Neutrophils represent the major effector cells of innate immunity. One mechanism for neutrophil‐mediated microbe killing is through the release of neutrophil extracellular traps (NETs). NET formation ...(NETosis) involves the fusion of granule and nuclear contents, which are then released in the extracellular space to capture and kill microbes. Chlorinated fatty acids (2‐ClFAs) are a product of neutrophil activation as a result of the myeloperoxidase (MPO) product, HOCl, targeting plasmalogen phospholipids. In this study, we examined the role of 2‐ClFA as an important lipid mediator of NETosis. Treating human peripheral blood neutrophils with physiological levels of 2‐ClFAs led to the formation of NETs, characterized by MPO association with DNA and neutrophil elastase (NE) redistribution to the perinuclear area. NETs induced by 2‐ClFA treatment were as efficient in killing bacteria as those produced by stimulating neutrophils with phorbol myristate acetate (PMA), a potent activator of NETosis. Like PMA, 2‐ClFA‐induced NETosis is calcium‐ and protein arginine deiminase 4 (PAD4)‐dependent. Interestingly, while PMA‐induced NETosis requires neutrophil activation, 2‐ClFAs initiate the NETosis process without neutrophil activation and degranulation. Furthermore, unlike PMA, 2‐ClFA elicits NETosis in bone‐marrow derived neutrophils from MPO deficient mice. Overall, 2‐ClFA may have an important role in directing neutrophil fate following activation, mediating the production on NETs and providing a new pharmacological target to either enhance bacterial killing or reduce host injury during inflammation.
Support or Funding Information
This study was supported by research funding from the National Institute of Health R01 GM‐115553 to D.A.F.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Background
Hepatic infiltration of polymorphonuclear leukocytes (PMNs) is a key driver of pathogenesis in non‐alcoholic steatohepatitis (NASH). Lipotoxicity arising from elevated free fatty acids in ...NASH is postulated to advance PMN activation and recruitment. Myeloperoxidase (MPO), a PMN‐derived enzyme mechanistically linked to oxidative stress (ROS) in inflammation, may contribute to this feedback. Chlorinated fatty acids (2‐ClFAs) are an MPO by‐product produced when HOCl targets plasmalogen phospholipids. 2‐CIFAs induce PMN activation, adhesion and ROS mediated cellular damage. In human patients, a strong positive correlation between MPO expressing cells and severity of NASH has been observed. However, mouse macrophages lack MPO and this is a critical limitation of current models. As a result, the contribution of MPO to NASH remains poorly understood.
Methods
To investigate the role of MPO in NASH, C57Bl/6J mice expressing macrophage transgenic human MPO (hMPOTg) were fed a 40% high fat (HF) diet for 10 weeks. At the end of the study biomarkers of hepatocellular damage and PMN infiltration were measured. Alterations in lipid species were detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS) lipidomics in order to elucidate contributing lipids to NASH.
Results
Here we show hMPOTg is associated with significant alteration in the hepatic lipidome, increased infiltration of PMNs, levels of MPO lipid by‐products and exacerbated hepatic steatosis. HF fed hMPOTg mice showed greater weight gain and hepatomegaly. Increased infiltration of PMN in hMPOTg HF livers was supported by immunohistochemistry and PCR analysis of PMN biomarkers. Significantly higher triglyceride levels were detected in hMPOTg HF fed plasma and livers. Major hepatic lipid species of cholesterol esters, phosphatidylcholine, and phosphatidylserine were also significantly altered. Systemic pro‐inflammatory 2‐CIFA levels were increased in HF fed hMPOTg.
Conclusion
Together, our results highlight the significant contribution of hMPO to hepatic lipid homeostasis. MPO is currently exploited as a biomarker and therapeutic target in cardiovascular disease. Expanding our understanding of MPO may extend the benefits of similar therapeutic strategies to patients suffering from NASH and metabolic syndrome.
Graphical : The Role of Macrophage Derived Myeloperoxidase in Nonalcoholic Steatohepatitis (NASH)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
Two interventions known to cause profound changes in cellular metabolism and redox enzyme expression/activity in heart and skeletal muscle are exercise (EX), and high‐fat, high sugar ...“Western pattern” diet (HFHS). To determine whether heart and skeletal muscle are differentially impacted by HFHS diet and EX, Sprague‐Dawley rats were fed either standard chow (CON) or a HFHS diet for 12 weeks (HFHS). A sub‐group of HFHS rats exercise‐trained 5 days/week for 2 hours/day (HFHS+EX). Following these interventions, mitochondrial H
2
O
2
emission (mH
2
O
2
) and Ca
2+
tolerance (mCa
2+
) supported by pyruvate + malate + succinate, and O
2
consumption (mO
2
) supported by glutamate+malate (GM) and palmitoyl‐carnitine (PC) was determined in permeabilized red gastrocnemius (RG) and cardiac fibers. HFHS diet increased mH
2
O
2
~2‐fold, and HFHS+EX decreased in RG (P<0.05) compared to CON. Surprisingly, the HFHS diet decreased mH
2
O
2
~2‐fold in heart compared to CON (P<0.05), and was even further decreased in HFHS+EX. Maximal PC‐supported mO
2
increased in RG in both HFHS and HFHS+Ex compared to CON (P<0.05), but in heart this was only increased in HFHS+EX. Maximal GM‐supported mO
2
decreased in both RG and heart with HFHS, and this decrease was prevented in HFHS+EX. Total mCa
2+
increased only with HFHS+EX in both RG and heart compared to CON. These findings suggest that heart and skeletal muscle mitochondria are differentially affected by diet and exercise, which point to adaptations in cellular redox state. (Supported by NIH grant HL098780 to EJA)
