Journal: Journal of Inflammation Research

Article Title: IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

doi: 10.2147/JIR.S368352

Figure Lengend Snippet: IFN-γ and LPS cooperatively induce CCL2 in monocytic cells/macrophages. THP-1 monocytic cells, primary monocytes, and THP-1-derived macrophages were treated for 24h with IFN-γ (10 ng/mL) alone or in combination with LPS (10 ng/mL). Total RNA was extracted and Ccl2 mRNA expression was quantified by real-time RT-PCR. CCL2 protein was measured in cell supernatants using commercial ELISA kit. All data are expressed as mean ± SEM (n ≥ 3). Group means between two data sets were compared using Student’s t -test and those of more than two data sets were compared using one-way ANOVA with post-hoc Tukey’s test. All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001). Elevated CCL2 gene ( A ) and secreted protein ( B ) expression is shown in THP-1 monocytic cells co-stimulated with IFN-γ and LPS compared to cells treated with IFN-γ and LPS alone. Primary monocytes ( C ) also show increased CCL2 secreted protein expression after co-stimulation with IFN-γ and LPS compared to stimulation with either IFN-γ or LPS. In addition to monocytic cells and primary monocytes, THP-1-derived macrophages co-stimulated with IFN-γ and LPS also display elevated expression of CCL2 transcripts ( D ) and secreted protein ( E ). ( F and G ) THP-1 cells were primed with IFN-γ and LPS, separately, followed by LPS and IFN-γ treatment, respectively, for 24h. Ccl2 mRNA and protein expression was quantified and statistically analyzed as described above. The data show that only the IFN-γ priming followed by LPS stimulation led to elevated CCL2 mRNA ( F ) and secreted protein ( G ) expression in monocytic cells compared to controls stimulated with IFN-γ or LPS alone.

Article Snippet: CCL2-secreted protein levels were measured in the supernatants of THP-1 cells stimulated with IFN-γ (10 ng/mL), LPS (10 ng/mL), alone or in combination, using human CCL2 DuoSet ELISA kit and following the manufacturer’s instructions (Cat. # DY 279, R&D Systems Inc.).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

doi: 10.2147/JIR.S368352

Figure Lengend Snippet: Synergistic expression of CCL2 is dependent on STAT1. ( A ) THP-1 monocytic cells were transfected with control/scrambled siRNA or STAT1 siRNA and incubated for 40h. Total RNA was extracted and real-time RT-PCR was performed to measure STAT1 mRNA expression. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2 −ΔΔCT method. Relative STAT1 mRNA expression was expressed as fold expression over average of control (scrambled siRNA) gene expression. All data are expressed as mean ± SEM (n ≥ 3) and group means between two data sets were compared using Student’s t -test. All p-values < 0.05 were considered significant. The data show significant suppression of STAT1 mRNA expression in cells transfected with STAT1 siRNA compared to control siRNA transfected cells (**p< 0.01). ( B and C ) STAT1-deficient cells were treated for 24h with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) and Ccl2 mRNA ( B ) and protein ( C ) expression was determined using real-time RT-PCR and ELISA, respectively. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2 −ΔΔCT method. Relative CCL2 mRNA expression was expressed as fold change over average of control (vehicle treatment) gene expression. All data are expressed as mean ± SEM (n ≥ 3) and group means between two data sets were compared using Student’s t -test. The data show significant suppression of ( B ) CCL2 mRNA (***p< 0.001) and ( C ) CCL2 secreted protein (**p< 0.01) in cells co-stimulated with IFN-γ and LPS as compared to those stimulated with IFN-γ or LPS alone. ( D ) Western blot showing phosphorylation of STAT1 after IFN-γ (10 ng/mL) treatment over time indicates the optimal STAT1 phosphorylation at 120 min.

Article Snippet: CCL2-secreted protein levels were measured in the supernatants of THP-1 cells stimulated with IFN-γ (10 ng/mL), LPS (10 ng/mL), alone or in combination, using human CCL2 DuoSet ELISA kit and following the manufacturer’s instructions (Cat. # DY 279, R&D Systems Inc.).

