NET Gain for Sepsis Research: A New Approach to Assess Neutrophil Function in Patients.

Neutrophil extracellular trap (NET) formation, a feature of neutrophils that involves extracellular release of a DNA web with attached histones and proteolytic enzymes, plays a critical role in the immune response to infection by trapping and preventing the dissemination of pathogens (1, 2). However, it is now well recognized that the release of NETs can also contribute to tissue injury in several pathologic conditions, including acute lung injury (3), thrombosis (4), and sepsis (5). Thanks to decades of research, we now have a deep understanding of the characteristics of NETs and subsequent effects on different organ systems in experimental models. However, there is still a gap in our knowledge regarding how we can use this information to improve clinical outcomes, especially during a critical illness. Although many assays have been developed to detect circulating NET components, including cell-free DNA, MPO (myeloperoxidase), and histones (6–8), these markers of already released NETs are not specific and may not always correlate with disease severity or outcomes. Furthermore, they are subject to degradation and clearance, which limits their potential to provide meaningful clinical information.

In an elegant study in this issue of the Journal, Abrams and colleagues (pp. 869–880) developed a novel assay to test the potential of plasma from patients with sepsis to stimulate healthy human neutrophils to release NETs (9). They then used this approach to prospectively test the association between plasma NET-forming capacity and clinical outcomes of ICU patients with sepsis. Using this new method, the authors discovered that the NET-forming capacity of plasma was independently associated with disease severity, the development of disseminated intravascular coagulation, organ injury, and mortality during critical illness. Importantly, the NET-forming capacity of plasma does not seem to be dependent on the neutrophil donor, and no plasma from healthy donors stimulated NETs. The assay procedure is relatively simple and straightforward, assuming that someone with the necessary expertise in neutrophil isolation, immunofluorescence, and microscopy, as well as fresh donor neutrophils, would be available when needed. An inability to meet these requirements could be a potential shortcoming of the assay. In addition, the time requirement of the assay (at least 4 h for stimulation of neutrophils, in addition to sample collection, neutrophil isolation, staining, and imaging) may not necessarily be an improvement from the predictive scoring mechanisms already in place, especially considering that the investigators found no significant improvement in the predictive capacity of this assay compared with Acute Physiology and Chronic Health Evaluation (APACHE) II or Sequential Organ Failure Assessment (SOFA). Nevertheless, this outside-the-box approach could provide additional insight into patient outcomes, as well as underlying pathological processes during a critical illness.

When they further investigated the NET-forming capacity of individual plasma samples, the authors identified IL-8 as a key component. In fact, blocking IL-8 using an antibody or receptor antagonist, or downstream mitogen-activated protein kinase signaling, removed the ability of patient plasma to induce NETs, although IL-8 levels alone could not predict disseminated intravascular coagulation or mortality. This finding is of particular interest because IL-8 receptor antagonists are currently being tested in clinical trials. Although these antagonists seem to be well tolerated in healthy humans (10) and have shown promise in murine models of sepsis (11), it remains to be determined how they will fare in critically ill patients. Of potential concern, IL-8 receptor antagonists can block the signaling of several ligands in multiple cell types, which could result in off-target effects during systemic inflammation. Nonetheless, this study has brought the importance of IL-8–induced NET formation during critical illness to light, and elicits further investigation into targeting this pathway therapeutically.

Looking beyond critical illness, this assay has the potential to be used for broader applications, as NETs are known to play a role in various diseases, including autoimmune disease (12), diabetes (13), atherosclerosis (14), and cancer (15). It would be interesting to determine whether this assay could assist in the early detection of some of these more chronic conditions or help improve outcomes. Expanding the possibilities of this approach even further, it would be worthwhile to consider whether additional functional measures (i.e., other functions of neutrophils, such as respiratory burst, or other cell types) could be tested using patient plasma samples to predict clinical outcomes. This unique way of thinking has the potential to be far-reaching.

The novel approach of using NETs as predictive biomarkers raises a few important questions. First, does NET formation cause worse outcomes during critical illness, or is it solely an indicator of enhanced inflammation? If NET formation contributes to disease progression, is it possible to intervene to inhibit or reverse the outcome? What other factors in plasma contribute to NET formation, and do these factors differ among patients or pathological stimuli? Given that therapies targeting cytokines and NETs have shown varied results, this assay could potentially help to inform the use of specific therapies based on a patient’s own plasma sample, resulting in a more personalized, targeted approach. The capacity of this approach to predict complications of disease might also improve prevention strategies for higher-risk patients. Importantly, this strategy has the potential to reveal new therapeutic targets using human clinical data, complementing studies of therapeutic targets discovered using preclinical animal models. In conclusion, although the novel approach proposed by Abrams and colleagues, which uses the NET-forming capacity of plasma to predict patient outcomes in critical illness, does not provide a direct measure of NETs or NET-induced injury, it is a great step toward understanding the role of NETs in sepsis and may help to inform potential therapies for critical illness and patient care in the ICU.