Extracellular Myeloperoxidase Research Articles

Calcium-sensing receptor and NF-κB pathways in TN breast cancer contribute to cancer-induced cardiomyocyte damage via activating neutrophil extracellular traps formation.

Cardiovascular disorders are commonly prevalent in cancer patients, yet the mechanistic link between them remains poorly understood. Because neutrophil extracellular traps (NETs) have implications not just in cardiovascular diseases (CVD), but also in breast cancer (BC), it was hypothesized to contribute to CVD in the context of oncogenesis. We established a mouse model using nude mice to simulate liver metastasis of triple-negative BC (TNBC) through the injection of MDA-MB-231 cells. Multiple imaging and analysis techniques were employed to assess the cardiac function and structure, including echocardiography, HE staining, Masson staining, and transmission electron microscopy (TEM). MDA-MB-231 cells underwent treatment with a CaSR inhibitor, CaSR agonist, and NF-κB channel blocker. The phosphorylation of NF-κB channel protein p65 and the expression and secretion of IL-8 were assessed using qRT-PCR, Western Blot, and ELISA, respectively. In addition, MDA-MB-231 cells were co-cultured with polymorphonuclear neutrophils (PMN) under varying conditions. The co-localization of PMN extracellular myeloperoxidase (MPO) and DNA were observed by cellular immunofluorescence staining to identify the formation of NETs. Then, the cardiomyocytes were co-cultured with the above medium that contains NETs or not, respectively; the effects of NETs on cardiomyocytes apoptosis were perceived by flow cytometry. The ultrastructural changes of myocardial cells were perceived by TEM, and ELISA detected the levels of myocardial enzyme (LDH, MDA and SOD). Overall, according to our research, CaSR has been found to have a regulatory role in IL-8 secretion in MDA-MB-231 cells, as well as in the formation of NETs by PMN cells. These findings suggest CaSR-mediated stimulation in PMN can lead to increased NETs formation and subsequently to cytotoxicity in cardiomyocytes, which potentially via activation of the NF-κB signaling cascade of BC cell.

Myeloperoxidase Inhibition in Heart Failure With Preserved or Mildly Reduced Ejection Fraction: SATELLITE Trial Results

BackgroundInflammation is a key driver of heart failure with preserved left ventricular ejection fraction. AZD4831 inhibits extracellular myeloperoxidase, decreases inflammation, and improves microvascular function in preclinical disease models. Methods and ResultsIn this double-blind phase 2a study (Safety and Tolerability Study of AZD4831 in Patients With Heart Failure [SATELLITE]; NCT03756285), patients with symptomatic heart failure, left ventricular ejection fraction of ≥40%, and elevated B-type natriuretic peptides were randomized 2:1 to once-daily oral AZD4831 5 mg or placebo for 90 days. We aimed to assess target engagement (primary end point: myeloperoxidase specific activity) and safety of AZD4831. Owing to coronavirus disease 2019, the study was terminated early after randomizing 41 patients (median age 74.0 years, 53.7% male). Myeloperoxidase activity was decreased by more than 50% from baseline to day 30 and day 90 in the AZD4831 group, with a placebo-adjusted decreased of 75% (95% confidence interval, 48, 88, nominal P < .001). No improvements were noted in secondary or exploratory end points, apart from a trend in Kansas City Cardiomyopathy Questionnaire overall summary score. No deaths or treatment-related serious adverse events occurred. AZD4831 treatment-related adverse events were generalized maculopapular rash, pruritus, and diarrhea (all n = 1). ConclusionsAZD4831 inhibited myeloperoxidase and was well tolerated in patients with heart failure and left ventricular ejection fraction of 40% or greater. Efficacy findings were exploratory owing to early termination, but warrant further clinical investigation of AZD4831. Lay SummaryFew treatments are available for patients with the forms of heart failure known as heart failure with preserved or mildly reduced ejection fraction. Current treatments do not target inflammation, which may play an important role in this condition. We tested a new drug called AZD4831 (mitiperstat), which decreases inflammation by inhibiting the enzyme myeloperoxidase. Among the 41 patients in our clinical trial, AZD4831 had a good safety profile and inhibited myeloperoxidase by the expected amount. Results mean we can conduct further trials to see whether AZD4831 decreases the symptoms of heart failure and improves patients’ ability to participate in physical exercise.

