Inhibition of IL-6 in the LCWE Mouse Model of Kawasaki Disease Inhibits Acute Phase Reactant Serum Amyloid A but Fails to Attenuate Vasculitis

Figures

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  Figure 1. Proteomics analysis of cardiac tissue using the LCWE induced mouse model of KD. (A) Hierarchical one-minus Pearson’s clustering. “LCWE” indicates LCWE-injected mice (n=13), “Control” indicates PBS injected mice (n=8), and “Treated” indicates LCWE injected mice treated with anakinra (n=5). (B) Heatmap of fold change for proteins found differentially expressed (FC > 2, p<0.05) between LCWE-injected mice and PBS controls or between anakinra-treated LCWE and untreated LCWE mice. (C) Top regulators (ranked by z-score) identified by IPA Upstream Analyses of proteins differentially expressed (FC > 2, p<0.05) between KD mice and PBS controls. D) STRING Gene Ontology Biological Process analyses of proteins differentially expressed (FC > 2, p<0.05) between KD mice and PBS controls.

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  Figure 2. STAT3 expression in the LCWE-induced model of vasculitis and response to anakinra treatment. (A) Representative images of immunohistochemistry staining for STAT3 in murine heart of PBS control, LCWE injected or LCWE injected mice treated with anakinra. (B) STAT3 staining score in PBS control, LCWE injected and LCWE injected mice treated with anakinra. (C) Representative images of pSTAT3 immunofluorescence staining in murine heart tissue from PBS control and LCWE injected mice. (D) Quantification of pSTAT3 immunofluorescence staining in murine heart tissue. (E) Western blot of pSTAT3 and STAT3 in murine heart tissue from PBS control and LCWE injected mice. (F) Quantification of pSTAT3/STAT3 ratio from Western Blot. Data was analyzed by one-way ANOVA with Tukey’s multiple testing comparison (B) or Student’s t test (D, F). * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 3. IL-6 expression in the LCWE-induced KD mouse model. (A) Volcano Plots demonstrating differential expression of Stat3, Il6, Csf3, Tgfb1, Ifng, Il10, Vegfa and Egf in the abdominal aorta of PBS vs. LCWE treated mice and LCWE vs. LCWE + anakinra treated mice. Csf2 was not detected. Red represents FDR < 0.05 and FC > 2, blue represents FDR < 0.05 and FC < 2, green represents FDR > 0.05 and FC < 2. (B) Serum levels of IL-6 in PBS, LCWE and LCWE + anakinra treated mice. Data was analyzed by one-way ANOVA with Tukey’s multiple testing comparison (B). ***p < 0.0001.

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  Figure 4. STAT3 inhibition fails to attenuate LCWE-induced KD vasculitis. (A) Schematic of the experimental design. (B) Representative images of pSTAT3 immunofluorescence staining in murine heart tissue from LCWE-injected mice treated with vehicle control of Stattic. (C) Quantification of pSTAT3 immunofluorescence staining in murine heart tissue. (D, E) Representative H&E stained heart sections (D) and heart vessel inflammation scores (E) of LCWE-injected WT mice treated with either vehicle control or the STAT3 inhibitor Stattic at 2 weeks post-LCWE injection. Scale bars, 500µm. (F) Pictures of the abdominal aorta of LCWE-injected WT mice treated with either vehicle control or Stattic at 2 weeks post-LCWE injection. (G, H) Maximal abdominal aorta diameter (G) and abdominal aorta area (H) of LCWE-injected WT mice treated with either vehicle control or Stattic at 2 weeks post-LCWE injection. Results are representative of two independent experiments (n = 6-7 mice per group). Data was analyzed by Student’s t test. ** p < 0.01.

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  Figure 5. Blocking IL-6R signaling fails to attenuate LCWE-induced KD vasculitis. (A) Schematic of the experimental design. (B) SAA concentration in serum from PBS or LCWE mice treated with isotype control or anti-IL6R antibody. (C, D) Representative H&E stained heart sections (C) and heart vessel inflammation scores (D) of LCWE-injected WT mice treated with either isotype control or anti-IL6R antibody at 1-week post-LCWE injection. Scale bars, 500µm. (E) Pictures of the abdominal aorta area of LCWE-injected WT mice treated with either isotype control or anti-IL6R antibody at 1-week post-LCWE injection. (F, G) Maximal abdominal aorta diameter (F) and abdominal aorta area (G) of LCWE-injected WT mice treated with either isotype control or anti-IL6R antibody at 1-week post-LCWE injection. Results are from two independent experiments combined (n = 13 mice per group). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple testing comparison (B) or Student’s t test (D, F, G). ****p < 0.0001.