Epub ahead of print February 16, 2016 - doi:10.1189/jlb.3RU0615-256R

Review

Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems Jiansheng Huang,* Amber Milton,* Robert D. Arnold,* Hui Huang,† Forrest Smith,* Jennifer R. Panizzi,† and Peter Panizzi*,1 *Department of Drug Discovery and Development, Harrison School of Pharmacy; and †Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama, USA RECEIVED JUNE 16, 2015; REVISED DECEMBER 19, 2015; ACCEPTED JANUARY 11, 2016. DOI: 10.1189/jlb.3RU0615-256R

ABSTRACT Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis. J. Leukoc. Biol. 99: 000–000; 2016.

Abbreviations: 5-HT = 5-hydroxytryptamide, ADHP = 10-acetyl-3,7dihydroxyphenoxazine, APF = 2-[6-(49-amino)phenoxy-3H-xanthen-3-on9-yl]benzoic acid, BODIPY = boron-dipyrromethene, CPH = 1-hydroxy-3carboxy-2,2,5,-tetramethyl-pyrrolidine hydrochloride, DEPMO = 5(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide, DHR123 = dihydrorhodamine 123, DMPO = 5,5-dimethyl-1-pyrroline N-oxide, EC = endothelial cell, (continued on next page)

0741-5400/16/0099-0001 © Society for Leukocyte Biology

PHYSIOLOGIC IMPORTANCE OF MPO FUNCTION MPO, a heme-containing peroxidase in myeloid cells (PMNs and monocytes), has a central role in the development of the nascent inflammatory response and the perpetuation of chronic inflammation in diseases such as RA. From the initial trigger (i.e., damage or infection), patrolling monocytes [1], mast cells [2], and dendritic cells [3] respond to the acute injury almost immediately by increasing vascular permeability through histamine release and simultaneous establishment of chemokine and chemoattractant gradients required to arrest circulating leukocytes (see Fig. 1). A hallmark PMN burst occurs when responding leukocytes crawl to a slow roll through interaction of leukocytederived L-selectin and endothelial E-selectin and P-selectin [4]. In the mouse, PMN and other leukocytes essentially stop as the burden of the drag exerted on the cell overcomes blood flow pressures through, in part, binding of LFA-1 and Mac1 (CD11b) with newly exposed ICAM on the activated EC surface (ECICAM1). This progress occurs near the damage and just before diapedesis [5]. Once at the inflammatory site, PMN, and later PMN-signaled monocytes, begin the task of clearing foreign material (i.e., microbes) or cell debris [4]. To combat pathogens in the area, PMNs ensnare microbes through exocytosis of chromosomal material called neutrophil extracellular traps enabling phagocytosis of ensnared pathogens by neighboring PMNs [6, 7]. At the same time, there is probably an increased extracellular concentration of MPO in the local microenvironment as a direct result of MPO contributed by apoptotic PMNs that have sought to ensnare the microbes or debris in their chromosomal neutrophil extracellular traps. Because MPO makes up 30% of the total cellular PMN protein content, the resulting HOCl wave should effectively sanitize the area [8]. PMNs also experience an influx of K+ [9] and an increased pH [10], which awakens dormant proteases [11]. Given this, the engulfed pathogens ultimately face harsh pH conditions in the

1. Correspondence: Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, 3211-J Walker Building, Auburn, AL 36849, USA. E-mail: [email protected]

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Copyright 2016 by The Society for Leukocyte Biology.

atherosclerosis [17–21], and even some cancers [22, 23]. Therefore, MPO is considered a druggable target with small-molecule inhibitors already identified and more expected for limiting ROS/RNS production. In preclinical study, several MPO inhibitors have limited inflammation in diseases, including thioxanthine-2 in chronic obstructive pulmonary disease studies [15], PF-1355 in vasculitis and glomerulonephritis [24], and INV315 in atherosclerosis [25]. In this review, we will mainly discuss the recent, emerging tools for in vivo assessment of MPO as a target with the goal of monitoring disease progression, tracking PMN migration, and detecting inflammatory sites for preclinical or clinical applications.

WHAT SETS MPO APART FROM OTHER PEROXIDASES?

