Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells *

doi: 10.1074/jbc.M113.464230

Figure Lengend Snippet: Transient expression of WT MMP-8 causes an up-regulation of IL-6 and IL-6 protein and mRNA expression that is dependent on the catalytic activity of MMP-8. A , MCF-7 cells were transiently transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8 ( EA ). Top panel , FACS cytokine bead array measurements of IL-6 and IL-8 protein levels in serum-free conditioned media collected 24 h after transfection. Lower panel , RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 mRNAs in cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B , Western blot analysis of WT MMP-8 and E198A MMP-8 expression in transfected MCF-7 cells. C , RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 mRNAs in SK-BR-3 cells transiently transfected with pcDNA4 empty vector, WT MMP-8, and E198A mutant MMP-8. D , RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 mRNAs in HMT-3552 S1 cells transiently transfected with pcDNA4 empty vector, WT MMP-8, and E198A mutant MMP-8. CL, cell lysate; CM, conditioned media.

Article Snippet: Conditioned media collected after 24 h serum starvation were used to assess the total levels of IL-6, IL-8, and TNFα using specific ELISAs (eBiosciences, San Diego, CA) following the guidelines of the manufacturer.

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells *

doi: 10.1074/jbc.M113.464230

Figure Lengend Snippet: MMP-8, IL-6, and IL-8 exist as part of an interconnected regulatory circuit. A , RNA quantification by real-time TaqMan RT-PCR of IL-6 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. B , RNA quantification by real-time TaqMan RT-PCR of IL-8 mRNA levels in WT MMP-8 overexpressing MDA-MB-231 cells after siRNA knockdown of IL-6 or IL-8 for 48 h. C , RNA quantification by real-time TaqMan RT-PCR of endogenous MMP-8 in naïve, non-transfected MDA-MB-231 cells treated with recombinant human IL-6 and IL-8 for 24 h. *, p < 0.05; **, p < 0.01;***, p < 0.001; ns , not significant.

Article Snippet: Conditioned media collected after 24 h serum starvation were used to assess the total levels of IL-6, IL-8, and TNFα using specific ELISAs (eBiosciences, San Diego, CA) following the guidelines of the manufacturer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells *

doi: 10.1074/jbc.M113.464230

Figure Lengend Snippet: Long-term stable MMP-8 overexpression also causes an up-regulation of IL-6 and IL-6 protein and mRNA expression that is dependent on the catalytic activity of MMP-8. Two independent clones of MDA-MB-231 cells stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8 were analyzed. Top panel , ELISA measurements of IL-6 and IL-8 protein levels in serum-free conditioned media collected 24 h after serum starvation. Lower panel , RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 mRNAs in the same cells. Both wild-type MMP-8 clones 1 and 2 were different compared with all other cells by at least p < 0.05 or p < 0.01 unless shown otherwise *, p < 0.05; **, p < 0.01; ***, p < 0.001. EA, E198A mutant MMP-8.

Article Snippet: Conditioned media collected after 24 h serum starvation were used to assess the total levels of IL-6, IL-8, and TNFα using specific ELISAs (eBiosciences, San Diego, CA) following the guidelines of the manufacturer.

Techniques: Over Expression, Expressing, Activity Assay, Clone Assay, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells *

doi: 10.1074/jbc.M113.464230

Figure Lengend Snippet: Both IL-6 and IL-8 up-regulation occurs via NF-κB, but IL-8 is also negatively regulated by TGFβ and PAR-2. A , ELISA measurements of IL-6 and IL-8 protein levels after incubation of MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant ( EA ) MMP-8 after incubation with 10 μ m BAY 11-7082, an NF-κB inhibitor, for 48 h. DMSO , dimethyl sulfoxide. B , ELISA measurements of IL-6 and IL-8 protein levels after incubation of MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8 after incubation with 10 μ m SB431542, a TGFβR1 inhibitor, for 48 h. C , RNA quantification by real-time TaqMan RT-PCR of PAR-2 in MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8. D , top panel , Western blot analysis and RNA quantification by real-time TaqMan RT-PCR of PAR2 to confirm the knockdown of PAR-2 after siRNA knockdown for 48 h in wild-type MMP-8 overexpressing MDA-MB-231 clonal isolate 1. Center panel , RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 in cells after PAR-2 siRNA knockdown. Bottom panel , ELISA measurement on serum-free conditioned media of IL-6 and IL-8 protein levels after PAR-2 siRNA knockdown. *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns , not significant. E , schematic showing the relationships between MMP-8, IL-6, and IL-8 in a self-reinforcing loop and pathways that may contribute to this system in breast cancer cells. On acute exposure to catalytically active WT MMP-8, breast cancer cells up-regulate the expression of IL-6 and IL-8 ( black lines and arrows ). IL-6 is also able to induce MMP-8 expression. Red lines and arrows show the pathways that are evident in breast cancer cells that adapt to long-term expression of WT MMP-8. Not shown on the diagram is that IL-6 and IL-8 expression is also dependent on NF-κB signaling, which may be activated by PAR-2 as well as other signaling pathways, including autocrine loops resulting from IL-6 and IL-8 induction.

