A hypolipoprotein sepsis phenotype indicates reduced lipoprotein antioxidant capacity, increased endothelial dysfunction and organ failure, and worse clinical outcomes

Sepsis is a prevalent, morbid and costly condition. In 2017, there were an estimated 48.9 million cases worldwide, with 11 million sepsis-related deaths accounting for 19.7% of all global deaths [1]. In the United States, there are approximately 1.7 million annual sepsis cases with an overall mortality of nearly 20% [2‐4]. {Formatting Citation} Sepsis patients who survive hospital admission frequently experience poor quality-of-life and increased long-term mortality [5, 6]. Nearly one-third of sepsis patients develop a state of chronic critical illness (CCI), defined as intensive care unit (ICU) stay ≥ 14 days with continued organ dysfunction. They are characterized by lean muscle wasting, cachexia, and a 1-year mortality approaching 50% [6‐8]. CCI patients require high levels of post-discharge care, and most of them are discharged to skilled nursing facilities or long-term acute care hospitals. Studying chronic drivers of inflammation and dysregulated immunity is key to understanding these poor outcomes.

Lipids and lipoproteins play an important role in sepsis pathogenesis. Studies have shown that high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) are protective in sepsis [9, 10]. First, HDL has antioxidant and anti-inflammatory functions mediated by paraoxonase 1 (PON-1, an HDL-associated esterase that protects lipids from oxidation) and apolipoprotein A-I (Apo A-I) [11, 12]. ApoA-I has been shown to suppress both neutrophil and monocyte activation [13‐15]. HDL has also been shown to downregulate inflammatory pathways in sepsis via the transcriptional regulator ATF3 [16]. Second, both HDL-C (via ApoA-I) and LDL-C bind and clear bacterial toxins during sepsis [11, 17‐23]. Third, HDL has direct endothelial protective effects during sepsis [24, 25].

Changes in lipid and lipoprotein levels, structure, and function may affect their ability to protect against sepsis. Barlage and colleagues demonstrated several lipoprotein differences between survivors and non-survivors in sepsis including that non-survivors had lower levels of total cholesterol, LDL-C, HDL-C, Apo-A-I, and Apo-B at all time points (day 1, 4, and 11) [26]. ApoA-I levels were an independent predictor of mortality in their cohort after adjusting for other factors, and HDL-C and ApoA-I levels were depressed and stayed the same or decreased in non-survivors, compared with survivors in whom levels gradually increased over the course of their hospitalization. Barlage and colleagues also found that HDL mediators of monocyte activity such as Apo-CI (which stimulates the response to LPS by macrophages), were reduced in non-survivors. More recently, others have shown a close clinical correlation between early HDL-C levels in sepsis and severity of organ failure or survival [27, 28], that HDL-C levels correlate inversely with pro-inflammatory cytokines including TNF-α, IL-6, IL-10 [28], and that cholesterol levels three days after sepsis admission do not return to pre-sepsis baseline levels [29]. Reasons for reduced HDL-C levels in sepsis may include decreased hepatic synthesis, consumption as part of the systemic response to infection, or increased clearance via scavenger receptor B1 [30].

Genetic studies designed to evaluate for causality identified an important link between genetically determined HDL-C levels and decreased risk of hospitalizations for infectious disease, lower odds of outpatient antibiotic usage, and reduced risk of mortality from sepsis, but not for LDL-C, or triglycerides [31, 32]. A rare missense variant in the cholesteryl ester transfer protein (CETP) gene was also recently identified and linked with significant reductions in HDL-C. Carriers of this allele had decreased survival, more severe organ failure and reduced 28-day survival.

We have shown that HDL becomes pro-inflammatory and dysfunctional (Dys-HDL) in sepsis, and that Dys-HDL correlates with and predicts organ failure in sepsis [33‐35]. Changes in HDL composition may partly explain the presence of Dys-HDL in sepsis. Some of these changes are due to increased levels of acute phase proteins such as serum amyloid A (SAA) which can displace ApoA-I and contribute to HDL’s pro-inflammatory state and may also contribute to reduced HDL-C levels [36, 37]. Also, HDL particle size is increased in septic patients compared to similarly ill non-septic ICU patients [38]. Larger HDL particles are less functional, and less mature than smaller, more dense HDL.