Figure 1. Biology of PMNs in response to injury and inflammation. Localized overexpression of E-selectin and P-selectin on activated endothelial cells slow the PMN roll upon the endothelium via leukocyte-derived L-selectin. Responding PMNs transmigrate through the endothelial cells after LFA-1 hooks intercellular adhesion molecule 1 (ICAM-1) and arrive at the site of damage just before diapedesis caused by the increased vascular permeability from histamine released from the mast cells. PMNs undergo phagocytosis of the invasive microbes once they arrive at the infection site. In addition, MPO is secreted from the patrolled PMNs to produce the potent antimicrobial reagent HOCl in response to infection.

vacuoles [12], degradation of cell wall components by PMN proteases (i.e., cathepsin G and elastase), and oxidation of DNA and proteins by the powerful HOCl oxidant generated by the prolific MPO-H2O2 system [13]. In humans, MPO has become a biomarker for heightened inflammation because of its role in the production of oxidized DNA and protein adducts [14]. For example, ROS and RNS production during oxidative stress has been linked to heightened MPO levels in chronic obstructive pulmonary disease [15], RA [16],

It is generally accepted that there are 2 distinct peroxidase superfamilies. One superfamily comprises all plant, fungal, and bacterial peroxidases, and the other comprises the mammalian peroxidases [26]. One primary difference between mammalian peroxidases (e.g., MPO, LPO, EPO, and thyroid peroxidase) and nonmammalian peroxidases (e.g., horseradish peroxidase, bacterial catalase-peroxidase, lignin peroxidase, and ascorbate peroxidase) is the presence of specific linkages between the respective heme prosthetic group and the main peroxidase protein scaffold [26]. There are generally 2 ester bond linkages, but in the case of MPO, the presence of an additional unique sulfonium linkage distorts the heme from its normal planar state and allows MPO to produce HOCl. Generally, all peroxidases have an active-site heme group that contains a central Fe atom that maintains its oxidation state (i.e., reactivity) through coordination with a distal His residue. MPO has a ferric heme, MPO-Fe(III), which is oxidized to a short-lived intermediate, termed Compound I (half life ;100 ms [27]), by reacting with a 100–10,000-fold lower relative concentration of H2O2, leading to generation of a ferryl porphyrin p cation radical, MPO-FEðIVÞ ¼ O þp (Equation 1) [28, 29]. MPO can regenerate its ferric state in 2 consecutive 1-electron steps with a classic peroxide electron donor (AH2) within a typical peroxidase catalytic cycle (Equations 2–3). The porphyrin radical is reduced to a ferryl heme, known as Compound II, in the first step (Equation 2). Compound II then is reduced back to ferric enzyme A by AH2 in the following step (Equation 3). At the same time, AH2 is oxidized to the free radical (AH ) [30, 31]. d

d

MPO2FeðIIIÞ1H2 O2 → Compound  I þ AH2 →

(continued from previous page) EPO = eosinophil peroxidase, EPR = electron paramagnetic resonance, Fe = iron, Gd = gadolinium, H2DCFDA = 29,79‑dichlorodihydrofluorescein diacetate, H2O2 = hydrogen peroxide, HC = heavy chain of myeloperoxidase, HOBr = hypobromous acid, HOCl = hypochlorous acid, HPF = 2-[6-(49hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, LPO = lactoperoxidase, MPO = myeloperoxidase, NO = nitric oxide radical, O22 = superoxide radical, 2 OCl = hypochlorite, OH = hydroxyl radical, ONOO2 = peroxynitrite, PDB = Protein Data Bank, PMN = polymorphonuclear neutrophil, RA = rheumatoid arthritis, ROS = reactive oxygen species, RNS = reactive nitrogen species d

d

2

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MPO2FeðIVÞ ¼ O Compound I

d

1p 

1H2 O

MPO 2 FeðIVÞ ¼ O þ AH Compound II

d

(1)

(2)

Compound  II þ AH2 →MPO 2 FeðIIIÞ þ AH þ H2 O

(3)

Compound  I þ Cl 2 →MPO 2 FeðIIIÞ þ HOCl

(4)

d

In the chlorination cycle of MPO, Cl2 ions are uniquely oxidized to HOCl by Compound I, and in the process, the MPO ferric state is regenerated (Equation 4).