Article Snippet: Conditioned media collected after 24 h serum starvation were used to assess the total levels of IL-6, IL-8, and TNFα using specific ELISAs (eBiosciences, San Diego, CA) following the guidelines of the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Selective activation of pro-anti-IL-1β antibody enhances specificity for autoinflammatory disorder therapy

doi: 10.1038/s41598-021-94298-y

Figure Lengend Snippet: Schematic illustration of pro-Canakinumab selective activation by MMP-9 cleavage and specific neutralization of IL-1β at the inflamed region in autoinflammatory diseases. We engineered the human IgG1 hinge as an Ab lock (i.e. EPKSCDKTHTCPPCP) in front of the antigen-binding site of Canakinumab (fully human anti-human IL-1β mAb) by using MMP-9 substrate peptide as linker to generate pro-Canakinumab. After the Ab lock is removed by the MMP-9 expressed in the disease region, the cleaved pro-Canakinumab is expected to be specifically activated, neutralize the local IL-1β antigen and reduce the systemic on-target toxicity during autoinflammatory disease treatment.

Article Snippet: After starving cells with serum free medium for 24 h, the medium was replaced with IL-1β (5 ng/mL, R&D Systems), and incubated with saline, Canakinumab (2 nM or 10 nM), pro-Canakinumab (2 nM or 10 nM), 100 μg/mL MMP-9 pre-incubated Canakinumab or 100 μg/mL MMP-9 pre-incubated pro-Canakinumab for 24 h at 37 °C.

Techniques: Activation Assay, Neutralization, Binding Assay

Journal: Scientific Reports

Article Title: Selective activation of pro-anti-IL-1β antibody enhances specificity for autoinflammatory disorder therapy

doi: 10.1038/s41598-021-94298-y

Figure Lengend Snippet: Generation and characterization of pro-Canakinumab. ( A ) Schematic of the Canakinumab (fully human anti-human IL-1β mAb) including (from the N to C terminus) the light chain (V L -C κ ) and heavy chain (V H -CH1 + CH2 + CH3) of Canakinumab that link with an internal ribosomal entry site (IRES). The pro-Canakinumab includes (from the N to C terminus) the IgG1 hinge (Ab lock) in front of the light chain (V L -C κ ) and heavy chain (V H -CH1 + CH2 + CH3) of Canakinumab, respectively, and these two fragments are also joined by an IRES. ( B ) SDS-PAGE analysis of protein A-purified Canakinumab and pro-Canakinumab: lane 1, molecular weight marker; lane 2, Canakinumab; lane 3, pro-Canakinumab. The black arrow indicates the heavy chain fragments of Canakinumab or pro-Canakinumab, respectively. The white arrow indicates the light chain fragments of Canakinumab or pro-Canakinumab, respectively. ( C ) The binding ability of Canakinumab (blue circles) and pro-Canakinumab (red squares) was assessed by IL-1β-based ELISA. The percentage of mean absorbance values (405 nm) of triplicate determinations are shown (n = 3). The bars indicate the SD.

Article Snippet: After starving cells with serum free medium for 24 h, the medium was replaced with IL-1β (5 ng/mL, R&D Systems), and incubated with saline, Canakinumab (2 nM or 10 nM), pro-Canakinumab (2 nM or 10 nM), 100 μg/mL MMP-9 pre-incubated Canakinumab or 100 μg/mL MMP-9 pre-incubated pro-Canakinumab for 24 h at 37 °C.

