The Chlorinated Lipidome Originating from Myeloperoxidase-Derived HOCl Targeting Plasmalogens: Metabolism, Clearance, and Biological Properties

Abstract

Myeloperoxidase produces the two-electron oxidant HOCl, which targets plasmalogen phospholipids liberating 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde has four known fates: 1) oxidation to 2-chlorofatty acid; 2) reduction to 2-chlorofatty alcohol; 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon. 2-Chlorofatty acid does not undergo conventional fatty acid β-oxidation due to the presence of the α-chlorinated carbon; however, 2-chlorofatty acid does undergo sequential ω-oxidation and β-oxidation from the ω-end, ultimately resulting in 2-chloroadipic acid urinary excretion. Recent studies have demonstrated that 2-chlorofatty acid clearance is increased by treatment with the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-chlorofatty acid has been shown to be a PPAR-α agonist, and thus accelerates its own clearance. The roles of 2-chlorofatty aldehyde and 2-chlorofatty acid on leukocyte and endothelial function have been explored by several groups, suggesting that chlorinated lipids induce endothelial cell dysfunction, neutrophil chemotaxis, monocyte apoptosis, and alterations in vascular tone. Thus, the chlorinated lipidome, produced in response to leukocyte activation, is a potential biomarker and therapeutic target to modulate host response in inflammatory diseases.

Graphical Abstract

Introduction: Peroxidases and Plasmalogens

Peroxidases, important enzymes expressed mainly in leukocytes, play important roles in immune responses and have been implicated as mediators in many disease states. Myeloperoxidase, eosinophil peroxidase, and thyroid peroxidase are capable of producing oxidants that target both microbe and host molecules. In the 1990s, Boeynaems and co-workers [1, 2] reported that reactive iodinating species produced by thyroid peroxidase target the vinyl ether bond of plasmalogens, liberating 2-iodohexadecanal. These seminal findings, coupled with the evolving interest in the role of myeloperoxidase in cardiovascular disease, provided the rationale for the Ford group to discover myeloperoxidase-derived chlorinated plasmalogen oxidation products [3]. Furthermore, this research led to the discovery of brominated plasmalogen oxidation products under conditions of hypobromous acid (HOBr) generation by either myeloperoxidase or eosinophil peroxidase [4, 5].

During activation, neutrophils degranulate and release granular proteins, including myeloperoxidase (MPO), the major component of primary granules [6]. MPO catalyzes the conversion of hydrogen peroxide (H₂O₂) and chloride to hypochlorous acid (HOCl), which has significant antimicrobial activity during infection [7–11]. MPO-deficient human neutrophils show an enhanced phagocytic capability, accompanied by a slower bactericidal rate compared to healthy human neutrophils [12, 13]. MPO-deficient patients display an enhanced susceptibility to various infections, in particular to Candida yeast [14]. Additional data, however, indicate compensatory mechanisms can function in the absence of MPO [15]. Despite the powerful antimicrobial role of MPO, MPO-deficient humans do not display significantly higher susceptibility to microbial infections [16].

MPO activity can have detrimental effects under pathological conditions. Numerous studies have reported the contribution of MPO to the development of cardiovascular disease and other inflammatory conditions [17–19]. The presence of active MPO in atherosclerotic arteries was first shown by Daugherty et al. [20], and the role of MPO-derived oxidation products has received considerable attention [21–27]. Studies in the Kettle laboratory investigated different physiological targets of MPO [28]. The MPO halogenation step can utilize chloride, bromide, or thiocyanate, leading to the production of the respective hypohalous acids. Although thiocyanate was shown to be the preferential substrate for MPO, hypothiocyanous acid is less reactive than HOCl with host and microbe biomolecules. In fact, there is no evidence to indicate that hypothiocyanous acid promotes halogenation of lipids [28]. This observation is in agreement with studies carried out in the Ford laboratory, which failed to detect hypothiocyanite-mediated plasmalogen oxidation products.

Albert et al. demonstrated in 2001 that MPO-derived HOCl targets the vinyl ether bond of plasmalogens [3]. Plasmalogens are a class of membrane phospholipids that contain a vinyl ether bond (masked aldehyde) at the sn-1 position. These phospholipids are abundant in leukocytes, endothelial cells, smooth muscle cells, cardiac muscle, and neurons [29–33]. Both Gross et al. [31] and Post et al. [34] showed plasmalogen enrichment in plasma membranes, while others revealed enrichment in lipid rafts and endoplasmic reticulum [35, 36]. Although the liver does not contain significant levels of plasmalogens, Vance et al. revealed that hepatocytes can synthesize plasmalogens that are exported with very low density lipoprotein [37]. Despite the abundance of plasmalogens in mammalian cells and tissues, their biological role is not completely understood.

Multiple roles for plasmalogens have been proposed including their unique biophysical packing into membranes, their enrichment with arachidonic acid, and the targeting of vinyl ether bonds by oxidants. Plasmalogens, most notably choline-containing plasmalogen, pack into membranes differently when compared to diacyl phosphatidylcholine, with the aliphatic chains more tightly packed and the polar head groups extending perpendicular to the plane of the membrane [38–40]. Hale and Ford subsequently proposed that this configuration supported the functional activity of the cardiac sarcolemmal sodium-calcium exchanger with plasmenylcholine potentially serving as a boundary lipid [41, 42]. Plasmalogens are also a storage depot for arachidonic acid, which can be liberated by phospholipases and used to produce oxylipids, such as prostacyclin and thromboxanes [30, 43]. The last proposed role for plasmalogens (i.e., targeting of the exposed vinyl ether bond by oxidants) is a key component of this review since chlorinated lipids are produced by the oxidant, HOCl, targeting plasmalogens.

Historically, targeting the vinyl ether bond of plasmalogens by free radicals, such as peroxyl radicals, metal ions, and singlet oxygen, was thought to be a cytoprotective sink [44]. More specifically, Zoeller et al. suggested the vinyl ether bond in ethanolamine plasmalogen is particularly sensitive to oxidation [45]. Vance also suggested vinyl ether oxidation of plasmalogens may protect against lipoprotein oxidation, reducing uptake by macrophages and foam cell formation [46]. However, although once believed to be an anti-oxidant, plasmalogens are also the precursors of the chlorinated lipidome, a toxic class of oxidized lipids.

Production and Metabolism of 2-Chlorofatty Aldehyde

HOCl oxidation of the sn-1 vinyl ether bond of plasmalogen phospholipids liberates the first member of the chlorinated lipidome, 2-chlorofatty aldehyde (2-ClFALD), as well as the corresponding lysophospholipid [3]. Using negative ion chemical ionization detection and gas chromatography-mass spectrometry methods, coupled with the use of a synthetic deuterated internal standard, Thukkani et al. developed a sensitive method to quantify 2-ClFALD at levels in the femtomole range [47]. As shown in Figure 1, 2-ClFALD has four known fates: 1) oxidation to 2-chlorofatty acid (2-ClFA) and subsequent incorporation into esterified complex lipids; 2) reduction to 2-chlorofatty alcohol (2-ClFOH); 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon.

Figure 1.

Summary of the production and four metabolic fates of 2-chlorofatty aldehyde (2-ClFALD), the first member of the chlorinated lipidome. 2-ClFA, 2-chlorofatty acid; 2-ClFOH, 2-chlorofatty alcohol.

Figure 2.

Summary of the clearance of 2-chlorofatty acid in the liver. 2-ClFA, 2-chlorofatty acid; 2-ClDCA, 2-chlorodicarboxylic acid; 2-ClAdA, 2-chloroadipic acid.

The metabolism of chlorinated lipids was first demonstrated using radiolabeled and stable isotope-labeled 2-ClFALD in neutrophils and endothelial cells [48]. 2-ClFALD is readily oxidized to the metabolite, 2-ClFA, by intracellular fatty aldehyde dehydrogenase [48–50]. Another metabolite of 2-ClFALD is formed by reduction to 2-ClFOH [50]. These biosynthesized metabolites are released into the extracellular space rather than accumulate within the cell. Although 2-ClFOH is the most abundant metabolite, 2-ClFA and 2-ClFALD elicit multiple detrimental biological properties, which are highlighted in this review. It should be noted that the potential biological roles of 2-ClFOH remain to be explored.

