Characterization of Immunomodulatory Activities of Honey

Dec 12, 2014 - (15) Likewise, honey also inhibited mitogen-activated T cell .... Cells were spun at 300g for 15 min at 4 °C. Pellet was resuspended i...
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Characterization of Immuno-modulatory Activities of Honey Glycoproteins and Glycopeptides M. Ahmed Mesaik, Nida Dastagir, Nazim Uddin, Khalid Rehman, and M. Kamran Azim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505131p • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 17, 2014

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Characterization

of

Immuno-modulatory

Activities

of

Honey

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Glycoproteins and Glycopeptides

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M. Ahmed Mesaik@1,2, Nida Dastagir@1, Nazim Uddin1, Khalid Rehman1 and M. Kamran Azim1*

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1

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Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. 2Faculty of

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Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala

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Lumpur, Malaysia.

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*

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Address for correspondence: Dr. M. Kamran Azim, International Center for Chemical and

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for

Corresponding author; @These authors contributed equally.

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Biological Sciences, University of Karachi, Karachi-75270, Pakistan.

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Tel. +9221-111222292 ext. 236

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Fax +9221-3498018

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Email: [email protected]; [email protected]

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ABSTRACT

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Recent evidences suggest an important role for natural honey in modulating immune response.

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To identify active components responsible, we investigated the immuno-modulatory properties

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of glycoproteins and glycopeptides fractionated from Ziziphus honey. Honey proteins/peptides

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were fractionated by size exclusion chromatography into 5 peaks with molecular masses in the

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range of 2-450 kDa. The fractionated proteins exhibited potent, concentration-dependent

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inhibition of reactive oxygen species production in zymosan-activated human neutrophils (IC50

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6-14 ng/mL) and murine macrophages (IC50 2-9 ng/mL). Honey proteins significantly suppressed

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the nitric oxide production by LPS-activated murine macrophages (IC50 96-450 ng/mL).

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Moreover, honey proteins inhibited the phagocytosis latex beads macrophages. The production

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of proinflammatory cytokines IL-1β and TNF-α by human monocytic cell line in presence of

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honey proteins was analyzed. Honey proteins did not affect the production of IL-1β, however

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TNF-α production was significantly suppressed. These findings indicated that honey

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glycoproteins and glycopeptides significantly interfere with molecules of the innate immune

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system.

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Keywords: Honey, glycoprotiens, immunomodulatory, phagocytosis, oxidative burst

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INTRODUCTION

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Natural honey is a well-known neutraceutical which is mainly composed of fructose, glucose,

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sucrose and maltose. Small amount of oligosaccharides, proteins, amino acids, minerals, trace

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elements, vitamins, aroma compounds and polyphenols are also presentreviewed in 1. Honey exerts a

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number of pharmacological effects including antimicrobial, immunomodulatory, prebiotic,

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antinematodal, anti-inflammatory and anti-nociceptive activities1-9. It showed both bacteriostatic

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and bactericidal potentials against pathogenic bacteria including drug-resistant strains5. Honey

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was found to exert anti-inflammatory effects via inhibiting (a) thrombin induced reactive oxygen

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species (ROS) production6 and (b) myeloperoxidase dependent oxidative burst by professional

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phagocytes7. It inhibited the nitric oxide production in carrageenen induced inflammatory rat

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modal10. In addition, Manuka, Pasture and Jelly bush honeys were found to reduce reactive

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oxygen species (ROS) production11 and stimulate monocytes (MonoMac-6 cell line) to produce

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tumor necrosis factor–α (TNF-α), Interleukin (IL)–1β and IL-6, which are known to play a vital

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role in tissue repair12. The effect of honey on cytokine production was found to be associated

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with a 5.8 kDa component isolated from manuka honey which interacted with TLR-413. Natural

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honey exhibited anti-tumor activity by inhibiting the growth of Lewis Lung Carcinoma/2 cells14.

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Immuno-stimulatory effect of honey on antibody production against thymus-dependent (sheep

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red blood cells) and thymus-independent (E. coli) antigens in mice has also been observed15.

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Likewise, honey also inhibited mitogen activated T cells proliferation in vitro16. Moreover,

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honey regulates the homeostasis by inhibiting the human platelet aggregation and blood

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coagulation proteins17.

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Natural honeys have low protein content which varies from type to type. Characterization of

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honey proteins from various sources was found to be valuable in determining the floral and 3 ACS Paragon Plus Environment

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geographical origin18. A number of reports pointed out immuno-modulatory potential of honey

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proteins. Four immunologically characterized IgE-binding glycoproteins (belongs to major royal

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jelly protein-1 (MRJP-1) family) were identified in honey and N-linked glycosylation in these

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proteins played an important role in binding of IgE19,21. Ultracentrifugation of royal jelly (RJ)

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revealed that the MRJP1, the most abundant protein of RJ present in monomeric (55 kDa) as

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well as oligomeric (ca 450 kDa) forms20. Honey contains MRJP1 protein as well which exerts its

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immuno-stimulatory effect by stimulating TNF-α production in murine peritoneal macrophages

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and the protein moiety of this glycoprotein was found be responsible for this property22.

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Recently, highly glycosylated Arabinogalactan proteins (AGPs) with molecular mass of ~110

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kDa have been characterized from New Zealand Kanuka honey23. AGPs are proteoglycans which

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contain type II arabinogalatans and 2-10% covalently attached protein that is typically rich in

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hydroxyproline and serine. AGPs are compact molecules consisting of a protein core to which

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highly branched polysaccharide chains are attached24. AGPs are found in the female sexual

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tissues of flowers, and thus could be a potential source of AGPs in honey. AGPs and type II

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arabinogalactans from different sources have been shown to exert immuno-modulatory

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properties25. As a continuation of our research, we have elaborated the role(s) of honey

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glycoproteins and glycopeptides in modulation of innate immune system.

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MATERIALS AND METHODS

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FRACTIONATION AND CHARACTERIZATION OF HONEY PROTEINS

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Honey samples: Monofloral Ziziphus species honey samples produced by Apis mellifera in bee

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farms located in Himalayan and Pothohar plateau regions in northern Pakistan were used during

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this study.

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Ammonium Sulphate Precipitation: Honey samples were diluted in buffer A (20 mM Tris-

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HCl, 150 mM NaCl, and 0.05% sodium azide, pH 7.4) and centrifuged at 2500 xg for 20 min at 4

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o

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processed for ammonium sulphate precipitation at 4 oC with 80% saturation. The solution was

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kept on stirrer for 1-2 hours followed by centrifugation at 8300 xg at 4 oC. The pellets were

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dissolved in buffer and stored at -20 oC till use.

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Gel Filtration Chromatography (GFC): Gel filtration chromatography crude honey proteins

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was carried out by HiloadTM 16/60 SuperdexTM 200 Prep Grade column (GE Healthcare,

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Uppsala, Sweden) with buffer A as mobile phase. Chromatography was done using AKTA

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FPLC system (GE Healthcare, Uppsala, Sweden) at the flow rate of 1 mL/min flow rate and

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fractions of 2 mL were collected. The resultant five chromatographic peaks were termed as GFC-

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P1 to GFC-P5. Fractions containing high molecular mass peak, GFC-P1 were pooled and treated

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with 8M urea (Sigma-Aldrich, Steinheim, Germany) followed by re-chromatography in the same

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conditions. The peaks GFC-P1 to GFC-P3 were concentrated by ultrafiltration (MWCO 10 kDa,

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Millipore, Darmstadt, Germany). Whereas peaks GFC-P4 and GFC-P5 was freeze dried

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(ALPHA 1-2/LD plus, Suarlee, Belgium) and resuspended in buffer A. Protein estimation was

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carried out by modified Lowry’s protein assay kit (Thermo Scientific, Rockford, USA)26. The

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molecular masses of proteins were estimated by Gel Filtration Calibration Kit HMW (GE

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Healthcare BioSciences AB, Sweden) which contains five proteins and Blue dextran 2000

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(Ovalbumin Mr 43,000, Conalbumin Mr 75,000, Aldolase Mr 158,000, Ferritin Mr

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440,000,Thyroglobulin Mr 669,000 and Blue dextran 2000 Mr 2,000,000).

