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Epigallocatechin-3-Gallate Inhibition of Myeloperoxidase and Its Counter-Regulation by Dietary Iron and Lipocalin 2 in Murine Model of Gut Inflammation

Open ArchivePublished:March 08, 2016DOI:https://doi.org/10.1016/j.ajpath.2015.12.004
      Green tea-derived polyphenol (−)-epigallocatechin-3-gallate (EGCG) has been extensively studied for its antioxidant and anti-inflammatory properties in models of inflammatory bowel disease, yet the underlying molecular mechanism is not completely understood. Herein, we demonstrate that EGCG can potently inhibit the proinflammatory enzyme myeloperoxidase in vitro in a dose-dependent manner over a range of physiologic temperatures and pH values. The ability of EGCG to mediate its inhibitory activity is counter-regulated by the presence of iron and lipocalin 2. Spectral analysis indicated that EGCG prevents the peroxidase-catalyzed reaction by reverting the reactive peroxidase heme (compound I:oxoiron) back to its native inactive ferric state, possibly via the exchange of electrons. Further, administration of EGCG to dextran sodium sulfate–induced colitic mice significantly reduced the colonic myeloperoxidase activity and alleviated proinflammatory mediators associated with gut inflammation. However, the efficacy of EGCG against gut inflammation is diminished when orally coadministered with iron. These findings indicate that the ability of EGCG to inhibit myeloperoxidase activity is one of the mechanisms by which it exerts mucoprotective effects and that counter-regulatory factors such as dietary iron and luminal lipocalin 2 should be taken into consideration for optimizing clinical management strategies for inflammatory bowel disease with the use of EGCG treatment.
      Green tea (Camellia sinensis, Theaceae) contains a plethora of antioxidant polyphenols that may promote better health.
      • Kim H.S.
      • Quon M.J.
      • Kim J.A.
      New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate.
      Among those polyphenols, the four major catechins (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin, (−)-epicatechin-3-gallate, and (−)-epicatechin are the most widely studied for their potential therapeutic properties. Indeed, EGCG, which accounts for about 40% of total polyphenol content in green tea, was reported to have preventive and therapeutic properties against a spectrum of diseases that include metabolic syndrome,
      • Bose M.
      • Lambert J.D.
      • Ju J.
      • Reuhl K.R.
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      • Yang C.S.
      The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.
      inflammatory bowel diseases,
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      Green tea polyphenols and sulfasalazine have parallel anti-inflammatory properties in colitis models.
      infectious diseases,
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      Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea.
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      Green tea epigallocatechin-3-gallate (EGCG) and other flavonoids reduce Alzheimer's amyloid-induced mitochondrial dysfunction.
      and cancers.
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      Turmeric and green tea: a recipe for the treatment of B-chronic lymphocytic leukemia.
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      Epigallocatechin gallate inhibits the proliferation of colorectal cancer cells by regulating Notch signaling.
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      A prospective phase II trial of EGCG in treatment of acute radiation-induced esophagitis for stage III lung cancer.
      Inflammatory bowel diseases (IBDs), including Crohn disease and ulcerative colitis, currently affect approximately 1.5 million individuals in the United States and 2.2 million individuals in Europe.
      • Ananthakrishnan A.N.
      Epidemiology and risk factors for IBD.
      In IBD, persistent elevated concentrations of proinflammatory cytokines, including IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF), cause neutrophils to continue infiltrating the intestinal mucosa, resulting in IBD flares and tissue damage.
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      • Parkos C.A.
      The role of neutrophils during intestinal inflammation.
      EGCG was shown to protect against weight loss, reduce histologic damage, and increase survival in the dextran sodium sulfate (DSS)-induced acute murine colitis model.
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      • Kucharzik T.
      • Lugering A.
      Green tea polyphenol epigallocatechin-3-gallate shows therapeutic antioxidative effects in a murine model of colitis.
      Likewise, long-term oral administration of EGCG was shown to reduce proinflammatory cytokines such as TNF, interferon γ, and IL-6 in the colonic mucosa of DSS-induced colitic mice.
      • Oz H.S.
      • Chen T.
      • de Villiers W.J.
      Green tea polyphenols and sulfasalazine have parallel anti-inflammatory properties in colitis models.
      However, the molecular mechanisms by which EGCG protects against gut inflammation are not completely elucidated.
      Neutrophils, the most abundant circulating white blood cells, are the first responder cell type during inflammation, infection, and injury. These cells infiltrate the intestinal mucosa to reach the site of insult, subsequently undergo respiratory burst, and release myeloperoxidase (MPO; E.C.1.11.2.2). MPO is a heme-containing enzyme that uses hydrogen peroxide (H2O2) and halide ions to generate hypohalous acid, a potent oxidizing and antimicrobial agent.
      • Klebanoff S.J.
      • Kettle A.J.
      • Rosen H.
      • Winterbourn C.C.
      • Nauseef W.M.
      Myeloperoxidase: a front-line defender against phagocytosed microorganisms.
      MPO is located within the neutrophils with the siderophore-binding innate immune protein called lipocalin 2 [(Lcn2; alias neutrophil gelatinase-associated lipocalin (NGAL)]. As part of the host response to inflammation, Lcn2 is released by both immune cells and intestinal epithelia,
      • Borregaard N.
      • Cowland J.B.
      Neutrophil gelatinase-associated lipocalin, a siderophore-binding eukaryotic protein.
      serving both as an epithelial growth factor
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      • Barasch J.
      Dual action of neutrophil gelatinase-associated lipocalin.
      and a chelator of bacterial siderophores, specifically enterobactin (Ent) with a high affinity (0.4 nmol/L).
      • Goetz D.H.
      • Holmes M.A.
      • Borregaard N.
      • Bluhm M.E.
      • Raymond K.N.
      • Strong R.K.
      The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.
      Thus, Lcn2 can deprive bacteria of iron and prevent their proliferation. Recently, we demonstrated that Ent is a potent inhibitor of MPO but loses its inhibitory properties when bound to Lcn2.
      • Singh V.
      • Yeoh B.S.
      • Xiao X.
      • Kumar M.
      • Bachman M.
      • Borregaard N.
      • Joe B.
      • Vijay-Kumar M.
      Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.
      Similar to Ent, EGCG was shown to reduce MPO activity in vitro
      • Nakano T.
      • Masuda M.
      • Suzuki T.
      • Ohshima H.
      Inhibition by polyphenolic phytochemicals and sulfurous compounds of the formation of 8-chloroguanosine mediated by hypochlorous acid, human myeloperoxidase, and activated human neutrophils.
      and to form complexes with iron and Lcn2 in vitro.
      • Bao G.H.
      • Xu J.
      • Hu F.L.
      • Wan X.C.
      • Deng S.X.
      • Barasch J.
      EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.
      However, a detailed mechanism of EGCG-mediated MPO inhibition, the extent to which iron- and host-derived Lcn2 influence EGCG bioactivity, and the in vivo functionality of these in vitro observations remain unknown.
      Iron is an essential trace metal that is required by both the host and the gut microbiota for their biological functions. Most circulating iron in the host are bound to proteins, although a small fraction remains unbound and is regarded as the labile or catalytic iron.
      • Kruszewski M.
      Labile iron pool: the main determinant of cellular response to oxidative stress.
      These unbound iron molecules are highly reactive and readily participate in Fenton reaction in which free ferrous iron (Fe2+) react with H2O2 to produce ferric iron (Fe3+).
      • Fenton H.J.H.
      Oxidation of tartaric acid in presence of iron.
      Fe3+ then reacts with H2O2 in the Haber-Weiss reaction to generate hydroxyl radicals and Fe2+ that again participate in the redox cycle.
      • Haber F.
      • Weiss J.
      Über die Katalyse des Hydroperoxydes (On the catalysis of hydroperoxide).
      The resulting cyclical overproduction of free radicals leads to increasing oxidative stress and damage, which contribute to disease such as IBD. Indeed, iron supplementation was shown to aggravate inflammation in murine models of colitis.
      • Oldenburg B.
      • Koningsberger J.C.
      • Van Berge Henegouwen G.P.
      • Van Asbeck B.S.
      • Marx J.J.
      Iron and inflammatory bowel disease.
      • Seril D.N.
      • Liao J.
      • Ho K.L.
      • Warsi A.
      • Yang C.S.
      • Yang G.Y.
      Dietary iron supplementation enhances DSS-induced colitis and associated colorectal carcinoma development in mice.
      Although EGCG was previously shown to be capable of binding and reducing Fe3+ to Fe2+,
      • Bao G.H.
      • Xu J.
      • Hu F.L.
      • Wan X.C.
      • Deng S.X.
      • Barasch J.
      EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.
      the effect of iron on the efficacy of EGCG in treating IBD remains unexplored.
      In this study, we report the potent inhibitory property of EGCG on two similar heme-containing peroxidases, MPO and lactoperoxidase (LPO; EC: 1.11.1.7)
      • Sharma S.
      • Singh A.K.
      • Kaushik S.
      • Sinha M.
      • Singh R.P.
      • Sharma P.
      • Sirohi H.
      • Kaur P.
      • Singh T.P.
      Lactoperoxidase: structural insights into the function, ligand binding and inhibition.
      in vitro and provide evidence to demonstrate that EGCG also inhibits MPO activity in vivo in the inflamed gut. The inhibitory properties of EGCG are abrogated in the presence of iron and/or when EGCG complexes with murine Lcn2 or its human ortholog NGAL. Spectral analysis with the use of LPO-H2O2 as a model system revealed that EGCG prevented the peroxidase-catalyzed reaction, suggesting that its antioxidative properties may be the underlying mechanism of EGCG-mediated MPO/LPO inhibition. Collectively, our study documents a novel in vivo mechanism for the protective bioactivity of EGCG during intestinal inflammation as being, at least in part, because of the inhibition of MPO, a process that is counter-regulated by iron and Lcn2.