Initial studies quantifying the amount of cellular 2-ClFALD and its lipid metabolites did not account for the total amount of exogenous 2-ClFALD administered to cells [48, 51]. Thus, the ability of 2-ClFALD to react with other cellular components, such as proteins, was examined. 2-ClFALD has since been shown to react with amino groups and proteins [51–53]. Formation of 2-ClFALD adducts with lysine and ethanolamine glycerophospholipids reveals the reactivity of 2-ClFALD with primary amines via Schiff base reaction, accompanied by loss of chloride, as indicated by stabilization with sodium cyanoborohydride [52]. Furthermore, glutathione acts as a nucleophile by attacking the α-chlorinated carbon of 2-ClFALD, yielding a GSH-aldehyde adduct [53]. Duerr et al. detected these adducts in cell culture, activated primary human neutrophils, and an in vivo model of inflammatory arthritis [53]. Nusshold and co-workers also identified multiple proteins that 2-ClFALD reacts with, but the mechanism of binding is currently unknown [51]. In these studies, samples were reduced with sodium borohydride, indicating the proposed modifications may be partially mediated by the formation of Schiff base adducts.

Chlorinated lipids have been identified in multiple biological systems and linked to several inflammatory diseases. Primary human neutrophils and monocytes produce both 2-ClFALD and 2-ClFA after activation [47, 54]. In these activated leukocytes, 2-ClFALD appears first following activation. After a peak at 30 minutes, 2-ClFALD levels decrease due to its metabolism and reactivity with amines, thiols and proteins. Both 2-ClFA and 2-ClFOH production increase over time. In fact, 2-ClFA is the major stable metabolite of the chlorinated lipidome detected in biological systems. 2-ClFA levels approach 20 μM in activated neutrophils and monocytes, which may be suggestive of the concentration at the site of chlorinated lipid production at the leukocyte-endothelial interface [50, 55].

It is important to note that 2-ClFA present in vivo is found both as a free fatty acid as well as in esterified lipid pools. The detected concentration of 2-ClFA in the circulation during inflammatory conditions ranges from low nanomolar levels to nearly 100 nM. The nanomolar ranges in the systemic blood likely reflect much higher levels of 2-ClFA found at sites of inflammation where neutrophils are activated. In a rat model of lipopolysaccharide (LPS)-mediated endotoxemia, plasma 2-ClFA levels tripled compared to naïve rats [49]. Additionally, mice exposed to a sub-lethal dose of chlorine gas have plasma 2-ClFA levels greater than 100 nM [56]. For both LPS-treated rats and mice exposed to chlorine gas, a substantial level of plasma 2-ClFA is in the esterified pool [49, 56]. Furthermore, exogenous treatment (i.p. injection of rats) with 2-ClFA results in its appearance in the plasma as both free and esterified forms, with the esterified form having a longer half-life [49]. This data suggest that 2-ClFA may associate with complex lipid pools, such as cholesteryl esters and triacylglycerols, but these esterified forms of 2-ClFA remain to be elucidated. As discussed in further detail in following sections, the chlorinated lipidome is produced in vivo under inflammatory conditions and may be an important biomarker in inflammatory pathologies involving MPO activation, such as atherosclerosis and sepsis.

Clearance of Chlorinated Lipids

The clearance pathway of the stable metabolite of the chlorinated lipidome, 2-ClFA, has been elucidated. The presence of chlorine at the α-carbon prevents 2-ClFA from undergoing fatty acid β-oxidation. As with conventional fatty acids that are not β-oxidized by fatty acyl dehydrogenase, 2-ClFA can be ω-oxidized and subsequently β-oxidized from the ω-end [49]. ω-Oxidation is initiated by ω-hydroxylation of the ω-methyl group. The resultant alcohol is subsequently oxidized to a carboxylic acid yielding the end-product, 2-chlorodicarboxylic acid. The newly formed dicarboxylic acid is shortened via β-oxidation from the unblocked ω-end (no chlorine modifications at the ω-end) and excreted as a 6-carbon chlorinated dicarboxylic acid, 2-chloroadipic acid (2-ClDCA), in the urine. Mass spectrometric analyses of HepG2 cells and media identified three chlorinated dicarboxylic acid products in this process: 2-chlorohexadecane-(1,16)-dioic acid, 2-chlorotetradecane-(1,14)-dioic acid, and 2-chloroadipic acid [49]. Stable isotope-labeled 2-ClFA was used to confirm that these compounds were, in fact, ω-oxidation catabolites of 2-ClFA. 2-Chloroadipic acid was found elevated in the urine of LPS-treated rats [49].