Techniques: Expressing, Transfection, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Inflammation Research

Article Title: IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

doi: 10.2147/JIR.S368352

Figure Lengend Snippet: H3K27 acetylation levels at different sites of CCL2 promotor region. ( A ) THP-1 monocytic cells were treated with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) for 4 h and the treatment with vehicle alone served as control. Cell lysates were used for determination of H3K27 acetylation by Western blotting. Briefly, cell lysates were resolved using 12% SDS-PAGE and blots were probed with rabbit anti-human H3K27 antibody (1:1000 dilution) at 4°C overnight. Blots were washed, incubated for 2h with HRP-conjugated secondary antibody (1:2500 dilution), and immunoreactive bands were developed and visualized using ChemiDoc™ MP Imaging Systems. ( B ) Western blot band densities were quantified and data were expressed as mean ± SEM (n = 3) values which were compared for various treatments using one-way ANOVA and post-hoc Tukey’s test. All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001). The data show increased H3K27 acetylation (H3K27 ac) in the cells stimulated with IFN-γ or IFN-γ+LPS compared to cells stimulated with LPS only (***p< 0.001). ( C ) The schematic diagram of CCL2 gene promotor region is shown. ( D – I ) THP1 cells were stimulated with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) by incubation for 24h at 37°C. Chromatin immunoprecipitation was done on cell lysate as described in methodology using antibodies specific to H3K27, Histone H3, and normal rabbit IgG for overnight at 4°C. H3K27 acetylation levels induced by treatments compared to control (vehicle) were detected by qPCR using primers specific to the closest regions of transcription start site at the CCL2 promotor. Data (mean ± SEM, n = 3) were expressed as fold enrichment levels and were compared for different treatments against control using one-way ANOVA with post-hoc Tukey’s test. All p-values < 0.05 were considered significant (*p< 0.05 and **p< 0.01).

Article Snippet: CCL2-secreted protein levels were measured in the supernatants of THP-1 cells stimulated with IFN-γ (10 ng/mL), LPS (10 ng/mL), alone or in combination, using human CCL2 DuoSet ELISA kit and following the manufacturer’s instructions (Cat. # DY 279, R&D Systems Inc.).

Techniques: Western Blot, SDS Page, Incubation, Imaging, Chromatin Immunoprecipitation

Journal: Journal of Inflammation Research

Article Title: IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

doi: 10.2147/JIR.S368352

Figure Lengend Snippet: Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate synergy between IFN-γ and LPS for the CCL2 production. THP-1 monocytic cells were treated with anacardic acid (HATs inhibitor; 50 μM) overnight or with TSA (HDACs inhibitor; 25 nM) for 6 h, followed by treatments with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) for 24h, and the treatment with vehicle alone served as control. Ccl2 mRNA expression was determined by real-time RT-PCR and target gene expression were normalized to GAPDH expression. Relative changes in Ccl2 gene expression were calculated using 2 −ΔΔCT method and expressed as fold change over its expression in control (vehicle treatment). CCL2 secreted protein expression was detected by ELISA as described in materials and methods. All data were expressed as mean ± SEM values (n = 3) and group means between two data sets were compared using Student’s t -test. All p-values < 0.05 were considered significant (**p<0.01, ***p<0.001). The data show the reduced expression of CCL2 ( A ) mRNA (***p<0.001) and ( B ) secreted protein (**p<0.01) in the cells that were treated with HAT-inhibitor anacardic acid before co-stimulation with IFN-γ and LPS as compared to similarly stimulated cells that were not pre-treated with anacardic acid. Interestingly, increased expression of CCL2 ( C ) mRNA (**p<0.01) and ( D ) secreted protein (***p<0.001) were observed in the cells that were treated with HDAC-inhibitor TSA before stimulation with LPS only as compared to similarly stimulated cells that were not pre-treated with TSA, suggesting that TSA priming could mimic effect of and substitute for IFN-γ in cooperativity with LPS.

Article Snippet: CCL2-secreted protein levels were measured in the supernatants of THP-1 cells stimulated with IFN-γ (10 ng/mL), LPS (10 ng/mL), alone or in combination, using human CCL2 DuoSet ELISA kit and following the manufacturer’s instructions (Cat. # DY 279, R&D Systems Inc.).

Techniques: Expressing, Quantitative RT-PCR, Targeted Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Primer sequences, annealing temperatures, and cycles used for semiquantitative PCR