Myeloperoxidase PET Imaging Tracks Intracellular and Extracellular Treatment Changes in Experimental Myocardial Infarction.

Myeloperoxidase (MPO) is a highly oxidative, pro-inflammatory enzyme involved in post-myocardial infarction (MI) injury and is a potential therapeutic target. While multiple MPO inhibitors have been developed, the lack of an imaging reporter to select appropriate patients and assess therapeutic efficacy has hampered clinical development. Thus, a translational imaging method to detect MPO activity non-invasively would help to better understand the role MPO plays in MI and facilitate novel therapy development and clinical validation. Interestingly, many MPO inhibitors affect both intracellular and extracellular MPO, but previous MPO imaging methods can only report extracellular MPO activity. In this study, we found that an MPO-specific PET imaging agent (18F-MAPP) can cross cell membranes to report intracellular MPO activity. We showed that 18F-MAPP can track the treatment effect of an MPO inhibitor (PF-2999) at different doses in experimental MI. The imaging results were corroborated by ex vivo autoradiography and gamma counting data. Furthermore, extracellular and intracellular MPO activity assays revealed that 18F-MAPP imaging can report the changes induced by PF-2999 on both intracellular and extracellular MPO activities. These findings support 18F-MAPP as a translational candidate to noninvasively report MPO activity and accelerate drug development against MPO and other related inflammatory targets.

Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

Arterial myeloperoxidase in the detection and treatment of vulnerable atherosclerotic plaque: a new dawn for an old light.

Intracellular myeloperoxidase (MPO) plays a specific role in the innate immune response; however, upon release into the extracellular space in the setting of inflammation, drives oxidative tissue injury. Extracellular MPO has recently been shown to be abundant in unstable atheroma and causally linked to plaque destabilization, erosion, and rupture, identifying it as a potential target for the surveillance and treatment of vulnerable atherosclerosis. Through the compartmentalization of MPO's protective and deleterious effects, extracellular MPO can be selectively detected using non-invasive molecular imaging and targeted by burgeoning pharmacotherapies. Given its causal relationship to plaque destabilization coupled with an ability to preserve its beneficial properties, MPO is potentially a superior translational inflammatory target compared with other immunomodulatory therapies and imaging biomarkers utilized to date. This review explores the role of MPO in plaque destabilization and provides insights into how it can be harnessed in the management of patients with vulnerable atherosclerotic plaque.

Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer\u2019s disease

IntroductionNeutrophil accumulation is a well-established feature of Alzheimer’s disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain.Materials and methodsWe used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD.ResultsThere was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature.ConclusionOur report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.

The Synthetic Myeloperoxidase Inhibitor AZD3241 Ameliorates Dextran Sodium Sulfate Stimulated Experimental Colitis.

Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p<0.05) and improved clinical score (9 fold p<0.05; a score comprising the time-dependent assessment of stool consistency and extent of rectal bleeding), loss of colonic crypts (p<0.001), preserved surface epithelium (p<0.001) and enhanced expression of the transcription factor Nrf-2 (regulator of antioxidants) and enhanced expression of the downstream antioxidant response element haeoxygenase-1 (HO-1) in the colon tissue. Also, the concentration of fecal hemoglobin and the myeloperoxidase specific oxidative damage biomarker 3-chlorotyrosine in the colon were significantly decreased in the presence of AZD3241. This latter result was consistent with AZD3241 inhibiting MPO activity in vitro. Overall, AZD3241 ameliorated the course and severity of experimental colitis through ameliorating MPO derived tissue damage and could be considered a potential therapeutic option, subject to further validation in chronic IBD models.

