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