Techniques: SDS Page, Purification, Molecular Weight, Marker, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Selective activation of pro-anti-IL-1β antibody enhances specificity for autoinflammatory disorder therapy

doi: 10.1038/s41598-021-94298-y

Figure Lengend Snippet: Selective activation of pro-Canakinumab by MMP-9 treatment. Canakinumab or pro-Canakinumab (25 nM) were incubated with 4 μg recombinant MMP-9 for 0, 15, 30 and 60 min, respectively. ( A ) The light chain molecular weight of the Canakinumab or active and inactive pro-Canakinumab were detected with HRP-conjugated anti-human IgG Fab Ab by Western blot and ( B ) the IL-1β binding ability of Canakinumab and pro-Canakinumab with or without MMP-9 treatment were analyzed by IL-1β-coated ELISA (n = 3). ( C ) The IL-1β antigen coated 96-well plate was also incubated with different concentrations of Canakinumab (blue circles), pro-Canakinumab (green upward triangle), Canakinumab pre-incubated with MMP-9 (red squares) or pro-Canakinumab pre-incubated with MMP-9 (pink downward triangles), respectively, and then the IL-1β binding ability was analyzed by ELISA. The percentage of mean absorbance values (405 nm) of triplicate determinations are shown (n = 3). The bars indicate the SD.

Article Snippet: After starving cells with serum free medium for 24 h, the medium was replaced with IL-1β (5 ng/mL, R&D Systems), and incubated with saline, Canakinumab (2 nM or 10 nM), pro-Canakinumab (2 nM or 10 nM), 100 μg/mL MMP-9 pre-incubated Canakinumab or 100 μg/mL MMP-9 pre-incubated pro-Canakinumab for 24 h at 37 °C.

Techniques: Activation Assay, Incubation, Recombinant, Molecular Weight, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Selective activation of pro-anti-IL-1β antibody enhances specificity for autoinflammatory disorder therapy

doi: 10.1038/s41598-021-94298-y

Figure Lengend Snippet: The neutralizing effect of pro-Canakinumab on IL-1β downstream signaling. Luciferase activity of NF-κB promoter constructs (pNF-κB-Luc) transiently transfected into HEK293 cells with or without treatment with IL-1β (10 ng/mL). IL-1β neutralizing ability was analyzed by incubating cells with saline (gray), Canakinumab (orange), pro-Canakinumab (blue), MMP-9 pre-incubated Canakinumab (pink) or MMP-9 pre-incubated pro-Canakinumab (purple) and the luciferase activity was detected through a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. IL-1β treatment was used as a control for reporter activity. The results are expressed as fold changes of luciferase activity (relative light unit, RLU) over those of internal control Renilla–Luc reporter plasmid. All data are the mean ± SD of triplicate independent experiments (n = 3). Statistical significance was calculated by t test with NS, no significance; * P < 0.05.

Article Snippet: After starving cells with serum free medium for 24 h, the medium was replaced with IL-1β (5 ng/mL, R&D Systems), and incubated with saline, Canakinumab (2 nM or 10 nM), pro-Canakinumab (2 nM or 10 nM), 100 μg/mL MMP-9 pre-incubated Canakinumab or 100 μg/mL MMP-9 pre-incubated pro-Canakinumab for 24 h at 37 °C.

Techniques: Luciferase, Activity Assay, Construct, Transfection, Saline, Incubation, Reporter Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: Selective activation of pro-anti-IL-1β antibody enhances specificity for autoinflammatory disorder therapy

doi: 10.1038/s41598-021-94298-y

Figure Lengend Snippet: Effect of pro-Canakinumab on IL-1β-induced IL-6 expression. Human lung carcinoma cell line A549 (1.2 × 10 5 ) was treated with IL-1β (5 ng/mL, gray), and incubated with serum free medium (SFM) (black), Canakinumab (orange), pro-Canakinumab (blue), MMP-9 pre-incubated Canakinumab (pink) or MMP-9 pre-incubated pro-Canakinumab (purple) for 24 h. The supernatant was collected and the expression level of IL-6 was detected by human IL-6 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. All data are the mean ± SD of triplicate independent experiments (n = 3). Statistical significance was calculated by t test with * P < 0.05; ** P < 0.01; *** P < 0.001 when compared between each indicated group.

Article Snippet: After starving cells with serum free medium for 24 h, the medium was replaced with IL-1β (5 ng/mL, R&D Systems), and incubated with saline, Canakinumab (2 nM or 10 nM), pro-Canakinumab (2 nM or 10 nM), 100 μg/mL MMP-9 pre-incubated Canakinumab or 100 μg/mL MMP-9 pre-incubated pro-Canakinumab for 24 h at 37 °C.

