Myeloperoxidase Inhibitor Research Articles

The Potential Role of Matrix Metalloproteinases 8 and 9 and Myeloperoxidase in Predicting Outcomes of Bacterial Meningitis of Childhood.

Background Matrix metalloproteinases (MMPs) and myeloperoxidase (MPO) contribute to the inflammatory cascade in the cerebrospinal fluid (CSF) during bacterial meningitis. We determined levels of MPO, MMP-8, MMP-9, and tissue inhibitor of metalloproteinase- (TIMP-) 1 in the CSF of children with bacterial meningitis and investigated how these inflammatory mediators relate to each other and to the disease outcomes. Methods Clinical data and the diagnostic CSF samples from 245 children (median age eight months) with bacterial meningitis were obtained from a clinical trial in Latin America in 1996–2003. MMP-9 levels in the CSF were assessed by zymography, while MMP-8, MPO, and TIMP-1 concentrations were determined with immunofluorometric and enzyme-linked immunosorbent assays. Results MPO correlated positively with MMP-8 (rho 0.496, P < 0.001) and MMP-9 (rho 0.153, P = 0.02) but negatively with TIMP-1 (rho -0.361, P < 0.001). MMP-8 emerged as the best predictor of disease outcomes: a CSF MMP-8 concentration above the median increased the odds of death 4.9-fold (95% confidence interval 1.8–12.9). Conclusions CSF MMP-8 presented as an attractive prognostic marker in children with bacterial meningitis.

Inhibiting myeloperoxidase prevents onset and reverses established high-fat diet-induced microvascular insulin resistance.

A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.

Myeloperoxidase Molecular MRI Reveals Synergistic Combination Therapy in Murine Experimental Autoimmune Neuroinflammation.

Background Despite advances in immunomodulatory agents, most current therapies for multiple sclerosis target lymphocytes or lymphocytic function. However, therapy response may be less than optimal due to demyelination and axonal damage caused by myeloid cells. Purpose To determine if myeloperoxidase (MPO) molecular MRI can evaluate whether combination therapy targeting both lymphoid and myeloid inflammation can improve autoimmune neuroinflammation compared with either drug alone, even at suboptimal doses. Materials and Methods Four groups of 94 female mice (8-10 weeks old) were induced with experimental autoimmune encephalomyelitis (EAE) from August 2, 2016, to March 30, 2018, and divided into saline control (n = 22), 4-aminobenzoic acid hydrazide (ABAH) therapy group (n = 19), glatiramer acetate (GA) therapy group (n = 22), and combination therapy group (n = 31). Mice were administered suboptimal doses of ABAH, an irreversible inhibitor of MPO; GA, a first-line multiple sclerosis drug; both ABAH and GA; or saline (control). Mice were imaged with bis-5-hydroxytryptamide-diethylenetriaminepentaacetate gadolinium (hereafter, MPO-Gd) MRI. One-way analysis of variance, two-way analysis of variance, Kurskal-Wallis, and log-rank tests were used. P < .05 was considered to indicate statistical significance. Results The combination-treated group showed delayed disease onset (day 11.3 vs day 9.8 for ABAH, day 10.4 for GA, day 9.9 for control; P < .05) and reduced disease severity (clinical score during the acute exacerbation period of 1.8 vs 3.8 for ABAH, 3.1 for GA, 3.9 for control; P < .05). The combination-treated group demonstrated fewer MPO-positive lesions (30.2 vs 73.7 for ABAH, 64.8 for GA, 67.2 for control; P < .05), smaller MPO-positive lesion volume (16.7 mm3 vs 65.2 mm3 for ABAH, 69.9 mm3 for GA, 66.0 mm3 for control; P < .05), and lower intensity of MPO-Gd lesion activation ratio (0.7 vs 1.9 for ABAH, 3.2 for GA, 2.3 for control; P < .05). Reduced disease severity in the combination group was confirmed at histopathologic analysis, where MPO expression (1779 vs 2673 for ABAH, 2898 for GA; P < .05) and demyelination (5.3% vs 9.0% for ABAH, 10.6% for GA; P < .05) were ameliorated. Conclusion Myeloperoxidase molecular MRI can track the treatment response from immunomodulatory drugs even if the drug does not directly target myeloperoxidase, and establishes that combination therapy targeting both myeloid and lymphocytic inflammation is effective for murine autoimmune neuroinflammation, even at suboptimal doses. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Walczak in this issue.