Given the complexity of lipid and lipoprotein metabolism in sepsis but strong associations with important clinical outcomes, our objectives were two-fold: (1) to confirm the predictive ability of lipids and lipoproteins for the clinically important outcomes of rapid recovery, CCI, and early death previously established by our group, and (2) determine the contribution of lipids and lipoproteins to sepsis heterogeneity. We hypothesized that lipids and lipoproteins would be predictive of poor outcomes, a composite of CCI and early death, from sepsis. To advance our understanding of complex metabolic, endothelial, and inflammatory relationships beyond predicting outcomes, we used use unsupervised clustering methods to phenotype sepsis patients using lipid and lipoprotein data.

There were 172 septic patients enrolled in the study with at least one of two lipid measures upon enrollment (HII or PON-1), in addition to a lipid panel for cholesterol levels (Fig. 1—Enrollment Flow Diagram). Table 1 displays demographics, disease severity and comorbidity measures. The median age was 61 years (IQR 51–70); most patients were male, and most self-reported as White, followed by Black. After adjusting for multiple comparisons, comorbidity burden was similar across groups by Charlson Score. Enrollment SOFA and Apache II scores were significantly worse in early death and CCI patients. Early death patients had the highest SOFA scores at presentation (10, IQR 9–13), followed by CCI (7, IQR 5–10), and rapid recovery (4, IQR 2–6) (Table 1). Most patients (67%, 114/172) had rapid recovery, while 23% (41/172) developed CCI, and 10% (17/172) had early death.

Urinary tract infections were the most common source of sepsis, followed by intra-abdominal and pulmonary infections (Additional file 8: Table 1). Urinary tract infections were most frequent in rapid recovery patients while pulmonary infections were most common among CCI patients. There were no significant differences in intravenous fluid administration between groups, though there was a trend towards more fluids in early death patients at 24 h (Additional file 9: Table 2). There were also no significant differences in time to antibiotic administration. Mechanical ventilation duration and vasopressor usage were highest among early death and CCI patients compared to rapid recovery. Early death and CCI patients were more likely to be mechanically ventilated at enrollment. Poor disposition rates were higher for CCI than rapid recovery, and all mortality end points were higher for CCI (39% dead at 1 year). One-fourth (43/172) of all patients had died by one year.

In this study, we demonstrated the predictive ability of lipids and lipoproteins for important clinical outcomes as previously defined by our group. We demonstrated that ApoA-I and LDL-C levels were predictive of the development of CCI or early death, in addition to mechanical ventilation, vasopressor use, and Charlson Comorbidity Score. Using unsupervised clustering, we were then able to phenotype sepsis patients into Hypolipoprotein and Normolipoprotein phenotypes, with the Hypolipoprotein phenotype being characterized by lower lipoprotein levels (lower HDL-C and ApoA-I), and increased endothelial dysfunction (ICAM-1) as well as higher SOFA scores. These findings were validated in an independent replication cohort of ED patients with sepsis or septic shock.

Our finding that ApoA-I and LDL-C, in addition to mechanical ventilation use, vasopressor use, and Charlson Score being predictive of CCI and early death, is not entirely new. Pavlou et al. as well as Zou et al. have previously shown that decreasing ApoA-I levels are associated with poor outcomes in sepsis [51, 52]. And others have shown the prognostic ability of LDL-C [53]. In this study, we used a diverse cohort of sepsis patients that contained both community-acquired and hospital-acquired sepsis from two settings at two hospitals.

