Citrullinated Histones as Emerging Host Defence Biomarkers: Understanding NETosis at the Bedside with the Backdrop of COVID-19

Neutrophils are a vital part of the host immune defence system that helps to control infection and modify inflammation conventionally by phagocytosis, degranulation, and secretion of reactive oxygen species.^1,2 Recent studies showed an increased role of neutrophils in the secretion of neutrophil extracellular traps (NETs).¹ Neutrophils secrete histones, proteins, DNA, granular enzymes, etc., forming weblike structures that form a trap for offending pathogens and inflammations, a process called NETosis.¹ It helps in immune regulation, tissue repair, and host defence in good health. However, when produced excessively or cleared inadequately, they can lead to endothelial damage, alveolar injury, immunothrombosis, and multiorgan dysfunction.^3–5 Several recent reviews have underlined this dual behavior of NETs, noting that their inflammatory and thrombotic effects form the basis of acute respiratory distress syndrome (ARDS), microvascular damage, and the procoagulant state typical of severe COVID-19.^4,5 This phenomenon is also implicated in autoimmune diseases like lupus and rheumatoid arthritis, sepsis, cancer metastasis, and thrombotic disorders, highlighting its broad relevance as both a biomarker and therapeutic target.^6–8

In COVID-19, the recent study by Kumar et al.,⁹ showed that elevated NETosis has been strongly linked to severe illness, and there is a temporal relationship with progression of disease to the severe stage. The authors present a prospective cohort study evaluating the association of circulating NETosis markers like citrullinated histone H3 (Cit-H3), myeloperoxidase (MPO), and neutrophil elastase with COVID-19 severity. Their findings strengthen the emerging evidence that NET-driven pathways contribute meaningfully to COVID-19 disease progression.^3,10

Multiple studies have consistently shown elevated NETs markers, including Cit-H3, MPO-DNA complexes, and cell-free DNA, in severe COVID-19.^3,10 Mechanistic reviews have demonstrated that excessive NET formation is triggered not only by cytokine storms but also by direct neutrophil sensing of viral RNA.³ The resulting histone-rich structures are profoundly cytotoxic and prothrombotic, activating platelets and coagulation cascades while perpetuating vascular injury.⁴ Kumar et al.⁹ explore three NET biomarkers with differing degrees of specificity. Cit-H3 represents peptidylarginine deiminase 4 (PAD4) mediated histone modification and is widely considered the most direct indicator of true NETosis.^1,3 Myeloperoxidase and elastase, while integral NET components, may also be released through non-NETotic neutrophil activation, which can reduce their discriminatory utility in clinical settings.¹⁰ This study's results align well with these mechanistic expectations. Among the three markers, Cit-H3 alone reliably distinguished severe from nonsevere disease. Myeloperoxidase displayed no significant difference from controls, and elastase, although elevated in COVID-19 patients, failed to differentiate between disease categories. So, of the three discussed, Cit-H3 may be the most stable and clinically useful biomarker of NETosis.

The study by Kumar et al.⁹ employs a prospective design with biomarker sampling at clinically relevant time points (at admission and at defined deterioration events), thereby enhancing temporal interpretability. The authors adopt the least absolute shrinkage and selection operator (LASSO) regression for biomarker selection, an important step in avoiding overfitting when dealing with multiple correlated inflammatory markers and a smaller sample. Their model identifies Cit-H3 and C-reactive protein (CRP) as independent predictors of severity. The appearance of CRP alongside Cit-H3 highlights the potential complementary relationships between traditional inflammatory markers and NET-specific surrogates. Unlike CRP, which reflects general systemic inflammation, NETosis reflects a specific neutrophil-mediated thrombo-inflammatory mechanism that is not captured by conventional acute-phase reactants.^3,4 Receiver operating characteristic curve (ROC) analysis demonstrates a fair predictive capability for Cit-H3, with sensitivities and specificities of approximately 74 and 64%, respectively, at a defined cut-off. With respect to this study, these markers cannot be diagnostic in isolation, but they can complement clinical assessment.

The study is single-centered, with a relatively small sample size, which may limit generalizability across diverse populations, variants, and healthcare systems. In our experience, there were variable immune responses to COVID-19 due to the comorbidities, viral strain differences, prior exposures, and vaccination status, all of which may influence NET formation. However, this information is lacking in the current study. While the study includes deterioration-linked sampling, it does not provide detailed longitudinal trajectories of NET markers over the course of illness. Persistent NETosis may contribute to chronic inflammation and immune dysregulation beyond the acute phase, which results in long COVID.^11,12

The use of nonhospitalized controls may introduce spectrum bias. Critical illness itself can induce neutrophil activation and NET-associated changes. A comparison with hospitalized or critically ill non-COVID controls might have more accurately reflected disease-specific effects. Emerging data show that impaired NET clearance, rather than absolute NET formation, may be a crucial determinant of microvascular thrombosis and autoantibody generation.^6,12 Incorporating NET degradation markers such as DNase activity or anti-NET antibodies could greatly enrich our understanding of NET dynamics. Myeloperoxidase assays, while widely used, often suffer from specificity issues, as MPO elevation can reflect multiple neutrophil activation pathways unrelated to NETosis.¹⁰

The findings of this study underscore the value of Cit-H3 as a biomarker that indicates a fundamental immunopathological process driving severe illness. With validation in larger, multicenter cohorts, Cit-H3 may have a role as a risk-stratification adjunct in hospitalized patients, particularly in conjunction with established clinical and laboratory markers.⁹ Several NET-modulating strategies like recombinant DNase I, PAD4 inhibitors, and agents targeting neutrophil chemotaxis are under investigation, thereby NETosis can also be a guide to treatment.^3,11 As evidence accumulates linking NET persistence to long COVID, early identification of individuals with excessive NETosis could offer targets for follow-up or intervention beyond the acute period.¹² We are also curious whether NETosis is actually an epiphenomenon in some patients, where it is merely reflecting disease severity rather than driving it.

In conclusion, the present study adds clinical evidence to the expanding body of knowledge surrounding NETosis in COVID-19. Citrullinated histone H3 emerges as a promising biomarker that reflects mechanistic disease drivers while demonstrating practical prognostic value. Clinically, NETosis has potential as a predictor of patients at risk for severe complications, a predictive marker of poor outcomes when levels remain high, and a dynamic marker to track disease progression or regression based on the rising or falling titres, respectively. The findings need to be confirmed in large-scale studies, and the cut-off point needs to be standardized. Current limitations include a lack of standardized assays and variability across disease stages. Future directions involve integrating NETosis into multimodal diagnostic panels with CRP, D-dimer, and cytokines, as well as exploring therapies such as DNase or PAD4 inhibitors to mitigate NET-mediated pathology.

Orcid

Kandaswamy Natarajan https://orcid.org/0000-0002-2557-3695

Natesh Prabu R https://orcid.org/0000-0003-4458-5410