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican and CCL2 mRNA levels in monocytes from patients with systemic sclerosis and healthy controls . Quantitative PCR analysis. Relative mRNA expression levels were calculated as a ratio of mRNA levels of the genes of interest to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars in the graph denote the mean. Differences between the groups were analyzed by Mann-Whitney U test. SSc, systemic sclerosis.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican V0 and CCL2 protein levels in monocyte culture supernatants . Versican V0 and CCL2 protein levels in monocyte culture supernatants derived from systemic sclerosis (SSc) patients and healthy controls. (A) Representative immunoblot evaluating versican V0 protein expression. Monocyte culture supernatants were concentrated, truncated, and applied to immunoblots. (B) Versican V0 protein levels in monocytes from 14 patients with SSc and 11 healthy controls, semiquantitatively measured by densitometry. (C) CCL2 protein levels in monocytes from 16 patients with SSc and 13 healthy controls. CCL concentration in culture supernatants was measured by an ELISA. Each bar in the graph denotes the mean. Results from the two groups were compared by Mann-Whitney U test.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Derivative Assay, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Formation of CCL2 and versican complex in circulating monocytes . (A) CCL2 was incubated with plastic plates coated with chondroitin sulfate (CS) or vehicle. Bound CCL2 was recovered and subjected to immunoblots (lanes 2 and 3, respectively). Lane 1, untreated CCL2 as a positive control. A representative result from three experiments is shown. (B) Versican and CCL2 cellular localization in monocytes from an systemic sclerosis (SSc) patient, assessed by multi-color immunocytostaining: upper panel, CCL2 (green), versican (red), and their merged image; lower panel, goldin-97 (green), versican (red), and their merged image. Nuclei were counterstained with TO-PRO3 (blue). A representative result from three independent experiments is shown. Original magnification, ×600.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Incubation, Western Blot, Positive Control

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: CCL2 chemoattractant activity promoting monocyte migration, with or without chondroitin sulfate binding . Circulating monocytes derived from healthy controls (A, C, E) and systemic sclerosis (SSc) patients (B, D, F). (A, B) CCL2-induced monocyte migration in the presence or absence of chondroitin sulfate (CS) coating. Lower chambers of a TransWell ® double-chamber system (Corning Incorporated, Corning, NY, USA) were coated with CS or vehicle, and CD14 + monocytes were applied to the upper chambers. (C, D) CCL2-induced monocyte migration on plastic plates precoated with serial concentrations of CS. (E, F) CCL2-induced monocyte migration on CS-coated plastic plates in the presence of anti-CCL2 mAb or control IgG. Relative monocyte migration was calculated as a percentage of migration in a control experiment using vehicle-coated wells without CCL2. All experiments were carried out in duplicate; the mean and standard deviation of three measurements is shown. Results from the two groups were compared using a Mann-Whitney U test. A representative result from four independent experiments is shown.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Activity Assay, Migration, Binding Assay, Derivative Assay, Control, Standard Deviation, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Binding to chondroitin sulfate chains protects CCL2 from protease-mediated degradation . (A) CCL2 was pre-incubated with chondroitin sulfate (CS) or vehicle, and treated with a series of proteases including elastase, cathepsin G, and trypsin. A representative immunoblot from three different experiments shows a band corresponding to intact CCL2 (10 kDa). (B) Amount of intact CCL2 in individual wells precoated with CS or vehicle, and subsequently treated with a series of proteases. Mean and standard deviation of three independent measurements is shown. The quantity of intact CCL2 in individual samples was expressed as a percentage of the quantity of CCL2 on wells precoated with CS and not treated with protease.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Binding Assay, Incubation, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Microglial and Astrocyte priming in the APP/PS1 model of Alzheimer’s Disease: increased vulnerability to acute inflammation and cognitive deficits

doi: 10.1101/344218

Figure Lengend Snippet: (A-C) Chemokine secretion in microglia and astrocyte supernatants, 6h after IL-1β challenge (2.5 ng/mL) measured by ELISA for CCL2, CXCL1 and CXCL10. Two way ANOVA, *p<0.001, compared to astrocyte control; #p<0.001, compared to microglia IL-1β. (D) Light microscopy labelling of CCL2 in WT and Tg mice, 2h after i.c. challenge with IL-1β (10 ng) or saline. (E) Confocal imaging of Tg+IL-1β group for CCL2 (red; 594nm) and GFAP (green; 488nm). Iba-1 (green; 488nm) is captured in the inset. (F) Light microscopy labelling of CXCL1. (G) Light microscopy labelling of CXCL10. Insets show detail of positive vascular and astrocytic cells for the different chemokines.

Article Snippet: For confocal microscopy, sections were labelled with the following combinations: 6E10 (1:1000; Biolegend 803001) with an Alexa Fluor 488 anti-mouse (1:800; Invitrogen) together with Iba-1 (1:2000; Abcam ab5076) Alexa Fluor 594 anti-goat (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse (1:800; Invitrogen) in combination with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse and GFAP with an Alexa Fluor 488 anti-rabbit; GFAP (1:2000; Dako Z0334) with an Alexa Fluor 594 anti-rabbit (1:800; Invitrogen) together with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit; GFAP with an Alexa Fluor 488 anti-rabbit in combination with CCL2 (1:200; R&D AF-479-NA) with Alexa Fluor 594 anti-goat; Iba-1 (1:2000; Abcam ab5076) with Alexa Fluor 594 anti-goat.