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Huang et al. MPO and imaging neutrophil function

DETECTION OF MPO-DERIVED ROS/RNS IN ISOLATED CELLS AND IN VITRO ASSAYS To study MPO in any complex mixture, there must be an effective means to differentiate between MPO-dependent ROS/ RNS products and other reactive products. As such, a number of sensors have been developed to meet this need, such as OH, singlet oxygen, O22 , ONOO2, and 2OCl. A summary of these sensors is shown in Table 1. This section will focus on introducing most of these sensors, attributing unique properties that drive their utility, and imparting their relative limitations. Generally, the cellular redox homeostasis is maintained by a series of checks and balances between the production of ROS/ RNS and the antioxidant system, wherein superoxide dismutase produces H2O2 from O22 , and this is counteracted by the activity of catalase that degrades H2O2 into H2O. Other antioxidants act as general scavengers, such as NADPH, glutathione, thioredoxin, and peroxiredoxin [44], which further modulate the levels of certain oxidants. Up-regulated NADPH oxidases increase the relative O22 levels, contributing to certain pathologies, such as cardiac hypertrophy, fibrosis, ischemic stroke, and neurodegenerative diseases [45]. Central to this and important to the discussion here is that MPO functions to generate the potent oxidants HOCl from Cl2 and H2O2, while generating other ROSs (e.g., OH, singlet oxygen, and ONOO2) [46]. Given the relative importance of the redox state of the catalytic Fe in MPO, the use of EPR or spin resonance spectroscopy has become a sensitive method for in vitro assessment of the inhibitoror substrate-altered redox state [47]. Some probes for this application include DMPO for trapping O22 [48], DEPMPO for MPO and MPO-derived oxidants [49], 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine for the spin trapping of ONOO2 and O22 [48], CPH for stable trapping O22 [47], 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6tetramethylpiperidine for detection of mitochondrial O22 [50], and 3,5-dibromo-4-nitrosobenzenesulfonic acid for trapping NO , ONOO2, and O22 in biologic systems [51]. The distinct advantage of the CPH probe is that it generates a more-stable NO with a longer half-life, which provides for a better detection window, whereas the use of nitrone spin traps, such as DMPO and DEPMPO, produce the more-unstable intermediate O22 [47]. In addition, CPH probes are membrane permeable and can provide insights into the generation of ROS across both extracellular and intracellular membranes, allowing cytosol concentrations to be compared with those in the mitochondria [52]. There are also cell-permeable, fluorogenic ROS/RNS sensors, such as H2DCFDA and DHR123, which are oxidized by ROO , ONOO2, and 2OCl [53]. H2DCFDA is converted into 29,79dichlorofluorescin by intracellular esterases within the cell [54]. Apart from the relative nonselectivity to ROS species and oxidants, both cytochrome c and heme peroxidases catalyze the oxidation of 29,79-dichlorofluorescein and DHR123 [55]. NO is able to oxidize another fluorescein-based probe 4-amino-5-methylamino- 29,79difluorescein to the fluorescent dye benzotriazole via oxidative cycloaddition reactions [56, 57]. The hydroethidine-based probe mito-SOX can detect O22 in the mitochondria, whereas dihydroethidium can detect O22 generally within cells [58]. These d

Figure 2. Surface representation of the active site of the MPO monomer. PDB numbers: 3F9P, the light chain is shown in pink, and the heavy chain is shown in blue. There are 3 linkages between the heme and protein through Asp94 on the light chain and Glu242 and Met243 on the heavy chain (highlighted as yellow). The catalytically essential residues Gln91, His95, and Arg239 (highlighted as red) compose the distal heme pocket; in the proximal site, the heme iron is coordinated through the His336 imidazole residue (highlighted as turquoise).