      Materials and Methods

       Reagents

      Human MPO, human NGAL, and murine Lcn2 were procured from R&D Systems (Minneapolis, MN). EGCG was obtained from two sources: Sigma-Aldrich (St. Louis, MO) and Taiyo Green Power (Jiangsu, China). Ferric chloride, H2O2, and 4-aminobenzoic acid hydrazide (ABAH; a known inhibitor of MPO
      • Kettle A.J.
      • Gedye C.A.
      • Hampton M.B.
      • Winterbourn C.C.
      Inhibition of myeloperoxidase by benzoic acid hydrazides.
      • Kettle A.J.
      • Gedye C.A.
      • Winterbourn C.C.
      Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.
      ) were procured from Sigma-Aldrich. Bovine milk LPO was purchased from Worthington Biochemical Corp (Lakewood, NJ). Guaiacol (2-methoxyphenol) and ascorbic acid were obtained from Alfa Aesar (Ward Hill, MA). Reagent grade DSS (mol. wt. = 36,000–50,000; lot no. M8667) was obtained from MP Biomedicals, LLC (Solon, OH). Bleomycin sulfate was procured from Biotang (Lexington, MA). K12 Escherichia coli strain was acquired from The E. coli Genetic Stock Center, Yale University (New Haven, CT).

       Mice

      C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in-house in the animal facility at The Pennsylvania State University. All animal experiments were approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University.

       MPO and LPO in Vitro Assay

      Human MPO and bovine milk LPO were reconstituted with 0.1 mol/L potassium phosphate buffer (pH 6.0) and stored at −80°C. MPO (12.5 μg/mL, final concentration) or LPO (25 μg/mL, final concentration) were preincubated with various concentrations of EGCG with or without iron or Lcn2/NGAL for 5 minutes at room temperature in 20-μL reaction volume. The reaction was initiated on the addition of 30 μL of 2.2 mmol/L H2O2 and 50 μL of 100 mmol/L guaiacol. This assay is based on the principle that peroxidase oxidizes guaiacol in the presence of H2O2 to a chromophore 3,3′-dimethoxy-4,4′-biphenylquinone.
      • Capeillere-Blandin C.
      Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol transient species as a mediator of NADPH oxidation.
      The change in absorbance at 470 nm was measured at 1-minute intervals over a period of 10 minutes. The reactions were performed in 96-well plates (Corning, Corning, NY) in triplicate with appropriate vehicles throughout the study at pH 6.0 and 25°C. One unit of MPO or LPO activity is defined as the amount that increases absorbance at 470 nm by one per minute at 25°C, calculated from the initial rate of reaction with the use of guaiacol as the substrate. The final concentrations of MPO and LPO used were determined to be equivalent to 0.20 to 0.25 unit of activity.

       In Vitro Bacterial Killing Assay

      In vitro bacterial killing assay was performed as described by Atosuo and Lilius.
      • Atosuo J.T.
      • Lilius E.M.
      The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils.
      Briefly, the MPO reaction mixture was prepared by first adding 5 μg/mL MPO with or without EGCG at various concentrations in sterile phosphate-buffered saline (PBS). The concentration of chloride ion in PBS is estimated to be approximately 0.138 mol/L, which serves as the substrate required by MPO for the generation of hypohalous acid. Hydrogen peroxide (30 μmol/L) was then added to the reaction mixture and incubated at room temperature for 5 minutes. Finally, E. coli (K12 strain) grown overnight in Lysogeny broth media at 37°C with shaking at 250 rpm were added to the reaction mixture (2.0 × 106 colony-forming unit per mL). After 30 minutes of incubation at room temperature, the reaction mixture was serially diluted and plated on nonselective Lysogeny broth agar plates in triplicates. Plates were incubated overnight at 37°C, and bacterial colony-forming units on plates were quantified. All indicated concentrations above represent final system concentrations.

       Spectral Analysis

      LPO (1 mg/mL) was reconstituted in 500 μL of 0.1 mol/L phosphate buffer (pH 6.0) and incubated with either vehicle, 50 μmol/L EGCG, or 50 μmol/L ABAH for 5 minutes. The reaction was initiated at room temperature on the addition of 30 μmol/L H2O2. Spectra at 250 to 500 nm were recorded every 10 seconds with the use of CARY50BIO UV-Visible Spectrophotometer. Each spectrum represents an average of six scans taken in 1 second. All indicated concentrations above represent final system concentrations.

       CAS Assay

      Chrome Azurol S (CAS) agar plates were prepared according to protocol outlined by Schwyn and Neilands.
      • Schwyn B.
      • Neilands J.B.
      Universal chemical assay for the detection and determination of siderophores.
      CAS remains blue in color when complexed with iron but turns orange when iron is chelated by iron chelators/siderophores. Briefly, 1 μL of EGCG mixed with or without ferric chloride at indicated concentrations were placed on CAS plate and monitored for orange halo formation. Aferric Ent (Sigma-Aldrich) was used as a positive control.