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: IL17A mRNA expression in paraffin-embedded (A) sub-macular choroid button and (B) macula. (C) IL17RC mRNA expression in paraffin-embedded macula. (D) IL17RC mRNA expression in macula vs. periphery of 3 donors. (E, F) Macular IL17A and IL17RC mRNA were verified in paraformaldehyde-fixed fresh frozen tissue. (G) Immunohistochemical detection of IL17A and IL17RC in paraffin-embedded macular sections. Isotype controls lacked primary antibodies. (H) Comparison of macular and peripheral IL17A immunostains. For box plots: center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; width of the boxes is proportional to the square root of the sample size; data points are plotted as open circles; sample numbers are indicated beneath each column. GA = geographic atrophy; nAMD = neovascular AMD; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; IS/OS = inner/outer segment; RPE = retinal pigment epithelium. *: P<0.05; **: P<0.005; ***:P<0.0001.

Article Snippet: Cells were serum-starved without antibiotics for 24 h and treated for 48 h with recombinant IL17A (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Immunohistochemical staining, Comparison, Software

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) Confocal microscopy of ARPE-19 treated 48h with IL17A. Nuclear signal of cleaved Caspase-3 (arrowheads). Blue = DAPI. Scale bar = 50 µm. Graphical representation of confocal data presented to the right of the panel. (B) ARPE-19 ultrastructure cultured without (i–ii) and with (iii–vi) 10 ng/ml IL17A for 48h. Indicated are healthy mitochondria (i, white arrow), lipid deposits (iii, iv, blue arrowheads), autophagosomes (iv, green arrowhead), mitochondrial damage (iii, iv, vi, yellow arrowheads), pyknotic nuclear clumps (v, black arrowhead), and cytoplasmic necrosis (vi, red arrowhead). (C) ARPE-19 vs. COS-7 viability after 48h treatment with IL17A. (D) Relative expression of IL17 receptors in ARPE-19 vs. COS-7. Data are presented as mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001; ****: P<0.0001.

Article Snippet: Cells were serum-starved without antibiotics for 24 h and treated for 48 h with recombinant IL17A (R&D Systems, Minneapolis, MN).

Techniques: Confocal Microscopy, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) IL17RC knockdown by 0.04 µM siRNA. IL17RC was measured by qPCR at 24h, 48h, and 72h post-transfection relative to a universal mRNA standard. (B) Relative viability of ctrl or IL17RC siRNA-transfected ARPE-19 with 100 ng/ml IL17A. (C) Confocal microscopy of ctrl or IL17RC siRNA-transfected ARPE-19 treated with or without IL17A. Nuclear localization of Caspase-3 (arrowheads). Graphical representation of the Caspase:DAPI ratio are indicated to the right of the images. Blue = DAPI. Scale bar = 50 µm. *: P<0.05; **: P<0.005; ****: P<0.0001. Data are presented as mean ± SEM.

Article Snippet: Cells were serum-starved without antibiotics for 24 h and treated for 48 h with recombinant IL17A (R&D Systems, Minneapolis, MN).

Techniques: Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) Retinal Il17a transcripts as a function of animal age. (B) Fundoscopic results 2 months post-intervention. (C) A2E quantification. For box plots: center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; width of the boxes is proportional to the square root of the sample size; data points are plotted as open circles. (D) Histopathological findings: sIL17R preserved photoreceptor IS/OS thickness compared to EV (asterisks) and maintained thickness of the ONL. EV retinas showed RPE degeneration (red arrow). Dystrophic rd8 lesions are indicated (black arrows). (E) Abundant lipofuscin in EV but not in sIL17R RPE (yellow arrowheads, upper left and right). Mitochondria (m) are damaged and disorganized within EV RPE (lower left) but are viable and organized in sIL17R RPE (lower right). EV RPE show extensive vacuolization, undigested OS and poor basal infoldings (lower left). (F) Western blot of downstream Il17 signaling. EV and sIL17R neuroretinas were treated with 50 ng/ml Il17a for 0, 5, or 15 min ex vivo . Relative Signal Intensities are presented of phosphorylated proteins relative to total protein to the right of the blots. NFL = nerve fiber layer; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; IS/OS = inner/outer segment; RPE = retinal pigment epithelium *: P<0.05; **: P<0.005; ****: P<0.00001.

Article Snippet: Cells were serum-starved without antibiotics for 24 h and treated for 48 h with recombinant IL17A (R&D Systems, Minneapolis, MN).

Techniques: Software, Western Blot, Ex Vivo