Recent studies demonstrated that 2-ClFA clearance is accelerated by treatment with the peroxisome proliferator-activated receptor (PPAR)-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation [57]. While primary hepatocytes isolated from wild-type mice treated with WY14643 display accelerated 2-ClFA ω-oxidation, those from PPAR-α^−/− mice did not respond to treatment with WY14643 to accelerate 2-ClFA ω-oxidation. To assess the effects of PPAR-α activity in vivo, wild-type and PPAR-α^−/− mice were injected with stable-isotope labeled 2-ClFA. Mass spectrometric analyses demonstrated that PPAR-α^−/− mice had higher levels of 2-ClFA in the plasma and lower levels of 2-chloroadipic acid in the urine compared to wild-type mice [57]. These results indicate that, in the absence of PPAR-α activity, catabolism of 2-ClFA is reduced, resulting in accumulation in systemic blood. Further analysis also revealed increased levels of 2-ClFA in the liver of PPAR-α^−/− mice, suggesting that catabolism of 2-ClFA occurs in hepatocytes. Interestingly, further studies conducted in both HepG2 and primary murine hepatocytes showed that 2-ClFA functions as a lipid ligand for PPAR-α [57]. A reporter gene system was used to identify 2-ClFA as a stimulator of PPAR-α activity. These findings were confirmed in primary murine hepatocytes treated with 2-ClFA, where an increase in the expression of PPAR-α target genes was observed [57].

Taken together, these results indicate that circulating 2-ClFA is catabolized by hepatic ω-oxidation under the direct influence of PPAR-α activity in the liver and 2-chlorofatty acid is a PPAR-α ligand that enhances its own catabolism (Figure 2). Ultimately, the short-chain 2-chlorodicarboxylic acid, 2-chloroadipic acid, is excreted in the urine, likely through the high uptake organic acid transporter of the proximal tubules of the kidneys.

Biological Properties of Chlorinated Lipids

The roles of 2-ClFALD and 2-ClFA on leukocyte and endothelial function have been explored by several groups, highlighting the potential implications of chlorinated lipids in inflammatory diseases. This review emphasizes the impact of chlorinated lipids on endothelial dysfunction, neutrophil chemotaxis, monocyte apoptosis, and vascular tone (Figure 3).

Figure 3.

Biological Roles of the Chlorinated Lipidome

Endothelial Dysfunction

Chlorinated lipids elicit endothelial dysfunction at both the blood-brain barrier and in human coronary artery endothelial cells. Using primary brain microvascular endothelial cells and LPS-treated mice, Ullen and co-workers demonstrated 2-ClFALD increases the permeability of the blood-brain barrier [51, 58, 59]. Inhibition of MPO or downstream mitogen-activated protein kinase (MAPK) signaling pathways protect against barrier dysfunction in vitro. Furthermore, LPS-induced blood-brain barrier dysfunction in MPO-deficient mice is significantly reduced compared to wild-type mice. The endothelial dysfunction is attributed to morphological changes in tight and adherens junctions [59]. Nusshold and co-workers recently demonstrated that 2-ClFALD binds to and likely modifies cytoskeletal proteins important for tight junction formation and stability [51]. Additionally, these studies suggest that 2-ClFALD-protein adduct localization to the Golgi apparatus and the endoplasmic reticulum is important in 2-ClFALD-elicited changes in blood-brain barrier function.

Recently, Hartman et al. identified 2-ClFA as a mediator of dysfunction at the endothelial barrier [60]. In contrast to 2-ClFALD, 2-ClFA localizes to Weibel-Palade bodies in human coronary artery endothelial cells [60]. Weibel-Palade bodies are endothelial cell-specific storage granules containing multiple key bioactive molecules, including P-selectin, angiopoietin-2, and von Willebrand factor [61, 62]. 2-ClFA elicits the release of Weibel-Palade body contents (angiopoietin-2 and von Willebrand factor) into the extracellular space, as well as increased surface expression of P-selectin and other adhesion molecules [60]. Functionally, the release of von Willebrand factor and increase in adhesion molecule surface expression leads to the increased adherence of platelets and leukocytes to the endothelium. Additionally, angiopoietin-2 acts as a Tie-2 receptor antagonist, destabilizing the cell-to-cell contacts that maintain the endothelial barrier [63]. 2-ClFA-induced release of angiopoietin-2 contributes to the dysfunction of the endothelial barrier, as seen in both human coronary artery endothelial cells and human lung microvascular endothelial cells [60]. Furthermore, plasma levels of 2-ClFA correlate with plasma angiopoietin-2 levels in human sepsis patients [64]. These studies suggest that another mechanism responsible for 2-ClFALD elicited endothelial dysfunction could be mediated through 2-ClFALD oxidation to 2-ClFA and the subsequent effects of 2-chlorofatty acid on Weibel-Palade body mobilization.