Abstract 1514: Myeloid cell derived myeloperoxidase links cellular innate immunity to inhibition of NF-κB signaling in melanoma tumor cells

Abstract Myeloid-derived cells are considered the primary mediators of the cellular innate immune system where myeloperoxidase (MPO) is the major enzyme present in these cells. MPO is essential for fighting infections but little is known about the role of MPO in cancer. Using the murine melanoma cell line, B16F10, in an immune-competent orthotopic tumor model, tumor growth was compared in wild type (MPO+/+) and syngeneic MPO-deficient host (MPO-/-) mice. Survival studies demonstrated that B16F10 tumors grew slower in MPO+/+ animals (mean survival 27.4 days ± 4.5 days) compared to MPO-/- animals (mean survival 23.4 days ± 1.6 days; p ≤ 0.005). Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4ABAH), we pharmacologically mimicked the MPO-/- phenotype by continuously dosing MPO+/+ animals. B16F10 tumors grew faster in MPO+/+ animals treated with 4ABAH (mean survival 23.8 days ± 2.5 days; p ≤ 0.017). Utilizing intravital imaging with skinfold window chamber animal models, we evaluated in real-time the recruitment of MPO-expressing myeloid-derived cells during melanoma progression in MPO+/+ and MPO-/- animals. B16F10 tumor NF-κB signaling, and MPO-mediated activation from immune infiltrates were imaged simultaneously using a multi-spectral, multi-modal imaging strategy. Because NF-κB signaling is a central coordinator of the immune system and cancer development, the dynamics of NF-κB signaling in B16F10 tumor in vivo were assessed using a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase reporter enabling real-time bioluminescent imaging and quantitative monitoring of NF-κB transcriptional activation. Mean NF-κB transcriptional activation within the tumor compartment in MPO-/- mice was 42.2% ± 1.6% greater compared to MPO+/+ animals. Intravital microscopy demonstrated heterogeneous activation levels of NF-κB within tumor cells in MPO+/+ animals. To explore whether the spatial heterogeneity was a consequence of myeloid-derived cell distribution within the microenvironment, myeloid-derived cells were labeled in vivo using an i.v. injection of fluorophore-labeled dextran and fluorophore-labeled αGr1 antibody. Remarkably, tumor cells in contact with myeloid cells in vivo demonstrated decreased NF-κB transcriptional activation compared to tumor cells not in contact with myeloid cells. These in vivo studies demonstrate that MPO-expressing myeloid-derived cells suppress NF-κB transcriptional activation within B16F10 tumors in a tight spatially-localized and proximity-dependent manner where MPO, expressed broadly by myeloid-derived cells, contributes to host protection and decreased tumor progression. We demonstrate that MPO-expressing myeloid-derived cells function as an anti-tumor component of the cellular innate immune response during early melanoma progression in a NF-κB-dependent manner. Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloid cell derived myeloperoxidase links cellular innate immunity to inhibition of NF-κB signaling in melanoma tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1514.

Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner

Tuberculosis remains a big threat, with 1.6 million deaths in 2017, including 0.3 million deaths among patients with HIV. The risk of developing active disease increases considerably during an HIV coinfection. Alveolar macrophages are the first immune cells to encounter the causative agent Mycobacterium tuberculosis, but during the granuloma formation other cells are recruited in order to combat the bacteria. Here, we have investigated the effect of efferocytosis of apoptotic neutrophils by M. tuberculosis and HIV-coinfected macrophages in a human in vitro system. We found that the apo­ptotic neutrophils enhanced the control of M. tuberculosis in single and HIV-coinfected macrophages, and that this was dependent on myeloperoxidase (MPO) and reactive oxygen species in an autophagy-independent manner. We show that MPO remains active in the apoptotic neutrophils and can be harnessed by infected macrophages. In addition, MPO inhibition removed the suppression in M. tuberculosis growth caused by the apoptotic neutrophils. Antimycobacterial components from apoptotic neutrophils could thus increase the microbicidal activity of macrophages during an M. tuberculosis/HIV coinfection. This cooperation between innate immune cells could thereby be a way to compensate for the impaired adaptive immunity against M. tuberculosis seen during a concurrent HIV infection.