       Fe2+-Dependent Bleomycin-Induced DNA Damage Assay

      The bleomycin assay was performed as outlined by Burkitt et al
      • Burkitt M.J.
      • Milne L.
      • Raafat A.
      A simple, highly sensitive and improved method for the measurement of bleomycin-detectable iron: the ‘catalytic iron index’ and its value in the assessment of iron status in haemochromatosis.
      with the following modifications. EGCG (1 mmol/L) or ascorbic acid (1 mmol/L) was added to a mixture that contained calf 50 μg/mL thymus DNA (Sigma-Aldrich), 50 mU/mL bleomycin sulfate, 0.1 mol/L Tris/HCl (Sigma-Aldrich), 5 mmol/L MgCl2·6H2O (Sigma-Aldrich) in water treated with Chelex 100 (Sigma-Aldrich). The reaction was initiated by adding the mixture to a series of Fe3+ concentrations prepared in 96-well solid black plate (Corning). For control, the mixture was prepared as above but without Fe3+. After incubation at 37°C for 1 hour, the reaction was stopped by adding 0.1 mmol/L EDTA (Sigma-Aldrich). The fluorescent DNA-intercalating agent ethidium bromide (0.1 mmol/L; Sigma-Aldrich) was added to the mixture. Fluorescence readings were then taken (excitation 510 nm; emission 590 nm). In principle, the conversion of Fe3+ to Fe2+ is detected as bleomycin-induced DNA damage that is proportional to the amount of available Fe2+. Damage to DNA corresponds to the loss in the ethidium bromide–enhanced fluorescent signal. Therefore, the percentage of DNA damage was calculated by comparing the ethidium bromide–enhanced fluorescence readings from the sample group with the control group. All indicated concentrations above represent final system concentrations.

       DSS-Induced Colitis in Mice

      In experiment 1, 8-week-old male C57BL/6J mice (n = 4) were administered 1.6% DSS in drinking water over a period of 7 days. The induction of colonic inflammation was confirmed via fecal occult blood, diarrhea, and loss in body weight. On day 6, mice were treated with 25 or 50 mg EGCG/kg body weight by oral gavage. On day 7, mice were given similar doses of EGCG and euthanized 3 hours later via carbon dioxide asphyxiation.
      In experiment 2, 8-week-old male C57BL/6J mice (n = 4) were treated with 1.6% DSS (in drinking water), and 5 mg EGCG/kg body weight by oral gavage once daily for 7 days. Mice were euthanized 3 hours after the last dose of EGCG.
      In experiment 3, 8-week-old male C57BL/6J mice (n = 4) were administered DSS as described above and then treated with either 50 mg EGCG/kg body weight alone or EGCG + iron [EGCG (50 mg/kg body weight) coadministered with iron in a molar ratio of 1:6] on day 6 and day 7 (24 and 3 hours before euthanasia, respectively).
      In all experiments, mice in the control groups received only regular drinking water (without DSS) and given PBS (vehicle) oral gavage. Blood samples were collected at the time of euthanasia in a BD microtainer (Becton Dickinson, Franklin Lakes, NJ), via retro-orbital plexus. Hemolysis-free serum samples were obtained after centrifugation and stored at −80°C until further use. Colons were flushed gently with PBS to remove fecal matter, weighed, and collected for MPO assay. Briefly, colons were homogenized in 0.5% cetyltrimethyl ammonium bromide in potassium buffer (pH 6.0), freeze-thawed three times, sonicated, and centrifuged.
      • Castaneda F.E.
      • Walia B.
      • Vijay-Kumar M.
      • Patel N.R.
      • Roser S.
      • Kolachala V.L.
      • Rojas M.
      • Wang L.
      • Oprea G.
      • Garg P.
      • Gewirtz A.T.
      • Roman J.
      • Merlin D.
      • Sitaraman S.V.
      Targeted deletion of metalloproteinase 9 attenuates experimental colitis in mice: central role of epithelial-derived MMP.
      Clear supernatant fluids were collected and used for MPO assay as described above.

       Cytokine Assays

      Fecal Lcn2, serum Lcn2, and serum keratinocyte-derived chemokine CXCL1 (KC) were measured by duoset enzyme-linked immunosorbent assay kits from R&D Systems according to the manufacturer's protocol.
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.

       Quantitative Reverse Transcription-PCR

      Mouse distal colons were collected in RNAlater (Sigma-Aldrich) and stored at −80°C. RNA was extracted from colon tissue with the use of Trizol reagent (Sigma-Aldrich) as described in the manufacturer's protocol. DSS is known to interfere with PCR reactions; therefore, we purified RNA with the use of the lithium chloride procedure as described in Viennois et al.
      • Viennois E.
      • Chen F.
      • Laroui H.
      • Baker M.T.
      • Merlin D.
      Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.
      Purified RNA (0.5 μg) was used to synthesize cDNA for quantitative real-time RT-PCR (RT-qPCR) with the use of SYBR green (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer's protocol. The following primers were used to assess gene expression: KC
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      forward, 5′-TTGTGCGAAAAGAAGTGCAG-3′, and reverse, 5′-TACAAACACAGCCTCCCACA-3′; Lcn2
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      forward, 5′-AAGGCAGCTTTACGATGTACAGC-3′, and reverse, 5′-CTTGCACATT-GTAGCTGTGTACC-3′; TNF
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      forward, 5′-ACTCCAGGCGGTGCCTATGT-3′, and reverse, 5′-AGTGTGAGGGTCTGGGCCAT-3′; nitric oxide synthase 2
      • Chen G.H.
      • Osterholzer J.J.
      • Choe M.Y.
      • McDonald R.A.
      • Olszewski M.A.
      • Huffnagle G.B.
      • Toews G.B.
      Dual roles of CD40 on microbial containment and the development of immunopathology in response to persistent fungal infection in the lung.
      forward, 5′-TTTGCTTCCATGCTAATGC-GAAAG-3′, and reverse, 5′-GCTCTGTTGAGGTCTAAAGGCTCCG-3′; and 36B4
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      forward 5′-TCCAGGCTTTGGGCATCA-3′, and reverse, 5′-CTTTATTCAGCTGCACATCACTCAGA-3′. 36B4 was used to normalize relative mRNA expression with the use of 2ΔΔCT method. Fold change was determined by comparison with the untreated control group.

       Histology and Immunohistochemistry

      After euthanasia, Swiss rolls of colons were fixed overnight in 10% buffered formalin solution and stored in 70% ethanol. Colons were processed for paraffin embedding, and serial paraffin sections (5 μm) were collected and stained with hematoxylin and eosin. For neutrophil staining, the paraffin-embedded colon sections were stained with rat anti-mouse antibody specific to neutrophil marker Ly6G (NIMP-R14; Abcam, Cambridge, UK) with the use of a diaminobenzidine peroxidase substrate kit from Vector Laboratories (Burlingame, CA) according to the manufacturer's instruction. An appropriate negative isotype control (rat IgG2b; Abcam) was included to omit nonspecific staining. To estimate neutrophil infiltration into the colonic mucosa, at least four images (size: 2592p × 1944p) of NIMP-R14–stained tissue were obtained at ×100 magnification. The images were analyzed with the use of ImageJ software version 1.49 (NIH, Bethesda, MD; http://imagej.nih.gov/ij), and a scale of 5 pixels/μm was set on the basis of the image legend. The mucosa layer was defined as the area between the muscularis mucosae and the luminal surface of the colon. Diaminobenzidine–NIMP-R14 positive-stained cells were counted and expressed as neutrophils/mm.
      • Bose M.
      • Lambert J.D.
      • Ju J.
      • Reuhl K.R.
      • Shapses S.A.
      • Yang C.S.
      The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.
      Histologic scoring on hematoxylin and eosin–stained colon sections was performed as in Cooper et al.
      • Cooper H.S.
      • Murthy S.N.
      • Shah R.S.
      • Sedergran D.J.
      Clinicopathologic study of dextran sulfate sodium experimental murine colitis.
      Briefly, colonic inflammation was graded for intestinal lesions, inflammation, mucosa, and percentage of affected area in the scale of 0 to 4, corresponding to the degree of increasing severity.