Using our unsupervised clustering approach, we identified, and verified in an independent replication cohort, a Hypolipoprotein phenotype of sepsis that is represented by lower levels of key lipoproteins, and is associated with increased endothelial dysfunction, organ failure, and poorer outcomes. In the derivation cohort, the Hypolipoprotein phenotype had significantly lower total cholesterol, HDL-C, LDL-C, PON-1, ApoA-I, and higher ICAM-1, indicative of more severe endothelial dysfunction. The low levels of PON-1 and ApoA-I also indicate the reduced antioxidant and anti-inflammatory capacity of patients in the Hypolipoprotein phenotype. Notably, the derivation cohort had higher cardiovascular SOFA scores, which contributed to overall higher severity of organ failure. Similarly, the replication cohort also had lower levels of HDL-C, ApoA-I, and higher ICAM-1. Additionally, the Hypolipoprotein phenotype in the replication cohort had significantly higher hepatic SOFA scores compared to the Normolipoprotein phenotype. One reason for the observed difference in hepatic dysfunction between derivation and replication cohorts is that severe hepatic failure was an exclusion for the derivation cohort, but not for the replication cohort. The exclusion of patients with severe hepatic failure and lipoprotein metabolic disorders, may have resulted in the exclusion of patients prone to low or abnormal lipoprotein levels.

Though our sample size may have limited our ability to detect more nuanced phenotypes in these analyses, it suggests that lipoprotein pathobiology may contribute to sepsis outcome heterogeneity. In our independent replication cohort, we were able to validate our two phenotypes and their associated outcomes. Further, combined with our multivariable regression results, our clustering results provide additional evidence supporting the relationship between lipoprotein dysregulation and morbid outcomes from sepsis.

Our biomarker correlation matrices provide insight into the biological underpinnings of aforementioned findings. The correlation matrices demonstrated strong, inverse relationships between HDL-C, ApoA-I, LDL-C, total cholesterol, and PON-1 with endothelial dysfunction markers but a positive correlation with triglycerides. This may indicate increased endothelial dysfunction as lipid levels and antioxidant capacity decrease in sepsis due to degradation, consumption, or transfer of lipids or lipoproteins to other moieties. This finding is supported by prior studies that have shown that HDL-C has potent endothelial protective effects, and may reflect loss of HDL’s endothelial protective effects in sepsis [24, 25, 54, 55]. HII was also shown to be positively correlated with MPO and MCP-1, both of which are measures of oxidation and inflammation, and likely reflects increased oxidized lipids in sepsis [56, 57]. In addition, the inverse association of nearly all the cholesterol measures with the inflammatory markers IL-6 and IL-8, as well as the correlation between HDL-C and ApoA-I with IL-10, GM-CSF, MPO, and TNFα is notable, as it demonstrates that HDL-C and ApoA-I may be more affected by the oxidizing effects of MPO and other inflammatory markers (TNF, IL-10) than other lipids. Finally, in addition to the reduction in cholesterol levels that has been previously described [53, 58, 59], our study demonstrated that PON-1 and ApoA-I levels were significantly higher in patients who rapidly recovered compared to CCI or early death, indicating reduced lipoprotein antioxidant capacity in patients with poor outcomes. Unsurprisingly, measures of endothelial dysfunction (E-selectin and ICAM-1) and inflammatory markers (IL-8, IL-6, and IP-10) also demonstrated significant differences between rapid recovery, CCI, and early death groups. IL-6, which stimulates acute phase proteins including serum amyloid A, and has been previously demonstrated to displace up to 45% of ApoA-I from circulating HDL-C in sepsis was also elevated, and may contribute to Dys-HDL pathogenesis [37, 60].

In summary, the results of our unsupervised clustering analysis and biomarker profiling suggest that reduced levels of protective lipids (HDL-C, ApoA-I, and LDL-C) are associated with increased endothelial dysfunction (E-selectin, ICAM-1) and worse outcomes. The reduction in these protective lipoproteins is also associated with increased inflammatory markers, HDL oxidation (elevated HII), and reduced PON-1 activity.

Here, we demonstrated that a Hypolipoprotein phenotype of sepsis, represented by lower levels of HDL-C, ApoA-I, and reduced PON-1 activity is associated with increased endothelial dysfunction, organ failure, and poorer outcomes compared to a Normolipoprotein phenotype. We showed that HDL-C and ApoA-I specifically have important associations with markers of endothelial dysfunction and have strong discriminative ability for poor versus favorable outcomes in a diverse sepsis cohort using classic and machine learning analytic approaches. Future studies should focus on the mechanisms by which lipids and lipoproteins influence sepsis heterogeneity and outcomes.