are shown. Scale bars: 100 μm. (C) Correlation between Ly6G⁺ and Ly6G ⁺ 3HCit⁺ (NET) area as shown in (A). (D) Ly6G⁺ splenic neutrophils were isolated and the cleaved and pro-form of IL-1β was assessed by Western blot. Unedited gels are shown in Supplementary material online, Figure S18. Cleaved IL-1β was quantified relative to β-actin (E) and the pro-form of IL-1β (F). n = 12. Each data point represents an individual mouse. n = 15. *P < 0.05, by t-test.