Plasma receptor interacting protein kinase-3 levels are associated with acute respiratory distress syndrome in sepsis and trauma: a cohort study

Critical illness precipitated by sepsis or trauma is characterized by a dysregulated immune response that may result in organ dysfunction and consequent death or long-term morbidity [1‐4]. Despite strides in early management strategies [5, 6], organ dysfunction syndromes such as the acute respiratory distress syndrome (ARDS) often complicate the early clinical trajectory of these patients and pose a significant barrier to improving outcomes. Disappointing results of trials for immune-targeted and other pharmacologic therapies have prompted interest in better understanding clinically relevant molecular pathways in ARDS through human translational study [7].

Necroptosis, a caspase-independent form of regulated cellular necrosis mediated by receptor interacting serine/threonine-protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) [8, 9], has recently been implicated as a key cell death modality in tissue and animal models of organ injury [10‐13]. Studies by our group and others have shown that lipopolysaccharide- and red blood cell-induced lung injury is attenuated by inhibition of necroptosis [10, 13]. This form of cell death could have particular relevance to ARDS. Unlike apoptosis, necroptosis results in plasma membrane rupture and release of damage-associated molecular patterns (DAMPs) that drive tissue injury; distinct from conventional descriptions of necrosis, necroptosis induction and execution is highly regulated [9, 14‐18]. Therefore, necroptosis may represent a novel potential therapeutic target for prevention or treatment of acute organ dysfunction.

Few studies to date have investigated necroptosis activation in critically ill patients. After finding that RIPK3 was released by cultured lung endothelial cells undergoing red blood cell-induced necroptosis, we reported an association of plasma RIPK3 with RBC transfusions and mortality in 37 sepsis patients [13]. We subsequently showed that plasma RIPK3 measured 48 h after presentation was associated with RBC transfusions and acute kidney injury (AKI) in 80 trauma patients [19]. Recent reports in medical ICU populations described higher plasma RIPK3 levels in mechanically ventilated and non-surviving patients [20, 21]. These studies provided limited information on the relationship of plasma RIPK3 with ARDS. Further, it remains unclear whether plasma RIPK3 is an accurate marker of expression of RIPK3 or execution of necroptosis in injured organs.

We sought to address these knowledge gaps using cohorts of critically ill sepsis and trauma patients as well as in vivo animal experiments. We hypothesized that plasma RIPK3 levels would be associated with ARDS in both sepsis and trauma populations, independent of patient-level characteristics. We also hypothesized that in a mouse model of systemic inflammation, plasma RIPK3 levels would correlate with lung tissue expression of the necroptosis mediators RIPK3 and MLKL. Secondarily, we sought to build on our prior reports [13, 19] by determining the association of plasma RIPK3 levels with AKI and mortality in larger cohorts of sepsis and trauma patients and by determining patient characteristics associated with plasma RIPK3. We studied these populations based on two considerations: first, sepsis and trauma are common critical illness syndromes with high rates of organ dysfunction; and second, sepsis and trauma are both characterized by a dysregulated immune response, ischemia-reperfusion injury, and treatment with blood product transfusions, all relevant for translation of existing pre-clinical studies of necroptosis [12, 13, 16, 18].

Detailed descriptions of human cohort and experimental animal studies, including the STROBE checklist, are in Additional file 1. Key methods are summarized here.

In this study, we demonstrated that the change in plasma RIPK3 concentration from presentation to 48 h was independently associated with ARDS in two major at-risk populations, sepsis and trauma and that both lung RIPK3 expression and plasma RIPK3 concentrations rose significantly in mice treated with systemic LPS alone or when given with an inhibitor of apoptosis. Despite a wealth of recent animal and tissue studies of necroptosis showing its potential relevance to multiple human syndromes including ARDS [9, 10, 15, 16], data in human populations remain limited [13, 19‐21, 37]. Our study provides the largest analysis to date of RIPK3 in ARDS, takes steps toward understanding the role of plasma RIPK3 as a marker of lung injury, and strengthens prior findings that plasma RIPK3 was associated with AKI and mortality. In the context of existing preclinical data [10, 13], these findings collectively suggest that necroptosis and other RIPK3-regulated pathways may be mechanistically important in ARDS and other acute organ dysfunction syndromes.

Since its description in 2009 [38], RIPK3-mediated necroptosis has emerged as a key mechanism in preclinical models of acute lung and renal injury [10, 13, 15]. The characteristic release of tissue-injurious DAMPs during necroptosis makes it of great interest as a driver, and therefore potential therapeutic target, of acute organ injury [13, 34, 39‐41]. In sepsis patients, Davenport et al. identified increased gene expression of RIPK3 and MLKL in circulating leukocytes as part of a molecular response subtype characterized by a two- to threefold mortality increase [42]. There are now reports of plasma RIPK3 associated with mortality [13, 19, 21], AKI [19, 37], and mechanical ventilation [20]. Studies on RIPK3 in ARDS, however, are limited, the largest being a subgroup analysis that included 24 patients with ARDS [20]. In cohorts with over three times that number of ARDS cases, we now show a convincing association of plasma RIPK3 with ARDS independent of relevant confounders. We also add novel findings about the time course of the RIPK3-ARDS association. While the rise in plasma RIPK3 over the first 48 h was clearly able to distinguish ARDS from non-ARDS cases, there was no signal that RIPK3 on presentation to the ED or trauma bay could predict ARDS, with similar findings for AKI and mortality. These results have potential implications for clinical utility: by 48 h, when ARDS is often already manifest, this biomarker may be most helpful to identify a subgroup with RIPK3 activation for possible targeted treatment. In fact, RIPK3 inhibitors have already shown protection against tissue injury in preclinical studies [35, 43], and efforts to translate these findings into effective therapies may be aided by understanding patient groups most likely to respond. For consideration of RIPK3 as an ARDS prediction or prevention tool, however, studies of serial early measurements would be needed to determine how rapidly after presentation the RIPK3-ARDS association becomes evident.

