Journal: Frontiers in Immunology

Article Title: Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis

doi: 10.3389/fimmu.2018.01612

Figure Lengend Snippet: Damage-associated molecular patterns (DAMPs) providing inflammatory signal to the receptors toll-like receptor (TLR) 2/4 and 9, are significantly increased in sera of patients with de novo autoimmune hepatitis (DAIH). Sera from liver transplanted patients: with DAIH ( n = 10), without DAIH (LTC) ( n = 19); healthy, non-transplanted children (HC) ( n = 29), and non-transplanted children with autoimmune hepatitis (AIH) ( n = 9), was subjected to (i) qPCR; (ii) ELISA. HEK cells that stably co-express a human TLR2, TLR4, or TLR9 gene and an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used to determine activation of TLRs from patient sera. (A,B) significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of cytosolic proteins associated with TLR 2/4 activation in sera of patients with DAIH compared to sera of LTC subjects (qPCR: p < 0.001 for ACTB; p < 0.001 for MT-ATP/complex I; p < 0.001 for Fibrinogen; p < 0.001 for HMGB1, p < 0.001 for HSP70; and p < 0.001 for HSP90, p = 0.05 for HSP60) (ELISA: p = 0.003 for Fibrinogen; p = 0.37 for HMGB1). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of mitochondrial, cytosolic, and functional proteins in sera of patients with DAIH compared to sera of HC subjects (qPCR: p < 0.001 for Fibrinogen, HMGB1, HSP70, HSP90, ACTB, and MT-ATP6, respectively, and p = 0.64 for HSP60). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of functional proteins in sera of patients with AIH compared to sera of HC subjects. [qPCR: p < 0.001 for ACTB (nuclear DNA); p = 0.002 for MT-ATP6 (mitochondrial DNA); p = 0.013 for Fibrinogen; p = 0.008, p = 0.005, and p = 0.008 for HSPs 60, 70 and 90, respectively] (ELISA: p = 0.043 for HMGB1; p = 0.11 for fibrinogen). *** p < 0.001, ** p < 0.01, * p < 0.05. (C,D) Activation of TLRs 2, 4, and 9 reporter cell lines by sera of patients with DAIH. DAIH vs. LTC: ( p = 0.47; TLR2) ( p = 0.008; TLR4) ( p = 0.02; TLR9). DAIH vs. HC: ( p = 0.17; TLR2) ( p < 0.001; TLR4) ( p = 0.001; TLR9). Minus sign: negative control, plus sign: positive control. Representative plate for three patients from each subject group and summary data. (E) Protein inhibition targeting heat shock protein, fibrinogen, and complexes I–III significantly reduced activation of TLR2/4 and TLR9 reporter cell lines, respectively [for TLR 9 reporter cell line inhibition: p = 0.03 for Rotenone (Complex 1), p = 0.03 for Thenoyltrifluoroacetone, TTFA (Complex II), p = 1.0 for Antimycin A (Complex III), p = 0.03 for Complexes I and II, p = 0.03 for Complexes I and III, p = 0.03 for Complexes II and III, p = 0.03 for Complexes I, II, and III; for TLR 4 reporter cell line inhibition: p = 0.06 for fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17- N -Allylamino-17-demethoxygeldanamycin, 17AAG, and p = 0.03 for both HSP90 and fibrinogen inhibitors; for TLR 2 reporter cell line inhibition: p = 0.09 for Fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17AAG, p = 0.06 for both HSP90 and fibrinogen inhibitors]. (HSP—heat shock protein; positive controls: FSL-1—synthetic diacylated lipoprotein, LPS—lipopolysaccharide, ODN 2006—CpG oligonucleotide). Summary data.

Article Snippet: Three Milliplex Map kits (EMD Millipore, Bilerica, MA, USA) were used to measure mitochondrial proteins in one assay (Complexes I–IV, Human Oxidative Phosphorylation Magnetic Bead Panel); heat shock proteins in another (Heat Shock Protein Magnetic Bead 5-Plex Kit); Fibrinogen protein in the third (Human Cardiovascular Disease Magnetic Bead Panel 3—Fibrinogen 1 Plex); and S100A8 and S100A12 in the fourth and fifth kit, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Activation Assay, Functional Assay, Negative Control, Positive Control, Inhibition