Neutrophil Biology

Chemotaxis is an essential mechanism for neutrophils to respond to infection or inflammation in tissues [65, 66]. A chemoattractant produced by host cells or microbes initiates the migration of neutrophils into tissue. Many lipid peroxidation products, including 4-hydroxynonenal and 4-hydroxytetradecenal, function as neutrophil chemoattractants in vitro [67]. Therefore, the impact of 2-ClFALD on neutrophil chemotaxis was examined. After a short incubation in a Boyden chamber with primary human neutrophils in the upper chamber and a potential chemoattractant in the lower chamber, 2-ClFALD was demonstrated to increase net migration of neutrophils [47]. Impressively, 2-ClFALD functions as a chemoattractant at concentrations as low as 90 nM. In contrast to 2-ClFALD, 2-ClFA was shown to not be a neutrophil chemoattractant [50]. The mechanism by which 2-ClFALD promotes neutrophil migration remains to be identified.

Ongoing studies in the Ford laboratory are focused on the role of 2-ClFA on neutrophil function. In addition to well-established antimicrobial properties, neutrophils have been recently described to form neutrophil extracellular traps (NETs), extracellular chromatin decorated with granular proteases and enzymes [68]. MPO was recently described to be critical for NETs formation [69, 70]; therefore, the role of MPO-derived chlorinated lipids in NETs formation was examined. 2-ClFA impacts the fate of activated neutrophils by initiating NETs formation, independently from neutrophil activation. These data suggest a novel role for 2-ClFA as a lipid mediator of NETs formation, a mechanism found to be responsible for cell toxicity and tissue damage in many inflammatory diseases [71].

Monocyte Apoptosis

Human monocytes contain MPO and produce chlorinated lipids after activation as previously described [54]. Monocyte-derived HOCl can target extracellular lipoproteins, leading to 2-ClFALD associated with low density lipoproteins [54]. Further studies are required to investigate the importance of chlorinated lipids associated with lipoproteins in vivo. In contrast to the function of 2-ClFA in endothelial cells and neutrophils, 2-ClFA accumulation in primary human monocytes induces apoptosis [55]. Further studies revealed that 2-ClFA increases caspase-3 activity, poly (ADP-ribose) polymerase (PARP) cleavage, and C/EBP homologous protein (CHOP) protein expression [55]. 2-ClFA-induced apoptosis is attributed to the endoplasmic reticulum stress response, as indicated by an increase in CHOP expression, X-box binding protein-1 mRNA splicing, phosphorylation of eukaryotic initiation factor 2α, and glucose-regulated protein-78 expression. Additionally, 2-ClFA was shown to increase reactive oxygen species production in THP-1 cells [55]. To summarize, chlorinated lipids induce apoptosis through endoplasmic reticulum stress and reactive oxygen production in monocytes, which may lead to more broad implications in inflammatory diseases.

Vascular Tone

The impact of chlorinated lipids on vascular tone has recently been examined. Previous studies demonstrated that chlorinated lipids inhibit the activity of endothelial nitric oxide synthase (eNOS) in endothelial cells [72]. Furthermore, eNOS-dependent vasodilation of the aorta is inhibited after chlorine gas exposure in rats [73, 74]. Therefore, the impact of chlorinated lipids on eNOS-dependent vasodilation was tested. Twenty-four hours after a single intranasal administration of either 2-ClFALD or 2-ClFA, acetylcholine-induced vasodilation of the aorta, which is eNOS-dependent, was inhibited in mice [56]. These effects were not seen in mice treated with the non-chlorinated control fatty aldehyde or fatty acid. These studies suggest that chlorinated lipids (or their metabolic products) travel from the lung, likely in the circulation, to evoke profound effects in the vasculature and heart. Future studies need to examine the impact of locally-produced chlorinated lipids and their implications on peripheral tissues.