477-P: Inhibiting Myeloperoxidase (MPO) Prevents Onset and Reverses Established High-Fat Diet (HFD)-Induced Microvascular Insulin Resistance (IR)

HFD rapidly recruits neutrophils to insulin target tissues and induces microvascular IR. MPO is abundant in neutrophils, inhibits nitric oxide mediated vaso-relaxation, and associates with increased cardiovascular risk. AZD5904 (AstraZeneca) irreversibly inhibits MPO and is in clinical trials. MPO knockout, or chemical inhibition, improves glucose regulation in HFD-fed mice. Whether MPO affects muscle microvascular or metabolic IR in vivo is unknown. To test whether AZD5904 can prevent onset of HFD-induced vascular and metabolic IR, Sprague-Dawley rats were fed chow or a HFD (60% calories) for 2 weeks. HFD-fed rats received vehicle (n=8) or AZD5904 subcutaneously (n=10) via Alzet pump. Chow-fed rats (n=8) received vehicle alone. On day 15, we measured muscle microvascular perfusion in all rats by contrast-enhanced ultrasound, at 0, 60 and 120 minutes of a 2-hour euglycemic insulin clamp (3 mU/kg/minutes) and glucose disposal was determined. We next tested whether AZD5904 can reverse established vascular IR. Two groups of rats (n = 10, each) were fed the HFD for 2 weeks to induce vascular and metabolic IR, HFD was continued for 2 additional weeks while giving vehicle or AZD5904. Microvascular and metabolic insulin action was measured again using the insulin clamp. HFD for 2 weeks completely blocked insulin-mediated muscle capillary recruitment, decreased glucose utilization by 35%, and inhibited muscle Akt phosphorylation (P&amp;lt;0.05, for each measure). AZD5904 during HFD selectively and completely restored insulin-induced microvascular sensitivity (P&amp;lt;0.05). Furthermore, AZD5904 during the last 2 of 4 weeks of HFD feeding fully restored insulin-mediated microvascular perfusion (P&amp;lt;0.05) again without affecting glucose disposal. We conclude that inhibiting MPO selectively corrects vascular IR. This effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans. Disclosure E. Barrett: Research Support; Self; AstraZeneca. W. Chai: None. K.W. Aylor: None. L. Gan: Employee; Self; AstraZeneca. E. Michaëlsson: Employee; Self; AstraZeneca. Funding University of Virginia; AstraZeneca

Inhibition of MPO (Myeloperoxidase) Attenuates Endothelial Dysfunction in Mouse Models of Vascular Inflammation and Atherosclerosis.

Objective- Inflammation-driven endothelial dysfunction initiates and contributes to the progression of atherosclerosis, and MPO (myeloperoxidase) has been implicated as a potential culprit. On release by circulating phagocytes, MPO is thought to contribute to endothelial dysfunction by limiting NO bioavailability via formation of reactive oxidants including hypochlorous acid. However, it remains largely untested whether specific pharmacological inhibition of MPO attenuates endothelial dysfunction. We, therefore, tested the ability of a mechanism-based MPO inhibitor, AZM198, to inhibit endothelial dysfunction in models of vascular inflammation. Approach and Results- Three models of inflammation were used: femoral cuff, the tandem stenosis model of plaque rupture in Apoe-/- mice, and C57BL/6J mice fed a high-fat, high-carbohydrate diet as a model of insulin resistance. Endothelial dysfunction was observed in all 3 models, and oral administration of AZM198 significantly improved endothelial function in the femoral cuff and tandem stenosis models only. Improvement in endothelial function was associated with decreased arterial MPO activity, determined by the in vivo conversion of hydroethidine to 2-chloroethidium, without affecting circulating inflammatory cytokines or arterial MPO content. Mechanistic studies in Mpo-/- mice confirmed the contribution of MPO to endothelial dysfunction and revealed oxidation of sGC (soluble guanylyl cyclase) as the underlying cause of the observed limited NO bioavailability. Conclusions- Pharmacological inhibition of MPO is a potential strategy to limit endothelial dysfunction in vascular inflammation. Visual Overview- An online visual overview is available for this article.

