NLRP3 Deficiency Reduces Macrophage Interleukin-10 Production and Enhances the Susceptibility to Doxorubicin-induced Cardiotoxicity

Abstract

NLRP3 inflammasomes recognize non-microbial danger signals and induce release of proinflammatory cytokine interleukin (IL)-1β, leading to sterile inflammation in cardiovascular disease. Because sterile inflammation is involved in doxorubicin (Dox)-induced cardiotoxicity, we investigated the role of NLRP3 inflammasomes in Dox-induced cardiotoxicity. Cardiac dysfunction and injury were induced by low-dose Dox (15 mg/kg) administration in NLRP3-deficient (NLRP3(-/-)) mice but not in wild-type (WT) and IL-1β(-/-) mice, indicating that NLRP3 deficiency enhanced the susceptibility to Dox-induced cardiotoxicity independent of IL-1β. Although the hearts of WT and NLRP3(-/-) mice showed no significant difference in inflammatory cell infiltration, macrophages were the predominant inflammatory cells in the hearts, and cardiac IL-10 production was decreased in Dox-treated NLRP3(-/-) mice. Bone marrow transplantation experiments showed that bone marrow-derived cells contributed to the exacerbation of Dox-induced cardiotoxicity in NLRP3(-/-) mice. In vitro experiments revealed that NLRP3 deficiency decreased IL-10 production in macrophages. Furthermore, adeno-associated virus-mediated IL-10 overexpression restored the exacerbation of cardiotoxicity in the NLRP3(-/-) mice. These results demonstrated that NLRP3 regulates macrophage IL-10 production and contributes to the pathophysiology of Dox-induced cardiotoxicity, which is independent of IL-1β. Our findings identify a novel role of NLRP3 and provided new insights into the mechanisms underlying Dox-induced cardiotoxicity.

Figure 1. NLRP3 deficiency exacerbates Dox-induced cardiac injury.

(A,B) Echocardiography was performed in WT, NLRP3^−/−, and IL-1β^−/−mice 5 days after Dox (15 mg/kg) or vehicle (Veh) treatment. Two-dimensional M-mode echocardiograms are shown (A). Cardiac function (%FS) was assessed (B) (n = 6–10). (C–E) Mice were sacrificed 5 days after Dox or Veh treatment. (C) The heart sections were obtained and stained with HE. The vacuolated cardiomyocytes were quantified (n = 4 for each). (D) Plasma levels of CPK, CK-MB, and LDH were assessed (n = 5 for each). (E) Heart Bnp mRNA levels were assessed by real-time RT-PCR analysis (n = 4 for each). Data are expressed as the mean ± SEM. **p < 0.01. ^#p < 0.05 vs. Veh treatment (WT).

Figure 2. NLRP3 deficiency has no effect on inflammatory cell infiltration but decreases IL-10 production.

The heart samples were obtained from WT and NLRP3^−/− mice 5 days after Dox or vehicle treatment. (A) The heart sections were stained immunohistochemically for CD45. The number of CD45⁺ cells was quantified (n = 5 for each). (B) The number of leukocytes (CD45⁺ cells) in the heart was analyzed by flow cytometry (n = 5 for each). (C) The heart sections were stained immunohistochemically for CD68. The number of macrophages (CD68⁺ cells) was quantified (n = 5 for each). (D) The number of macrophages (CD11b⁺F4/80⁺ cells) in the heart was analyzed by flow cytometry (n = 5 for each). (E) The protein levels of IL-1β, TNF-α, and IL-10 in the heart were assessed (n = 5 for each). Data are expressed as the mean ± SEM. **p < 0.01.

Figure 3. NLRP3 in bone marrow cells contributes to Dox-induced myocardial injury.

BMT^{WT to WT}, BMT ^{WT to NLRP3−/−}, and BMT^{NLRP3−/− to WT} mice were developed, and treated with Dox or Veh 8 weeks after BMT. Echocardiography was performed 5 days after Dox or Veh treatment. Two-dimensional M-mode echocardiograms are shown (A). Cardiac function (%FS) was assessed (B) (n = 6–10). (C,D) Mice were sacrificed 5 days after Dox or Veh treatment. (C) The heart sections were obtained and stained with HE. The vacuolated cardiomyocytes were quantified (n = 4 for each). (D) Plasma levels of CPK and CK-MB were assessed (n = 5 for each). (E) Primary cardiomyocytes were isolated from WT and NLRP3^−/− mice, and treated with Dox (0.1–2.0 μM) for 24 h. Cytotoxicity was assessed by LDH activity (n = 6 for each). Data are expressed as the mean ± SEM. **p < 0.01, and ^#p < 0.05 vs. Veh treatment (BMT^{WT to WT}).

Figure 4. NLRP3 deficiency causes decreased IL-10 production in macrophages.

(A,B) Primary macrophages derived from WT and NLRP3^−/− mice were stimulated with LPS (30–300 ng/mL) or Pam3CSK4 (3–30 ng/mL) for 24 h at the concentrations indicated. The IL-10 protein levels in the supernatants were assessed. (C,D) Primary WT and NLRP3^−/− macrophages were treated with Dox (2 μM) or Veh for 24 h. The mRNA levels of Tlr4 and Tlr2 were assessed (n = 5 for each). (E) Primary WT and NLRP3^−/− macrophages were treated with Dox or Veh for the indicated periods. The Il10 mRNA levels were assessed (n = 5 for each). (F,G) Splenocytes isolated from WT and NLRP3^−/− mice were stimulated with LPS (3–10 ng/mL) or Pam3CSK4 (0.3–3 ng/mL) for 24 h at the concentrations indicated. Data are expressed as the mean ± SEM. **p < 0.01. ^#p < 0.05 vs. no LPS treatment (WT).

Figure 5. AAV-mediated IL-10 overexpression restores Dox-induced cardiotoxicity.

WT and NLRP3^−/− mice were injected intramuscularly with AAV1-GFP and AAV1-IL-10. (A) The IL-10 protein levels were assessed at 14 days after the injection (n = 8–9 for each). ND indicates not detected. (B–F) WT and NLRP3^−/− mice were treated with Dox or Veh 14 days after the AAV injection. (B,C) Echocardiography was performed 5 days after Dox or Veh treatment. Two-dimensional M-mode echocardiograms are shown (B). Cardiac function (%FS) was assessed (C) (n = 6–10). (D–F) Mice were sacrificed 5 days after Dox or Veh treatment. (D) The heart sections were obtained and stained with HE. The vacuolated cardiomyocytes were quantified (n = 4 for each). (E) Plasma levels of CPK, CK-MB, and LDH were assessed (n = 5 for each). (F) Heart Bnp mRNA levels were assessed by real-time RT-PCR analysis (n = 4 for each). Data are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01.