Several MPO crystal structures have been solved that demonstrate complex formations between the MPO enzyme and a variety of ligands and inhibitors, which has provided a wealth of insight into MPO catalysis and inhibitor binding. Substrates gain access to the proximal surface of the cavern, wherein the heme prosthetic group acts as floor of the MPO active site. A narrower substrate-binding chase leads from the enzyme and can be seen in the MPO isoform C structure (see PDB accession number 1LXP), MPO cocrystallized with cyanide and thiocyanate (1DNW), the complex of MPO with cyanide (1DNU), the thioxanthine-inhibited MPO (3ZS1), the ceruloplasmin-bound MPO (4EJX), and the MPO with aromatic hydroxamates (4C1M) [32–38]. As mentioned, the protoporphyrin IX macrocycle of MPO has 3 covalent bonds with the parent protein as shown in Fig. 2, and the planar distortion of the heme leads to the distant spectral signature of MPO [26]. These linkages correspond to the following: 1) an ester bond between the light chain of the myeloperoxidase Asp94 residue of the MPO light chain and the methyl side chain of pyrrole C, 2) another ester bond between glutamate on the heavy chain (HCGlu242) and the methyl side chain of pyrrole A, and 3) a vinyl sulfonium linkage between a heavy chain methionine (HCMet243) and pyrrole A [26]. Classic biochemical studies [26, 39] have proven this vinyl sulfonium bond between MPO and the pyrrole A causes a distinct Soret (also known as an absorbance) band formation at 430 nm. Met243 variants of MPO have diminished peroxidase activity compared with native MPO, and sitedirected disruption of this linkage causes the Soret band at 430 nm to be lost in favor of a 413-nm band, similar to that in absorbance spectra of LPO [26, 39]. Other Soret bands are responsible for the green coloration of the MPO protein, namely bands at 496, 570, 620, and 690 nm [40]. Our recent study found that some potent MPO inhibitors, like benzoic acid hydrazide and its analogs, cause disruption of these important ester linkages [41, 42]. In mammalian peroxidase, these linkages are thought to impart a level of resistance to the powerful oxidants that they produce, namely HOCl and HOBr, for MPO and LPO, respectively [43].

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d

d

d

d

d

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Journal of Leukocyte Biology 3

TABLE 1. A summary of the sensors for detection of MPO-derived oxidants and other ROSs/RNSs

Modality/ Agent EPR DMPO DEPMPO

Excitation l (nm)

Emission l (nm)

113.2





235.2





MW (Da)

Cell culture (in vitro)

Preclinical (in vivo)

OH, O22

U

3

OH, O22

U

3

U

3

U

3

U

3

U

3

Target d

d

2

CMH

201.3





CPH

223.7





mito‑TEMPO‑H

511.1





ONOO , O22 ONOO2, O22 O22

367.0





NO , ONOO ,

423.4

490

515

d

HPF

424.4

490

515

d

DAF-FM

412.4

495

515

NO

H2 DCFDA HKGreen-3 mito-SOX

487.3 613.1 759.7

495 520 510

527 535 580

DHR123

346.4

500

512.2 315.4 257.2 640.0

DBNBS Fluorescence APF

2

d

O22 ,

MPO

OH, ONOO2, and HOCl/Br

U

3

OH, ONOO2

U

3

U

3

OH, ROO , ONOO , HOCl ONOO2 O22 , heme peroxidases

U U U

3 3 3

536

ONOO2, NO2 , HOCl

U

3

520 518 571 614

541 605 585 676

HOCl O22 , heme peroxidases Heme peroxidases HOCl

U U U U

3 3 U U

423.3 (oxazine)

620

672

ONOO2, HOCl

U

U

41,000 510.5

785 —

810 470

O22

OH, O22 and H2O2

U U

U U

177.2



425

U

U

L-012

288.7



425

U

U

MCLA

291.7



465

U

U

Pholasin

34,600



Blue light

OH, ONOO2, O22 , HOCl and HOBr OH, ONOO2, HOCl and HOBr O22 , MPO-derived oxidants, ONOO2 OH, ONOO2, O22 and MPO

U

U

N/A N/A

— —

800 620

MPO-derived HOCl O22 , and MPO-derived oxidants

U U

U U

420/500

535

H2O2

U

U

553.7 (IR775S)

