Discussion
The serum metabolome of the WTC-exposed firefighters soon after 11 September 2001 is metabolically rich and empowered by a well-described cohort that strengthens associations in the investigation of WTC-LI and systemic pathology; however, these associations are limited in interpretation as our study’s samples are entirely collected within 200 days of WTC exposure. Although we can speculate on the metabolic environment prior to 11 September, inferences can only be made assuming that any individual component of MetSyn may not clinically change within such a short period of time. The state of health prior to exposure can only speculatively, but not definitively, be considered by review of the early blood draws post exposure.
Prior literature closely links concurrent metabolic disease and lung function. In contrast, we have previously shown that these associations of MetSyn and future lung function loss occur in a cohort with normal lung function prior to exposure. Thus, we suggest that the metabolic environment soon after exposure can trigger an inflammatory cascade that contributes to persistent and irreversible lung damage.
Although the metabolome reflects the complex interaction between many different parent cells, our data suggest that the most biologically active WTC-LI-associated metabolites reflect lipids and amino acids. Several of these have been identified as associated with broader categories of OAD, and, in line with our hypothesis, these metabolites clustered in patterns representative of their established mechanisms; however, other clusters contain novel metabolites. Overall, these metabolite clusters represent biologically plausible signalling cascades.
Our exclusively sphingolipid cluster, C4, highlighted the importance of these established inflammatory and metabolic mediators. Sphingosine 1-phosphate is a pleiotropic inflammatory mediator involved in immune cell trafficking and asthmatic hyper-reactivity induction.32 Elevation of sphingosine, a product of sphingosine 1-phosphate degeneration, in cases of WTC-LI may indicate decreased bioavailability of sphingosine 1-phosphate, leading to compromised vascular integrity.32 We saw a decrease in sphingosine 1-phosphate levels in cases of WTC-LI. Of interest is our finding that cases of WTC-LI also had significantly higher HR and SBP.
In contrast to the homogeneity of C4, the metabolites in C1 were far more diverse, including lipids from a variety of subpathways. The importance of lignoceroyl sphingomyelin (d18:1/24:0) proposes that ceramide, a building block of sphingosine and a metabolite of LDL and very-low-density lipoprotein, may be involved in WTC-LI pathogenesis. Sphingomyelins are involved in ceramide synthesis and metabolism. Ceramide is involved in the synthesis and degradation of ceramide 1-phosphate. Ceramide 1-phosphate triggers release of arachidonate (20:4n6), the third most important metabolite to class separation in this study and a key mediator in the inflammatory cyclooxygenase (COX) pathway.32 Arachidonate (20:4n6), being the primary substrate in eicosanoid production, is involved in immune cell recruitment.33 34 Additionally, eicosanoids are pivotal in inflammatory pathogenesis. Moreover, changes in ceramide 1-phosphate correlate directly with changes in sphingosine and sphingosine 1-phosphate, suggesting that ceramide 1-phosphate is a precursor of sphingosine 1-phosphate.32 Together, these data elucidate interconnected pathways involved in early response to WTC-exposure that differentiate well cases of WTC-LI and controls based on the metabolome years pre diagnosis.
Chronic inflammation has been identified as a probable mediator of loss of lung function after exposure to WTC-PM. We know that metabolic dysfunction is not only a predictor of WTC-LI but also causes chronic inflammation. Azelate, a saturated FA in C1, was important to class separation. Saturated FAs activate TLR-4 in a mechanistic link between systemic inflammation and obesity, a key component of metabolic dysfunction.32 This activation of TLR-4 induces ceramide biosynthetic genes required for TLR-4-dependent insulin resistance.32 Glucose, a marker of insulin resistance, is a predictor of WTC-LI in the entire WTC-exposed firefighter cohort.
Notably, several classes of FAs were revealed by this analysis. In C1, lignoceroyl sphingomyelin (d18:1/24:0) clustered with polyunsaturated fatty acids (PUFA)s including arachidonate (20:4n6), and FAs, monohydroxy, as well as phospholipids. By examination of figure 5, it becomes apparent that phospholipid and sphingolipid metabolism may be linked by FA metabolism. While little is known about the specific FAs and phospholipids identified in this analysis, we do know the inflammatory roles of the PUFA arachidonate (20:4n6). The metabolites correlated with arachidonate (20:4n6) may also be related to immune cell recruitment. Evidence of this relationship is the central cell membrane role of phospholipids and sphingolipids, that sphingosine 1-phosphate is a potent pro-inflammatory mediator and neutrophil chemotactant, and that elevated sphingolipids may be indicative of an increased, disproportionate response to perturbation. The information presented here warrants mechanistic research into this potential pathway as a therapeutic target to ameliorate derailed inflammatory responses.
Figure 5Pathway visualisation. Metabolic pathways of sphingolipids and phospholipids reveal pathways involving key metabolites, and that sphingolipid metabolism is linked with phospholipid metabolism by long-chain fatty acid metabolism. Node size correlates to fold change (red—up, blue—down, cases of World Trade Center Lung Injury/control).