Neutrophils as a Source of Factors Increasing Duration of the Inflammatory Phase of Wound Healing in Patients with Type 2 Diabetes Mellitus

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as the pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the duration of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the severity of lower extent damage: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). At admission to hospital, T2DM patients had significantly higher (р < 0.05) levels of blood glucose and glycated hemoglobin (groups 1−3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1−3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1−3) and erythrocyte glutathione peroxidase activity (groups 2 and 3) than in the control group (healthy volunteers). The length of hospital stay after surgery corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r = 0.72, p < 0.05). Leukocytic intoxication index depended on wound area (r = 0.59, р < 0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The correlation found can be attributed to the increase in extracellular MPO and NETs due to the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

Ischaemic stroke and the recanalization drug tissue plasminogen activator interfere with antibacterial phagocyte function

BackgroundStroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE.Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here.MethodsEx vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy.ResultsMPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release.r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA.ConclusionsIntracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies.r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.

Placental histology and neutrophil extracellular traps in lupus and pre-eclampsia pregnancies

ObjectiveSystemic lupus erythematosus (SLE) is associated with increased risk of adverse pregnancy outcomes, including pre-eclampsia, particularly in association with antiphospholipid antibody syndrome (APS). While significant placental abnormalities are expected in...

Impaired intracellular signaling, myeloperoxidase release and bactericidal activity of neutrophils from patients with alcoholic cirrhosis

Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP). Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists. fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release. We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

Oxidation of calprotectin by hypochlorous acid prevents chelation of essential metal ions and allows bacterial growth: Relevance to infections in cystic fibrosis

Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.

Effects of docosahexaenoic supplementation and in vitro vitamin C on the oxidative and inflammatory neutrophil response to activation.

We studied the effects of diet supplementation with docosahexaenoic (DHA) and in vitro vitamin C (VitC) at physiological concentrations on oxidative and inflammatory neutrophil response to phorbol myristate acetate (PMA). Fifteen male footballers ingested a beverage enriched with DHA or a placebo for 8 weeks in a randomized double-blind study. Neutrophils were isolated from blood samples collected in basal conditions at the end of nutritional intervention. Neutrophils were cultured for 2 hours at 37°C in (a) control media, (b) media with PMA, and (c) media with PMA + VitC. PMA induces neutrophil degranulation with increased extracellular myeloperoxidase and catalase activities, nitric oxide production, expression of the inflammatory genes cyclooxygenase-2, nuclear factor κβ, interleukin 8 and tumor necrosis factor α, and interleukin 6 production. DHA diet supplementation boosts the exit of CAT from neutrophils but moderates the degranulation of myeloperoxidase granules induced by PMA. VitC facilitates azurophilic degranulation of neutrophils and increases gene expression of myeloperoxidase induced by PMA. VitC and DHA diet supplementation prevent PMA effects on inflammatory gene expression, although together they do not produce additional effects. DHA diet supplementation enhances antioxidant defences and anti-inflammatory neutrophil response to in vitro PMA activation. VitC facilitates neutrophil degranulation but prevents an inflammatory response to PMA.

Modulating myeloperoxidase‐induced endothelial damage by a carbon monoxide‐releasing molecule, CORM‐3 (146.9)

Previous work has demonstrated that carbon monoxide releasing molecules (CORMs) suppress inflammation, however, the mechanisms are not well understood. Neutrophil (PMN)‐derived myeloperoxidase (MPO) is known to play a significant role in the pathophysiology of numerous inflammatory disorders through: 1) direct peroxidation and 2) producing hypohalous acids, (e.g. hypochlorous acid). Thus, an overwhelming MPO‐dependent production of oxidants in the sub‐endothelial space after PMN degranulation, contributes to tissue damage and propagating inflammation.We hypothesized that CORM‐3 interferes with MPO’s catalytic activity, and thereby mitigates endothelial inflammatory damage.In vitro assays were used to study MPO catalytic activity inhibition. Further, live cell in vitro assays were used to investigate endothelial damage through measuring reactive oxygen species (ROS) and cellular permeability.Our results indicate that CORM‐3 inhibits total MPO activity as well as MPO’s peroxidation and halogenation cycles. Cellular assays determined that ROS production and cellular permeability are decreased when extracellular MPO is treated with CORM‐3. In all assays inactivated CORM‐3 (iCORM‐3) was significantly less effective than CORM‐3.We conclude that CORM‐3 may mitigate inflammatory damage partly through inhibiting MPO activity.Grant Funding Source: Supported by Heart and Stroke Foundation, HSFO‐393 and LHRI IRF‐025‐09

Endogenous myeloperoxidase is a mediator of joint inflammation and damage in experimental arthritis.