680

838

ONOO2, HOCl

U

U

707.2, 788.7





MPO

U

U

HKOCl-1 DHE ADHP SNAPF Quenched nanoparticle Oxazine nanoparticle LS601R-PEG40 Lucigenin BLI Luminol

CRET Luminol-QD800 MCLA- BP-AF594 FRET HyPer sensor Photo-acoustic NIR light-absorbing RSPN1 MRI Gd-bis-5-HT-DTPA /Gd- 5-HTDOTA

52,000

d

×

d

2

d

d

d

d

d

Abbreviations: BLI, bioluminescence imaging; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine; CRET, chemiluminescence resonance energy transfer; DAF-FM, 4-amino-5-methylamino- 29,79-difluorescein; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid; DHE, dihydroethidium; DTPA, diethylenetriaminepentaacetic acid; SPN, semiconducting polymer nanoparticle; mito-TEMPO-H, 1-hydroxy-4-[2-triphenylphosphonio)acetamido]-2,2,6,6-tetramethylpiperidine; N/A, not applicable.

probes are not directly used for the detection of MPO-derived products but can be used to confirm the oxidant generated or present in a complex mixture. Other ROS, cell-permeable, fluorogenic probes include APF and HPF, which undergo O-dearylation oxidation to form highly 4

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fluorescence products in the presence of OH, ONOO2, and 2OCl [57, 59]. HPF can selectively detect the presence of OH and ONOO2, whereas 2OCl can also oxidize APF. MPO-derived HOCl and EPO-derived HOBr were specifically detected in PMNs and eosinophils by the combination of APF and HPF, respectively d

d

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Huang et al. MPO and imaging neutrophil function

[59, 60]. Other oxidation-mediated probes include HKGreen-3, which detects ONOO2 in vitro or in RAW 264.7 macrophages, generating fluorescent N-methylrhodol by an N–dearylation reaction [61]. Similarly, a BODIPY-based fluorescent probe, HKOCl-1, detects HOCl from the formation of benzoquinone from p‑methoxyphenol [61, 62]. A red-shifted fluorogenic sensor of peroxidase activity is ADHP, also known as Amplex Red, which has a strict H2O2 dependency, allowing H2O2 to be used as a trigger for inhibitor studies, whereby the enzyme and the compound can be allowed to reach equilibrium before initiation of the chemical reaction (Fig. 3A). Previously, we used stopped flow spectroscopy and the ADHP substrate to study MPO inhibition by benzoic acid hydrazide and its analogs [41, 42]. ADHP has also been used in tissue-based assays for determining the presence of MPO activity, as a surrogate for assessing inflammation [63]. There is evidence that ADHP can undergo autoradical formation under prolonged exposure to excitation energy [58]. The BODIPY-based fluorescent probe HKOCl-1, specific for the detection of MPO-derived HOCl, has never, to our knowledge, been used apart from the original reported study. Significant effort exists toward adapting these probes for use in intravital PMN recruitment and transmigration studies [64–66].

STRATEGIES FOR IMAGING MPO ACTIVITY IN PRECLINICAL MOUSE MODELS Biodistribution studies indicate that there are static spatial and temporal differences in expression of mammalian peroxidases throughout host cells and tissues. Mobile immune cells are also an excellent source of stored peroxidases because these enzymes are sequestered in granules, as is the case for MPO and EPO [67]. In response to stimuli, PMNs flood injured tissue with MPO to catalyze the production of the disinfectant HOCl. Quantification of MPO