As with previously discussed clusters, C2 contains some well-understood metabolites and some with unexplored roles. The most important metabolite to class separation is n2-acetyllysine, a lysine metabolite; lysine is an essential amino acid involved in the formation of protein and acetylation of lysine residues plays a key role in epigenetic modulation of inflammatory responses, creating bromodomain docking sites.35 In murine macrophages, pharmacological inhibition of a bromodomain attenuated nuclear factor-κB-directed production of nitric oxide and interleukin-6.35
Other essential amino acids presented in C2, namely several leucine, isoleucine and valine metabolites. These branched-chain amino acids (BCAAs) are hypothesised to be biologically active in respiratory illness due to their high anabolic capacity.36 Attenuated blood BCAA concentrations have been observed in patients with chronic obstructive pulmonary disease (COPD).37–39 Dietary supplementation with BCAAs ameliorates COPD-related respiratory muscle weakness and weight loss, and low serum BCAA concentration has been identified in COPD cohorts.37 40 41 This is the first data suggesting low serum BCAA concentration may predispose to PM-related lung disease in our WTC FDNY cohort.
One well-known metabolite found in C3, VMA, is indicative of epinephrine and norepinephrine release (both known stress hormones). Epinephrine is responsible for increased release of FAs and is a potential explanation of raised levels of FAs, such as propionylcarnitine and azelate (nonanedioate) (C3), in cases of WTC-LI. The pleiotropic inflammatory transcription factor c-Jun N-terminal kinases (JNK) is activated by exposure to free FAs and is involved in the signalling cascade of the receptor for advanced glycation end products (RAGE).42 We are currently investigating the roles of RAGE and JNK in WTC-LI.23 43 44
As hypothesised, this analysis can identify biologically plausible signalling pathways relevant to the development of WTC-LI. While established mediators such as sphingolipids were reviewed, several potentially novel mediators, including FAs, lysine, BCAAs (in their predispositional nature), glutamine and stress hormone metabolites, have been elucidated. Some of our findings, especially the COX mediators, sphingolipids and BCAAs, mimic similar findings in COPD and pollutant-exposed cohorts. The similarities observed between the metabolome of this select group of WTC-exposed firefighters and that of other cohorts with inflammatory disease support generalisability to OAD cohorts, and support validity of findings in this select group of WTC-exposed firefighters (additional discussion of metabolites identified in the clustering can be found in the supplement).
Our study has several limitations. The metabolome provides only a contemporaneous snapshot of small organic compounds present in serum. As such, our ability to single out only WTC-LI-pathogenesis-related metabolites is limited; however, we attempted to mitigate the prevalence of spurious discoveries through implementation of machine learning algorithms. Given the limited sample size of the current study, confounders have been relatively well controlled by selection from a homogeneous subject pool adhering to numerous criteria. Assuringly, the groups significantly differ in few metabolic biomarkers, only SBP, HR and BMI; other metabolic biomarkers and biomarkers of MetSyn are not significantly different between groups. Effects due to baseline BMI differences were mitigated by case definition based on percent predicted values. Additional unknown effects due to differences in exposure and duration of exposure are also minimised by the absence of significant differences in these variables between cases of WTC-LI and controls in subjects with metabolome assessed. Because distant history of smoking can predispose to non-resolving inflammation, our primary distinction is between ‘ever’ and ‘never’ smokers. Only never-smokers were included in this analysis; the metabolomic profile of ever-smoking WTC-exposed subjects remains unknown. Additionally, further profiling, including genomic, of this cohort must be performed to understand WTC-LI susceptibility.
It is important to note that any machine learning model is specific to its training data. However, the bootstrap-aggregating procedure followed by RF helps avoid overfitting that may typically be produced by a high variable-to-observation ratio. Simply put, the potential for false discovery is decreased by the high numbers of decision trees grown. Another aspect of this analysis is its strength in identifying intrinsic data patterns. While these patterns do not establish causal relationships, we assess model validity by out-of-bag classification procedures and support findings with pertinent literature. Due to the lack of an independent validation cohort, our findings have not been validated yet, but we acknowledge that this type of external validation would significantly strengthen the replicability and generalisability of our findings and methods.
Findings from this study have identified several metabolic pathways that may contribute to the pathogenesis of WTC-LI and similar lung injury-related conditions. The clusters differentiating WTC-exposed subjects with and without lung injury were primarily composed of lipid and protein metabolites, and therefore diet may be an important modifiable determinant of WTC-LI risk.
Dietary FAs may be able to alter the lipidomic signature that characterised the WTC-LI subjects. Specifically, a diet that is low in saturated fat intake and has a low omega-6-to-omega-3 FA ratio may help to correct high ceramide and the imbalance in phospholipid-derived long-chain PUFA metabolites (eg, arachidonate, docosahexaenoylcholine), which could have downstream beneficial effects on inflammatory and insulin signalling pathways.45–47
Diet may also help address the altered amino acid profile seen in WTC-LI subjects. In patients with advanced lung disease (COPD), BCAA supplements have been found to increase BCAA concentrations and improve health outcomes;38 48 however, in COPD, inadequate intake of energy, protein and BCAAs likely contributes importantly to the low BCAA concentrations. Although there is no data yet available on the dietary intakes of WTC-LI subjects, based on their sociodemographic background and prevalence of adiposity-based chronic disease and cardiometabolic risk factors, we assume that they tend to consume adequate energy and follow a typical Western diet, which, especially for males, tends to be high in protein.49
Importantly, it remains unclear to what extent, if any, diet contributes to the metabolomic signature of WTC-LI subjects, and what is the optimal diet for preventing, managing and treating WTC-LI and similar lung injuries. Additional research, including randomised clinical trials, is needed to examine the impact of diet and other modifiable lifestyle behaviours on WTC-LI progression.