Activated neutrophil carbamylates albumin via the release of myeloperoxidase and reactive oxygen species regardless of NETosis

Objective: An anti-carbamylated albumin antibody was detected in rheumatoid arthritis (RA) patients, and its presence was associated with serum myeloperoxidase (MPO) levels, as we reported previously. Since MPO is a key enzyme for carbamylation and is released by neutrophil extracellular traps (NETs), we aimed to demonstrate that NETosis induces carbamylation.Methods: Human neutrophils were isolated from a healthy donor, pre-treated with or without diphenyleneiodonium (DPI, an inhibitor for the generation of reactive oxygen species (ROS)), Cl-amidine (a peptidylarginine deiminase inhibitor), 4-aminobenzoic acid hydrazide (4-ABAH, an MPO inhibitor), or GW311616A (a neutrophil elastase (NE) inhibitor), and incubated for 8 h with or without phorbol 12-myristate 13-acetate (PMA). Proteins in the medium were collected and the carbamylation of albumin was evaluated by Western blotting.Results: The carbamylation of albumin was detected in the culture medium of activated neutrophils. NETosis was observed under the stimulation by PMA. DPI and 4-ABAH inhibited the carbamylation of albumin and NETosis. GW311616A inhibited NETosis, but not carbamylation. Neither carbamylation nor NETosis was inhibited by Cl-amidine.Conclusion: Activated neutrophils may carbamylate ambient albumin, and this is dependent on ROS and MPO, but does not require NETosis.

Targeting Plasminogen Activator Inhibitor‐1 Alleviates Inflammatory Responses Invoked by Sepsis or Chlorinated Lipid

Plasminogen activator inhibitor‐1 (PAI‐1) promotes leukocyte‐endothelium interactions and transmigration secondary to ischemia/reperfusion (I/R). Our previous work demonstrated that 2‐chlorohexadecanal (2‐ClHDA), derived from myeloperoxidase (MPO) produced hypochlorous acid (HOCL) targeting of plasmalogen phospholipids, may serve as a mediator of the inflammatory responses to sepsis. Given the fact that the septic inflammatory responses invoked by cecal ligation and puncture (CLP) can be reversed by an MPO inhibitor, we have postulated that sepsis‐induced inflammation may be mediated through MPO/HOCL/2‐ClHDA axis. However, the utility of drug targeting of PAI‐1 to inhibit inflammatory responses induced by CLP or 2‐ClHDA superfusion remains to be evaluated. We have begun to explore this issue by pretreating rats with PAI‐1 inhibitor, PAI‐039 (tiplaxtinin, 44 ug/kg) one hour prior to CLP or Sham surgery and then again one hour before beginning to take measurements. Six hours after surgery, inflammatory responses in four groups of Sprague‐Dawley rats (n=6) were quantified and compared on leukocyte‐endothelial cell interactions and mast cell degranulation in rat mesenteries using intravital microscopy. In other groups of rats pretreated with PAI‐039 one hour beforehand, the exposed mesenteries were superfused with 2‐ClHDA at 10 mM (a dose within the range of plasma concentrations reported for septic patients or rats) for ninety minutes, with inflammatory responses recorded at baseline, thirty, sixty and ninety minutes. The data showed that CLP or mesenteric superfusion with 2‐ClHDA induced marked increases in the numbers of rolling and firmly adherent leukocytes, and significant mast cell activation that were similar in magnitude in both models, and were prevented by pretreatment with the PAI‐1 inhibitor PAI‐039. Our results indicate that the increases in leukocyte rolling, leukocyte adhesion, and mast cell degranulation invoked by 2‐ClHDA superfusion mimicked the responses to CLP. Moreover, these proinflammatory responses appear to be dependent on PAI‐1.Support or Funding InformationSupported by NIH grant GM‐115553.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myeloperoxidase inhibition in mice alters atherosclerotic lesion composition.

Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture.

Propofol specifically reduces PMA-induced neutrophil extracellular trap formation through inhibition of p-ERK and HOCl

Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. AimsTo compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. Materials and methodsPMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. Key findingsThe neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. SignificanceThrough this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.

