Journal: Journal of Immunology Research

Article Title: Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MARCO

doi: 10.1155/2014/304180

Figure Lengend Snippet: Evaluation of the effects of doses of silica and IL-1 β production in AM from WT and MARCO −/− mice. (a) Dose response to silica-stimulated IL-1 β release from AM cocultured with endotoxin (LPS at 20 ng/mL) 24 hrs was measured by ELISA. Mean ± SEM IL-1 β from cultured macrophages where filled circle ⬤ indicates MARCO −/− AM and open circle ⚪ indicates C57BL/6 WT AM. Double asterisk ** indicates P < 0.01 compared to WT control at the corresponding concentration, n = 4 per experimental group. (b) IL-1 β release from WT AM with MARCO Ab. AM were pretreated with or without MARCO Ab (5 μ g/mL) then exposed to silica (100 μ g/mL) in the presence or absence of LPS (20 ng/mL) 24 hrs. Mean ± SEM IL-1 β release from cultured macrophages was analyzed by ELISA. Triple asterisk *** indicates P < 0.001 compared to control at the corresponding treatment, n = 4 per experimental group. Unstimulated cells were used as control.

Article Snippet: For determining specificity of MARCO in IL-1 β release experiment, AM from WT mice were treated with MARCO Ab (polyclonal goat IgG, rmMARCO; aa 70–518 assession number: Q60754, Catalog number: AF2956 obtained from R&D Systems, Minneapolis, MN) 30 min prior to exposure to silica or endotoxin for 24 hr.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Concentration Assay

Journal: Journal of Immunology Research

Article Title: Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MARCO

doi: 10.1155/2014/304180

Figure Lengend Snippet: Effect of MARCO on NLRP3 activation using IL-1 β as an indicator and OVA as signal 1. IL-1 β release from WT and MARCO −/− AM in response to silica (100 μ g/mL) cocultured with OVA (10 mg/mL) and with or without cathepsin B inhibitor (10 μ M CA-074-Me). Mean ± SEM IL-1 β from cultured macrophages where white bars indicate WT AM and grey bars indicate MARCO −/− AM. Asterisk * indicates P < 0.05 compared to WT, n = 3 per experimental group.

Article Snippet: For determining specificity of MARCO in IL-1 β release experiment, AM from WT mice were treated with MARCO Ab (polyclonal goat IgG, rmMARCO; aa 70–518 assession number: Q60754, Catalog number: AF2956 obtained from R&D Systems, Minneapolis, MN) 30 min prior to exposure to silica or endotoxin for 24 hr.

Techniques: Activation Assay, Cell Culture

Journal: Journal of Immunology Research

Article Title: Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MARCO

doi: 10.1155/2014/304180

Figure Lengend Snippet: Pro-IL-1 β mRNA expression in WT and MARCO −/− AM analyzed using RT-PCR. White bars indicate WT AM and grey bars indicate MARCO −/− AM. Fold change in pro-IL-1 β expression was compared to unstimulated control. The data represent one experiment with 2 replications.

Article Snippet: For determining specificity of MARCO in IL-1 β release experiment, AM from WT mice were treated with MARCO Ab (polyclonal goat IgG, rmMARCO; aa 70–518 assession number: Q60754, Catalog number: AF2956 obtained from R&D Systems, Minneapolis, MN) 30 min prior to exposure to silica or endotoxin for 24 hr.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: Altered DNA methylation underlies monocyte dysregulation and innate exhaustion memory in sepsis

doi: 10.1101/2023.08.30.555580

Figure Lengend Snippet: (A) scRNA-seq UMAP feature plots for transcription factor Tcf7l2 motif enrichment and gene expression in WT and Ticam2 -/- BMMCs following PBS control or repetitive LPS stimulation. Numbers indicate different cell clusters, as outlined in . (B) qRT-PCR for key exhaustion gene expression in BMMCs under PBS control or repetitive LPS stimulation in the presence of different concentrations of Wnt Agonist 1. Expression levels were normalized to the geometric mean of Ube2l3 , Oaz1 , and Nktr . Data points represent independent experiments, with mean expression +/- STD indicated (n = 3-6; one-way ANOVA with Sidak’s multiple comparisons test; **** p-adj. < 0.0001; *** < 0.001; ** < 0.01; * < 0.05; ns not significant; differences are not significant unless otherwise specified; see Appendix for exact p-values). (C) Flow cytometry mean fluorescence intensity (MFI) for exhaustion (CD38, MARCO, PD-L1, CXCR2) and macrophage (F4/80) markers relative to PBS control. Box plots indicate median MFI values (n = 5-7; one-way ANOVA with Sidak’s multiple comparisons test). (D) Gating strategy (top) and population statistics (bottom) for non-classical (Ly6C low ), intermediate (Ly6C int ), and classical (Ly6C high ) monocyte subtypes in cultured BMMCs (n = 6-13 ; one-way ANOVA with Sidak’s multiple comparisons test). (E) NAD+ levels in cultured BMMCs normalized to total protein levels (n = 3-6; one-way ANOVA with Sidak’s multiple comparisons test). (F) Correlation heatmap for average DNA methylation levels at key exhaustion loci in cultured BMMCs. (n = 3-9 for each condition). (G) UCSC browser track views of average DNA methylation at the Plac8 promoter (chr5:100,572,230-100,572,607) and Cebpa/g distal enhancer (chr7:35,064,805-35,065,304), and Tcf7l2 intron (chr19:55,768,986-55,769,585) regions in cultured BMMCs. ENCODE annotated promoters (red) and enhancers (orange) are depicted in the bottom track.

