Myeloperoxidase FA
Myeloperoxidase is the major peroxidase in neutrophils and is responsible for the generation of reactive oxidants. This involves an oxidase cycle in which H2O2 is used to catalyze the conversion of MPO-Fe(IV) to hypohalous acid (HOCl), reactive halide species such as HOBr and HOSCN, and singlet oxygen [1,2,3,4,5].
These inflammatory oxidants are critical in the pathogenesis of several diseases, including cancer, atherosclerosis, renal disease, lung injury, and multiple sclerosis.
1. Activation of Neutrophils
In response to tissue-based inflammatory stimuli, polymorphonuclear neutrophils cross the vascular barrier to enter sites of inflammation. They also initiate the production of cytokines and other mediators that activate the immune system [110]. As such, MPO is one of the best prognostic markers of inflammation and oxidative stress in diseases such as tuberculosis, asthma, rheumatoid arthritis, chronic sinusitis, and other autoimmune disorders. It has also been implicated in a number of other health conditions, including ischemic heart disease and acute coronary syndromes.
Although MPO is widely known for its pathological role in inflammation, the molecular mechanisms regulating these effects are still not well understood. However, evidence is emerging for non-canonical functions of MPO, both ROS-generation dependent and independent.
To examine the ability of MPO to activate neutrophils, a variety of cellular and genetic approaches have been used. These include zymosan-stimulated neutrophils and mouse splenocytes. In addition, in vitro and in vivo experiments have shown that MPO deficiency increases proinflammatory cytokine production by phagocytes.
For example, zymosan-stimulated neutrophils from MPO-deficient mice secreted more CD11b (Figure 1A). These findings suggest that MPO may play an important regulatory role in regulating neutrophil activation and function under certain conditions, although the precise mechanism is unknown.
The activation of circulating neutrophils is a key aspect of immune function that requires a significant energy expenditure. The bone marrow MPO FA produces 2 x 1011 neutrophils per day, which represents a considerable metabolic investment. As such, MPO is a potential target for therapeutics that may limit excessive neutrophil phagocytosis and reduce the risk of recurrent infections.
MPO also has a direct impact on fibrosis and tumor development. In murine models of lung carcinogenesis, MPO KO mice were significantly smaller than WT animals, demonstrating that MPO may play a critical role in tumor growth and progression. Inhibition of MPO with N-acetyl lysyltyrosylcysteine amide reduced tumor size and prevented development of butylated hydroxytoluene-driven lung cancer.
In addition, MPO has a catalytic activity that allows it to oxidize aromatic compounds. This can exacerbate inflammatory and oxidative damage by producing reactive oxygen species (ROS) that are toxic to tissue cells. These ROS damage the DNA and proteins of a cell, which contributes to disease progression.
2. Reactive Oxygen Species (ROS)
The reactive oxygen species (ROS) produced by MPO FA are a vital component of the immune response against microorganisms. These ROS are involved in many biological processes, including signal transduction, hormone biosynthesis and destruction of intracellular pathogens. In addition, excessive levels of ROS can contribute to a variety of diseases and are associated with oxidative stress, which is often correlated with certain chronic conditions such as chronic granulomatous disease or infection-driven apoptosis in neutrophils [2,3,4,5].
The primary ROS species are the superoxide anion (O2*-), hydrogen peroxide (H2O2) and hydroxyl radical (HO*). These species are highly reactive due to an unpaired electron. H2O2 and HO* can react with organic substrates to form intermediates, which may be capable of producing other ROS subspecies. These include O2*-, hydroxyl radicals and singlet oxygen.
Reactive oxygen species can be generated in a number of ways, most notably by redox reactions in various cell organelles or by primary enzyme function. In macrophages, ROS production is largely mediated by the NADPH oxidase (NOX) system. It has been demonstrated that the absence of this enzyme in gp91phox-/- mice leads to the development of chronic granulomatous disease.
Another source of intracellular ROS is the mitochondrial oxidative phosphorylation (OXPHOS) complexes. Cytochrome C from these complexes has been shown to support caspase activation for neutrophil apoptosis, an alternative cell fate to autophagy/NETosis. However, the specific enzymatic pathways that support this process are still unknown.
ROS can be produced in a number of different ways, most notably by the xanthine/xanthine oxidase system. This oxidase system generates a series of O2*- species in the cytoplasm, which dismutate to produce H2O2, HO* and other ROS species.
Furthermore, the Fenton reaction of H2O2 by ferric iron (Fe3+) leads to a reduction of H2O2 to HO* and *OH. The resulting reactive intermediates can be further reduced to produce other ROS species by a number of enzymes, including myeloperoxidase and oxidative phosphorylation.
The primary roles of ROS in the phagocytic cell are to trigger the activation of neutrophils and induce apoptosis. While the production of ROS has been linked to a proinflammatory response, it is now known that ROS can play important roles in directing cellular signaling in favor of an antimicrobial outcome.
