Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi.

BACKGROUND: Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. OBJECTIVES: We aimed to explore the contribution of PAD4 to EET formation. METHODS: We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. RESULTS: Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. CONCLUSION: As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

Neutrophil peptidylarginine deiminase 4 is essential for detrimental age-related cardiac remodelling and dysfunction in mice.

Mice fully deficient in peptidylarginine deiminase 4 (PAD4) enzyme have preserved cardiac function and reduced collagen deposition during ageing. The cellular source of PAD4 is hypothesized to be neutrophils, likely due to PAD4's involvement in neutrophil extracellular trap release. We investigated haematopoietic PAD4 impact on myocardial remodelling and systemic inflammation in cardiac ageing by generating mice with Padi4 deletion in circulating neutrophils under the MRP8 promoter (Ne-PAD4-/-), and ageing them for 2 years together with littermate controls (PAD4fl/fl). Ne-PAD4-/- mice showed protection against age-induced fibrosis, seen by reduced cardiac collagen deposition. Echocardiography analysis of structural and functional parameters also demonstrated preservation of both systolic and diastolic function with MRP8-driven PAD4 deletion. Furthermore, cardiac gene expression and plasma cytokine levels were evaluated. Cardiac genes and plasma cytokines involved in neutrophil recruitment were downregulated in aged Ne-PAD4-/- animals compared to PAD4fl/fl controls, including decreased levels of C-X-C ligand 1 (CXCL1). Our data confirm PAD4 involvement from circulating neutrophils in detrimental cardiac remodelling, leading to cardiac dysfunction with old age. Deletion of PAD4 in MRP8-expressing cells impacts the CXCL1-CXCR2 axis, known to be involved in heart failure development. This supports the future use of PAD4 inhibitors in cardiovascular disease. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.