Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents.

Heme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance. IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.

Oxidation of human plasma fibronectin by inflammatory oxidants perturbs endothelial cell function.

Dysfunction of endothelial cells of the artery wall is an early event in cardiovascular disease and atherosclerosis. The cause(s) of this dysfunction are unresolved, but accumulating evidence suggests that oxidants arising from chronic low-grade inflammation are contributory agents, with increasing data implicating myeloperoxidase (MPO, released by activated leukocytes), and the oxidants it generates (e.g. HOCl and HOSCN). As these are formed extracellularly and react rapidly with proteins, we hypothesized that MPO-mediated damage to the matrix glycoprotein fibronectin (FN) would modulate FN structure and function, and its interactions with human coronary artery endothelial cells (HCAEC). Exposure of human plasma FN to HOCl resulted in modifications to FN and its functional epitopes. A dose-dependent loss of methionine and tryptophan residues, together with increasing concentrations of methionine sulfoxide, and modification of the cell-binding fragment (CBF) and heparin-binding fragment (HBF) domains was detected with HOCl, but not HOSCN. FN modification resulted in a loss of HCAEC adhesion, impaired cell spreading and reduced cell proliferation. Exposure to HCAEC to HOCl-treated FN altered the expression of HCAEC genes associated with extracellular matrix (ECM) synthesis and adhesion. Modifications were detected on HCAEC-derived ECM pre-treated with HOCl, but not HOSCN, with a loss of antibody recognition of the CBF, HBF and extra-domain A. Co-localization of epitopes arising from MPO-generated HOCl and cell-derived FN was detected in human atherosclerotic lesions. Damage was also detected on FN extracted from lesions. These data support the hypothesis that HOCl, but not HOSCN, targets and modifies FN resulting in arterial wall endothelial cell dysfunction.

Cationic peroxidase from proso millet induces human colon cancer cell necroptosis by regulating autocrine TNF-α and RIPK3 demethylation.

A cationic peroxidase (POD) was purified from proso millet seeds (PmPOD) using ammonium sulfate fractionation, cation exchange, and size exclusion chromatography. The purified PmPOD showed toxicity to normal cells and tumor cells, but was more sensitive in HT29 cells. Furthermore, the mechanism driving HCT116 and HT29 cell death by PmPOD was the induction of receptor interacting protein kinase 1 (RIPK1)- and RIPK3-dependent necroptosis, independent of apoptosis. More importantly, PmPOD could induce tumor necrosis factor-α (TNF-α) production through transcriptional upregulation. In addition, PmPOD could restore RIPK3 expression in HCT116 cells via the demethylation of the RIPK3 genomic sequence. Taken together, these results suggest that two distinct mechanisms are involved in PmPOD-induced necroptosis: the autocrine production of TNF-α and the restoration of RIPK3 expression.

p-Nitrophenol toxicity to and its removal by three select soil isolates of microalgae: the role of antioxidants.

The nontarget effects, in terms of biochemical changes induced by p-nitrophenol (PNP) in three soil microalgae, Chlorella sp., Chlorococcum sp., and Heterochlamydomonas sp., and the PNP removal efficiency of these isolates, were determined. On exposure to 20 mg L(-1) PNP, Chlorella sp. showed greater activity of peroxidase, superoxide dismutase, and glutathione reductase as well as high contents of proline and carotenoids. While Heterochlamydomonas sp. exhibited higher levels of catalase and protein, Chlorococcum sp. produced greater amounts of malondialdehyde, a measure of lipid peroxidation, in the presence of PNP. Chlorella sp. tolerated PNP by producing large quantities of antioxidants coupled with less lipid peroxidation, while Chlorococcum sp. was susceptible, as evidenced by low antioxidant production and high lipid peroxidation. During 7-d exposure, Chlorella sp., Heterochlamydomonas sp., and Chlorococcum sp. were able to remove 39, 18, and 4% of 20 mg L(-1) PNP, respectively. The present results indicate that proline, carotenoids and malondialdehyde are the potential biomarkers for assessing PNP toxicity toward microalgae, and their response could be considered for differentiating tolerant and susceptible strains. Moreover, there is a clear correlation between PNP removal and antioxidant synthesis in microalgae on exposure to the pollutant.

Retinal pigment epithelium protection from oxidant-mediated loss of MMP-2 activation requires both MMP-14 and TIMP-2.

PURPOSE: Eyes with age-related macular degeneration (AMD) demonstrate accumulation of specific deposits and extracellular matrix (ECM) molecules under the retinal pigment epithelium (RPE). Metalloproteinases (MMP) are crucial regulators of basement membrane and ECM turnover. Accordingly, loss of RPE MMP activity most likely leads to excessive accumulation of collagen and other ECM, a potential mechanism for formation of deposits. A prior study showed that MMP-2 activity, but not pro-MMP-2 protein, decreases after RPE oxidative injury, indicating that oxidant injury disrupts the enzymatic cleavage of pro-MMP-2. Activation of MMP-2 requires the formation of a tri-molecular complex of pro-MMP-2, MMP-14, and tissue inhibitor of metalloproteinases (TIMP)-2. Therefore, a study was conducted to investigate the impact of oxidant injury on the interaction between these three molecules. METHODS: Human GFP-RPE cells were oxidant injured by transient exposure to H2O2 and myeloperoxidase, and the time course of recovery determined. Supernatants and cell lysates were collected for analysis of MMP-2, MMP-14, and TIMP-2 activity, mRNA and protein expression. In some studies, overexpression with either MMP-14 or TIMP-2 was performed to revert the cells to a preinjury phenotype. RESULTS: Transient injury resulted in a decrease of both MMP-14 and TIMP-2 activity and protein. Overexpression of each single molecule failed to prevent the injury-induced decrease of MMP-2 activity. In contrast, overexpression of MMP-14 together with the addition of exogenous TIMP-2 prevented the reduction of MMP-2 activation. CONCLUSIONS: Loss of MMP-2 activity after oxidant injury is caused by the downregulation of MMP-14 and TIMP-2. Overexpression of either MMP-14 or TIMP-2 alone before oxidant injury is not enough to prevent loss of MMP-2 activity. All three components of the tri-molecular complex must be present to preserve normal MMP-2 activity after oxidant injury.

Nonlethal oxidant injury to human retinal pigment epithelium cells causes cell membrane blebbing but decreased MMP-2 activity.

PURPOSE: This study was undertaken to determine whether transient or sustained nonlethal oxidant injury can induce RPE cell membrane blebbing and alter RPE expression of matrix metalloproteinase (MMP)-2 and type IV collagen, two molecules that are necessary for regulation of the turnover of the RPE basal lamina. METHODS: The ARPE-19 cell line stably expressing green fluorescent protein (GFP) targeted to the cell membrane was bleb injured by exposure to myeloperoxidase (MPO; 10 microunits) and H(2)O(2) (100 microM). Sustained (>6 hours) or transient (up to 6 hours) exposure to MPO/H(2)O(2) was evaluated. An MTS assay conversion and cell counts were used to detect cell viability. Supernatants and the cell homogenates were collected from cultured ARPE-19 to assess fluorescent GFP-derived blebs, MMP-2 protein by Western blot, MMP-2 activity by zymography, and type IV collagen accumulation by ELISA. Expression of MMP-2 was examined by real-time RT-PCR with total RNA. RESULTS: Both sustained and transient exposure of RPE cells to nonlethal oxidant injury upregulated blebbing and increased pro-MMP2 protein, but downregulated the MMP-2 activity released into the supernatant in a time-dependent manner. Only sustained oxidant injury for 24 hours induced an increase in collagen type IV. After removal of transient oxidant exposure, blebbing resolved and RPE MMP-2 activity and protein recovered to normal levels within 48 hours. CONCLUSIONS: Sustained or transient oxidant injury causes increased cell membrane blebbing but decreased activation of MMP-2. The findings lead to the hypothesis that blebs released in the absence of active MMP-2 may become trapped between the RPE and its basal lamina as sub-RPE deposits, possibly contributing to drusen formation in age-related macular degeneration. Also, the results lead to the postulation that oxidant injury disrupts the cell-specific surface proteases necessary to cleave and activate pro-MMP-2.

Oxygen in the alveolar air space mediates lung inflammation in acute pancreatitis.

During the early stages of acute pancreatitis, acute respiratory distress syndrome often occurs. This is associated with the release of proinflammatory mediators into the blood, but it remains unclear why these mediators induce inflammation especially in the lung. One of the first events occurring during the progression of acute pancreatitis is the induction of P-selectin expression in the endothelial cells of the lung. This expression has been associated with the generation of superoxide radicals by circulating xanthine oxidase. Because this enzyme needs molecular oxygen to perform the reaction, we have hypothesized that oxygen present in the alveolar space favors the generation of free radicals by xanthine oxidase and explains why P-selectin is expressed only in the lung. For this purpose, we evaluated the progression of the inflammatory process in rats with induced acute pancreatitis and one lung breathing nitrogen while the other lung continued breathing air. Acute pancreatitis was induced by intraductal administration of taurocholate and myeloperoxidase; P-selectin expression was measured 3 h after induction. Results indicated that, in the absence of oxygen in the alveolar space, the xanthine oxidase-dependent P-selectin expression did not occur and lung inflammation was significantly reduced.

Inhibition of PMN- and HOC1-induced vascular injury in isolated rabbit lungs by acetylsalicylic acid: a possible link between neutrophil-derived oxidative stress and eicosanoid metabolism?

Neutrophils are involved in the pathogenesis of acute lung injury. The neutrophil-derived enzyme myeloperoxidase (MPO) catalyzes the formation of the oxidant hypochlorous acid (HOCl). This study characterizes the effects of (A) continuous HOCl infusion, and (B) stimulated neutrophils on pulmonary circulation in an isolated rabbit lung model. Furthermore, the effect of cyclooxygenase inhibition by acetylsalicylic acid (ASA, 0.5 mM) on these effects was investigated. (A) Infusion of HOCl (in nmol min-1, groups: 0, 0+ASA, 1000, 1000+ASA, 2000, and 2000+ASA) into the isolated organ was started after a 45-min steady-state period (t=0). (B) Neutrophils (PMN group: 1480+/-323 and ASA group 1294+/-320 microliter-1) were added into the perfusate between (t=-45 min) and stimulated with FMLP (1 microM) after two 45-min steady-state periods (t=0). Perfusate MPO activity was measured at t=-90, -45, 0, 1, 2, 3, 5, 10, 15, 30, 60, and 90 min. For both groups, pulmonary artery pressure (PAP) and lung weight were continuously recorded and the capillary filtration coefficient (Kf,c in 10(-4) cm(3) s(-1) cm H2O(-1) g(-1) was calculated from the slope of weight gain after a hydrostatic challenge at t=-45, -15, 30, 60 and 90 min. (A) Continuous HOCl infusion (1000/2000 nmol min-1) evoked a significant increase in DeltaPAP and an up to 10-fold increase in Kf,c reaching the maximum extent of the observed effects significant earlier in the 2000 nmol min-1 group. ASA reduced DeltaPAPmax significantly to about 50% in corresponding groups and the increase in PAP and Kf,c occurred later in the ASA groups. (B) Neutrophil stimulation (PMN group/ASA group) evoked a rapid increase in DeltaPAP and MPO activity, while the changes in vascular permeability were rather moderate, but still significant. The release of MPO activity was similar in both groups. ASA significantly reduced the increase in DeltaPAP without affecting the release of MPO activity. Compared to baseline values, the preventive effects on vascular permeability increase reached level of significance as well. In summary, the described changes in pulmonary circulation caused by HOCl infusion or by neutrophil stimulation are significantly reduced by ASA. An involvement of cyclooxygenase products in the mediation of neutrophil-derived oxidative stress could be concluded.

Inhibition of tumor cell glutamine uptake by isolated neutrophils.

Antitumor activity of phorbol myristate acetate-(PMA) stimulated neutrophils was measured against CCRF-CEM cells. Neutrophils and tumor cells were incubated (a) as a suspension with continuous mixing to maximize the availability of oxygen or (b) after centrifugation as a pellet to maximize cell-cell contact. The cells were then incubated briefly as a suspension with [14C]glutamine under conditions that blocked further damage to the tumor cells. When cells were incubated as a suspension, inhibition of tumor-cell glutamine uptake was mediated by the myeloperoxidase/hydrogen peroxide/chloride system of stimulated neutrophils. Inhibition was blocked by adding catalase, an inhibitor of myeloperoxidase, or compounds that scavenge hypochlorous acid or chloramines. When cells were incubated as a pellet, a portion of the inhibition could not be blocked in this way, indicating that a nonoxidative mechanism contributed to inhibition. In both systems, inhibition of glutamine uptake was rapid and was obtained at effector-cell/target-cell ratios as low as 0.5:1. This inhibition was obtained under conditions that did not result in 51Cr release from cells labeled with [51Cr]-chromate, indicating that inhibition of glutamine uptake measured cytotoxicity rather than cytolysis. 51Cr release was observed only when cells were incubated together for an hour or more as a pellet at high E/T ratios. This cytolysis was mediated by the myeloperoxidase system, and a nonoxidative contribution to cytolysis was not observed. The results indicate that stimulated neutrophils are potent antitumor effectors cells when cytotoxicity rather than cytolysis is the measure of activity. Because glutamine is required for growth of many tumor cells, inhibition of glutamine uptake may represent a significant tumoristatic or tumoricidal effect.

Neutrophil-mediated nonoxidative tumor lysis stimulated by high concentrations of phorbol myristate acetate.

The mechanism of neutrophil-mediated lysis of tumor targets was investigated. Tumor lysis was directly related to the concentration of phorbol myristate acetate (PMA) used to stimulate PMNs. Lysis increased as the PMA concentration increased between 10(-7) and 10(-4) M. In contrast, the production of H2O2 plateaued between concentrations of 10(-5) and 10(-4) M. The K562 erythroleukemia cell, the target used in this study, was found to be relatively resistant to preformed H2O2, with an LD50 of 8.3 X 10(-3) M. Myeloperoxidase was not capable of enhancing K562 lysis. Although resistant to preformed H2O2, K562 lysis mediated by PMNs stimulated with 10(-7) M PMA was oxidative in nature. It was sensitive to inhibition by catalase and was not significant when PMNs from patients with chronic granulomatous disease were used. In contrast, PMN lysis stimulated by 10(-4) M PMA was nonoxidative in nature. The inhibitors catalase and superoxide dismutase had no effect on lysis, lysis was significant when the assay was performed in an anaerobic atmosphere, and PMNs from patients with chronic granulomatous disease were comparable to control PMNs in tumor lysis. A single-cell conjugate and cytotoxicity assay demonstrated that PMA was both able to increase the ability of PMNs to bind to tumor targets and to enhance their lysis of bound targets. These data indicate that PMNs are capable of achieving tumor lysis by nonoxidative pathways under certain conditions. The high-dose PMA model may be valuable as a tool for investigating these alternative mechanisms of tumor lysis.

The injurious effect of eosinophil peroxidase, hydrogen peroxide, and halides on pneumocytes in vitro.

Recent data suggest that eosinophils may cause lung injury. To determine if the eosinophil peroxidase (EPO)-hydrogen peroxide (H2O2)-halide system could mediate this injury, we added human EPO, H2O2 (or glucose and glucose oxidase as a continuous source of H2O2), and various halides to monolayers of 51Cr-labeled human A549 and rat type II pneumocytes. Cell lysis was measured as soluble 51Cr release. In initial experiments, EPO in solution did not induce lysis under these conditions. Therefore, in subsequent experiments, pneumocytes were preincubated with EPO for 15 minutes, washed to remove unbound enzyme, and then glucose, glucose oxidase, and the halides were added. EPO alone was not injurious, nor was the addition of glucose and glucose oxidase in the absence of EPO. In contrast, the combined addition of EPO, glucose, glucose oxidase, and chloride produced marked target-cell lysis. This effect was time and EPO dose dependent and was enhanced by the addition of iodide. Catalase and azide substantially inhibited the lysis produced by the EPO-H2O2-halide system, suggesting that EPO-catalyzed products of halide oxidation mediated this form of injury. Finally, the addition of eosinophil major basic protein at 10(-5) mol/L to EPO-coated pneumocytes incubated with glucose, glucose oxidase, and halides failed to enhance or inhibit lysis. We hypothesize that the EPO-H2O2-halide system may injure the lung in asthma and eosinophilic pulmonary syndromes.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

Oxidant membrane injury by the neutrophil myeloperoxidase system. II. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants.

The stimulated human neutrophil can damage a variety of target cells, and in some models, a mechanism involving secretion of myeloperoxidase and H2O2 has been demonstrated. We explored the characteristics of this cell-cell interaction by using neutrophils and our recently described liposome model target cell system. Exposure of 51Cr-labeled liposomes to phorbol myristate acetate-stimulated human neutrophils resulted in release of 25 to 30% of the radioactivity. 51Cr release was abrogated by omission of the neutrophils, the phorbol ester or halide (iodide), replacement of the phorbol by an inactive congener, or addition of azide, cyanide, or catalase. Neutrophils from patients with hereditary absence of myeloperoxidase (MPO) or a failure of H2O2 formation (chronic granulomatous disease) did not cause liposome lysis unless purified MPO or a source of H2O2, respectively, was added. These data indicate that 51Cr release from liposomes is a consequence of the secretion of MPO and H2O2, which combine with extracellular halides to form a membrane lytic system. The influence of liposome composition on injury was then examined, with a focus on physiologically relevant lipid soluble antioxidants. Liposomes containing either alpha-tocopherol (0.33 to 1.67% of molar fraction of lipid) or beta-carotene (1.67% of molar fraction of lipid) were markedly resistant to lysis by the cellfree MPO-H2O2-chloride system. When the major structural lipid phosphatidyl choline was replaced by dipalmitoyl phosphatidyl choline, a synthetic phospholipid with no oxidizable double bonds, the resultant liposomes were totally resistant to lysis by the MPO-H2O2-chloride system. The addition of iodide to this system (i.e., both chloride and iodide present) changed the pattern of protection dramatically in that alpha-tocopherol and beta-carotene were no longer protective and the resistance of dipalmitoyl phosphatidyl choline liposomes was partial rather than complete. In contrast to iodide, the addition of bromide or thiocyanate did not have a major effect on the protection by antioxidants. Finally, we demonstrated protection by alpha-tocopherol or dipalmitoyl phosphatidyl choline against liposome lysis by phorbol-activated neutrophils. These studies illustrate the use of model phospholipid membranes in the characterization of oxygen-dependent cell-mediated cytotoxicity. Activated neutrophils lyse liposome targets through a MPO-dependent mechanism. Target properties, especially the content of lipid-soluble antioxidants, have a marked influence on susceptibility to lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxic effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on Mycobacterium leprae.

Mycobacterium leprae are killed by myeloperoxidase (or eosinophil peroxidase), H2O2, and a halide, thus suggesting a mechanism for their destruction by peroxidase-containing phagocytes.