Citrullination of histone tails (histones H3, H4 and H2A) is a widely studied nucleosome post-translational modification (PTM) in immunology research, and has been linked to numerous diseases, including cancer, autoimmunity, and thrombosis1, 2. Citrullination of histone H3 (H3Cit) is particularly well studied owing to available affinity reagents. H3Cit occurs in very specific contexts; most notably, H3Cit is established in activated neutrophils and plays a crucial role in the development of Neutrophil Extracellular Traps (NETs). NETs are webs of decondensed chromatin and granular proteins, which are released by neutrophils during the process of NETosis to help capture invading pathogens for degradation1, 2. However, there is increasing evidence that aberrant production of NETs, outside of active infection, contributes to disease development.
H3Cit is a fascinating mark at the intersection of chromatin structure and immunology. Here, we will cover the basics of H3Cit and NETs, including a brief summary of their pathogenic role in disease.
Neutrophils and NETs
In order to understand NETs and NETosis, it is important to know the role of neutrophils in immunology. Neutrophils are the most abundant leukocyte in the human body and are known for their phagocytic abilities. They are often referred to as “granulocytes” due to the presence of dense granules in the cytoplasm, which contain antimicrobial enzymes. As an integral part of the innate immune system, their primary function is to defend the host against bacterial and fungal infection. They are recruited to infected tissues by way of the vascular endothelium, where they become fully activated in response to inflammatory cytokines and/or the surface proteins of the pathogens (e.g. lipopolysaccharide or LPS)3.
Activated neutrophils employ several mechanisms to dispose of pathogens and prevent further infection, including phagocytosis, degranulation, and generation of reactive oxygen species (ROS). One of the most interesting methods involves neutrophil formation and expulsion of NETs. This dense network of granular proteins and chromatin creates a web that traps and kills bacteria, fungi, and other extracellular invaders, neutralizing infections1, 2.
NETosis is the term commonly used to describe NET extrusion from the cell. Precisely how and under what circumstances NETs are formed and released is a hotly debated topic in the field, but it does seem partly related to the type of stimulus used for neutrophil activation. For a thorough review of the pathways related to NETosis, we refer to articles by Jorch and Kubes (2017)1, Papayannopoulos (2018)2, and Konig and Andrade (2016)4.
NETosis pathways and PAD4
As indicated above, there are a variety of potential mechanisms and stimuli that induce NETosis and work to eliminate pathogens or emerging infections. These pathways can be divided into two general categories4-6:
- Suicidal (or lytic) NETosis, which results in cell death and NET release. This form of NETosis occurs when cultured neutrophils are stimulated with PMA, calcium ionophores, physiological stimuli (e.g. IL-8), bacteria, fungi, and autoantibodies (e.g. systemic lupus erythematosus)7-11.
- Vital (or non-lytic) NETosis, in which neutrophils release NETs without cell death. This process is associated with rapid responses following treatment of neutrophils with TLR ligands or bacterial products12-14.
Importantly, these two outcomes are due to far more than two mechanisms, and thus are a major source of discussion in the field4-6.
For the purposes of this blog, we will be focusing on PAD4-related suicidal NETosis (for brevity, PAD4-NETosis), which represents a central mechanism leading to altered NET formation in human disease1, 2.
In this version of NETosis, stimulation of neutrophils activates the peptidyl arginine deiminase PAD4 to citrullinate chromatin11, 15, 16. PAD4 is a calcium-dependent enzyme that converts a positively charged arginine to a neutral citrulline, resulting in a net loss of positive charge at the modified residue17. This altered charge profoundly impacts chromatin structure, promoting extensive decondensation, which is essential for NET development. At the conclusion of this pathway, the plasma membrane ruptures, and NETs are released into the extracellular space.
Other enzymes also become activated and contribute to NET formation in this process, most notably myeloperoxidase (MPO) and neutrophil elastase (NE). However, MPO and NE are also involved in PAD4-independent mechanisms of suicidal NETosis18, 19, complicating analysis of this pathway.
PAD4-induced H3Cit is a direct and specific readout of this form of NETosis and is an emerging biomarker candidate for multiple human pathologies (see below).
