Original articleIntracardiac administration of neutrophil protease cathepsin G activates noncanonical inflammasome pathway and promotes inflammation and pathological remodeling in non-injured heart
Introduction
Inflammation is a hallmark of chronic heart failure (HF) initially triggered by nonimmune modes of cardiac injury, such as myocardial infarction (MI), genetic mutations, and mechanical stress (e.g., pressure overload) [1,2]. Signals in the stressed or injured myocardium triggers the activation of inflammatory signaling pathways, which lead to complement activation, reactive oxygen species (ROS) generation and cytokine/chemokine upregulation [1,2]. These upregulated chemokines promote leukocyte extravasation to the injured myocardium to remove dead cells and extracellular matrix (ECM) debris and prepare the area for cardiac repair. However, defects in the resolution of inflammation or an excessive inflammatory response have been associated with increased myocyte death and adverse cardiac remodeling and function, thus leading to HF [1]. Several clinical and experimental data implicate neutrophils as the dominant immunological cell type involved in the acute inflammatory response subsequent to myocardial injury that progresses into the chronic inflammatory reactions seen in HF [3,4]. Herein neutrophilic release of chemoattractants such as monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), or stromal cell-derived factor (SDF)-1α and upstream regulators of chemotaxis, such as interleukin (IL)-1β increases tissue infiltration of monocytes and T-cells after injury [4,5]. However, neutrophils also produce high levels of serine proteases in response to inflammatory mediators, and the role of these proteases in cardiac inflammation, remodeling and function is less well understood.
Inflammatory serine proteases (ISPs) including cathepsin G (Cat.G), elastase, proteinase 3 and chymase are known mainly for their function in the intracellular degradation of pathogens. Their extracellular release upon leukocyte activation is traditionally regarded as the primary reason for tissue damage at the sites of inflammation. Cat.G in particular, has functionality which extends beyond ECM protein degradation and exerts multiple physiological effects through its enzymatic activity including antimicrobial activity, vasoregulation, and cytokine processing [6]. Mice deficient in multiple ISPs (Cat.G, elastase, proteinase-3) exhibit reduced inflammatory reactions and are highly protected from the development of several autoimmune conditions [[7], [8], [9]]. Furthermore, in vivo studies utilizing a dual inhibitor of Cat.G and chymase demonstrated a reduced amount of cardiac inflammation and an improvement in cardiac function following cardiac ischemia-reperfusion injury in comparison to vehicle-treated animals [10]. This suggests that Cat.G and/or chymase could serve as important amplifiers of inflammation, but the mechanisms to achieve this remain obscure.
The functional role of ISPs, such as elastase and chymase, in cardiac remodeling has mainly been examined in settings of acute inflammation following myocardial injury or stress associated with significant myocyte loss and neutrophil infiltration [11,12]. However, in these studies the role of ISPs on inflammation was obscured by the other confounding pro-inflammatory factors associated with myocardial injury, such as cell death and inflammatory cell infiltration. In this study, we evaluated the effects of Cat.G in the initiation and amplification of the inflammatory response and on cardiac remodeling in a non-stressed or uninjured rat heart to minimize the confounding factors presented by the inflammatory response associated with myocardial injury [13,14]. We utilized a complementary technique of in vivo cardiac transfer of Cat.G and found that enhanced local myocardial concentrations of Cat.G induced processing and activation of key cytokines involved in the initiation of the inflammatory response that resulted in pathological cardiac remodeling in absence of prior myocardial stress or injury.
Section snippets
Animal preparation
Male Sprague-Dawley rats (8–9 weeks, 250–300 g) were administered a bolus of either vehicle or endotoxin free human neutrophil-derived Cat.G (1 mg/kg; MP Biomedicals, cat. no. 02191344; Solon, OH) using a previously described catheter based technique [15]. Briefly, rats were anesthetized with pentobarbital (50 mg/kg) intraperitoneally (i.p.) and placed on a ventilator. The chest was entered from the left side through the third intercostal space. A 26 G catheter was advanced from the apex of the
Increased Cat.G levels in the myocardium enhance inflammation
Neutrophil infiltration into the injured myocardium has been linked to early inflammatory responses subsequent to cell death [3]. However, the role of ISPs in cardiac inflammation and remodeling remains unclear. We assessed whether Cat.G alone is sufficient to promote changes in absence of myocardial injury. Rats were injected with a bolus of Cat.G (1 mg/kg) or its vehicle into the heart via a catheter-based technique to achieve generalized cardiac delivery during transient occlusion of the
Discussion
The current study provides compelling evidence that Cat.G can trigger a potent chemotactic response in the absence of myocardial injury and cell death that results in increased accumulation of neutrophils, macrophages, and eventually mast cells. The chemotactic activity of Cat.G was associated with the processing and activation of the early response cytokines, IL-1β and IL-18, independently of NLRP-3/1 signaling pathway. As a result, Cat.G-treated hearts show increased ECM degradation and
Integrity of research and reporting
Animal studies have been approved by the Animal Care and Use Committee of Temple University and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
The manuscript does not contain clinical studies or patient data.
Declaration of competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the National Institutes of Health (HL360338 and HL360343 for AS) and 5T32HL091804 for SM).
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