       Statistical Analysis

      All in vitro experimental results were reproduced in at least three independent experiments performed in triplicates. The in vivo mice experiments were individual studies with sample size of 4 mice. All values in the results are expressed as means ± SEM. The significance of difference between different groups was determined by one-way analysis of variance, followed by post hoc Sidak/Dunnet multiple comparison test in case of three or more groups and unpaired Student t-test in case of two groups. P < 0.05 was considered as significant. GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA) was used to calculate statistical significance.

      Results

       EGCG Inhibits Heme Peroxidases in a Dose-Dependent Fashion

      EGCG was previously demonstrated to exhibit antioxidant and anti-inflammatory properties,
      • Kim H.S.
      • Quon M.J.
      • Kim J.A.
      New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate.
      although the exact mechanisms underlying such properties are not clearly understood. In this study, we tested whether ECGC could affect the bioactivity of neutrophil MPO, a pro-oxidant enzyme associated with acute inflammation. ABAH, which is a known MPO inhibitor,
      • Kettle A.J.
      • Gedye C.A.
      • Hampton M.B.
      • Winterbourn C.C.
      Inhibition of myeloperoxidase by benzoic acid hydrazides.
      • Kettle A.J.
      • Gedye C.A.
      • Winterbourn C.C.
      Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.
      was used as the positive control. EGCG inhibited MPO activity in vitro in a dose-dependent manner (Figure 1A). EGCG also dose dependently inhibited the activity of LPO (Figure 1B), a heme-containing peroxidase with properties similar to MPO.
      • Sharma S.
      • Singh A.K.
      • Kaushik S.
      • Sinha M.
      • Singh R.P.
      • Sharma P.
      • Sirohi H.
      • Kaur P.
      • Singh T.P.
      Lactoperoxidase: structural insights into the function, ligand binding and inhibition.
      In addition, EGCG mediated its inhibition on MPO activity even after the MPO reaction was initiated (Figure 1C). The ability of EGCG to inhibit MPO and LPO activity was observed over a range of physiologically relevant pH (5.0 to 7.4; data not shown) and temperatures (25 to 40°C; data not shown).
      Figure thumbnail gr1
      Figure 1EGCG inhibits and LPO activity. A and B: EGCG or ABAH was preincubated at the indicated final concentrations with either 12.5 μg/mL MPO or 25 μg/mL LPO for 5 minutes in 20-μL reaction before adding 30 μL of 2.2 mmol/L H2O2 and 50 μL of 100 mmol/L guaiacol. Line graph represents EGCG dose-dependent inhibition of MPO (A) and LPO (B) activity. C: Line graph represents inhibition of MPO activity by 50 μmol/L EGCG or 50 μmol/L ABAH which was added at indicated time points after MPO activity was initiated. For the in vitro bacterial killing assay, indicated final concentrations of EGCG were preincubated with 5 μg/mL MPO for 5 minutes, and then 30 μmol/L H2O2 was added to the mixture. K12 Escherichia coli (2 × 106 CFU/mL) was added to the reaction mixture and incubated for 30 minutes at room temperature. D: Percentage of bacteria killing by MPO was based on bacteria that were serially diluted from the reaction, plated on nonselective Lysogeny broth plates, and quantified as bacterial colony-forming units after overnight incubation at 37°C. Results are expressed as means ± SEM. Assays were performed in triplicates and are representative of three independent experiments. P < 0.05. ABAH, 4-aminobenzoic acid hydrazide; EGCG, (−)-epigallocatechin-3-gallate; H2O2, hydrogen peroxide; LPO, lactoperoxidase; MPO, myeloperoxidase.
      The end product of MPO-catalyzed reaction, hypohalous acid, acts as a potent antibacterial agent by halogenating bacterial cell membrane components. To confirm that EGCG-mediated inhibition could reduce the generation of hypohalous acid by MPO, we measured the viability of K12 E. coli exposed to MPO + H2O2 in the presence or absence of EGCG. Our study found that EGCG dose dependently reduced MPO + H2O2-mediated bacterial killing compared with the control (Figure 1D), suggesting that EGCG could prevent cellular damage induced by MPO and its end products.

       Fe3+-Bound EGCG Fails to Inhibit Heme Peroxidase Activity

      Because EGCG is known to chelate iron,
      • Reznichenko L.
      • Amit T.
      • Zheng H.
      • Avramovich-Tirosh Y.
      • Youdim M.B.
      • Weinreb O.
      • Mandel S.
      Reduction of iron-regulated amyloid precursor protein and beta-amyloid peptide by (−)-epigallocatechin-3-gallate in cell cultures: implications for iron chelation in Alzheimer's disease.
      we next asked whether the presence of iron could affect the ability of EGCG to inhibit MPO and LPO. First, we tested whether the EGCG used in this study is iron free and could bind to iron. EGCG chelated iron from CAS plate which resulted in a distinct orange halo formation (Figure 2A). Ent was used as a positive control. We conducted this experiment with EGCG procured from two different sources, and the results were consistent (data not shown), confirming that the EGCG used in this study is iron free. However, this halo-forming effect was lost in the presence of increasing concentrations of ferric iron (Figure 2A). When EGCG was mixed with high concentration of ferric iron, we observed the presence of a black-colored precipitate in the solution and also on the CAS plate, suggesting that EGCG is capable of reacting with iron and likely converts Fe3+ to Fe2+ iron.
      • Nakagawa H.
      • Wachi M.
      • Woo J.T.
      • Kato M.
      • Kasai S.
      • Takahashi F.
      • Lee I.S.
      • Nagai K.
      Fenton reaction is primarily involved in a mechanism of (−)-epigallocatechin-3-gallate to induce osteoclastic cell death.
      To ascertain this possibility, we performed a modified bleomycin-detectable iron assay to evaluate the redox reactions of EGCG with iron. Ascorbic acid, as a reducing agent and a positive control, reduces ferric iron and induces oxidative DNA damage even at a low iron concentration. Compared with ascorbic acid, EGCG catalyzed the conversion of Fe3+ to Fe2+ only in the presence of higher Fe3+ concentration, beginning with the molar ratio EGCG:Fe3+ of 1:1 (Figure 2B). More importantly, the presence of EGCG:Fe3+ in the molar ratio of 1:5 and 1:10 significantly mitigated the ability of EGCG to inhibit MPO and LPO activity (Figure 2, C and D), which highlight ferric iron as a potential counter-regulator of EGCG function.
      Figure thumbnail gr2
      Figure 2Ferric iron-bound EGCG loses its ability to inhibit MPO and LPO activity. A: EGCG was preincubated with or without Fe3+ at indicated molar ratio and then 1 μL of the mixture was placed on CAS plate. Formation of orange halo indicates that EGCG chelated the iron from the CAS plate. Ent and FeCl3 were used as positive and negative control, respectively. In the bleomycin-detectable Fe2+ assay, EGCG or ascorbic acid (positive control) was incubated with indicated doses of Fe3+ in the reaction mixture for 1 hour at 37°C. B: Line graphs represent the conversion of Fe3+ to Fe2+, which was quantitated as Fe2+-dependent bleomycin-induced DNA damage. C and D: Bar graphs represent inhibition of MPO (C) and LPO (D) activity by 50 μmol/L EGCG that was preincubated with Fe3+ for 5 minutes at the indicated molar ratio before the assay was initiated. Results are expressed as means ± SEM. Assays were performed in a 96-well plate in triplicates and are representative of three independent experiments. P < 0.05. CAS, chrome Azurol S; EGCG, (−)-epigallocatechin-3-gallate; Ent, enterobactin; Fe2+, free ferrous iron; Fe3+, ferric iron; FeCl3 iron(III) chloride; LPO, lactoperoxidase; MPO, myeloperoxidase.