levels can indicate overall inflammatory burden. Chronic inflammation leads to prolonged signal production and constant cellular recruitment, and, as such, both plasma and synovial fluids taken from patients with RA have increased MPO levels compared with healthy controls [16]. In addition, genetic production of MPO deficienctmice attenuated RA severity in mouse models without altering circulating cytokine levels [68], indicating that MPO is a critical mediator of joint inflammation and damage in experimental RA. Cytokine levels are not altered in these mice, which may show the lack of MPO-dependent feedback signaling, but that is conjecture at this stage. Although use of the aforementioned ROS/RNS probes seems compelling, their hydrodynamic and pharmacokinetic properties limit their utility in animal studies. Their narrow imaging window is driven by their enhanced washout kinetics and reduced circulation half-life. For example, solid tumors and regions of inflammation are known to be hyperpermeable to microparticles and nanoparticulates via enhanced permeability and retention properties [69]. Currently, there are already several in vivo imaging methods available, such as MRI of MPO-induced oligomerization of chelated Gd containing serotonin analogs [70], fluorescence imaging of MPO-induced release of oxazine probes from a nanoparticle scaffold [71], and MPO-induced chemiluminescence using luminol (Sigma-Aldrich, St. Louis, MO, USA) [72, 73]. Previously, bioluminescence imaging of MPO activity was successfully conducted in spontaneous, large, granular lymphocytic tumors in Gzmb:Tax mice with intraperitoneal administration of luminol [72]. On the surface, these results are not surprising because PMNs infiltrated these tumors. This “pseudo” in vivo Western blot is an interesting tool for studies of MPO activity but is limited to superficial sites because of photon scattering and the absorptive properties of blood and tissue [74]. This methodology is employed in Fig. 4. In this example, intraperitoneal injection of

Figure 3. Fluorescence reaction for measuring MPO activity. (A) General mechanism of ADHP oxidation in a proposed 2-step reaction, whereby the MPO–H2O2 complex generates 2 ADHP radicals that undergo a subsequent enzyme-independent dismutation reaction to complete formation of 1 resorufin and 1 ADHP molecule. (B) Classic biochemical assays are possible using absorbance changes caused by the MPO–H2O2 system and the use of new fluorogenic probes, such as ADHP. Pictures are cuvettes containing increasing concentrations of the MPO–H2O2 system, as indicated, at a static ADHP level (40 mM). (C) Stopped-flow progress curves of resorufin generation by MPO (23 nM) initiated by the addition of H2O2 (22 mM) for a series of given ADHP concentrations (adapted from Huang et al. [41]).

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luminol was used to detect MPO activity in inflammation sites associated with an acute cutaneous infection (Fig. 4B). This method has some limitations in that luminol has an extremely short half-life (20 min) in mice [72] and is 90% cleared by the kidney and excreted through the urine [75]. Another limitation is that this imaging method can only be used as a paninflammation-type sensor because it does not discriminate between sterile inflammation and active infection [76]. This process is highly H2O2-dependent, and without an optimal H2O2 concentration available to MPO, the light production is severely affected (Fig. 4C). To overcome the depth limitation of the luminol-based and normal fluorescent reflectance methods, hybrid techniques were developed that combines a near-infrared–emitting quantum dot, tethered to a light-production engine, by way of the MPO-mediated oxidation of the luminol payload [73]. This probe, in an inflammatory environment, allows for bioluminescence-resonance energy transfer to occur between the oxidized luminol and the probe. Another option available for in vivo probe sensors is to push to longer wavelengths to abrogate tissue interference, which was the case for the APF-derived, near-infrared dye sulfonaphthoaminophenyl fluorescein, used to assess HOCl production from human whole blood and advanced atherosclerotic plaques [77]. Previously, we were also able to modulate the poor pharmacokinetics of these small-molecule ROS/RNS probes by attaching an activatable, nearinfrared oxazine probe to the surface of an iron-oxide nanoparticle (increasing the half-life to ;9 h) [71]. In this case, the probe was oxidized and released from the particle only in the presence of HOCl or peroxynitrite [71]. Another report of similar conceptual design is the quenched nanoparticle LS601R-PEG40, a nonfluorescent

Figure 4. In vivo monitoring of MPO activity in mice. (A) Experimental design of the imaging study is shown. (B) A representative image of a mouse 6 d after s.c. injection of Streptococcus pyogenes (3 3 1010 CFU in phosphate-buffered saline) in the upper back of the animal shown in white light (right image) and bioluminescence imaging (left image). A yellow dotted circle indicates the region of interest. (C) An example of the hydrogen peroxide (H2O2)-dependence of MPO in vivo activity is shown. Albino C57BL/6 mice injected with 180 nM of MPO with either 400 mM of H2O2 (site 1), 2 mM of H2O2 (site 2), 4 mM of H2O2 (site 3), and 40 mM of H2O2 (site 4) were added to the matrigel (100 ml) before s.c. implant. (B–C) To visualize the MPO activity, an i.p. injection of luminol (Sigma-Aldrich, 3 mg/kg) was given 10 min before luminescence imaging using the IVIS Lumina XRMS system (PerkinElmer, Waltham, MA, USA). The Institutional Animal Care and Use Committee of Auburn University approved all animal procedures.

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hydrocyanine probe conjugated with PEG40, which can be oxidized to fluorescent LS601 in the presence of various ROSs [78]. Shifts are also being seen in the in vivo methods used to collect this ROS/RNS information, namely, the advent of the photoacoustic or optoacoustic imaging modalities. One such probe used in this way was IR775S, a cyanine dye derivative sensitive to ONOO2 and HOCl oxidation and used in a variety of inflammatory states [79]. Another option used in the preclinical setting is MRI, which allows for the capture of noninvasive, anatomic information, while gradient modulation can lead to differential relaxation profiles and probe visualization. An example of this is the MPO-dependent polymerization of 5-HT chelated to Gd, namely Gd-bis-5-HTdiethylenetriaminepentaacetic acid [70, 80]. As a polymer, the sensor has an enhanced magnetic resonance signal, which is prolonged up to 120 mins with the MPO-Gd probe in matrigel experiments, mice brains, and the infarct zone in mice [81–85]. This probe has been used extensively for in vivo inflammatory models that have been established in such a way as to prove the MPO-dependent nature of the reporter using MPO-deficient mice [81–83].

WHAT DOES THE FUTURE HOLD FOR MONITORING MPO ACTIVITY AND PMN RECRUITMENT NONINVASIVELY? New, emerging technology will allow for the development of better optical ROS/RNS sensors that take advantage of the spectrum of near-infrared wavelengths to limit potential interference and scatter from blood and tissue during in vivo animal imaging studies. To complement this, there are ongoing attempts to develop the next generation of probes for in vivo imaging of MPO, which, hopefully, will have better pharmacodynamic properties for in vivo work to improve the detectability of MPO by increasing the signal-to-noise ratio. Besides new probe chemistry, improved imaging hardware has already begun to provide better resolution for researchers using optical imaging, especially with the advent of photoacoustic/optoacoustic techniques [86–88]. Distinct from fluorescence, strong absorbing dyes are desired because the thermal expansion of the probe causes a resultant and proportional ultrasound signal [89, 90]. The benefit of these technologies over the standard reflectancefluorescence imaging is that they reduce the signal scattering as the light passes through tissue because sound waves are used instead of light for the later portion of the imaging strategy [91].

CONCLUDING REMARKS Imaging MPO activity and assessing the imaging of this important peroxidase remains an active area of research because of its central role in inflammation. In designing new MPO inhibitors, the physiologic role of the enzyme, the elements that lead to HOCl generation, the gamut and specificity of secondary ROS/ RNS reporter probes, and the lead compounds that ultimately cause toxicity in vivo must be understood. Often the best inhibitor for MPO is not the safest (e.g., sodium azide is a potent MPO inhibitor but toxic when applied to an animal). Taken together, a more-holistic approach can be implemented, which would provide a streamlined approach for screening positive traits to accelerate new anti-inflammatory drug designs.

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Huang et al. MPO and imaging neutrophil function

AUTHORSHIP J.H., A.M., J.R.P., and P.P. wrote this review. R.D.A., H.H., and F.S. edited the review and made critical comments throughout. J.H., A.M., and P.P. performed the experiments needed to demonstrate the luminol-based detection of MPO activity.

ACKNOWLEDGMENTS Supported, in part, by the U.S. National Institutes of Health through grants provided by the National Heart, Lung, and Blood Institute (Grant R01HL114477 to P.P.), the National Institute of Biomedical Engineering and Imaging (Grant R01EB EB016100 to R.D.A.), and Auburn University Research Initiative Cancer Graduate Fellowship (to J.H.).

DISCLOSURES

The authors declare no conflicts of interest.

REFERENCES

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KEY WORDS: bioluminescence noninvasive imaging neutrophil chemotaxis reactive oxygen species reactive nitrogen species •







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Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems.

Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we...
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