Myeloperoxidase (MPO) is implicated as a local mediator of tissue damage when released extracellularly in many chronic inflammatory diseases. The purpose of this study was to explore the role of endogenous MPO in experimental rheumatoid arthritis (RA). K/BxN serum-transfer arthritis was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO(-/-) ) mice, and disease development was assessed. MPO activity was measured in joint tissues from mice with or without K/BxN arthritis. Collagen-induced arthritis (CIA) was induced in WT and MPO(-/-) mice, and disease development and immune responses were examined. MPO expression was assessed in synovial biopsy samples from patients with active RA, and the effect of MPO on synovial fibroblasts was tested in vitro. MPO was up-regulated in the joints of mice with K/BxN arthritis, and MPO deficiency attenuated the severity of the disease without affecting circulating cytokine levels. In CIA, MPO(-/-) mice had enhanced CD4+ T cell responses and reduced frequency of regulatory T cells in the lymph nodes and spleen, as well as augmented interleukin-17A and diminished interferon-γ secretion by collagen-stimulated splenocytes, without an effect on circulating anticollagen antibody levels. Despite enhanced adaptive immunity in secondary lymphoid organs, CIA development was attenuated in MPO(-/-) mice. Intracellular and extracellular MPO was detected in the synovium of patients with active RA, and human MPO enhanced the proliferation and decreased the apoptosis of synovial fibroblasts in vitro. MPO contributes to the development of arthritis despite suppressing adaptive immunity in secondary lymphoid organs. This suggests distinct effects of local MPO on arthritogenic effector responses.

Measuring myeloperoxidase activity in biological samples.

BackgroundEnzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.Principal findingsWe found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.ConclusionWe validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.

Modulating Myeloperoxidase‐Induced Endothelial Permeability by a Carbon Monoxide‐Releasing Molecule, CORM‐3

Previous work has demonstrated that CORM‐derived carbon monoxide (CO) supresses inflammation. However, the mechanisms are not well understood. Neutrophil (PMN)‐derived myeloperoxidase (MPO) is known to play a significant role in the pathophysiology of numerous inflammatory disorders through: 1) direct peroxidation and 2) producing hypohalous acids, particularly hypochlorous acid. Thus, an overwhelming MPO‐dependent production of oxidants in the sub‐endothelial space after PMN degranulation, contributes to tissue damage and propagating inflammation.We hypothesized that CORM‐3‐derived CO interferes with MPO's catalytic activity, and thereby mitigates endothelial inflammatory damage.In vitro assays were used to study the inhibition of MPO's catalytic activity. Further, live cell in vitro assays were used to investigate endothelial damage through measuring reactive oxygen species (ROS) and cellular permeability.Our results indicate that CORM‐3 inhibits total MPO activity as well as MPO's peroxidation and halogenation cycles. Cellular assays determined that ROS production and cellular permeability are decreased when extracellular MPO is treated with CORM‐3. In all assays inactivated CORM‐3 (iCORM‐3) was significantly less effective than CORM‐3.We conclude that CO may mitigate inflammatory damage partly through inhibition of MPO activity. Support: HSFO‐393 and LHRI IRF‐025–09

The role of myeloperoxidase and myeloperoxidase–antineutrophil cytoplasmic antibodies (MPO-ANCAs) in the pathogenesis of human MPO-ANCA-associated glomerulonephritis

It is well known that antineutrophil cytoplasmic antibodies (ANCAs) are pathogenic and have a diagnostic value for ANCA-associated vasculitis. We demonstrated that a rise in myeloperoxidase (MPO)-ANCA titers during remission is often predictive of a future relapse in MPO-ANCA-associated vasculitis. Pathological examination of renal biopsies indicated that not only MPO-ANCAs, but also extracellular MPO, an in situ immune complex composed of MPO and MPO antibodies, may play important roles in the pathogenesis of glomerular capillary injury in MPO-ANCA-associated vasculitis.