A versatile imaging platform with fluorescence and CT imaging capabilities that detects myeloperoxidase activity and inflammation at different scales.

Aberrant innate immune response drives the pathophysiology of many diseases. Myeloperoxidase (MPO) is a highly oxidative enzyme secreted by activated myeloid pro-inflammatory immune cells such as neutrophils and macrophages, and is a key mediator of the damaging innate immune response. Current technologies for detecting MPO activity in living organisms are sparse and suffer from any combination of low specificity, low tissue penetration, or low spatial resolution. We describe a versatile imaging platform to detect MPO activity using an activatable construct conjugated to a biotin moiety (MPO-activatable biotinylated sensor, MABS) that allows monitoring the innate immune response and its modulation at different scales and settings.Methods:We designed and synthesized MABS that contains MPO-specific and biotin moieties, and validated its specificity and sensitivity combining with streptavidin-labeled fluorescent agent and gold nanoparticles imaging in vitro and in vivo in multiple mouse models of inflammation and infection, including Matrigel implant, dermatitis, cellulitis, cerebritis and complete Fraud's adjuvant (CFA)-induced inflammation.Results: MABS MPO imaging non-invasively detected varying MPO concentrations, MPO inhibition, and MPO deficiency in vivo with high sensitivity and specificity. MABS can be used to obtain not only a fluorescence imaging agent, but also a CT imaging agent, conferring molecular activity information to a structural imaging modality. Importantly, using this method on tissue-sections, we found that MPO enzymatic activity does not always co-localize with MPO protein detected with conventional techniques (e.g., immunohistochemistry), underscoring the importance of monitoring enzymatic activity.Conclusion:By choosing from different available secondary probes, MABS can be used to create systems suitable to investigate and image MPO activity at different scales and settings.

Platelet Activation Is Induced &lt;i&gt;via&lt;/i&gt; Myeloperoxidase Production and Anti-Myeloperoxidase Antibodies Primed by Thrombin

In addition to their central role in hemostasis and thrombosis, platelets involve in inflammatory responses. Anti-neutrophil cytoplasmic autoantibodies (ANCAs) directed to myeloperoxidase (MPO-ANCAs) are the main serological markers of ANCA-associated vasculitis. The current study demonstrated that MPO-ANCAs caused normal human platelets to activate, primed by low doses of thrombin. Purified immunoglobulin G, MPO-ANCAs and their F(ab')2 fragments evidently increased the CD62P expression of platelets compared with controls. The existence of MPO in platelets was confirmed with mass spectrometry technology, after analyzing the F(ab')2 fragment pull-downed proteins. After thrombin priming, platelets not only expressed MPO on the surface with an activation-dependent manner, but also released the enzyme to extracellular space. These effects markedly enhanced the activation of platelets. Additionally, there was no apparently increased CD62P-expression on platelets in MPO-ANCAs group compared with controls when adding MPO inhibitor, proving that primed platelets have ANCA antigen at their surfaces interacting with the autoantibodies. These findings suggest a previously unappreciated character for platelets as immune cells and the autoantibodies-induced activation in an acute inflammatory disease. Funding: This study was supported by the grant from the National Key Research and Development Program (No. 2016YFC0906102), three grants from the National Natural Science Fundation of China (No. 81870478, 81425008 and 81621092) and the grant of University of Michigan Health System and Peking University Health Sciences Center Joint Institute for Translational and Clinical Research, and Program of Changjiang Scholar (BMU2017CJ002). Declaration of Interest: All the authors declared no competing interests. Ethical Approval: The study was in compliance with the Declaration of Helsinki and was approved by the ethics committees of Peking University First Hospital.