Journal: Frontiers in Immunology

Article Title: Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis

doi: 10.3389/fimmu.2018.01612

Figure Lengend Snippet: Damage-associated molecular patterns (DAMPs) providing inflammatory signal to the receptors toll-like receptor (TLR) 2/4 and 9, are significantly increased in sera of patients with de novo autoimmune hepatitis (DAIH). Sera from liver transplanted patients: with DAIH ( n = 10), without DAIH (LTC) ( n = 19); healthy, non-transplanted children (HC) ( n = 29), and non-transplanted children with autoimmune hepatitis (AIH) ( n = 9), was subjected to (i) qPCR; (ii) ELISA. HEK cells that stably co-express a human TLR2, TLR4, or TLR9 gene and an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used to determine activation of TLRs from patient sera. (A,B) significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of cytosolic proteins associated with TLR 2/4 activation in sera of patients with DAIH compared to sera of LTC subjects (qPCR: p < 0.001 for ACTB; p < 0.001 for MT-ATP/complex I; p < 0.001 for Fibrinogen; p < 0.001 for HMGB1, p < 0.001 for HSP70; and p < 0.001 for HSP90, p = 0.05 for HSP60) (ELISA: p = 0.003 for Fibrinogen; p = 0.37 for HMGB1). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of mitochondrial, cytosolic, and functional proteins in sera of patients with DAIH compared to sera of HC subjects (qPCR: p < 0.001 for Fibrinogen, HMGB1, HSP70, HSP90, ACTB, and MT-ATP6, respectively, and p = 0.64 for HSP60). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of functional proteins in sera of patients with AIH compared to sera of HC subjects. [qPCR: p < 0.001 for ACTB (nuclear DNA); p = 0.002 for MT-ATP6 (mitochondrial DNA); p = 0.013 for Fibrinogen; p = 0.008, p = 0.005, and p = 0.008 for HSPs 60, 70 and 90, respectively] (ELISA: p = 0.043 for HMGB1; p = 0.11 for fibrinogen). *** p < 0.001, ** p < 0.01, * p < 0.05. (C,D) Activation of TLRs 2, 4, and 9 reporter cell lines by sera of patients with DAIH. DAIH vs. LTC: ( p = 0.47; TLR2) ( p = 0.008; TLR4) ( p = 0.02; TLR9). DAIH vs. HC: ( p = 0.17; TLR2) ( p < 0.001; TLR4) ( p = 0.001; TLR9). Minus sign: negative control, plus sign: positive control. Representative plate for three patients from each subject group and summary data. (E) Protein inhibition targeting heat shock protein, fibrinogen, and complexes I–III significantly reduced activation of TLR2/4 and TLR9 reporter cell lines, respectively [for TLR 9 reporter cell line inhibition: p = 0.03 for Rotenone (Complex 1), p = 0.03 for Thenoyltrifluoroacetone, TTFA (Complex II), p = 1.0 for Antimycin A (Complex III), p = 0.03 for Complexes I and II, p = 0.03 for Complexes I and III, p = 0.03 for Complexes II and III, p = 0.03 for Complexes I, II, and III; for TLR 4 reporter cell line inhibition: p = 0.06 for fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17- N -Allylamino-17-demethoxygeldanamycin, 17AAG, and p = 0.03 for both HSP90 and fibrinogen inhibitors; for TLR 2 reporter cell line inhibition: p = 0.09 for Fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17AAG, p = 0.06 for both HSP90 and fibrinogen inhibitors]. (HSP—heat shock protein; positive controls: FSL-1—synthetic diacylated lipoprotein, LPS—lipopolysaccharide, ODN 2006—CpG oligonucleotide). Summary data.

Article Snippet: Three Milliplex Map kits (EMD Millipore, Bilerica, MA, USA) were used to measure mitochondrial proteins in one assay (Complexes I–IV, Human Oxidative Phosphorylation Magnetic Bead Panel); heat shock proteins in another (Heat Shock Protein Magnetic Bead 5-Plex Kit); Fibrinogen protein in the third (Human Cardiovascular Disease Magnetic Bead Panel 3—Fibrinogen 1 Plex); and S100A8 and S100A12 in the fourth and fifth kit, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Activation Assay, Functional Assay, Negative Control, Positive Control, Inhibition