Article Snippet: For Panel 2, cells were stained with antibodies against Ly6C (Pacific Blue; Biolegend #128014), CD11b (BV650; BD Biosciences #563402), Ly6g (PE-Cy5.5; Elabscience #E- AB-F1108I), CXCR2 (Alexa Fluor 488; R&D Systems # FAB2164G), CD68 (APC-Fire750; Biolegend #137041), CD172a (BV510; BD Biosciences #740159), CX3CR1 (PE-Cy7; Biolegend #149016), F4/80 (BV711; BD Biosciences #565612), CD38 (BV750; BD Biosciences #747103), PD-L1 (BV421; BD Biosciences #564716), MARCO (Alexa Fluor 647; R&D Systems #FAB29561R), FAS (BV605; Biolegend #152612), MHCII (PerCP; Biolegend #107624), CD168 (PE; Novus #NBP1-76538PE), and TREM2 (Alexa Fluor 700; R&D Systems #FAB17291N).

Techniques: Gene Expression, Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Cell Culture, DNA Methylation Assay

Journal: Journal of Neuroinflammation

Article Title: The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

doi: 10.1186/1742-2094-8-11

Figure Lengend Snippet: Bacterial supernatants of Neisseria meningitidis induced MARCO expression in primary rat astrocytes and meningeal cells . Astrocytes (A and B), microglia (C and D) and meningeal cells (E and F) were incubated with bacterial supernatants of Gram-positive bacteria Streptococcus pneumoniae (SP) or Gram-negative bacteria Neisseria meningitidis (NM) for 0, 6, 12 and 24 h. FPRL1 or MARCO mRNA expression was analyzed using SYBR green real-time RT-PCR and results were compared to the untreated sample. GAPDH (housekeeping gene) was used as an internal control. The data were assessed from three independent experiments in triplicate. An asterisk indicates a significant difference (**, p < 0.001; *, p < 0.05) compared to control as determined by ANOVA followed by the Bonferroni test.

Article Snippet: The following primary antibodies were used for this study: rabbit anti-MARCO antibody (1:100; Santa Cruz, USA), rabbit anti-FPRL1 antibody (1:100; sc-18191, Santa Cruz, USA), goat anti-rCRAMP antibody (1:100; sc-34170, Santa Cruz, USA), and rabbit anti-LL-37 antibody (1:100; ab64892, Abcam, Cambridge, UK).

Techniques: Expressing, Incubation, Bacteria, SYBR Green Assay, Quantitative RT-PCR, Control

Journal: Journal of Neuroinflammation

Article Title: The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

doi: 10.1186/1742-2094-8-11

Figure Lengend Snippet: Neisseria meningitides bacterial supernatants induced an increase of MARCO immunoreactivity in astrocytes . Primary rat astrocytes and microglia were fixed and labelled with anti-FPRL1 and anti-MARCO antibodies. FPRL1 (A) and MARCO (B) protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.

Article Snippet: The following primary antibodies were used for this study: rabbit anti-MARCO antibody (1:100; Santa Cruz, USA), rabbit anti-FPRL1 antibody (1:100; sc-18191, Santa Cruz, USA), goat anti-rCRAMP antibody (1:100; sc-34170, Santa Cruz, USA), and rabbit anti-LL-37 antibody (1:100; ab64892, Abcam, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining

Journal: Journal of Neuroinflammation

Article Title: The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

doi: 10.1186/1742-2094-8-11

Figure Lengend Snippet: Strongly increased MARCO expression in astrocytes after Neisseria meningitides infection in an infant rat model of meningitis . Coronal brain sections from rats infected with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were fixed at defined time-points after infection and immunolabeled using anti-MARCO (green) and anti-glial fibrillary acidic protein (anti-GFAP) antibodies to identify astrocytes with nuclear counterstaining (blue). Sections were examined by double fluorescence microscopy. The figures show representative results from one of three independent experiments. Scale bar = 200 μm for overview and 20 μm for the other images. (B) Shows a detail of (A) . Please note that the detail shows a strong colocalization between GFAP and MARCO in the sub-cortical layer after NM infection.

Article Snippet: The following primary antibodies were used for this study: rabbit anti-MARCO antibody (1:100; Santa Cruz, USA), rabbit anti-FPRL1 antibody (1:100; sc-18191, Santa Cruz, USA), goat anti-rCRAMP antibody (1:100; sc-34170, Santa Cruz, USA), and rabbit anti-LL-37 antibody (1:100; ab64892, Abcam, Cambridge, UK).

Techniques: Expressing, Infection, Immunolabeling, Fluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

doi: 10.1186/1742-2094-8-11

Figure Lengend Snippet: Inhibition of FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation . siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F) . Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).

Article Snippet: The following primary antibodies were used for this study: rabbit anti-MARCO antibody (1:100; Santa Cruz, USA), rabbit anti-FPRL1 antibody (1:100; sc-18191, Santa Cruz, USA), goat anti-rCRAMP antibody (1:100; sc-34170, Santa Cruz, USA), and rabbit anti-LL-37 antibody (1:100; ab64892, Abcam, Cambridge, UK).

Techniques: Inhibition, Expressing, Phospho-proteomics, Control, Transfection, SYBR Green Assay, Quantitative RT-PCR, Incubation, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

doi: 10.1186/1742-2094-8-11

Figure Lengend Snippet: FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells . For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B) , hFPRL1- (C) , and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H) . Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J) , hFPRL1- (K) , and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.

Article Snippet: The following primary antibodies were used for this study: rabbit anti-MARCO antibody (1:100; Santa Cruz, USA), rabbit anti-FPRL1 antibody (1:100; sc-18191, Santa Cruz, USA), goat anti-rCRAMP antibody (1:100; sc-34170, Santa Cruz, USA), and rabbit anti-LL-37 antibody (1:100; ab64892, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Transfection, Expressing, Western Blot, Control, Inhibition, Activity Assay