Mouse LIMR3/CD300f is a negative regulator of the antimicrobial activity of neutrophils

Leukocyte mono-immunoglobulin-like receptor (LMIR)/CD300 proteins comprise a family of immunoglobulin-like receptors that are widely expressed on the immune cell surface in humans and mice. In general, LMIR3/CD300f suppresses the inflammatory response, but it can occasionally promote it. However, the precise roles of LMIR3 in the function of neutrophils remain to be elucidated. In the present study, we investigated LMIR3 expression in mature and immature neutrophils, and evaluated the effects of LMIR3 deficiency in mouse neutrophils. Our results indicated that bone marrow (BM) neutrophils expressed LMIR3 on their cell surface during cell maturation and that surface LMIR3 expression increased in response to Pseudomonas aeruginosa infection in a TLR4/MyD88-dependent manner. LMIR3-knockout (KO) neutrophils displayed significantly increased hypochlorous acid production, and elastase release, as well as significantly augmented cytotoxic activity against P. aeruginosa and Candida albicans; meanwhile, inhibitors of elastase and myeloperoxidase offset this enhanced antimicrobial activity. Furthermore, LMIR3-KO mice were significantly more resistant to Pseudomonas peritonitis and systemic candidiasis, although this may not be entirely due to the enhanced activity of neutrophils. These results demonstrate that LMIR3/CD300f deficiency augments the antimicrobial activity of mouse neutrophils.

Trigonelline therapy confers neuroprotection by reduced glutathione mediated myeloperoxidase expression in animal model of ischemic stroke

AimStroke is devastating with a limited choice of intervention. Many pharmacological entities are available but none of them have evolved successfully in counteracting the multifaceted molecular alterations following stroke. Myeloperoxidase (MPO) has been reported to play an important role in neuroinflammation following neurodegenerative diseases. Therefore, using it as a therapeutic target may be a strategy to confer neuroprotection in stroke. Trigonelline (TG), a plant alkaloid has shown neuroprotective effects in the past. Here we explore its neuroprotective effects and its role in glutathione mediated MPO inhibition in ischemic stroke. MethodsAn in silico study was performed to confirm effective TG and MPO interaction. An in vitro evaluation of toxicity with biochemical estimations was performed. Further, in vivo studies were undertaken where rats were treated with 25, 50 and 100 mg/kg TG or standard MPO inhibiting drug4‑Aminobenzoic hydrazide (4‑ABH) at 60 min prior, post immediate and an hour post 90 min of middle cerebral artery occlusion (MCAo) followed by 24 h reperfusion. Rats were evaluated for neurodeficit and motor function tests. Brains were further harvested for infarct size evaluation, biochemical analysis, and western blot experiments. Key findingsTG at 100 mg/kg dose i.p. administered immediately post ischemia confers neuroprotection by reducing cerebral infarct with improvement in motor and neurodeficit scores. Furthermore, elevated nitrite and MDA levels were also found to be reduced in brain regions in the treated group. TG also potentiated intrinsic antioxidant status and markedly inhibited reduced glutathione mediated myeloperoxidase expression in the cortical brain region. SignificanceTG confers neuroprotection by reduced glutathione mediated myeloperoxidase inhibition in ischemic stroke.

Potent Triazolopyridine Myeloperoxidase Inhibitors.

Myeloperoxidase (MPO) generates reactive oxygen species that potentially contribute to many chronic inflammatory diseases. A recently reported triazolopyrimidine MPO inhibitor was optimized to improve acid stability and remove methyl guanine methyl transferase (MGMT) activity. Multiple synthetic routes were explored that allowed rapid optimization of a key benzyl ether side chain. Crystal structures of inhibitors bound to the MPO active site demonstrated alternate binding modes and guided rational design of MPO inhibitors. Thioether 36 showed significant inhibition of MPO activity in an acute mouse inflammation model after oral dosing.

34 - The role of myeloperoxidase in hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary disease of premature neonates. While ventilation with supplemental O2 is required for development and survival of the premature neonate, it increases the risk of BPD. Both hyperoxia-induced oxidative stress and transient hypoxia resulting from apneic events increase recruitment and activation of myeloid cells to the lungs. Activated myeloid cells release myeloperoxidase (MPO), which oxidizes chloride ions to hypochlorous acid causing oxidative damage and cell death. If myeloid cell recruitment, activation, and MPO release contribute to the pathogenesis of BPD, then inhibiting MPO oxidant production should protect against hyperoxia-induced lung injury. N-acetyl lysyltyrosylcysteine amide (KYC) is a tripeptide inhibitor of MPO that prevents the generation of toxic oxidants by shuttling high energy peroxy radicals in the iron-heme active site into the glutathione pathway. In order to test the hypothesis that inhibiting MPO prevents hyperoxia-induced lung damage, Sprague-Dawley neonatal rat pups were exposed to ten days (P1-P10) of hyperoxia (>90% O2) and injected twice daily with either 5 mg/kg KYC or vehicle control (PBS). KYC treatment reduced myeloid cell counts and MPO and Cl-Tyr levels in the lungs of hyperoxic neonatal pups. Additionally, KYC-treated pups had increased microvascular and alveolar complexity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels. These data provide evidence that myeloid cell recruitment and activation and MPO-induced oxidative damage are significant contributors to hyperoxia-induced lung injury in the developing lungs, and that KYC is an effective inhibitor of MPO-dependent oxidative damage to neonatal lungs.