Techniques: Enzyme-linked Immunosorbent Assay, Light Microscopy, Imaging

Journal: bioRxiv

Article Title: Microglial and Astrocyte priming in the APP/PS1 model of Alzheimer’s Disease: increased vulnerability to acute inflammation and cognitive deficits

doi: 10.1101/344218

Figure Lengend Snippet: Exogenous i.c. administration of LPS in normal mice induces activation of microglial cells to produce regulated secretion of IL-1β and this IL-1β may activate astrocytes to release chemokines such as CCL2, CXCL1 or CXCL10. However, in APP/PS1 mice, LPS stimulates more robust production and processing of IL-1β from microglia primed by Amyloid pathology. Astrocytes primed by amyloid pathology also respond in an exaggerated way to exogenous IL-1β, producing enhanced levels of chemokines. If levels of IL-1, arising endogenously from microglial stimulation, are already exaggerated this will produce a further amplification upon stimulating astrocytes that are hypersensitive to IL-1. These exaggerated responses also appear to affect many other astrocytic transcripts involved in innate immune function as shown by fold-change expression in comparison to WT+saline.

Article Snippet: For confocal microscopy, sections were labelled with the following combinations: 6E10 (1:1000; Biolegend 803001) with an Alexa Fluor 488 anti-mouse (1:800; Invitrogen) together with Iba-1 (1:2000; Abcam ab5076) Alexa Fluor 594 anti-goat (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse (1:800; Invitrogen) in combination with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit (1:800; Invitrogen); 6E10 with an Alexa Fluor 633 anti-mouse and GFAP with an Alexa Fluor 488 anti-rabbit; GFAP (1:2000; Dako Z0334) with an Alexa Fluor 594 anti-rabbit (1:800; Invitrogen) together with IL-1β (1:50; Peprotech 500-P51) with an Alexa Fluor 488 anti-rabbit; GFAP with an Alexa Fluor 488 anti-rabbit in combination with CCL2 (1:200; R&D AF-479-NA) with Alexa Fluor 594 anti-goat; Iba-1 (1:2000; Abcam ab5076) with Alexa Fluor 594 anti-goat.

Techniques: Activation Assay, Amplification, Expressing

Journal: Nature Communications

Article Title: TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis

doi: 10.1038/ncomms15395

Figure Lengend Snippet: ( a , b ) Naive WT or Fn14-deficient mice were injected with rTWEAK or IgG, administered once s.c. After 4 days expression of mRNA for indicated chemokines was measured in skin biopsies. ( a , left) RNAseq heatmap of chemokine expression from 3 to 4 animals/group. (right) Mean (AV) fold increase and SD calculated from RNAseq data of the comparisons of rTWEAK into WT versus Fn-deficient mice, rTWEAK versus IgG into WT mice, and rTWEAK versus no injection into WT mice. ( b ) qPCR mRNA expression of indicated chemokines relative to GAPDH. Combined results from two experiments. Each data point one mouse. ( c – f ) naive WT mice were injected with rTWEAK or IgG (s.c.) twice weekly over 14 days as in , with either a combination of blocking antibodies to CCL2, CCL5 and CCL7, or a combination of control antibodies (i.p). Combined results from two experiments. ( c ) Masson's trichrome staining of representative skin sections. Scale bar, 200 μm. ( d , e ) Quantification of epidermal and dermal thickness. ( f ) Total numbers of CD45 + cells and SigF + CD45 + CD11b + Ly6G − eosinophils from skin biopsies. ( g , h ) WT and TWEAK-deficient animals were treated with HDM for 23 days ( g ) and IMQ for 8 days ( h ) and mRNA for indicated chemokines assessed in skin, relative to GAPDH expression. Combined from two experiments. Each data point one mouse. Bars represent mean values. The Mann–Whitney U -test was performed to compare the groups. Asterisks indicate P <0.05. qPCR, quantitative PCR.

Article Snippet: In some experiments, animals injected with rTWEAK were treated i.p. with a combination of 50 μg hamster-α-mouse CCL2 (clone 2H5, Bioxcell, West Lebanon, NH), 50 μg rat-α-mouse CCL5 (clone 53405, R&D systems, Minneapolis, MN, USA) and 20 μg goat-α-mouse CCL7 (AF-456, R&D systems) twice weekly, or with a cocktail of the respective control antibodies twice weekly, throughout the experiment.

Techniques: Injection, Expressing, Blocking Assay, Control, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction