Histone Citrullination, NETs and NETosis : Linking Chromatin to Inflammatory Responses and Disease

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.


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!

Fill out my online form


  1. Jorch SK et al. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med 23, 279-87 (2017). PMID:28267716
  2. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 18, 134-47 (2018). PMID:28990587
  3. Mayadas TN et al. The multifaceted functions of neutrophils. Annu Rev Pathol 9, 181-218 (2014). PMID:24050624
  4. Konig MF et al. A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination. Front Immunol 7, 461 (2016). PMID:27867381
  5. Yang H et al. New Insights into Neutrophil Extracellular Traps: Mechanisms of Formation and Role in Inflammation. Front Immunol 7, 302 (2016). PMID:27570525
  6. Masuda S et al. NETosis markers: Quest for specific, objective, and quantitative markers. Clin Chim Acta 459, 89-93 (2016). PMID:27259468
  7. Garcia-Romo GS et al. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med 3, 73ra20 (2011). PMID:21389264
  8. Kenny EF et al. Diverse stimuli engage different neutrophil extracellular trap pathways. Elife 6, (2017). PMID:28574339
  9. Takei H et al. Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis. J Leukoc Biol 59, 229-40 (1996). PMID:8603995
  10. Brinkmann V et al. Neutrophil extracellular traps kill bacteria. Science 303, 1532-5 (2004). PMID:15001782
  11. Neeli I et al. Histone deimination as a response to inflammatory stimuli in neutrophils. J Immunol 180, 1895-902 (2008). PMID:18209087
  12. Clark SR et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13, 463-9 (2007). PMID:17384648
  13. Yipp BG et al. NETosis: how vital is it? Blood 122, 2784-94 (2013). PMID:24009232
  14. Yipp BG et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18, 1386-93 (2012). PMID:22922410
  15. Li P et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med 207, 1853-62 (2010). PMID:20733033
  16. Wang Y et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184, 205-13 (2009). PMID:19153223
  17. Wang Y et al. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306, 279-83 (2004). PMID:15345777
  18. Douda DN et al. SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A 112, 2817-22 (2015). PMID:25730848
  19. Neeli I et al. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release. Front Immunol 4, 38 (2013). PMID:23430963
  20. Saffarzadeh M et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 7, e32366 (2012). PMID:22389696
  21. Villanueva E et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol 187, 538-52 (2011). PMID:21613614
  22. Kolaczkowska E et al. Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature. Nat Commun 6, 6673 (2015). PMID:25809117
  23. Zuo Y et al. Neutrophil extracellular traps in COVID-19. JCI Insight 5, (2020). PMID:32329756
  24. Knight JS et al. Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases. Front Immunol 3, 380 (2012). PMID:23248629
  25. Kroot EJ et al. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum 43, 1831-5 (2000). PMID:10943873
  26. Vencovsky J et al. Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis. Ann Rheum Dis 62, 427-30 (2003). PMID:12695154
  27. Khandpur R et al. NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med 5, 178ra40 (2013). PMID:23536012
  28. Willis VC et al. N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis. J Immunol 186, 4396-404 (2011). PMID:21346230
  29. Seri Y et al. Peptidylarginine deiminase type 4 deficiency reduced arthritis severity in a glucose-6-phosphate isomerase-induced arthritis model. Sci Rep 5, 13041 (2015). PMID:26293116
  30. Papadaki G et al. Neutrophil extracellular traps exacerbate Th1-mediated autoimmune responses in rheumatoid arthritis by promoting DC maturation. Eur J Immunol 46, 2542-54 (2016). PMID:27585946
  31. Cools-Lartigue J et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest, (2013). PMID:23863628
  32. Rayes RF et al. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects. JCI Insight 5, (2019). PMID:31343990
  33. Albrengues J et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science 361, (2018). PMID:30262472
  34. Demers M et al. Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology 5, e1134073 (2016). PMID:27467952
  35. Park J et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Sci Transl Med 8, 361ra138 (2016). PMID:27798263
  36. Erpenbeck L et al. Neutrophil extracellular traps: protagonists of cancer progression? Oncogene 36, 2483-90 (2017). PMID:27941879
  37. Thalin C et al. Quantification of citrullinated histones: development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma. J Thromb Haemost, (2020). PMID:32654410
  38. Thalin C et al. Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer. PLoS One 13, e0191231 (2018). PMID:29324871
  39. Demers M et al. NETosis: a new factor in tumor progression and cancer-associated thrombosis. Semin Thromb Hemost 40, 277-83 (2014). PMID:24590420
  40. Martinod K et al. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A 110, 8674-9 (2013). PMID:23650392
  41. Brill A et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 10, 136-44 (2012). PMID:22044575
  42. Demers M et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A 109, 13076-81 (2012). PMID:22826226
  43. Fuchs TA et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 107, 15880-5 (2010). PMID:20798043
  44. Mauracher LM et al. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. J Thromb Haemost 16, 508-18 (2018). PMID:29325226
  45. Thalin C et al. NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation. Thromb Res 139, 56-64 (2016). PMID:26916297
  46. Martinod K et al. Thrombosis: tangled up in NETs. Blood 123, 2768-76 (2014). PMID:24366358
  47. Louzada ML et al. Risk of recurrent venous thromboembolism according to malignancy characteristics in patients with cancer-associated thrombosis: a systematic review of observational and intervention studies. Blood Coagul Fibrinolysis 22, 86-91 (2011). PMID:21245746
  48. Elyamany G et al. Cancer-associated thrombosis: an overview. Clin Med Insights Oncol 8, 129-37 (2014). PMID:25520567
  49. Middleton EA et al. Neutrophil Extracellular Traps (NETs) Contribute to Immunothrombosis in COVID-19 Acute Respiratory Distress Syndrome. Blood, (2020). PMID:32597954
  50. Barnes BJ et al. Targeting potential drivers of COVID-19: Neutrophil extracellular traps. J Exp Med 217, (2020). PMID:32302401