Chlorinated Lipids in Disease

The presence of chlorinated lipids in biological tissues and their potential role in inflammation is evident. Chlorinated lipids have been shown to accumulate in activated human neutrophils and monocytes [54]. Chlorinated lipids accumulate in multiple mouse models of inflammation, including LPS-induced endotoxemia, Sendai virus infection, and K/BxN arthritis [49, 50, 53]. Additionally, the role of chlorinated lipids has been examined in rat myocardial ischemia injury since repair mechanisms associated with ischemia include neutrophil infiltration into ischemic zones [75–78]. 2-ClFALD accumulates in rat infarcted myocardium [79]. Furthermore, 2-ClFALD treatment of isolated perfused rat hearts results in decreased heart rate and coronary flow rate, indicating a potential deleterious role of 2-ClFALD in heart function [79]. Additional studies have shown 2-ClFALD increases over 1400-fold in human atherosclerotic lesions [80]. Collectively, these studies underscore potentially important roles of MPO-derived chlorinated lipids in cardiovascular disease.

Chlorinated lipids have been investigated in human lupus. Serum 2-ClFA levels were assessed specimens from the Study of Lupus Vascular and Bone Long-term Endpoints (SOLVABLE), a study designed to determine the risk of subclinical and clinical cardiovascular disease in systemic lupus erythematosus (SLE) [81]. Results revealed that serum 2-ClFA levels are elevated in SLE patients, but are not associated with cardiovascular risk in either SLE or non-SLE subjects. One deficit of this study, however, was the statistical analyses in the subgroups of subjects with cardiovascular disease versus no evidence of cardiovascular disease may have been underpowered. It was noted that serum 2-ClFA levels were elevated, albeit not statistically significant, in the non-SLE subjects with cardiovascular disease compared to the non-SLE subjects without cardiovascular disease.

A more recent study investigated plasma levels of 2-ClFA in human patients with sepsis enrolled in the Molecular Epidemiology of SepsiS in the ICU (MESSI) cohort [64]. In this study, plasma was collected at admission to the ICU for analyses of multiple biomarkers. Human sepsis patients had elevated levels of free 2-ClFA levels in the plasma compared to healthy, non-septic controls. Furthermore, levels of 2-ClFA associated with 30-day mortality. Plasma MPO, used as a marker of neutrophil activation, was not associated with 30-day mortality but did associate with 2-ClFA levels. Additionally, neutropenic sepsis patients have significantly lower levels of plasma 2-ClFA compared to sepsis patients with normal levels of neutrophils. Interestingly, 2-ClFA significantly correlates with acute respiratory distress syndrome (ARDS), which is characterized by widespread inflammation and damage to the lungs [64]. Furthermore, the addition of 2-ClFA levels to the Acute Physiology and Chronic Health Examination III (APACHE) score improved the prediction for ARDS. Collectively, these studies suggest levels of plasma 2-ClFA may have value as a predictor of sepsis outcomes and in particular ARDS-caused mortality.

Plasma markers of neutrophil-endothelial dysfunction in the microcirculation were also related to plasma 2-ClFA levels in the MESSI cohort [64]. Plasma levels of 2-ClFA also associate with plasma levels of angiopoietin-2, E-selectin, and soluble thrombomodulin. These findings prompted further studies to examine a direct mechanistic link between chlorinated lipids and endothelial dysfunction. In both human lung microvascular endothelial cells and human coronary artery endothelial cells, chlorinated lipids increase the surface expression of selectins and the release of angiopoietin-2 to contribute to endothelial dysfunction [64].

As discussed previously, chlorinated lipids play a key role in multiple biological processes including endothelial permeability barrier, neutrophil biology, monocyte biology, and vascular tone. These studies, using human sepsis samples, highlight the potential role of chlorinated lipids as both a biomarker of inflammatory disease and a mediator of disease. Future studies will pinpoint the mechanisms by which chlorinated lipids elicit their detrimental effects, and these mechanistic studies may provide new rationale for the development of therapeutics to combat these harmful consequences of the innate immune system.

Current Topics and Future Directions

MPO-derived HOCl can oxidize many biological molecules with different target localization and reaction kinetics. The most reactive target molecules for HOCl contain sulfhydryl residues, and based only on kinetic studies, the vinyl ether bond of plasmalogens is a secondary target for HOCl reactions [82–84]. However, following degranulation, MPO tends to adhere to the plasma membrane due to its positively charged surface. Thus, it is likely that MPO-derived HOCl has access to the vinyl ether bonds of plasmalogens, which are accessible at the hydrophilic plane of biological membranes. In contrast, it should be appreciated that alkenes are kinetically less reactive than the vinyl ether bond, and aliphatic alkenes are imbedded in the hydrophobic domain of membranes and may not be accessed by HOCl [83, 85]. The likelihood of reactivity of plasmalogens with HOCl in biological systems is also increased since the plasma membranes of neutrophils, as well as endothelial cells, vascular smooth muscle cells, cardiac myocytes, and brain cells, are enriched with plasmalogens, which provide an abundance of vinyl ether targets [86].