3. Inhibition of Neutrophil Migration
Myeloperoxidase (MPO) is a pro-inflammatory biomarker that contributes to the development of several diseases, such as rheumatoid arthritis, asthma, neurodegenerative disorders, diabetic retinopathy, liver disease and transplant rejection. MPO production is triggered by various stimuli, including cytokines/chemokines and activated neutrophils. The enzymatic activity of MPO involves the formation of reactive oxygen species (ROS), such as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals, and the nitrosylation of proteins and lipoproteins. In addition, MPO oxidizes various types of lipids and peptides, such as phosphatidylcholines, lipoproteins, and bile acids, to form various compounds that can cause tissue damage [26].
MPO is a cytotoxic molecule with multiple pro-inflammatory and protective activities. It enables the recruitment of neutrophils to inflamed sites MPO FA through different mechanisms that result in oxidative stress, cell death and tissue damage. It also modulates immune and inflammatory responses by inhibiting apoptosis and inhibiting the release of inflammatory cytokines.
Neutrophil migration is mediated by the interaction of MPO with the b2 integrin CD11b and is dependent on activation of chemokine/cytokine receptors or TLR9 and release of MPO from primary granules. In addition to facilitating the migration of neutrophils, MPO evokes activation and degranulation, generates survival cues, and inhibits neutrophil apoptosis through non-enzymatic actions on CD11b.
To evaluate the role of MPO in NET extrusion, MPO- and MPO-ANCA-primed C5a neutrophils were treated with ISO-1 to induce a respiratory burst and the release of oxygen radicals. After treatment with ISO-1, lactoferrin amounts in MPO- or PR3-primed neutrophils were significantly decreased compared to non-primed cells.
Moreover, MPO-primed MIF-primed neutrophils were found to have higher mean fluorescence intensity values and significantly elevated respiratory bursts in contrast to non-primed cells. These findings are consistent with the idea that MPO is an important mediator of the respiratory burst in neutrophils. In addition, MPO FA inhibits oxygen radical production in C5a-primed neutrophils treated with patient-derived ANCA-positive IgG (Fig. 1).
These results suggest that MPO may have an important function in the regulation of neutrophil trafficking and that a multistep event is required to allow neutrophil adhesion and migration into inflamed tissue. However, further research is needed to understand the underlying molecular mechanism.
4. Antimicrobial Activity
MPO FA has been shown to have a wide range of antimicrobial activity against different pathogens. These effects are mediated through the production of reactive oxygen species and other antimicrobial compounds, such as hydrogen peroxide, hypothiocyanate, and hydroxyl radicals, which kill bacteria through different mechanisms.
These reactive molecules are also known to have a strong influence on the progression of various inflammatory and oxidative stress-related diseases such as rheumatoid arthritis, diabetes, cardiovascular disease, and cancer. Moreover, they are the main antimicrobial compounds released by white blood cells during immune responses to foreign bodies or in response to tissue injuries and inflammation.
The activity of MPO FA is highly dependent on the cellular environment and the conditions at the site of infection. This is a key factor in its ability to elicit strong antimicrobial effects, especially against gram-positive bacteria such as Staphylococcus aureus and Staphylococcus pneumoniae. In addition, MPO FA can also be targeted by certain drugs and cytotoxic agents to kill cells and prevent them from becoming resistant to antimicrobial therapy.
In contrast, some other microorganisms produce MPO-inhibiting compounds that evade this innate defense mechanism and prevent them from being killed by MPO-mediated cytotoxicity. For example, Staphylococcus aureus has a protein called staphylococcal peroxidase inhibitor (SPIN) that binds directly to MPO and inhibits its action. This is a protective mechanism against MPO-mediated bacterial killing and has been shown to be effective in protecting the bacterium from further MPO-mediated cell death.
To explore the antimicrobial activity of ITC, we determined the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against representative Gram-positive and Gram-negative strains, including E. coli, P. aeruginosa, and S. aureus, by using broth microdilution assays. We found that ITC exhibited MICs in the range of 7.8-31.3 mg ml-1 and MBCs in the range of 31.3-250 mg ml-1, and that it was capable to elicit a bactericidal effect against all the test strains and to effectively eradicate mono- and dual-species biofilms.
We also performed a standardized quantitative suspension test to evaluate the influence of LPO on the lactoperoxidase-thiocyanate-hydrogen peroxide system’s antimicrobial effectiveness against Streptococcus mutans and Candida albicans and found that both thiocyanate and H2O2 were able to reduce bacterial and fungal growth, respectively, when the LPO concentration was above physiological levels. However, despite the significant increase in bacterial inhibition, the antimicrobial effectiveness of the combined ITC system was not significantly enhanced by LPO compared to that of the corresponding systems without this additional antimicrobial compound.