NETs and related products have been shown to be involved in both acute and chronic inflammation and tissue damage [29–31]. In sepsis, excessive NET formation can trigger the release of pro-inflammatory cytokines, activate coagulation pathways, and impair microcirculatory function, ultimately leading to tissue damage and organ failure [21, 32]. In addition to sepsis, NETs have been implicated in the pathogenesis of other inflammatory and autoimmune diseases [15, 33, 34]. The role of NETs in chronic inflammation is thought to be related to their ability to induce tissue damage and activate immune cells, leading to a sustained inflammatory response. The advent of the standardized NuQ H3.1® assay for automated and standardized quantification of nucleosomes has opened up new avenues for further exploration and research in this field. To our knowledge, we report the first study that explores nucleosomes H3.1 in a substantial cohort of septic shock patients who simultaneously exhibit a severe inflammatory response and profound alterations of immune cellular parameters.
With the present study, we provide several important results. The first one is to demonstrate that circulating nucleosome H3.1 level was markedly increased in patients upon admission. In addition, and as expected, we found positive correlation between nucleosome H3.1 concentrations, interleukin 6, a pro-inflammatory cytokine, and neutrophils the responsible cells for NETs formation. Interestingly, we observed an increased correlation over time with neutrophils, suggesting that after initial tissue aggression, which could generate intranuclear material by different mechanisms, the production of nucleosomes is increasingly dependent on residual activation of neutrophils. This point would require additional investigations to further explore this finding.
The second one is to show that higher nucleosome concentrations were significantly associated with SOFA and severity scores and, most importantly, with both 5- and 28-day mortality in an independent manner. Indeed, when we focused on the upper quartile of nucleosome values, this association remained significant in multivariate analysis, indicating that nucleosome levels alone can provide valuable clinical information. In contrast, significant differences in interleukin-6 (IL-6) levels between survivors and non-survivors disappeared after multivariate analysis. However, Kaplan Meir analysis demonstrated that, when associated, higher values of both nucleosome and IL-6 at admission identified a subset of patients who died very rapidly. Therefore, nucleosomes may contribute to the establishment of a hyper-inflammatory phenotype (along with other inflammatory markers), which could guide clinicians toward very aggressive anti-inflammatory strategies, such as personalized corticosteroid use.
A third important finding was the persistent high nucleosome values observed over time. Even at the end of the first week, these values remained largely above the control values. Of particular significance was the association between the Day 6–8 nucleosome values and 28-day mortality (supplemental Fig. 1). Given that higher nucleosome values were linked to lower Day 6–8 mHLA-DR (Fig. 2), the question arises as to whether nucleosome and persistence of inflammation contribute to delayed immunosuppression. The clearance mechanisms for NETs are not completely understood. In experimental infection models, NETs have been observed to persist for several days and are believed to be broken down by plasma nuclease DNAse I. However, even after DNA degradation, NETs can still persist, indicating the involvement of additional clearance mechanisms. One possible mechanism could be the scavenging properties of monocytes which are known to be impaired in sepsis [35]. Further research is needed to explore the potential causal relationship between nucleosomes and low mHLA-DR levels.
Overall, the current findings support previous research indicating that various compounds released during NET formation, such as histones, circulating cell-free DNA, and myeloperoxidase, are associated with mortality or negative outcomes in septic patients [30, 31, 36, 37]. However, none of these compounds, including nucleosomes, are, by themselves, specific to NETs and can also be released in response to other tissue damage. Thus, numerous mediators have the potential to serve as biomarkers of NET formation. Therefore, the analytical aspects of measuring these compounds, particularly in clinical research, need to be considered. Currently, ELISA assays and home-made protocols are the main methods for measuring NET products. As such, these tests present several analytical challenges to clinical deployment, such as manual performance, preparation procedures, the need for skilled technicians, limited access, and poor standardization. As a result, the poor reproducibility and reliability of these assays pose significant challenges to their practical use in clinical settings. Therefore, an automated NET assay may provide a more standardized and reliable approach to investigating NET formation in clinical settings.
Beyond its interest as biomarker, the involvement of NETs and related products in various inflammatory and immune-mediated diseases highlights the potential of targeting these pathways for the development of novel therapeutic strategies. In this context, elevated nucleosomes may indicate a treatable trait. Clinical development of inhibitors to directly target NET formation, has already been started [21, 28, 38, 39]. In addition, therapeutic plasmapheresis (i.e., the selective extra corporeal removal of NET) is under investigation (NCT04749238). In case of favorable preliminary results, nuclesosome levels would constitute an obvious candidate to guide individualized therapy as companion biomarker.
This study has some limitations that need to be addressed. Firstly, the cohort used in the study consisted of critically ill septic shock patients with a high mortality rate. Therefore, the results of the study cannot be generalized to all septic patients. Secondly, this study was conducted retrospectively using the IMMUNOSEPSIS cohort, which was primarily aimed at monitoring immunosuppression (NCT02803346). Thus, a prospective study, including longer follow-up, should be designed to specifically explore nucleosome, along with other relevant markers, to obtain more in-depth information regarding its potential as a biomarker and/or as an actor of delayed immune deregulation in sepsis.