Although much progress has been made in the understanding and characterization of chlorinated lipids, many questions remain unanswered. For instance, HOCl targeting of plasmalogens leads to two 2-ClFALD products (and subsequent metabolites) with 16- or 18-carbon chains. However, the majority of the aforementioned studies are based on the use of 16-carbon chlorinated lipids, leaving the role of the 18-carbon chlorinated lipids unknown. Moreover, 2-ClFALD and 2-ClFA can be released by the neutrophils and taken up by surrounding cells, such as endothelial cells and hepatocytes. Recent studies demonstrated that 2-ClFA localizes to Weibel-Palade bodies in endothelial cells, which are endothelial cell-specific organelles. The subcellular localization in other cells and the relevance of subcellular localization in cells needs to be investigated.

Table 1 (at end of file) shows a summary of the levels of chlorinated lipids that have been reported. It should be noted that data shown in Table 1 reflect many biological specimens and normalization for comparisons can be quite different, which likely reflects the nature of the specimen of interest. For example, in diseased tissues, edema should be considered as a potential artifact by determining the wet to dry tissue weight. Additionally, under some conditions chlorinated lipid levels are normalized to lipid phosphorous (indicated as inorganic phosphate in Table 1). Future studies examining a cellular response to chlorinated lipids should consider the physiological levels of chlorinated lipids to determine an appropriate experimental concentration. As previously mentioned, systemic plasma levels are in the 1–100 nM range but at the site of activated neutrophils, both 2-ClFA and 2-ClFALD levels reach 10–90 μM. Thus, these levels should be considered relevant physiological concentrations at the site of inflammation, whereas offsite concentrations (e.g., that in systemic blood) may be as high as 100 nM. Interestingly, for endothelial permeability, 2-ClFA effects on leakiness have been observed at concentrations as low as 100 nM [64].

Table 1.

Summary of Chlorinated Lipids Found in Biological Samples.

Chlorinated Lipid	Tissue/Cell	Chlorinated Lipid Level	Reference
α-Chlorofatty aldehyde	Human neutrophils	Not detected in unstimulated cells	[47]
		35 pmol/106 PMA-stimulated cells	[47]
	Human monocytes	0.4 pmol/106 unstimulated cells	[54]
		6.9 pmol/106 PMA-stimulated cells	[54]
	Human aorta	0.0015 pmol/nmol inorganic phosphate in normal tissue	[80]
		2.08 pmol/nmol inorganic phosphate in atherosclerotic tissue	[80]
	Rat heart	0.3 pmol/μmol inorganic phosphate with no surgical intervention	[79]
		4.5 pmol/μmol inorganic phosphate with 24 h LAD occlusion	[79]
		1.3 pmol/μmol inorganic phosphate in pericarditis model	[79]
	Mouse lung	0.075 pmol/mg in air-treated mice	[56]
		4.2 pmol/mg in mice, time 0 post-chlorine gas treatment	[56]
α-Chlorofatty acid	Human neutrophils	1.2 pmol/106 unstimulated cells	[50]
		13 pmol/106 PMA-stimulated cells	[50]
	Mouse BALF	0.0113 pmol/mL in healthy mice	[50]
		0.0453 pmol/mL in Sendai virus-treated mice	[50]
	Mouse plasma	0.8 pmol/mL in healthy mice	[56]
		1.4 pmol/mL in Sendai virus-treated mice	[50]
		1.8 pmol/mL in K/BxN mouse model of arthritis	[53]
		100 pmol/mL in mice, time 0 post-chlorine gas treatment	[56]
	Rat plasma	0.5 pmol/mL in control-treated rats	[49, 56]
		15 pmol/mL in rats, 12 hr post-chlorine gas treatment	[56]
		3.2 pmol/mL in LPS-treated rats	[49]
	Mouse lung	0.050 pmol/mg in air-treated mice	[56]
		4.8 pmol/mg in mice, time 0 post-chlorine gas treatment	[56]
	Rat lung	0.025 pmol/mg in air-treated rats	[56]
		0.8 pmol/mg in rats, time 0 post-chlorine gas treatment	[56]
	Human plasma	0.86 pmol/mL in healthy humans
		1.05 pmol/mL in Systemic Lupus Erthematosus patients	[81]
		0.77 pmol/mL in sepsis patients with neutropenia	[64]
		1.85 pmol/mL in sepsis patients without neutropenia	[64]
α-Chlorofatty alcohol	Human neutrophils	0.3 pmol/106 unstimulated cells	[50]
		2.3 pmol/106 PMA-stimulated cells	[50]