       Innate Immune Protein Lcn2 Prevents EGCG from Inhibiting Heme Peroxidases Activity

      One of the well-established functions of Lcn2 is to chelate the bacterial siderophore Ent, which is rich in phenolic groups.
      • Goetz D.H.
      • Holmes M.A.
      • Borregaard N.
      • Bluhm M.E.
      • Raymond K.N.
      • Strong R.K.
      The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.
      Because both MPO and Lcn2 are present in the neutrophils and are robustly expressed during intestinal inflammation,
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      we next asked whether Lcn2/NGAL can influence EGCG-mediated inhibition of MPO and LPO, especially because EGCG is also rich in phenolic groups. Human NGAL prevented EGCG from inhibiting MPO and LPO activity in a dose-dependent manner (Figure 3, A and B). Similar results were observed when the experiments were performed with Lcn2, which is the murine ortholog of the human NGAL (Figure 3, C and D).
      Figure thumbnail gr3
      Figure 3Human NGAL and its murine ortholog Lcn2 protect MPO and LPO from EGCG-mediated inhibition. EGCG (10 μmol/L) and indicated molar ratio of NGAL/Lcn2 were preincubated with either 12.5 μg/mL MPO or 25 μg/mL LPO for 5 minutes before the assay was initiated. A and B: Bar graphs represent inhibition of MPO (A) and LPO (B) activity by EGCG + NGAL at indicated molar ratio. C and D: Bar graph represents inhibition of MPO (C) and LPO (D) activity by EGCG preincubated with murine Lcn2 at indicated molar ratio. Results are expressed as means ± SEM. Assays were performed in a 96-well plate in triplicates and are representative of three independent experiments. P < 0.05. EGCG, (−)-epigallocatechin-3-gallate; Lcn2, lipocalin 2; LPO, lactoperoxidase; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin.

       EGCG Inactivates Heme Peroxidase-Catalyzed Reaction

      To elucidate the underlying mechanism of EGCG-mediated MPO/LPO inhibition, we performed spectral analysis of LPO-catalyzed reaction with H2O2 in the presence or absence of EGCG. Because of technical limitation and unavailability of MPO supplied at the amount required for experiments with spectral analysis, we therefore used LPO as our model system. Spectral analysis of LPO alone showed a λmax of 412 nm (Figure 4A), which is characteristic for the heme moiety in the enzyme.
      • Zederbauer M.
      • Furtmuller P.G.
      • Brogioni S.
      • Jakopitsch C.
      • Smulevich G.
      • Obinger C.
      Heme to protein linkages in mammalian peroxidases: impact on spectroscopic, redox and catalytic properties.
      The addition of EGCG or ABAH (positive control; known inhibitor of LPO and MPO
      • Kettle A.J.
      • Gedye C.A.
      • Hampton M.B.
      • Winterbourn C.C.
      Inhibition of myeloperoxidase by benzoic acid hydrazides.
      • Kettle A.J.
      • Gedye C.A.
      • Winterbourn C.C.
      Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.
      ) to LPO did not induce any change in the λmax, suggesting that EGCG and ABAH do not directly associate with the heme moiety of LPO or chelate heme Fe3+ (Figure 4, B and C). On the addition of H2O2 to LPO, the heme iron [Fe(III)] in LPO reacts with H2O2 to produce the LPO redox intermediate oxoiron [Fe(IV)+O] that has a λmax of 430 nm (Figure 4D). However, when H2O2 was added to LPO preincubated with EGCG, the λmax initially shifted from 412 nm to 430 nm, but then immediately reverted back to 412 nm (Figure 4E) in a fashion that is similar to that observed when ABAH was used instead of EGCG (Figure 4F). This suggests that the antioxidant EGCG is able to revert the redox reaction and to restore the native Fe(III) state to the heme of LPO, without directly interfering with the heme moiety of the peroxidase. After spectral analysis, we collected the reaction mixture and confirmed that the LPO incubated with EGCG is completely inactivated via LPO assay described previously (data not shown).
      Figure thumbnail gr4
      Figure 4EGCG prevents the formation of peroxidase-catalyzed hypohalous acid. To perform spectral analysis, 1 mg/mL LPO was incubated with either 50 μmol/L EGCG or 50 μmol/L ABAH for 5 minutes. Reaction was initiated at room temperature by adding 30 μmol/L H2O2. Spectra were recorded at 250 to 500 nm with each spectrum representing an average of six scans taken in 1.0 second. AF: Image represents LPO alone (A), LPO + EGCG (B), LPO + ABAH (C), LPO + H2O2 (D), LPO + EGCG + H2O2 (E), and LPO + ABAH + H2O2 (F). Arrows indicate the direction of spectral changes over time on the initiation of the reaction. Results are representative of three independent experiments. ABAH, 4-aminobenzoic acid hydrazide; EGCG, (−)-epigallocatechin-3-gallate; H2O2, hydrogen peroxide; LPO, lactoperoxidase.

       EGCG Inhibits MPO Activity in the Inflamed Gut of Colitic Mice

      We tested whether the EGCG-mediated inhibition of MPO in vitro could be replicated in the DSS-induced murine model of colitis. Treatment with EGCG (25 or 50 mg/kg body weight) dose dependently reduced colonic MPO activity compared with DSS-only control (Figure 5A). However, it is possible that the observed decrease in colonic MPO activity could be due to decreased neutrophil infiltration, secondary to other anti-inflammatory effects of EGCG. Hence, we examined the differences in neutrophil infiltration by immunohistochemistry. Although DSS-only treatment increased neutrophil infiltration compared with control, EGCG treatments did not significantly mitigate those effects (Figure 5, B and D) compared with the DSS-only treatment, demonstrating that the observed reduced colonic MPO activity was attributable to inhibition by EGCG and not due to changes in neutrophil infiltration. In this experiment, however, oral administration of only two doses of EGCG (24 and 3 hours before euthanasia) did not result in observable improvement in the inflamed colon histology (Figure 5C).
      Figure thumbnail gr5
      Figure 5EGCG inhibits colonic MPO activity in the inflamed gut of colitic mice. DSS-induced colitic mice were orally gavaged with 25 or 50 mg EGCG/kg bodyweight at 24 and 3 hours before euthanasia. Colons were collected and processed for MPO assay. AC: Bar graph represents the colonic MPO activity (A), neutrophil count (B), and colon histologic score (C). D: Images represent immunohistochemistry staining for neutrophil marker Ly6G. Results are expressed as means ± SEM. n = 4 DSS-induced colitic mice. P < 0.05. Original magnification: ×100 (D, upper row); ×400 (D, lower row, enlarged from corresponding boxed areas above.). DSS, dextran sodium sulfate; EGCG, (−)-epigallocatechin-3-gallate; MPO, myeloperoxidase.