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Reverse Phase HPLC of GFC Peaks: The gel chromatography peaks GFC-P1 and GFC-P5

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were subjected to reverse phase HPLC using C-18 column (Purospher STAR, RP-18 endcapped

C in order to remove particulates, particularly pollens. The supernatants were collected and

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(5 µm, 150-4.6, MERCK, Darmstadt, Germany). Column was equilibrated with 0.1% TFA and

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100 µL of filtered protein samples were applied with flow rate of 1.0 mL/min. Gradient elutions

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were made with acetonitrile as 5min/0%, 40min/70% and 45min/0%. Peaks were collected,

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concentrated and stored at -20oC till next use.

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Mass Spectrometry: MALDI-TOF analysis of peaks obtained by RPLC was carried out as

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follows. 0.5 µL of peptide suspension was mixed with 0.5 µL of matrix solution (Sinapinic acid

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in 1:1 0.1% TFA/Acetonitrile). The matrix/analyte solution was applied to sample plate. Mass

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spectra were recorded on a Bruker Ultraflex III TOF/TOF spectrometer. Two hundered shots of

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light from smart beam laser were irradiated to record mass spectra. Bioinformatics analysis of

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mass spectrometric data was done by using Glycosuite Database and GlycoMod tool of ExPASy

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server (www.expasy.org).

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IMMUNOMODULATORY ACTIVITY ASSAYS

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Isolation of Phagocytic Cells: Polymorph nuclear leukocytes (PMNLs) were collected from

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human blood by using ficoll paque gradient centrifugation. For this purpose, blood was drawn

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from healthy human volunteer (36-45 years old who did not receive any medication for one

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week). Blood was mixed with equal volume of lymphocyte separation medium (MP Biomedical,

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Inc, Kaysersberg, France) and hank’s balanced salt solution without calcium and magnesium

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(Sigma-Aldrich, Steinheim, Germany). The diluted blood was kept undisturbed at room

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temperature for 30-45 min. When red blood cells settled down, supernatant was layered onto 5

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mL of LSM in separate centrifuge tube and centrifuged at 400 g for 20 min at 4oC. Supernatant

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was discarded and pellet was resuspended in HBSS free from calcium and magnesium and

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washed at 300 g for 15 min at 4oC. In presence of RBCs contamination, RBCs were lysed with

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sterile distilled water; finally pellet was resuspended in HBSS containing calcium and

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magnesium. Cells viability was determined using trypan blue dye and cell count was adjusted to

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one million cells per mL.

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In addition, murine peritoneal macrophages were collected after injecting 10 mL sterile

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phosphate buffer saline (PBS) into mouse peritoneum (immunized with 1 mL sterile fetal bovine

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serum 72 hrs earlier). Cells were spun at 300 g for 15 min at 4oC. Pellet was resuspended in 1

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mL sterile PBS, viability was checked with trypan blue exclusion method (cells were >95%

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viable) and count was adjusted as one million cells per mL.

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Chemiluminescence Assay for Oxidative Burst: The effect of honey protein fractions on

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reactive oxygen species (ROS) production was measured on polymorph nuclear leucocytes

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(PMNLs) and mouse peritoneal macrophages. In brief, cells were plated in flat bottom 96-well

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plates and activated by serum opsonized zymosan-A (Sigma-Aldrich, Steinheim, Germany) at

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250 µg/mL final concentration. Proteins/peptides/glypeptides samples added at various

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concentrations (0.125-2.0 µg/mL) and plates were incubated for 20 min at room temperature.