Two independent research groups have shown changes in endothelial cells in response to chlorinated lipids (2-ClFALD and 2-ClFA) [58, 87]. However, another group has not seen similar deleterious effects of 2-ClFALD on smooth muscle cells [88]. There are several possibilities to account for these differences. First, 2-ClFALD reactivity with glutathione [53] may differ in these studies based on the cell type and cell culture media. Second, it is possible that proteins in cell culture media in these studies result in differences in reactivity of the 2-ClFALD with peptidylcysteines and other small molecules. Third, there may be disparate metabolism of 2-ClFALD in endothelial cells compared to smooth muscle cells. Finally, differences in these studies may simply reflect differences in sensitivity of endothelial cells to 2-ClFALD treatment compared to smooth muscle cells. For future experimentation with 2-ClFALD, investigators should be aware of potential metabolism, as well as reactivity with cell culture media containing proteins, that may render the 2-ClFALD concentration much less than that provided to the cells. Reactivity and metabolism of 2-ClFALD should always be considered in experimental design.

Conclusion

Myeloperoxidase-derived HOCl attack on invading microbes is a key feature of innate immunity but can have deleterious effects on host tissue. The vinyl ether bond of plasmalogen phospholipids is exposed for HOCl oxidation, leading to the production of the chlorinated lipidome. 2-ClFALD is the first chlorinated lipid liberated and is quickly metabolized to the stable metabolites, 2-chlorofatty acid and 2-chlorofatty alcohol. Additionally, 2-chlorofatty aldehyde can bind to and modify amino acids, small molecules, and proteins. Chlorinated lipids have been detected in biological systems and in models of inflammatory disease.

The unique metabolism of 2-ClFA is characterized by sequential ω-oxidation and β-oxidation from the ω-end with elimination as 2-chloroadipic acid in the urine. The clearance of 2-ClFA is increased by the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-ClFA has been shown to be a PPAR-α agonist and thus, accelerates its own clearance.

The role of MPO-derived 2-ClFALD and 2-ClFA on leukocyte and endothelial function have been explored by several groups, suggesting that these chlorinated lipids have unique and specific deleterious effects on the function of the endothelial cell, neutrophil, and monocyte, as well as vascular tone and cardiovascular function. Studies in human sepsis patients demonstrating the association of plasma 2-ClFA levels with 30-day mortality and ARDS highlight the potential for its use as a biomarker. Consequently, this novel chlorinated lipidome, produced in response to neutrophil activation, represents a potential therapeutic target to modulate inflammatory diseases. Future studies will elucidate the role of these metabolites as mediators of disease.

Highlights.

• Myeloperoxidase-derived chlorinated lipids are produced as deleterious products of inflammation.

• 2-Chlorofatty acid is present in vivo as a free fatty acid and esterified into complex lipid pools.

• 2-Chlorofatty acid is cleared in the urine after hepatic ω-oxidation and modulated by PPAR-α activity.

• Chlorinated lipids impact the function of neutrophils, monocytes, and endothelial cells.

• Chlorinated lipids are a potential biomarker and therapeutic target for inflammatory diseases.

Abbreviations

ARDS

acute respiratory distress syndrome

CHOP

C/EBP homologous protein

2-ClDCA

2-chlorodicarboxylic acid

2-ClFA

2-chlorofatty acid

2-ClFALD

2-chlorofatty aldehyde

2-ClFOH

2-chlorofatty alcohol

eNOS

endothelial nitric oxide synthase

H₂O₂

hydrogen peroxide

HOCl

hypochlorous acid

HOBr

hypobromous acid

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinases

MPO

myeloperoxidase

NETs

neutrophil extracellular traps

PARP

poly (ADP-ribose) polymerase

PMA

phorbol 12-myristate 13-acetate

PPAR

peroxisome proliferator-activated receptor

SLE

systemic lupus erythematosus