PAD4-NETosis in Disease
While PAD4-mediated histone citrullination and resulting NETs serve a therapeutic role in pathogen elimination, excessive NETs can also be pro-inflammatory20. When NETs stay in the body for an extended period of time, they cause tissue and even organ damage, and often stimulate production of autoantibodies21. Indeed, PAD4 NETosis is strongly implicated in these processes; for instance, PAD4-deficient mice display reduced NET formation and associated tissue damage in response to infection with S. aureus22.
Increased levels of PAD4-NETs and H3Cit are linked to a range of diseases, including autoimmunity, cancer, thrombosis, and even COVID-191, 2, 23. Below is a brief summary of how H3Cit / NETs contribute to diverse disease states, and their potential application in future liquid biopsy biomarker assays.
Autoimmunity – Rheumatoid Arthritis
PAD4-NETs are connected with multiple autoimmune diseases, particularly rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)1, 2. The research strongly suggests that this link may be pathogenic24. In RA, patients generate autoantibodies to H3Cit and other citrullinated proteins, and higher levels of autoantibodies to these targets are associated with more aggressive forms of disease25-27. It is not fully known how NETs and their autoantibodies contribute to autoimmunity1, although experiments in mouse RA models have suggested PAD4-NETs are important for establishing disease28-30.
The role of PAD4-NETs in cancer is a fascinating area of investigation. A foundational paper from Cools-Lartigue et al. demonstrated that NETs produced in mouse models of sepsis can capture circulating tumorigenic cells and promote their dissemination to other organs31. The function of NETs in cancer development and metastasis has been confirmed in additional studies32-34, and in one paper breast cancer cells were shown to induce NETs independently of infection35. Treatment with NET inhibitors or using PAD4 knockout mice can slow tumor growth32, 35, and PAD4 is an active area of cancer therapeutic and biomarker development36.
EpiCypher’s recent publication with collaborators at the Karolinska Institute and University of North Carolina37 provides a novel approach for quantitative detection of H3Cit, and validated previous studies showing that cancer patients exhibit increased H3Cit vs. healthy controls38, 39. (Look for another blog post on this paper soon!)
Thrombosis is an interesting target of histone citrullination research, as PAD4-NETs and H3Cit have been directly linked to thrombotic events in a variety of mouse models40-42, non-human primates43, and human cancer patients44, 45. The formation of NETs assists in thrombosis formation by providing a framework for blood to coagulate. Platelets and red blood cells stick to the protein-DNA complex, forming a clot, which can cause blockage or tissue damage46. Cancer patients are at particularly high risk of thrombotic events47, and thrombosis in cancer is associated with higher mortality48.
SARS-CoV-2 / COVID-19
NETosis has recently been linked to COVID-19. In a study published by Zuo et al., the blood sera of COVID-19 patients contained higher levels of PAD4-NETosis markers, including H3Cit and MPO-associated DNA, in comparison to healthy controls. In a separate study by Middleton et al., H3Cit positive neutrophils were detected in microthrombi within the lung of COVID-19 patients49. In both of these studies, levels of MPO and cell-free DNA correlated with COVID-19 severity, such that patients on ventilators and those who died displayed the highest levels of NET markers23, 49. Thus, the study of NETosis may provide insight into the pathology and treatment of critically ill COVID-19 patients50.
New Assays for H3Cit Are Needed
Histone citrullination and PAD4-dependent NETosis are related to a number of diseases, making reliable detection and accurate quantification of this PTM increasingly important to the field. In addition, H3Cit is a valuable marker for interrogating NETosis mechanisms, as it is a direct indicator of the PAD4-NETosis pathway. However, standardized approaches to quantify H3Cit have been lacking, making it difficult to characterize this mark and compare results across studies.
EpiCypher is a leader in quantitative chromatin assay development, antibody validation, and recombinant nucleosome technology, and with our most recent publication37, we are well-equipped to help scientists implement robust approaches to study histone citrullination. Stay tuned for more about this PTM, and to learn about EpiCypher’s efforts to develop a novel H3Cit assay using nucleosome-validated antibodies and recombinant modified designer nucleosomes for reliable assay quantification!
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