       EGCG Attenuates DSS-Induced Colitis in Mice

      Iron chelator deferoxamine was previously demonstrated to induce increased expression and secretion of proinflammatory chemokine IL-8 by intestinal epithelial cells.
      • Choi E.Y.
      • Kim E.C.
      • Oh H.M.
      • Kim S.
      • Lee H.J.
      • Cho E.Y.
      • Yoon K.H.
      • Kim E.A.
      • Han W.C.
      • Choi S.C.
      • Hwang J.Y.
      • Park C.
      • Oh B.S.
      • Kim Y.
      • Kimm K.C.
      • Park K.I.
      • Chung H.T.
      • Jun C.D.
      Iron chelator triggers inflammatory signals in human intestinal epithelial cells: involvement of p38 and extracellular signal-regulated kinase signaling pathways.
      Hence, we tested whether EGCG, also an iron chelator, would affect the basal expression of inflammatory markers in the gut of healthy mice. We orally administered 50 mg EGCG/kg body weight to 8-week-old male C57BL/6 mice at 24 and 3 hours before euthanasia and then cultured colon sections ex vivo in incomplete Dulbecco's modified Eagle's medium for 24 hours. However, we observed no difference in the levels of secreted Lcn2 and KC (murine homolog of human IL-8) in the culture media when comparing the untreated and EGCG-treated group (data not shown), indicating that EGCG treatment does not affect basal expression of inflammatory markers in healthy mice.
      Having established that EGCG could inhibit MPO activity in vivo, we next asked whether EGCG can attenuate intestinal inflammation as well. Although treatment with daily doses of 5 mg EGCG/kg body weight for 7 days did not prevent weight loss in colitic mice (Figure 6A); the treatment prevented splenomegaly and colon shortening that are associated with DSS-induced colitis (Figure 6, B–D). These mice displayed a less severe colitis as reflected by the significantly reduced levels of colonic MPO activity, serum KC, serum Lcn2, and fecal Lcn2 (Figure 6, E–H), all of which are sensitive markers of intestinal inflammation.
      • Chassaing B.
      • Srinivasan G.
      • Delgado M.A.
      • Young A.N.
      • Gewirtz A.T.
      • Vijay-Kumar M.
      Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation.
      Further, the colonic expression for proinflammatory genes that encode KC, Lcn2, nitric oxide synthase 2, and TNF were significantly reduced in EGCG-treated colitic mice (Figure 6, I–L). However, we did not observe significant histologic changes between colon of DSS-only and EGCG-treated group (Figure 6, M and N).
      Figure thumbnail gr6
      Figure 6EGCG attenuates DSS-induced colitis in mice. DSS-induced colitic mice were orally gavaged with daily dose of either phosphate-buffered saline (vehicle) or 5 mg EGCG/kg body weight for 7 days and then euthanized 3 hours after the last EGCG dose. AL: The following variables were analyzed: body weight (A), gross colon (B), spleen weight (C), colon length (D), colonic myeloperoxidase activity (E), serum KC (F), serum Lcn2 (G), and fecal Lcn2 (H). Quantitative real-time RT-PCR analysis was used to quantify mRNA expression of KC (I), Lcn2 (J), NOS2 (K), and TNF (L). mRNA values are represented as fold-change normalized to 36B4 housekeeping gene. M and N: Colon histologic score (M) and hematoxylin and eosin staining of colon Swiss rolls at 100× (upper panel) and 400× (lower panel) magnification (N). Results are expressed as means ± SEM. n = 4 DSS-induced colitic mice. P < 0.05. Original magnification: ×100 (N, upper row); ×400 (N, lower row, enlarged from the boxed areas in the corresponding panels above). DSS, dextran sodium sulfate; EGCG, (−)-epigallocatechin-3-gallate; KC, keratinocyte-derived chemokine CXCL1; Lcn2, lipocalin 2; NOS2, nitric oxide synthase 2; TNF, tumor necrosis factor.

       Oral Administration of Iron-Saturated EGCG Fails to Inhibit MPO in Vivo in Acute Colitis

      Our in vitro results demonstrated that iron-saturated EGCG failed to inhibit MPO- and LPO-catalyzed reactions. We next examined whether this effect could be recapitulated in vivo by treating DSS-induced colitic mice with either vehicle, 50 mg/kg body weight EGCG alone, or EGCG + iron in 1:6 molar ratio on day 6 and day 7 of DSS treatment. Consistent with our earlier observations, mice receiving EGCG-alone treatment exhibited loss in body weight (Figure 7A) but displayed a substantial alleviation of gut inflammation with protection against splenomegaly and colon shortening (Figure 7, B–D) compared with DSS control, EGCG + iron, and the iron-alone treated groups. Notably, the ability of EGCG to reduce colonic MPO activity was abrogated when EGCG was coadministered with iron (Figure 7, E and F). In addition, only the EGCG-alone group exhibited reduced levels of serum KC, serum Lcn2, fecal Lcn2, and reduced colonic expression of proinflammatory genes (encoding KC, Lcn2, and TNF), but this was not observed in the EGCG + iron group, which further confirmed that the presence of iron could counter-regulate the efficacy of EGCG treatment (Figure 7, G–L). However, we again did not observe EGCG-mediated amelioration of colitis at the histologic level (Figure 7, M and N), despite the significant reduction in markers associated with colitis (Figure 5, Figure 6 and 7). Such discrepancies are likely due to the rather low doses of EGCG used in this study.
      Figure thumbnail gr7
      Figure 7Oral administration of iron-saturated ECGC fails to mediate MPO inhibitory activity in vivo in mice with DSS-induced acute colitis. DSS-induced colitic mice were orally gavaged on day 6 and 7 (24 and 3 hours before euthanasia, respectively) with either phosphate-buffered saline (vehicle), 50 mg/kg body weight EGCG alone, or EGCG + iron (50 mg/kg bodyweight with iron co-administered at EGCG:iron molar ratio of 1:6). Control mice received only regular drinking water. AI: The following variables were analyzed: body weight (A), gross colon (B), spleen weight (C), colon length (D), MPO activity at the proximal colon (E) and distal colon (F), serum KC (G), serum Lcn2 (H), and fecal Lcn2 (I). Quantitative real-time RT-PCR analysis was used to quantify mRNA expression of KC (J), Lcn2 (K), and TNF (L). mRNA values are represented as fold-change normalized to 36B4 housekeeping gene. M and N: Hematoxylin and eosin staining of colon Swiss rolls (M) and their corresponding histologic score (N). Results expressed as means ± SEM. n = 4 DSS-induced colitic mice. P < 0.05. Original magnification: ×100× (M, upper row); ×400 (M, lower row). DSS, dextran sodium sulfate; EGCG, (−)-epigallocatechin-3-gallate; KC, keratinocyte-derived chemokine CXCL1; Lcn2, lipocalin 2; MPO, myeloperoxidase; TNF, tumor necrosis factor.

      Discussion

      Elevated levels of proinflammatory mediators and oxidative stress play a key role in the pathology of IBD. Numerous naturally occurring plant-derived polyphenols were shown to alleviate gut inflammation in IBD patients and animal models of colitis.
      • Furst R.
      • Zundorf I.
      Plant-derived anti-inflammatory compounds: hopes and disappointments regarding the translation of preclinical knowledge into clinical progress.
      One such plant-derived compound, EGCG, has been widely studied for its possible anticolitogenic and antioxidant properties. Even though EGCG was shown to negatively affect MPO activity in vitro,
      • Nakano T.
      • Masuda M.
      • Suzuki T.
      • Ohshima H.
      Inhibition by polyphenolic phytochemicals and sulfurous compounds of the formation of 8-chloroguanosine mediated by hypochlorous acid, human myeloperoxidase, and activated human neutrophils.
      its molecular mechanism of gut protection in vivo was not clearly elucidated. Herein, we demonstrate that EGCG potently inhibits MPO activity in a dose-dependent manner at physiologic pH and temperatures in vitro and also in vivo in the inflamed gut. Because of its ability to bind iron, we expected EGCG to be capable of chelating iron from the heme moiety of MPO and LPO, but this was not the case. Contrary to our initial hypothesis, EGCG mediates its antioxidant effect without directly interfering with the heme moiety of the peroxidase. Spectral analysis on EGCG-mediated inhibition of LPO, a heme-containing enzyme that has similar function to MPO, reveals that EGCG reverts the peroxidase radical intermediate oxoiron back to its native inactive state, possibly by donating electrons to oxoiron. Oxoiron is required to react with halide ion to generate hypohalous acid.
      • Zederbauer M.
      • Furtmuller P.G.
      • Brogioni S.
      • Jakopitsch C.
      • Smulevich G.
      • Obinger C.
      Heme to protein linkages in mammalian peroxidases: impact on spectroscopic, redox and catalytic properties.
      The reactivity of hypohalous acid to heme iron (including those in MPO) was shown to induce heme degradation and the release of free or reactive iron,
      • Maitra D.
      • Shaeib F.
      • Abdulhamid I.
      • Abdulridha R.M.
      • Saed G.M.
      • Diamond M.P.
      • Pennathur S.
      • Abu-Soud H.M.
      Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway.
      • Maitra D.
      • Byun J.
      • Andreana P.R.
      • Abdulhamid I.
      • Saed G.M.
      • Diamond M.P.
      • Pennathur S.
      • Abu-Soud H.M.
      Mechanism of hypochlorous acid-mediated heme destruction and free iron release.
      leading to a cascade of free radical generation. In lieu of this, the inactivation of oxoiron by EGCG would effectively negate the production of hypohalous acid by MPO and LPO. By limiting MPO activity, EGCG could play a role in preventing collateral tissue damage by hypohalous acid during acute intestinal inflammation.
      The beneficial effects of EGCG in treating gut inflammation were demonstrated in mouse models of colitis. In one study, EGCG-enriched diet was shown to induce down-regulation of inflammation-associated genes such as IL1B, TNFA, interferon γ gene, Toll-like receptor 2 gene, and CD14 in spontaneously colitic Mdr1a−/− mice.
      • Barnett M.P.
      • Cooney J.M.
      • Dommels Y.E.
      • Nones K.
      • Brewster D.T.
      • Park Z.
      • Butts C.A.
      • McNabb W.C.
      • Laing W.A.
      • Roy N.C.
      Modulation of colonic inflammation in Mdr1a(−/−) mice by green tea polyphenols and their effects on the colon transcriptome and proteome.
      It is thought that EGCG mediates its therapeutic effects by modulating gene expression via inhibition of various transcription factors, including NF-κB, Sp1, activating protein-1, STAT1, STAT3, and forkhead box O1.
      • Abboud P.A.
      • Hake P.W.
      • Burroughs T.J.
      • Odoms K.
      • O'Connor M.
      • Mangeshkar P.
      • Wong H.R.
      • Zingarelli B.
      Therapeutic effect of epigallocatechin-3-gallate in a mouse model of colitis.
      • Aktas O.
      • Prozorovski T.
      • Smorodchenko A.
      • Savaskan N.E.
      • Lauster R.
      • Kloetzel P.M.
      • Infante-Duarte C.
      • Brocke S.
      • Zipp F.
      Green tea epigallocatechin-3-gallate mediates T cellular NF-kappa B inhibition and exerts neuroprotection in autoimmune encephalomyelitis.
      • Chen A.
      • Zhang L.
      The antioxidant (−)-epigallocatechin-3-gallate inhibits rat hepatic stellate cell proliferation in vitro by blocking the tyrosine phosphorylation and reducing the gene expression of platelet-derived growth factor-beta receptor.
      • Dong Z.
      • Ma W.
      • Huang C.
      • Yang C.S.
      Inhibition of tumor promoter-induced activator protein 1 activation and cell transformation by tea polyphenols, (−)-epigallocatechin gallate, and theaflavins.
      • Kim H.
      • Sakamoto K.
      (−)-Epigallocatechin gallate suppresses adipocyte differentiation through the MEK/ERK and PI3K/Akt pathways.
      • Lee I.T.
      • Lin C.C.
      • Lee C.Y.
      • Hsieh P.W.
      • Yang C.M.
      Protective effects of (−)-epigallocatechin-3-gallate against TNF-alpha-induced lung inflammation via ROS-dependent ICAM-1 inhibition.
      • Townsend P.A.
      • Scarabelli T.M.
      • Pasini E.
      • Gitti G.
      • Menegazzi M.
      • Suzuki H.
      • Knight R.A.
      • Latchman D.S.
      • Stephanou A.
      Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis.
      • Yang F.
      • de Villiers W.J.
      • McClain C.J.
      • Varilek G.W.
      Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model.
      Aside from this, EGCG not only inhibits NADPH oxidase,
      • Nishikawa H.
      • Wakano K.
      • Kitani S.
      Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell.
      which produces reactive oxygen species (ROS), but also scavenges ROS and reduces oxidative stress.
      • Nanjo F.
      • Mori M.
      • Goto K.
      • Hara Y.
      Radical scavenging activity of tea catechins and their related compounds.
      • Lambert J.D.
      • Elias R.J.
      The antioxidant and pro-oxidant activities of green tea polyphenols: a role in cancer prevention.
      ROS is one of the main factors that contribute to IBD pathology, and its elimination by EGCG may alleviate gut inflammation. Hence, the ability of EGCG to inhibit MPO and other neutrophils' pro-oxidant enzymes could provide an additional layer of protection, which perhaps could be one of the primary effects of EGCG that leads to the secondary down-regulation of downstream proinflammatory responses.
      • Haegens A.
      • Heeringa P.
      • van Suylen R.J.
      • Steele C.
      • Aratani Y.
      • O'Donoghue R.J.
      • Mutsaers S.E.
      • Mossman B.T.
      • Wouters E.F.
      • Vernooy J.H.
      Myeloperoxidase deficiency attenuates lipopolysaccharide-induced acute lung inflammation and subsequent cytokine and chemokine production.
      Interestingly, EGCG is also demonstrated to inhibit neutrophil elastase and to impair the migration of neutrophils in response to the chemoattractant IL-8.
      • Dona M.
      • Dell'Aica I.
      • Calabrese F.
      • Benelli R.
      • Morini M.
      • Albini A.
      • Garbisa S.
      Neutrophil restraint by green tea: inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis.
      • Sartor L.
      • Pezzato E.
      • Garbisa S.
      (−)Epigallocatechin-3-gallate inhibits leukocyte elastase: potential of the phyto-factor in hindering inflammation, emphysema, and invasion.
      However, we did not observe a difference in the amount of neutrophils in the mucosa between the untreated and EGCG-treated colitic mice, despite the apparent reduction of colonic MPO activity in the latter. It is likely that the neutrophils were already present in mucosa before EGCG treatment or that the dosage of EGCG that we used in this study was insufficient to induce observable reduction in neutrophil infiltration. In addition, the low EGCG doses used in this study may also explain the lesser degree of protection against colitis compared with previous reports that used higher doses of EGCG.
      • Oz H.S.
      • Chen T.
      • de Villiers W.J.
      Green tea polyphenols and sulfasalazine have parallel anti-inflammatory properties in colitis models.
      • Bruckner M.
      • Westphal S.
      • Domschke W.
      • Kucharzik T.
      • Lugering A.
      Green tea polyphenol epigallocatechin-3-gallate shows therapeutic antioxidative effects in a murine model of colitis.
      • Abboud P.A.
      • Hake P.W.
      • Burroughs T.J.
      • Odoms K.
      • O'Connor M.
      • Mangeshkar P.
      • Wong H.R.
      • Zingarelli B.
      Therapeutic effect of epigallocatechin-3-gallate in a mouse model of colitis.
      • Ran Z.H.
      • Chen C.
      • Xiao S.D.
      Epigallocatechin-3-gallate ameliorates rats colitis induced by acetic acid.
      Regardless, the effectiveness of EGCG in treating IBD was shown to be comparable with the widely used anti-colitogenic drug sulfasalazine in the murine model of DSS-induced colitis.
      • Oz H.S.
      • Chen T.
      • de Villiers W.J.
      Green tea polyphenols and sulfasalazine have parallel anti-inflammatory properties in colitis models.
      Indeed, the potential use of EGCG to treat gut inflammation and other diseases has garnered a great deal of attention, leading to its current inclusion in human clinical studies.
