Interleukin-1β is crucial for the induction of coronary artery inflammation in a mouse model of Kawasaki disease

Abstract

Background: Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease in US children. Untreated, children may develop coronary artery aneurysms, myocardial infarction, and sudden death as a result of the illness. Up to a third of KD patients fail to respond to intravenous immunoglobulin, the standard therapy, and alternative treatments are being investigated. Genetic studies have indicated a possible role for interleukin (IL)-1β in KD. We therefore explored the role of IL-1β in a murine model of KD.

Methods and results: Using an established mouse model of KD that involves injection of Lactobacillus casei cell wall extract (LCWE), we investigated the role of IL-1β and caspase-1 (activated by the inflammasome and required for IL-1β maturation) in coronary arteritis and evaluated the efficacy of IL-1 receptor antagonist as a potential treatment. LCWE-induced IL-1β maturation and secretion were dependent on the NLRP3 inflammasome in macrophages. Both caspase-1-deficient and IL-1 receptor-deficient mice were protected from LCWE-induced coronary lesions. Injection of recombinant IL-1β into caspase-1-deficient mice restored the ability of LCWE to cause coronary lesions in response to LCWE. Furthermore, daily injections of the IL-1 receptor antagonist prevented LCWE-mediated coronary lesions up to 3 days after LCWE injection.

Conclusions: Our results strongly suggest that caspase-1 and IL-1β play critical roles in the development of coronary lesions in this KD mouse model, blocked by IL-1 receptor antagonist. Therefore, anti-IL-1β treatment strategies may constitute an effective, more targeted treatment of KD to prevent coronary lesions.

Figure 1

LCWE induces IL-1β in a NLRP3 and ASC dependent manner. (A)Bone marrow derived macrophages were stimulated with 10 μg/ml of LCWE for 12hrs and the level of IL-1β, TNF-α, or PGE₂ in the supernatants were determined by ELISA. (B, C) Bone marrow macrophages derived from Nlrp3^−/−, Asc^−/− or WT mice were stimulated with 10 μg/ml LCWE for 12hrs and IL-1β (B) and TNF-α (C) levels in the supernatants were determined by ELISA. Experiments were performed in triplicate. Data shown are mean±SE and were compared by use of Student's t test or one-way ANOVA with Tukey's post-hoc test (B and C). A probability value of P<0.05 was considered statistically significant. N.D. abbreviates `not detectable'.

Figure 2

Caspase-1 mice are protected from LCWE-induced vasculitis and coronary arteritis. C57BL6/J or Casp1^−/− mice were injected i.p. with 250 μg of LCWE and their hearts were harvested on day 14, (n=9). (A) Representative Hematoxylin and eosin (H&E)-stained heart sections and elastin/collagen-stained sections (B) are shown. The scale bar indicates 250 μm. Heart vessels inflammation score (C) and incidence (D) were evaluated as described in Material and Methods. Myocardial inflammation (E) was evaluated as described in Material and Methods. Data shown are mean±SE and were compared using the Mann-Whitney test (C and E) and Fisher's exact test for incidence (D). A probability value of P<0.05 was considered statistically significant.

Figure 3

Exogenous recombinant IL-1β reconstitutes LCWE-induced vasculitis and coronary arteritis in Casp1^−/− mice. C57BL6/J or casp-1^−/− mice were injected i.p. with LCWE and then treated with 10 ng of mouse recombinant IL-1β (i.p.) daily from day 0 to day 5. On day 7, hearts were harvested and analyzed. Representative H&E-stained sections are shown (A). The scale bar indicates 250 μm. Heart vessels inflammation score (B) and incidence (C) were evaluated (n=9 or12 per group). Data shown are mean±SE and were compared by one-way ANOVA with Tukey's post-hoc test and Fisher's exact test for incidence. A probability value of P<0.05 was considered statistically significant.

Figure 4

IL-1R deficient mice are protected from LCWE induced vasculitis and coronary arteritis. WT (A), Il1r1^−/− mice (B) were administrated LCWE (i.p.) and their hearts were harvested on day 14. H&E staining was performed and representative sections are shown (A and B). The scale bar indicates 250 μm. (C) Incidence was evaluated by use of Fisher's exact test (n=5 or 9). A probability value of P<0.05 was considered statistically significant.

Figure 5

IL-1 receptor antagonist (IL-1Ra) protects against LCWE induced vasculitis, coronary arteritis and myocarditis. Following LCWE injection, WT mice were administrated i.p daily with 500 μg IL-1Ra (from day -1 to day 5), 200 μg human TNF-α mAb (once on day 0) or same volume of PBS for control and hearts were harvested at day 7 for analysis. Representative H&E-stained sections are shown (A). The scale bar indicates 250 μm. Heart vessels inflammation score (B), incidence (C) and myocardium inflammation score (D) were evaluated for each group as mentioned in Methods. Data shown are mean±SE and were compared by the Kruskal-Wallis with Dunn's post-hoc test (B and D) and Fisher's exact test for incidence (C), (n=9). A probability value of P<0.05 was considered statistically significant.

Figure 6

IL-1Ra treatment can prevent LCWE induced vasculitis and coronary arteritis up to three days after LCWE injections. Following LCWE injection, groups ofWT mice were administrated daily (i.p.) with 500 μg IL-1Ra from different starting time points. Experimental schematic is shown (A). The hearts were collected at day 7 and analyzed H&E-staining (B). The scale bar indicates 250 μm. (C) The lesion size was measured to evaluate the effective inhibition by IL-1Ra administration. Data shown are mean±SE and were compared using one-way ANOVA with Dunnett's post-hoc test (n=5). The data was transformed using square root transformation prior to the analysis. All the groups were compared against the control group (no IL-1Ra). A probability value of P<0.05 was considered statistically significant. *P<0.05, ** : P<0.01, *** : P<0.001.

Figure 7

LCWE induces increased body temperature and inflammatory biological markers in the serum of mice. C57BL/6 WT mice were injected (i.p.) with 250μg of LCWE or same volume of PBS. (A) Rectal body temperature was measured as described in methods, (n=9). (B) Plasma PGE₂ levels were quantified by ELISA. Plasma from age matched C57BL/6J mice was used as control. (C) Plasma pentraxin 3 level were measured. Data shown are mean±SE and were compared using the repeated measures two-way ANOVA with Bonferroni's post-hoc test (A) or one-way ANOVA with Tukey's post-hoc test (B–E). A probability value of P<0.05 was considered statistically significant.