35 - Neutrophils in sickle cell disease: the missing candidate – myeloperoxidase

Sickle cell disease (SCD) is caused by a single point mutation of the β-globin gene that increases the polymerization of hemoglobin under hypoxia and subsequent sickling of red blood cells (RBCs). Sickled RBCs occlude capillaries, inducing ischemic attacks in the lungs of affected individuals. Increased hemolysis and repeated bouts of ischemia-reperfusion injury lead to chronic states of oxidative stress and inflammation. Previously, we reported that sickle disease increases neutrophil activation and release of myeloperoxidase (MPO), which generates toxic oxidants that impair vascular function in SCD mice. In the present study, we determine if N-acetyl lysyltyrosylcysteine amide (KYC), a tripeptide inhibitor of MPO prevents toxic oxidant production and oxidative damage to sickle RBCs in SCD mice. KYC treatment of SCD mice reduced NO2-Tyr and Cl-Tyr, both markers of MPO-mediated damage, on sickle RBC membranes. Interestingly, the number of sickled RBCs in the lungs in KYC-treated SCD mice were reduced, which correlated with a reduction in the number of occluded blood vessels in the lungs of perfused SCD mice. KYC treatment also reduced MPO deposition, Cl-Tyr formation, and IgG deposition in the interstitium of the lungs of SCD mice. These data indicate KYC reduces oxidative damage to sickle RBCs which decreases sickling, vaso-occlusion, and ischemic injury of the lungs in sickle animals. KYC-dependent reductions in MPO-dependent oxidative damage to the lungs correlated with decreased vascular leakage in the lungs of SCD mice and reductions in SCD-related lung pathology. In conclusion, inhibitors that target MPO, such as KYC, may be an effective strategy for reducing vaso-occlusive events in SCD.

Advanced Glycated End Products Alter Neutrophil Effect on Regulation of CD4+ T Cell Differentiation Through Induction of Myeloperoxidase and Neutrophil Elastase Activities.

CD4+ T cell subset imbalance plays an important role in the development of diabetic complications. Neutrophils have recently been known as the regulator of CD4+ T cell differentiation. However, whether neutrophils affect CD4+ T cell population in diabetes is still elusive. In this study, we investigated the effect of neutrophils stimulated with advanced glycated end products (AGEs), the marker of diabetes, on CD4+ T cell differentiation and its underlying mechanism. Our data showed that the cultural medium of healthy adult neutrophils treated with AGEs increased expressions of both Th1 (IFN-γ) and Th17 (IL-17) phenotypes and the transcription factors of Th1 (Tbet) and Th17 (RORγt) in naive CD4+T cells and CD4+CD25+FoxP3+ (Treg) T cells in vitro. Next, we found that AGEs induced the generations of myeloperoxidase (MPO) and neutrophil elastase (NE) in neutrophils; inhibition of MPO or NE attenuated the effect of AGE-stimulated neutrophils on CD4+ T cell bias. Furthermore, receptor for AGEs (RAGE) inhibitor interrupted AGE-induced MPO and NE expressions, but MPO and NE inhibitions did not change AGE-increased RAGE gene expression. These results suggested that AGEs drive the effect of neutrophils on CD4+ T cell differentiation into pro-inflammatory program through inducing MPO and NE productions in neutrophils, which is mediated by AGE-RAGE interaction.

The soluble curcumin derivative NDS27 inhibits superoxide anion production by neutrophils and acts as substrate and reversible inhibitor of myeloperoxidase

A water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-β-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼107 M−1 s−1) but not for Compound II (∼103 M−1 s−1) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-β-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-β-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO.