Changes in urinary glutathione sulfonamide (GSA) levels between admission and discharge of patients with cystic fibrosis.

There is an urgent need to develop sensitive, non-invasive biomarkers that can track airway inflammatory activity for patients with cystic fibrosis (CF). Urinary glutathione sulfonamide (GSA) levels correlate well with GSA levels in BAL samples and other markers of neutrophilic inflammation, suggesting that this biomarker may be suitable for tracking disease activity in this population. We recruited 102 children (median 11.5 years-old) and 64 adults (median 32.5 years-old) who were admitted to hospital for management of an acute pulmonary exacerbation and/or eradication of infectious agents such as Pseudomonas aeruginosa or Staphylococcus aureus. Our aim was to explore how urinary GSA levels changed across admission timepoints. Urine samples were collected at admission and discharge, and GSA measured by liquid chromatography with mass spectrometry. Paired admission-discharge results were compared using Wilcoxon signed-rank test. Paired admission-discharge samples were available for 53 children and 60 adults. A statistically significant difference was observed between admission-discharge for children and adults. Spearman's correlation analysis identified a correlation between urinary GSA levels and sex and S. aureus infection for children only. Our preliminary findings suggest that urinary GSA is responsive to the resolution of an acute pulmonary exacerbation and therefore warrants further studies in this population.

Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections.

Inside the phagosome: A bacterial perspective.

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.

Superoxide: The enigmatic chemical chameleon in neutrophil biology.

The burst of superoxide produced when neutrophils phagocytose bacteria is the defining biochemical feature of these abundant immune cells. But 50 years since this discovery, the vital role superoxide plays in host defense has yet to be defined. Superoxide is neither bactericidal nor is it just a source of hydrogen peroxide. This simple free radical does, however, have remarkable chemical dexterity. Depending on its environment and reaction partners, superoxide can act as an oxidant, a reductant, a nucleophile, or an enzyme substrate. We outline the evidence that inside phagosomes where neutrophils trap, kill, and digest bacteria, superoxide will react preferentially with the enzyme myeloperoxidase, not the bacterium. By acting as a cofactor, superoxide will sustain hypochlorous acid production by myeloperoxidase. As a substrate, superoxide may give rise to other forms of reactive oxygen. We contend that these interactions hold the key to understanding the precise role superoxide plays in neutrophil biology. State-of-the-art techniques in mass spectrometry, oxidant-specific fluorescent probes, and microscopy focused on individual phagosomes are needed to identify bactericidal mechanisms driven by superoxide. This work will undoubtably lead to fascinating discoveries in host defense and give a richer understanding of superoxide's varied biology.

A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.

Oxidation of bacillithiol during killing of Staphylococcus aureus USA300 inside neutrophil phagosomes.

Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low-molecular-weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH-deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase-derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms.

Formation of Calprotectin-Derived Peptides in the Airways of Children with Cystic Fibrosis.

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.

Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates large amounts of hydrogen peroxide (H2O2), which will be converted to hypothiocyanous acid (HOSCN) by lactoperoxidase (LPO) in the human respiratory tract. S. pneumoniae has been shown to be more resistant to HOSCN than some bacteria, and sensitizing S. pneumoniae to HOSCN may be a novel treatment strategy for combating this deadly pathogen. In this study we investigated the role of the low molecular weight thiol glutathione in HOSCN resistance. S. pneumoniae does not synthesize glutathione but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidized in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione (ΔgshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H2O2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δgor). This research highlights the importance of glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilization by S. pneumoniae may be a way to limit colonization and pathogenicity.

Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli. METHODS: Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay. RESULTS: Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA. CONCLUSION: Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.

Mycobacterium smegmatis Resists the Bactericidal Activity of Hypochlorous Acid Produced in Neutrophil Phagosomes.

Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothiol-deficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system.

Macrophage migration inhibitory factor inhibits neutrophil apoptosis by inducing cytokine release from mononuclear cells.

The chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) is a pivotal driver of acute and chronic inflammatory conditions, cardiovascular disease, autoimmunity, and cancer. MIF modulates the early inflammatory response through various mechanisms, including regulation of neutrophil recruitment and fate, but the mechanisms and the role of the more recently described MIF homolog MIF-2 (D-dopachrome tautomerase; D-DT) are incompletely understood. Here, we show that both MIF and MIF-2/D-DT inhibit neutrophil apoptosis. This is not a direct effect, but involves the activation of mononuclear cells, which secrete CXCL8 and other prosurvival mediators to promote neutrophil survival. Individually, CXCL8 and MIF (or MIF-2) did not significantly inhibit neutrophil apoptosis, but in combination they elicited a synergistic response, promoting neutrophil survival even in the absence of mononuclear cells. The use of receptor-specific inhibitors provided evidence for a causal role of the noncognate MIF receptor CXCR2 expressed on both monocytes and neutrophils in MIF-mediated neutrophil survival. We suggest that the ability to inhibit neutrophil apoptosis contributes to the proinflammatory role ascribed to MIF, and propose that blocking the interaction between MIF and CXCR2 could be an important anti-inflammatory strategy in the early inflammatory response.

Antimicrobial Activity of Neutrophils Against Mycobacteria.

The mycobacterium genus contains a broad range of species, including the human pathogens M. tuberculosis and M. leprae. These bacteria are best known for their residence inside host cells. Neutrophils are frequently observed at sites of mycobacterial infection, but their role in clearance is not well understood. In this review, we discuss how neutrophils attempt to control mycobacterial infections, either through the ingestion of bacteria into intracellular phagosomes, or the release of neutrophil extracellular traps (NETs). Despite their powerful antimicrobial activity, including the production of reactive oxidants such as hypochlorous acid, neutrophils appear ineffective in killing pathogenic mycobacteria. We explore mycobacterial resistance mechanisms, and how thwarting neutrophil action exacerbates disease pathology. A better understanding of how mycobacteria protect themselves from neutrophils will aid the development of novel strategies that facilitate bacterial clearance and limit host tissue damage.

Oxidation of bacillithiol by myeloperoxidase-derived oxidants.

Bacillithiol is a major low-molecular-weight thiol in gram-positive firmicutes including the human pathogen Staphylococcus aureus. Bacillithiol is regarded as an important defence mechanism against oxidants produced by the immune system, especially myeloperoxidase-derived hypochlorous acid (HOCl). However, it is unknown how fast BSH reacts with HOCl and what products are formed in the reaction. In the present study, we used sensitive MRM-based LC-MS methods to characterize the reaction of BSH with HOCl in cell-free solutions and in S. aureus. In the cell-free system, BSH formed predominantly the disulfide dimer (BSSB) at low mole ratios of HOCl and the sulfinic and sulfonic acids at higher oxidant concentrations. HOCl also promoted the formation of bacillithiol sulfonamide. In S. aureus, the oxidation pattern was similar except that a small proportion of BSH also formed mixed disulfides with protein thiols. Using competition with methionine, we determined the second-order rate constant for the reaction of HOCl with BSH to be 6 × 107 M-1s-1, which indicated a fast, near diffusion-controlled reaction. Other reactive halogen species, including hypothiocyanous acid (HOSCN), also produced bacillithiol sulfonamide, albeit to a smaller extent than HOCl. The sulfonamide was not produced by hydrogen peroxide, which instead formed BSSB. This study helps our understanding of BSH redox biology and provides tools for gauging the exposure of BSH-producing bacteria to oxidative stress.

Myeloperoxidase inhibition decreases morbidity and oxidative stress in mice with cystic fibrosis-like lung inflammation.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by severe bacterial infections, excessive neutrophilic inflammation and oxidative stress. The neutrophil enzyme myeloperoxidase (MPO), which produces hypochlorous acid, is associated with worse disease outcomes. Therefore, pharmacological inhibition of MPO in the airways has therapeutic potential. We investigated whether treating mice with an MPO inhibitor during pulmonary infection decreases oxidative stress and improves infection outcomes in mice with CF-like lung inflammation without impacting on bacterial clearance. METHODS: Transgenic ß-epithelial sodium channel (ßENaC)-overexpressing mice (n = 10) were infected with Burkholderia multivorans and treated twice daily with the MPO inhibitor AZM198 (125 µmol/kg) or vehicle administered by oral gavage for two days. Bodyweight was recorded daily. MPO activity, markers of oxidative stress, inflammatory cytokines and leukocytes numbers were measured in bronchoalveolar lavage fluid (BALF). Bacterial burden was determined in lung tissue homogenates. RESULTS: During the course of infection, mice treated with AZM198 lost less weight than vehicle-treated mice (p < 0.01). MPO activity and glutathione sulfonamide, a hypochlorous acid-specific glutathione oxidation product, were significantly lower in BALF from AZM198-treated mice (p < 0.05). The inflammatory cytokines CXCL1 and TNF-α in BALF and bacterial burden in the lung were not significantly different between treated and control mice. CONCLUSIONS: Orally administered AZM198 inhibits MPO activity in epithelial lining fluid. Blocking hypochlorous acid production in epithelial lining fluid during pulmonary infections through inhibition of MPO improves morbidity in mice with CF-like lung inflammation without interfering with clearance of bacteria. Pharmacological inhibition of MPO is an approach to limit destructive oxidative stress in cystic fibrosis lung disease in humans.

Ribose-cysteine protects against the development of atherosclerosis in apoE-deficient mice.

