NLRP3 inflammasome-driven IL-1β and IL-18 contribute to lipopolysaccharide-induced septic cardiomyopathy

https://doi.org/10.1016/j.yjmcc.2023.05.003Get rights and content

Highlights

  • NLRP3 inflammasome is required for the development of LPS-induced SCM.

  • IL-1β deficiency with AAV-mediated overexpression of IL-18BP prevented the development of LPS-induced SCM although deficiency of IL-1β, GSDMD, or IL-1α alone failed to prevent it.

  • Splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM and the production of IL-1β and IL-18.

Abstract

Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1β and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3−/− mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3−/− mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1β, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3−/− mice. LPS-induced SCM was also prevented in Casp1/11−/− mice, but not in Casp11mt, IL-1β−/−, IL-1α−/−, or GSDMD−/− mice. Notably, LPS-induced SCM was apparently prevented in IL-1β−/− mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1β and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.

Introduction

Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated host response to infection [1,2]. Despite significant advances in the diagnosis and treatment of sepsis, it continues to be the most common cause of death worldwide. Cardiac dysfunction (called septic cardiomyopathy [SCM]) is a common form of sepsis-induced organ dysfunction. SCM is characterized by acute, global, but reversible myocardial depression, and is associated with higher mortality and a poor outcome [3,4]. To date, many factors have been shown to mediate the development of SCM, including inflammation, mitochondrial dysfunction, oxidative and nitrosative stress, downregulation of the adrenergic pathway, abnormalities of intracellular calcium handling, and changes in the coronary microvasculature [3,4]. Specifically, it is well recognized that excessive inflammatory responses and cytokines play critical roles in the pathophysiology of SCM. For instance, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) have been reported to be elevated in the plasma of patients with sepsis, and directly cause cardiomyocyte depression [5]. However, the exact mechanism of how the inflammatory response and cytokines induce SCM in vivo has remained obscure.

The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and cysteine protease caspase-1 (Casp1), and contributes to inflammatory responses in various diseases, including cardiovascular diseases [6,7]. The formation of NLRP3 inflammasome causes auto-activation of Casp1 which leads to the processing of pro-IL-1β and pro-IL-18 to their active forms, resulting in inflammation. Activated Casp1 also processes gasdermin D (GSDMD) to N-GSDMD fragment, which drives pore formation that leads to pyroptosis. In addition to these direct downstream effectors, IL-1α production is also reported to be partially regulated by the NLRP3 inflammasome [8]. Regarding SCM, several experimental studies have suggested the potential roles of NLRP3 inflammasome [[9], [10], [11]]; however, it is still unclear which effectors are responsible for SCM. In the present study, we produced a mouse model of SCM injected with lipopolysaccharide (LPS). LPS (also known as endotoxin) is a Gram-negative bacterial wall product which, when injected, induces most of the pathological features of sepsis, including acute and reversible myocardial dysfunction [5]. Indeed, a previous study reported that the administration of endotoxin to human volunteers caused a decrease in left ventricular function [3]. Here, we investigated the roles of NLRP3 inflammasome and its downstream effectors by using mice deficient in NLRP3, Casp1/11, Casp11, IL-1β, GSDMD, and IL-1α as well as adeno-associated virus (AAV) vectors encoding IL-18 binding protein (AAV–IL-18BP), and demonstrated that both IL-1β and IL-18 driven by NLRP3 inflammasome contribute to the development of LPS-induced SCM.

Section snippets

Animal protocols

All experiments in this study were carried out in accordance with the Jichi Medical University Guide for Laboratory Animals (permit No. 20116–02). Wild-type (WT, C57BL/6 J background) mice were purchased from SLC Japan (Shizuoka, Japan). NLRP3−/−, caspase-1/11−/−, and IL-1β−/− mice were kindly provided by Drs. Vishava M. Dixit (Genentech, South San Francisco, CA), Hiroko Tsutsui (Hyogo Medical College, Japan), and Yoichiro Iwakura (Tokyo University of Science, Japan), respectively [[12], [13],

Deficiency of NLRP3 and Casp1/11 prevents LPS-induced cardiac dysfunction and damage

We first evaluated the survival times of WT mice injected with LPS (6 mg/kg), which is a commonly used model of SCM, and found that approximately 40% of WT mice died 48 h after LPS injection (Fig. 1A). In contrast, none of the NLRP3−/− mice died after LPS injection (p = 0.0129). To clarify this role further, we injected WT and NLRP3−/− mice with LPS and assessed cardiac function using echocardiography. Consistent with clinical features of SCM, cardiac function (determined by %FS) was

Discussion

Inflammation is believed to be one of the crucial mechanisms underlying SCM; however, its exact mechanism is not yet fully understood. In the present study, we demonstrated that NLRP3 inflammasome mediates LPS-induced cardiac dysfunction and damage in a mouse model of SCM. Although deficiency of Casp11, IL-1β, GSDMD, or IL-1α (downstream effectors of the NLRP3 inflammasome) alone failed to prevent the development of LPS-induced SCM, IL-1β deficiency with AAV-mediated overexpression of IL-18BP

Disclosures

None

Author contributions

K.F., T.Ka., and M.T. designed the study concept and experiment. K.F., T.Ka., T.Ko., N.Y., Y.M., and C.B. performed the experiments and analyzed the data; K.O. and N.T. provided mice; H.M. provided AAV vectors; T.M. and K.K. discussed the data and provided scientific advice. K.F., T.Ka., and M.T. wrote the manuscript.

Acknowledgments

We thank Drs. Tadashi Kasahara and Norihiko Takeda (Jichi Medical University) for providing invaluable suggestions, and Dr. Vishva M. Dixit (Genentech, CA), Dr. Shun'ichiro Taniguchi (Shinshu University, Japan), Hiroko Tsutsui (Hyogo Medical College, Japan), and Dr. Yoichiro Iwakura (Tokyo University of Science, Japan), for providing NLRP3 inflammasome-related gene-deficient mice. This study was supported by grants from the Japan Society for the Promotion of Science (JSPS) through a

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