      • Martin D.A.
      • Bolling B.W.
      A review of the efficacy of dietary polyphenols in experimental models of inflammatory bowel diseases.
      The efficacy of EGCG in human clinical trials remains somewhat inconclusive because EGCG treatments were found to aggravate gut inflammation rather than to alleviate the disease in several cases.
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      A review of the efficacy of dietary polyphenols in experimental models of inflammatory bowel diseases.
      Although it is not completely understood, many studies implicate that this could be due to the pro-oxidant properties of EGCG in the presence of Fe3+.
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      Green tea extract and (−)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities.
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      Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants.
      EGCG was previously shown to form complexes with Fe3+ and to participate in the Fenton reaction.
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      EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.
      The reduction of Fe3+ to Fe2+ accelerates the formation free hydroxyl radicals and ROS that are detrimental to the gut. Interestingly, high doses of EGCG were shown to induce Fenton reaction and to generate more ROS, whereas low doses of EGCG scavenges ROS in vitro.
      • Tian B.
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      Chemiluminescence analysis of the prooxidant and antioxidant effects of epigallocatechin-3-gallate.
      Accordingly, several animal studies also demonstrated that high doses of dietary EGCG tend to induce a pro-oxidative response that aggravates gut inflammation in vivo.
      • Martin D.A.
      • Bolling B.W.
      A review of the efficacy of dietary polyphenols in experimental models of inflammatory bowel diseases.
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      Deleterious effects of high concentrations of (−)-epigallocatechin-3-gallate and atorvastatin in mice with colon inflammation.
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      High-dose green tea polyphenols induce nephrotoxicity in dextran sulfate sodium-induced colitis mice by down-regulation of antioxidant enzymes and heat-shock protein expressions.
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      Although we did not observe the purported detrimental effects of EGCG in this study, our in vitro findings do demonstrate that iron at 1:1 molar ratio induces EGCG to switch from being an antioxidant into one that is pro-oxidant. In addition, the presence of excess/dysregulated iron can counter the beneficial effect of EGCG by additionally abrogating its ability to inhibit MPO and LPO activity. This suggests that the presence of iron-rich conditions could potentially alter EGCG bioactivity and may shift it from being an inhibitor of MPO into a harmful pro-oxidant. Hence, the consumption of diets rich in iron or oral iron supplements to treat iron deficiency-induced anemia in IBD at the same time as EGCG administration may be detrimental.
      Iron is an essential nutrient to most bacteria, excluding Lactobacillus and Borrelia species.
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      The hypoferremic conditions induced by acute inflammation in the gut may allow growth of bacteria that could better adapt to iron-poor conditions. The ability of EGCG to bind Fe3+ and its reduction to Fe2+ may have substantial influence over bacterial iron acquisition because bacterial siderophores have high affinity specific to Fe3+ but not Fe2+. In addition, EGCG can prevent Enterococcus faecalis biofilm formation
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      via EGCG-Fe2+ mediated hydroxyl radical formation. Further, the chelation of EGCG + Fe3+ binary complex by host antibacterial innate immune protein NGAL/Lcn2 may add another layer of complexity to the acquisition of iron by resident bacteria in the inflamed gut. However, the iron-binding and oxidative properties of EGCG may be beneficial in exerting antibacterial pressure in the inflamed gut and perhaps play a role in alleviating disease by modulating gut bacteria composition and growth. Yet, this may also result in a rapid decrease in beneficial bacteria and overgrowth of opportunistic pathogens (eg, E. coli), which can express Lcn2/NGAL-resistant stealth siderophores.
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      The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.
      Further studies are warranted, given our current limited knowledge on the interplay between EGCG and gut microbiota in the inflamed gut.
      A study by Barasch and colleagues
      • Bao G.H.
      • Xu J.
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      • Wan X.C.
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      • Barasch J.
      EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.
      demonstrated that NGAL could form a stable complex with iron-bound EGCG and could suppress its pro-oxidant properties in vitro, highlighting the first molecule of plant origin to form complexes with NGAL. Although the presence of NGAL may be beneficial in preventing the adverse pro-oxidative effect of EGCG, the EGCG sequestration by NGAL could also eliminate its therapeutic properties. As confirmed in our present study, both human NGAL and murine Lcn2 counter-regulate EGCG-mediated inhibition of MPO and LPO activity in a dose-dependent fashion. It would appear that NGAL inhibits EGCG optimally at a molar ratio NGAL:EGCG of 1:1, which is consistent with a previous report that one molecule of NGAL can bind to one molecule of EGCG.
      • Bao G.H.
      • Xu J.
      • Hu F.L.
      • Wan X.C.
      • Deng S.X.
      • Barasch J.
      EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.
      NGAL and its murine ortholog Lcn2 are known to be significantly elevated in the inflamed gut of humans
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      Rectal dialysate and fecal concentrations of neutrophil gelatinase-associated lipocalin, interleukin-8, and tumor necrosis factor-alpha in ulcerative colitis.
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      Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair.
      and mice,
      • Vijay-Kumar M.
      • Sanders C.J.
      • Taylor R.T.
      • Kumar A.
      • Aitken J.D.
      • Sitaraman S.V.
      • Neish A.S.
      • Uematsu S.
      • Akira S.
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      • Gewirtz A.T.
      Deletion of TLR5 results in spontaneous colitis in mice.
      • Vijay-Kumar M.
      • Wu H.
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      • Kolachala V.L.
      • Neish A.S.
      • Sitaraman S.V.
      • Gewirtz A.T.
      Activation of toll-like receptor 3 protects against DSS-induced acute colitis.
      • Vijay-Kumar M.
      • Wu H.
      • Jones R.
      • Grant G.
      • Babbin B.
      • King T.P.
      • Kelly D.
      • Gewirtz A.T.
      • Neish A.S.
      Flagellin suppresses epithelial apoptosis and limits disease during enteric infection.
      respectively. Therefore, NGAL/Lcn2 presents a possible counter-regulatory role that potentially mitigates the efficacy of EGCG in treating IBD. Future studies should consider the levels of circulating/mucosal NGAL/Lcn2 in designing an optimal dose of EGCG to be administered to patients with IBD.
      Overall, our study demonstrates that inhibition of MPO by EGCG serves as a mechanism by which EGCG confers protection against gut inflammation. Moreover, as shown in Figure 8, the beneficial functions of EGCG potentially depend on the environment in the gut (ie, dietary iron and luminal NGAL/Lcn2, both of which counter-regulate EGCG bioactivity). Thus, our data provide the experimental basis to take these counter-regulatory factors into consideration to further develop EGCG as a potential therapeutic agent to treat human IBD.
      Figure thumbnail gr8
      Figure 8Potential mechanism by which EGCG mediates antioxidant effects that could alleviate gut inflammation. During inflammation, neutrophils release a plethora of pro-oxidant enzymes; one such enzyme is MPO whose bioactivity is associated with flares of inflammatory bowel disease. EGCG potently inhibits the activity of MPO and therefore reduces the level of oxidative stress in the inflamed gut. However, the beneficial MPO inhibition by EGCG is counter-regulated by iron and host Lcn2/NGAL. Altogether, the present study unravels the complex regulation of EGCG on MPO activity and its counter-regulation by iron and host Lcn2/NGAL. EGCG, (−)-epigallocatechin-3-gallate; Fe3+, ferric iron; Lcn2, lipocalin 2; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin.

      Acknowledgments

      We thank Timothy Yosca (Pennsylvania State University) for technical assistance with spectral analysis.
      B.S.Y. and R.A.O. designed and performed experiments with in vitro assays, analyzed the data, and co-wrote the manuscript; V.S. performed spectral analysis; X.X. performed animal experiments and RT-qPCR analyses; M.J.K. performed histologic analysis in a blinded fashion and co-wrote the discussion; B.J. discussed data interpretations and co-wrote the manuscript; J.D.L. performed data analysis and co-wrote the discussion; M.V.-K. conceptually developed the project, designed the experiments, analyzed the data, and co-wrote the manuscript.

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