Ribose-cysteine is a synthetic compound designed to increase glutathione (GSH) synthesis. Low levels of GSH and the GSH-dependent enzyme, glutathione peroxidase (GPx), is associated with cardiovascular disease (CVD) in both mice and humans. Here we investigate the effect of ribose-cysteine on GSH, GPx, oxidised lipids and atherosclerosis development in apolipoprotein E-deficient (apoE-/-) mice. Female 12-week old apoE-/- mice (n = 15) were treated with 4-5 mg/day ribose-cysteine in drinking water for 8 weeks or left untreated. Blood and livers were assessed for GSH, GPx activity and 8-isoprostanes. Plasma alanine transferase (ALT) and lipid levels were measured. Aortae were quantified for atherosclerotic lesion area in the aortic sinus and brachiocephalic arch and 8-isoprostanes measured. Ribose-cysteine treatment significantly reduced ALT levels (p<0.0005) in the apoE-/- mice. Treatment promoted a significant increase in GSH concentrations in the liver (p<0.05) and significantly increased GPx activity in the liver and erythrocytes of apoE-/-mice (p<0.005). The level of 8-isoprostanes were significantly reduced in the livers and arteries of apoE-/- mice (p<0.05 and p<0.0005, respectively). Ribose-cysteine treatment showed a significant decrease in total and low density lipoprotein (LDL) cholesterol (p<0.05) with no effect on other plasma lipids with the LDL reduction likely through upregulation of scavenger receptor-B1 (SR-B1). Ribose-cysteine treatment significantly reduced atherosclerotic lesion area by >50% in both the aortic sinus and brachiocephalic branch (p<0.05). Ribose-cysteine promotes a significant GSH-based antioxidant effect in multiple tissues as well as an LDL-lowering response. These effects are accompanied by a marked reduction in atherosclerosis suggesting that ribose-cysteine might increase protection against CVD.

The nitroxide 4-methoxy-tempo inhibits the pathogenesis of dextran sodium sulfate-stimulated experimental colitis.

Inflammatory bowel disease (IBD) is a chronic condition characterised by leukocyte recruitment to the gut mucosa. Leukocyte myeloperoxidase (MPO) produces the two-electron oxidant hypochlorous acid (HOCl), damaging tissue and playing a role in cellular recruitment, thereby exacerbating gut injury. We tested whether the MPO-inhibitor, 4-Methoxy-TEMPO (MetT), ameliorates experimental IBD. Colitis was induced in C57BL/6 mice by 3% w/v dextran-sodium-sulfate (DSS) in drinking water ad libitum over 9-days with MetT (15 mg/kg; via i. p. injection) or vehicle control (10% v/v DMSO+90% v/v phosphate buffered saline) administered twice daily during DSS challenge. MetT attenuated body-weight loss (50%, p < 0.05, n = 6), improved clinical score (53%, p < 0.05, n = 6) and inhibited serum lipid peroxidation. Histopathological damage decreased markedly in MetT-treated mice, as judged by maintenance of crypt integrity, goblet cell density and decreased cellular infiltrate. Colonic Ly6C+, MPO-labelled cells and 3-chlorotyrosine (3-Cl-Tyr) decreased in MetT-treated mice, although biomarkers for nitrosative stress (3-nitro-tyrosine-tyrosine; 3-NO2-Tyr) and low-molecular weight thiol damage (assessed as glutathione sulfonamide; GSA) were unchanged. Interestingly, MetT did not significantly impact colonic IL-10 and IL-6 levels, suggesting a non-immunomodulatory pathway. Overall, MetT ameliorated the severity of experimental IBD, likely via a mechanism involving the modulation of MPO-mediated damage.

Exposure of Pseudomonas aeruginosa to bactericidal hypochlorous acid during neutrophil phagocytosis is compromised in cystic fibrosis.

Myeloperoxidase is a major neutrophil antimicrobial protein, but its role in immunity is often overlooked because individuals deficient in this enzyme are usually in good health. Within neutrophil phagosomes, myeloperoxidase uses superoxide generated by the NADPH oxidase to oxidize chloride to the potent bactericidal oxidant hypochlorous acid (HOCl). In this study, using phagocytosis assays and LC-MS analyses, we monitored GSH oxidation in Pseudomonas aeruginosa to gauge their exposure to HOCl inside phagosomes. Doses of reagent HOCl that killed most of the bacteria oxidized half the cells' GSH, producing mainly glutathione disulfide (GSSG) and other low-molecular-weight disulfides. Glutathione sulfonamide (GSA), a HOCl-specific product, was also formed. When neutrophils phagocytosed P. aeruginosa, half of the bacterial GSH was lost. Bacterial GSA production indicated that HOCl had reacted with the bacterial cells, oxidized their GSH, and was sufficient to be solely responsible for bacterial killing. Inhibition of NADPH oxidase and myeloperoxidase lowered GSA formation in the bacterial cells, but the bacteria were still killed, presumably by compensatory nonoxidative mechanisms. Of note, bacterial GSA formation in neutrophils from patients with cystic fibrosis (CF) was normal during early phagocytosis, but it was diminished at later time points, which was mirrored by a small decrease in bacterial killing. In conclusion, myeloperoxidase generates sufficient HOCl within neutrophil phagosomes to kill ingested bacteria. The unusual kinetics of phagosomal HOCl production in CF neutrophils confirm a role for the cystic fibrosis transmembrane conductance regulator in maintaining HOCl production in neutrophil phagosomes.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 µM, cross-linked calprotectin (5 µM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 µM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.