Original article
Intracardiac administration of neutrophil protease cathepsin G activates noncanonical inflammasome pathway and promotes inflammation and pathological remodeling in non-injured heart

https://doi.org/10.1016/j.yjmcc.2019.06.016Get rights and content

Highlights

  • Cathepsin G triggers leukocyte chemotaxis and proinflammatory responses in unstressed heart

  • Cathepsin G impairs survival signaling and promotes pathological remodeling in unstressed heart

  • Inflammatory proteases activate IL-1β and IL-18 in cardiomyocytes, independent of inflammasome

  • Inhibition of IL-1 signaling attenuates cathepsin G-induced adverse cardiac remodeling

Abstract

Background

Inflammatory serine proteases (ISPs) play an important role in cardiac repair after injury through hydrolysis of dead cells and extracellular matrix (ECM) debris. Evidence also suggests an important role of ISPs in the coordination of the inflammatory response. However, the effect of ISPs on inflammation is obfuscated by the confounding factors associated with cell death and inflammatory cell infiltration induced after cardiac injury. This study investigated whether neutrophil-derived cathepsin G (Cat.G) influences inflammation and remodeling in the absence of prior cardiac injury and cell death.

Methods and results

Intracardiac catheter delivery of Cat.G (1 mg/kg) in rats induced significant left ventricular (LV) dilatation and cardiac contractile dysfunction at day 5, but not at day 2, post-delivery compared to vehicle-treated animals. Cat.G delivery also significantly increased matrix metalloprotease activity and collagen and fibronectin degradation at day 5 compared to vehicle-treated rats and these changes were associated with increased death signaling pathways and myocyte apoptosis. Mechanistic analysis shows that Cat.G-treatment induced potent chemotactic activity in hearts at day 2 and 5 post-delivery, characterized by processing and activation of interleukin (IL)-1β and IL-18, stimulation of inflammatory signaling pathways and accumulation of myeloid cells when compared to vehicle-treated rats. Cat.G-induced processing of IL-1β and IL-18 was independent of the canonical NLRP-3 inflammasome pathway and treatment of isolated cardiomyocytes with inhibitors of NLRP-3 or caspase-1 failed to reduce Cat.G-induced cardiomyocyte death. Notably, rats treated with IL-1 receptor antagonist (IL-1Ra) show reduced inflammation and improved cardiac remodeling and function following Cat.G delivery.

Conclusions

Cat.G exerts potent chemoattractant and pro-inflammatory effects in non-stressed or injured heart in part through processing and activation of IL-1 family cytokines, subsequently leading to adverse cardiac remodeling and function. Thus, targeting ISPs could be a novel therapeutic strategy to reduce cardiac inflammation and improve cardiac remodeling and function after injury or stress.

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).

References (46)

  • E. Tchougounova et al.

    A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2

    J. Biol. Chem.

    (2005)
  • B.R. Lucchesi et al.

    The role of the neutrophil and free radicals in ischemic myocardial injury

    J. Mol. Cell. Cardiol.

    (1989)
  • J.A. Stewart et al.

    Cardiac mast cell- and chymase-mediated matrix metalloproteinase activity and left ventricular remodeling in mitral regurgitation in the dog

    J. Mol. Cell. Cardiol.

    (2003)
  • A. Sabri et al.

    Neutrophil cathepsin G promotes detachment-induced cardiomyocyte apoptosis via a protease-activated receptor-independent mechanism

    J. Biol. Chem.

    (2003)
  • N.G. Frangogiannis

    The inflammatory response in myocardial injury, repair and remodeling

    Nature

    (2014)
  • D.L. Mann

    Innate immunity and the failing heart: the cytokine hypothesis revisited

    Circ. Res.

    (2015)
  • S.D. Prabhu et al.

    The biological basis for cardiac repair after myocardial infarction

    Circ. Res.

    (2016)
  • O. Soehnlein et al.

    Neutrophils as protagonists and targets in chronic inflammation

    Nat Rev Immunol

    (2017)
  • W.M. Nauseef et al.

    Neutrophils at work

    Nat. Immunol.

    (2014)
  • O. Chertov et al.

    Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils

    J. Exp. Med.

    (1997)
  • A.M. Adkison et al.

    Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

    J. Clin. Invest.

    (2002)
  • Y. Hu et al.

    Dipeptidyl peptidase I regulates the development of collagen-induced arthritis

    Arthritis Rheum.

    (2005)
  • C.T. Pham

    Neutrophil serine proteases: specific regulators of inflammation

    Nat Rev Immunol

    (2006)
  • Cited by (7)

    • SERPINA3C ameliorates adipose tissue inflammation through the Cathepsin G/Integrin/AKT pathway

      2022, Molecular Metabolism
      Citation Excerpt :

      Because adipose SERPINA3C was found to play protective role against insulin resistance in our study, the involvement and mechanism of β1 Integrin in the regulation of adipose tissue metabolism by SERPINA3C was then investigated. β1 Integrin is known to form heterodimers with α5 Integrin to activate AKT signaling [44,46–51]. We examined α5 Integrin and β1 Integrin protein expressions in KO and AAV-SERPINA3C mice.

    View all citing articles on Scopus
    View full text