Background: Hepatic infiltration of polymorphonuclear leukocytes (PMNs) is a key driver of pathogenesis in non-alcoholic steatohepatitis (NASH). Lipotoxicity arising from elevated free fatty acids in ...NASH is postulated to advance PMN activation and recruitment. Myeloperoxidase (MPO), a PMN-derived enzyme mechanistically linked to oxidative stress (ROS) in inflammation, may contribute to this feedback. Chlorinated fatty acids (2-ClFAs) are an MPO by-product produced when HOCl targets plasmalogen phospholipids. 2-CIFAs induce PMN activation, adhesion and ROS mediated cellular damage. In human patients, a strong positive correlation between MPO expressing cells and severity of NASH has been observed. However, mouse macrophages lack MPO and this is a critical limitation of current models. As a result, the contribution of MPO to NASH remains poorly understood.Methods: To investigate the role of MPO in hepatosis C57Bl/6J mice expressing macrophage transgenic human MPO (hMPOTg) were fed a 40% high fat (HF) diet for 10 weeks. At the end of the study biomarkers of hepatocellular damage and PMN infiltration were measured. Alterations in lipid species were detected using electrospray ionization tandem mass spectrometry (ESI-MS/MS) lipidomics in order to elucidate contributing lipids to NASH.Results: Here we show hMPOTg is associated with significant alteration in the hepatic lipidome, increased infiltration of PMNs, levels of MPO lipid by-products and exacerbated hepatic steatosis. HF fed hMPOTg mice showed greater weight gain and hepatomegaly. Increased infiltration of PMN in hMPOTg HF livers was supported by immunohistochemistry and PCR analysis of PMN biomarkers. Significantly higher triglyceride levels were detected in hMPOTg HF fed plasma and livers. Major hepatic lipid species of cholesterol esters, phosphatidylcholine, and phosphatidylserine were also significantly altered. Systemic pro-inflammatory 2-CIFA levels were increased in HF fed hMPOTg.Conclusion: Together, our results highlight the significant contribution of hMPO to hepatic lipid homeostasis. MPO is currently exploited as a biomarker and therapeutic target in cardiovascular disease. Expanding our understanding of MPO may extend the benefits of similar therapeutic strategies to patients suffering from NASH and metabolic syndrome.
Consequences of oxidative stress may be beneficial or detrimental in physiological systems. An organ system’s position on the ‘hormetic curve’ is governed by the source and temporality of reactive ...oxygen species (ROS) production, proximity of ROS to moieties most susceptible to damage, and the capacity of the endogenous cellular ROS scavenging mechanisms. Most importantly, the resilience of the tissue (the capacity to recover from damage) is a decisive factor, and this is reflected in the disparate response to ROS in cardiac and skeletal muscle. In myocytes, a high oxidative capacity invariably results in a significant ROS burden which in homeostasis, is rapidly neutralized by the robust antioxidant network. The up-regulation of key pathways in the antioxidant network is a central component of the hormetic response to ROS. Despite such adaptations, persistent oxidative stress over an extended time-frame (e.g. months to years) inevitably leads to cumulative damages, maladaptation and ultimately the pathogenesis of chronic diseases. Indeed, persistent oxidative stress in heart and skeletal muscle has been repeatedly demonstrated to have causal roles in the etiology of heart disease and insulin resistance, respectively. Deciphering the mechanisms that underlie the divergence between adaptive and maladaptive responses to oxidative stress remains an active area of research for basic scientists and clinicians alike, as this would undoubtedly lead to novel therapeutic approaches. Here, we provide an overview of major types of ROS in striated muscle and the divergent adaptations that occur in response to them. Emphasis is placed on highlighting newly uncovered areas of research on this topic, with particular focus on the mitochondria, and the diverging roles that ROS play in muscle health (e.g., exercise or preconditioning) and disease (e.g., cardiomyopathy, ischemia, metabolic syndrome).
Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ...ACSL1 (Acsl1T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1T−/− hearts, control and Acsl1T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function.
Abstract only
Cardiac monoamine oxidase (MAO) has been shown to have an etiological role in heart failure due to the mitochondrial toxicity of the reactive oxygen (ROS) and catechol‐aldehyde species ...(RCS) produced by this enzyme. A causal role for MAO in cardiomyopathy with obesity and type 2 diabetes has been proposed due to the elevated sympathetic tone that occurs in these conditions, although this has never been investigated in either experimental or clinical models. We performed a cross‐species kinetic analysis of MAO isoform‐specific (A & B) oxidation of dopamine (DA), phenylethylamine (PEA), serotonin (5‐HT), norepinephrine (NE), and Tyramine (TYR) in heart tissue from nondiabetic/control (Ctl) and obese/diabetic (ObD) mice (n=12), rats (n=12), and humans (n=46) using MAO‐derived ROS production as index. In mouse hearts, maximal MAO‐B activity >> MAO‐A, in rat hearts MAO‐A >> MAO‐B, while in humans the isoform activity appears to be equivalent. Maximal MAO activity (both A & B) was significantly higher in ObD mouse and rat hearts with TYR and DA substrates compared with Ctl. ObD mouse hearts also had higher rates of MAO activity with PEA. In human myocardium, MAO activity (both A & B) was 1.5‐fold and 3‐fold higher in ObD patients compared to Ctl patients with NE and DA, respectively. Cardiac MAO‐B activity with 5‐HT also was significantly greater in ObD patients. These translational findings suggest that in obese/diabetic hearts, increased MAO activity and the ROS/RCS derived from it may contribute to cardiomyopathy, and imply that targeting cardiac MAO may have therapeutic potential.
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