For any such pathway-targeted applications, though, it is important to know to what degree plasma RIPK3 reflects underlying lung injury. We previously demonstrated that human vascular endothelial cells undergoing necroptosis release RIPK3 [13], but it remained unclear if in vivo circulating RIPK3 levels reflected RIPK3 expression in lung tissue. Our current finding that murine lung and plasma RIPK3 rose substantially and concomitantly in response to systemic LPS and LPS-ZVAD suggests that the RIPK3-ARDS association in sepsis and trauma patients could reflect increased expression and release of RIPK3 from injured lung tissue, injury that is not explained by apoptosis. While necroptotic cell death is one explanation for these findings, other RIPK3-dependent pathways may be involved: Lawlor et al. have shown that RIPK3 can promote inflammasome activation independent of MLKL and necroptosis [44]. Notably, we found that pMLKL, an intracellular mediator of necroptosis downstream of RIPK3, did not increase after LPS or LPS-ZVAD. These findings are also consistent with those of Siempos et al. in which RIPK3-deficient mice were protected from ventilator-induced lung injury while MLKL-deficient mice were not [20]. Further studies, potentially including testing of plasma pMLKL and other key cell death pathway mediators that may be involved in the RIPK3-ARDS association, are important if therapies targeting programmed necrosis are to be considered for acute lung injury.

Our study expands on smaller reports that plasma RIPK3 is associated with AKI in sepsis and trauma patients [19, 37]. We now demonstrate a RIPK3-AKI association robust to adjustment for relevant confounders and independent of ARDS. This lends further clinical relevance to multiple preclinical studies showing the importance of RIPK3 and necroptosis in acute renal injury [8, 12, 15, 37, 45]. It is highly plausible that programmed necrosis in the kidneys, as well as the lungs and other organs, results in elevated circulating RIPK3 levels in sepsis and trauma patients. It is also possible that circulating RIPK3 is itself a causal factor in multiple organ dysfunction, similar to well-established DAMPs like cell-free DNA. In either case, RIPK3 may identify a process of necroinflammation in which the release of a diverse groups of DAMPs by necrotic cells serve to propagate and sustain the inflammatory response [46]. For example, we previously found that the DAMP high-mobility group box 1 protein (HMGB1) released following transfusion-induced necroptosis primes mice to subsequent lung injury [13], and others showed that cigarette smoke-induced necroptosis and DAMP release increase airway inflammation [47]. The complexity of how necroptosis and other regulated necrosis pathways result in and interact with release of myriad DAMPs in vivo to promote inflammation, tissue injury, and multiple organ dysfunction remains inadequately understood. Clinically relevant animal models of sepsis and trauma may be best suited to clarify these knowledge gaps.

There are limited existing data on patient characteristics associated with RIPK3 levels [19, 20]. We found that severity of illness measures tracked with plasma ΔRIPK3 in both sepsis and trauma. In MESSI but not PETROS, this was true for presentation RIPK3 as well, possibly reflecting a greater delay from initial insult to ED presentation in sepsis patients, allowing more time for circulating RIPK3 to rise. In PETROS, variables including race, penetrating trauma, shock, crystalloid volume, and blood product transfusions were all significantly associated with ΔRIPK3. We have previously shown that RBCs can induce RIPK3 release from lung endothelial cells [13]. Transfused patients are also at increased risk of ARDS [30]. If RIPK3 proves to be a causal link, targeting RIPK3 pathways could be considered to reduce ARDS rates among the substantial number of trauma patients requiring transfusions.

Our study has several limitations. First, we did not have plasma samples at time points between presentation and 48 h. The kinetics of plasma RIPK3 in the early hours of sepsis and trauma remain unclear, as does the ability of RIPK3 at such time points to predict subsequent ARDS. While we showed in trauma patients that ΔRIPK3 was associated with ARDS developing after 48 h, future studies with serial RIPK3 measurements may provide more granular detail of the time-varying relationship of RIPK3 and ARDS during early critical illness to allow for more robust causal inference. Second, while our study is the largest analysis to date of RIPK3 and ARDS, our cohort sizes did not allow adjustment for all possible confounders without potentially overfitting the multivariable models. Third, while the significant rise in murine plasma RIPK3 concentrations in response to LPS and LPS-ZVAD mirrored that seen in lung tissue, larger studies would be needed to firmly establish a tight correlation of plasma and lung RIPK3 concentrations. Studies testing RIPK3 expression in other organs may help to determine whether plasma RIPK3 also reflects extra-pulmonary tissue expression. Fourth, the specificity of RIPK3 as a marker of necroptosis in the lung or other organs remains unknown in human populations. Future studies could include using tissue or fluid obtained from affected organs, such as bronchoalveolar lavage fluid or urine, to validate plasma RIPK3 as a non-invasive marker of necroptosis or other RIPK3-related pathways. Finally, whether clinically available tests reflecting cell death such as lactate dehydrogenase strongly correlate with RIPK3, and therefore could be used as surrogates, remains unclear but could be tested in future studies.

We demonstrated a significant association of plasma ΔRIPK3 with ARDS in critically ill sepsis and trauma patients and showed that both lung and plasma RIPK3 increased rapidly in mice injected with LPS-ZVAD. These findings extend prior preclinical studies and suggest that necroptosis and other RIPK3-dependent processes may be important mechanisms underlying ARDS in these two at-risk populations. Ongoing studies of this marker may prove useful in identifying novel molecular pathways to target for ARDS prevention and treatment.