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Screening of the anti-inflammatory and antioxidant activity of the conventional (CONV) and the three optimized (OPT1, OPT2, and OPT3) Olive Pomace (OP) extracts on TNF-α-induced cytokine release and UV-B-induced reactive oxygen species (ROS) production by HCE cells, respectively. For cell cytokine stimulation, cells were pre-treated with vehicle (0.4% ethanol) or 0.40 mg/mL of CONV, OPT1, OPT2, and OPT3 for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h ( A – C , black bars). Vehicle-treated-TNF-α stimulated cells and cells not stimulated with TNF-α ( A – C , white bars) were used as control. IL-6, IL-8, and IP-10 were measured in cell supernatants by a multiplex bead-based array. OPT3 inhibited all measured cytokines/chemokines ( A – C ), while CONV significantly decreased IL-6 ( A ). For the UV-B-induced ROS production, cells were pre-treated with vehicle (0.4% ethanol) or 0.05 mg/mL of CONV, OPT1, OPT2 and OPT3 for 1 h. Subsequently, cells were incubated with 10 μM H2DCF-DA solution for 30 min, and then treated with the treatments and exposed to 107.25 mJ/cm2 UV-B light ( D , black bars). After 1 h of incubation, intracellular fluorescence intensity was measured. Vehicle-treated-UV-B stimulated cells and cells not stimulated with UV-B ( D , white bars) were used as control. OPT2 and OPT3 decreased ROS levels significantly. Data are presented as picograms (pg) of cytokine/chemokine (for cell cytokine stimulation) or relative fluorescence units (RFUs) (for ROS production) normalized to micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, ** p < 0.01, compared with vehicle-treated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells. Vehicle: 0.4% EtOH.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Antioxidant Activity Assay, Multiplex Assay, Incubation, Fluorescence

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Effect of the conventional (CONV) and the selected optimized (OPT3) olive pomace (OP) extracts on TNF-α-induced cytokine release by HCE cells. Cells were pre-treated with CONV (0.05, 0.10, 0.20, 0.40, 0.60, and 0.80 mg/mL), OPT3 (0.005, 0.025, 0.050, 0.100, 0.200, and 0.400 mg/mL), or vehicle (0.4% ethanol) for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h (black squares). Vehicle-treated-TNF-α stimulated cells and cells not stimulated with TNF-α (white circles) were used as control. IL-6, IL-8, IP-10, and IL-1β were measured in cell supernatants by a multiplex bead-based array. TNF-α failed to stimulate IL-1β in the experiment performed for OPT3. CONV significantly decreased IL-6 levels from 0.40 mg/mL ( A ), IL-8 levels from 0.60 mg/mL ( B ), and IL-1β levels from 0.40 mg/mL ( D ). For TNF- α stimulated cells, IP-10 production was not decreased significantly by CONV. Nevertheless, no significant differences were observed between stimulated and non-stimulated cells at 0.80 mg/mL ( C ). OPT3 inhibited IL-6 ( E ), IL-8 ( F ), and IP-10 ( G ) secretion from 0.200 mg/mL significantly. Data are presented as picograms (pg) of cytokine/chemokine per micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with vehicle-treated-TNF-α stimulated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Multiplex Assay

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Effect of Hydroxytyrosol (HT) and Oleuropein (OL) on TNF-α-induced cytokine release by HCE cells. Cells were pre-treated with HT (1, 5, 10, 25, 50, and 100 μM for both cell lines, and 150 μM for HCE), OL (5, 10, 25, 50, 100, 150, 200, 250, and 300 μM) or vehicle (cell culture medium) for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h (black squares). Vehicle-treated-TNF-a stimulated cells and cells not stimulated with TNF-α (white circles) were used as control. IL-6, IL-8, IP-10, and IL-1β were measured in cell supernatants by a multiplex bead-based array. HT significantly decreased IL-6 ( A ), IL-8 ( Β ), IP-10 ( C ), and IL-1β ( D ) secretion at 50, 100, 100, and 150 μM, respectively. OL failed to inhibit any of the cytokines/chemokines measured ( E – H ). Data are presented as picograms (pg) of cytokine/chemokine per micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, ** p < 0.01, compared with vehicle-treated-TNF-α stimulated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Cell Culture, Multiplex Assay

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Effect of the conventional (CONV) and the selected optimized (OPT3) olive pomace (OP) extracts on TNF-α-induced cytokine release by IM-ConjEpi cells. Cells were pre-treated with CONV (0.05, 0.10, 0.20, 0.40, 0.60, and 0.80 mg/mL), OPT3 (0.005, 0.025, 0.050, 0.100, 0.200, and 0.400 mg/mL) or vehicle (0.4% ethanol) for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h (black squares). Vehicle-treated-TNF-α stimulated cells and cells not stimulated with TNF-α (white circles) were used as control. IL-6, IL-8, IP-10, and IL-1β were measured in cell supernatants by a multiplex bead-based array. CONV and OPT3 reduced IP-10 levels at 0.20 mg/mL ( C ) and 0.05 mg/mL ( G ), respectively. IL-8 levels at baseline cells were decreased by CONV and OPT3 at 0.10 mg/mL ( B ) and 0.025 mg/mL ( F ), respectively. No significant inhibition was observed for IL-6 and IL-1β, neither by CONV ( A , D , respectively) nor by OPT3 ( E , H , respectively). Data are presented as picograms (pg) of cytokine/chemokine per micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with vehicle-treated-TNF-α stimulated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Multiplex Assay, Inhibition

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Effect of Hydroxytyrosol (HT) and Oleuropein (OL) on TNF-α-induced cytokine release by IM-ConjEpi cells. Cells were pre-treated with HT (1, 5, 10, 25, 50, and 100 μM), OL (5, 10, 25, 50, 100, 150, 200, 250, and 300 μM), or vehicle (cell culture medium) for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h (black squares). Vehicle-treated-TNF-α stimulated cells and cells not stimulated with TNF-α (white circles) were used as control. IL-6, IL-8, IP-10, and IL-1β were measured in cell supernatants by a multiplex bead-based array. HT and OL reduced IP-10 levels at 25 μM ( C ) and 200 μM ( G ), respectively. No significant inhibition was observed for IL-6, IL-8, and IL-1β, neither by HT ( A , B , D , respectively) nor by OL ( E , F , H , respectively). Data are presented as picograms (pg) of cytokine/chemokine per micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, compared with vehicle-treated-TNF-α stimulated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Cell Culture, Multiplex Assay, Inhibition

Journal: Antioxidants

Article Title: Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium

doi: 10.3390/antiox10071150

Figure Lengend Snippet: Effect of the mixture of Hydroxytyrosol (HT) and Oleuropein (OL) on TNF-α-induced cytokine release by HCE ( A – D ) and IM-ConjEpi ( E – H ) cells. Cells were pre-treated with 5 μM of OL + 10 μM of HT, 5 μM of OL + 25 μM of HT, 5 μM of OL + 50 μM of HT, or vehicle (cell culture medium) for 2 h. Following this, they were stimulated with 25 ng/mL TNF-α in the presence of the treatments for 24 h. Vehicle-treated-TNF-α stimulated cells and cells not stimulated with TNF-α were used as control. IL-6, IL-8, IP-10, and IL-1β were measured in cell supernatants by a multiplex bead-based array. For HCE cells, 5 μM of OL + 10 μM of HT had a synergistic effect, decreasing IP-10 levels ( C ), whereas the decrease of IL-6 levels by 5 μM of OL + 50 μM of HT ( A ) can also be achieved by 50 μM HT alone. None of the mixtures tested were able to decrease IL-8 ( B ) or IL-1β ( D ) secretion significantly. For IM-ConjEpi cells, 5 μM of OL + 25 μM of HT reduce IP-10 production ( G ); however, this is also demonstrated by HT alone. None of the mixtures were able to inhibit IL-6 ( E ), IL-8 ( F ), or IL-1β ( H ) secretion significantly. Data are presented as picograms (pg) of cytokine/chemokine per micrograms (μg) of total protein for three independent experiments (performed in duplicate) ± SEM. * p < 0.05, *** p < 0.001, compared with vehicle-treated-TNF-α stimulated cells; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with control cells. Vehicle: Cell culture medium.

Article Snippet: For the cell-based assays, plastic culture flasks, plates, tips and pipettes, Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) + GlutaMax, DMEM/F12 without phenol red, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), human epithelial growth factor (EGF), human insulin, penicillin, streptomycin, and bicinchoninic acid (BCA) assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA); cytokine TNF-α from bioNova scientific (Fremont, CA, US); bovine insulin, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) and 5-methylphenazinium methyl sulfate (PMS) reagents, dimethyl sulfoxide (DMSO), and Milliplex Human Cytokine/Chemokine HCYTOMAG-60K-5 plex Magnetic Kit (IL-1beta, IL-6, IL-8/CXCL8, IL-17A, and IP-10) from Merck Life Science (St. Louis, MO, USA).

Techniques: Cell Culture, Multiplex Assay