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Luminol (3-Aminophthalhydrazide) (Alfa Aesar, Karlsruhe, Germany) was added at 1.5 µg/mL

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final concentration. Plates were immediately kept in the thermostated chamber of Luminoskan

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and chemluminescence kinetics of ROS production was monitored as relative light units [RLUs]

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for 50 min in Luminoskan (Labsystem, Helsinki, Finland) with repeated scan27.

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Nitric Oxide Assay: The murine macrophage cells (J774.2 cell line) were purchased from

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European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in complete DMEM

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(Sigma-Aldrich, Steinheim, Germany) supplemented with 10% heat inactivated fetal bovine

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serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. The effect of honey proteins on

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nitric oxide production was monitored according to Inoue et al.28. Briefly cells were seeded in a

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96-well plate as 1.8x105 cells/well and activated by 30 µg/mL of LPS (E. coli serotype 0111:B4,

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DIFCO Laboratories, Michigan, USA) for the production of nitric oxide. Protein samples were

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added at various concentrations and plate was incubated at 37 oC for 48 hours. Supernatants (50

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µL) from each well were mixed with an equal amount of griess reagent (0.1% naphthyl ethylene

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diamine dihydrochloride in distilled water and 1% sulfanilic acid in 5% H3PO4 in 1:1 ratio)

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(Fisher Scientific, Leicestershire, UK) and absorbance was recorded at 550 nm using Spectra

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Max plus 340 (Molecular Devices, CA, USA).

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Phagocytosis Assay: Phagocytosis assay was performed according to Foukas et al. with some

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modifications29. The murine macrophage cells (J774.2 cell line) were cultured in complete

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DMEM and seeded into 96-well tissue culture treated plate with 5x104 cells/well. Culture

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reaction plate was incubated in CO2 environment at 37 oC with 5% CO2 for 24 hour. Next day,

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latex beads (fluorescent coated carboxylated modified polystyrene beads, 1 µm, Sigma-Aldrich,

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Steinheim, Germany) were added as 100 beads/cell. Protein samples were added as 800 ng/mL

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final concentration and plate was further incubated for 24 hours at 37 oC in CO2 environment.

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Afterward, supernatant was removed and wells were washed twice by gently inverting the plate

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with 1X PBS. 1X trypan blue (50 µL) was added to each well and plate was kept at room

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temperature for 1 minute. The trypan blue was removed and fluorescence was recorded at 575-

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610 nm using fluorescence reader (Molecular Devices, CA, USA).

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Cytokine (TNF-α and IL-1β) Production Assay: THP-1 cell line (human monocytic leukemia

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cells) was purchased from European Collection of Cell Cultures (Salisbury, UK). Cells were

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cultured in 75 mm2 tissue culture flask with complete RPMI-1640 medium (MP Biomedical,

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Ohio, USA) supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L8 ACS Paragon Plus Environment

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glutamin, 5.5mM D-glucose, 50 µM β-mercaptoethanol, and 1% penicillin & streptomycin. Cells

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were seeded into a 24-well polystyrene sterile culture plate with 20 ng/mL PMA (SERVA,

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Heidelberg, Germany), which cause differentiation of monocytes to macrophages. The culture

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plate was incubated at 37 oC for 24 hours. Next day, cells were activated with 50 ng/mL LPS,

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protein samples were added in various concentrations and plate was incubated at 37 oC with 5%

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CO2. Supernatants were collected after 6 hours and level of TNF-α and IL-1β were estimated

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using ELISA kit (R & D System, Minneapolis, USA). Absorbance was recorded at 450 nm using

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microplate reader, (DIA Reader, Gmblt, Wr. Neudorf, Austria)30.

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Statistical Analysis: All experiments were run in triplicates and data were presented as

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statistical mean ± standard deviation (STD). A one-way Analysis of Variance (ANOVA) was

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applied to measure the significant difference between the results of test proteins and controls.

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Significance level was set as P