Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: CD5L -KO cells have reduced inflammatory state. Deletion of CD5L led to decrease in basal and LPS-induced cytokine mRNA levels. TNF and IL-1β levels in unstimulated cells were decreased nearly 300-fold, with average basal levels of 0.003 for knockout cells compared to the value of 1 set for the controls. A total of 2.5 × 10 5 cells in 24-well plates were differentiated using 50 ng/mL PMA for 48 hours. Stimulation was performed after 24 hours of rest in PMA-free media with 100 ng/mL LPS for 4 hours. Inflammatory cytokine expression levels were determined using qRT-PCR of RNAs isolated from cell lysates. Graphs are representative of at least three independent experiments. PMA, phorbol 12-myristate 13-acetate. (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Isolation

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: RORα is epistatic to CD5L . Induction of inflammatory TNF (A) and IL-1β (B) gene expression in CD5L/RORA DKO cells is similar to that observed in RORA deletion cells (n = 3, ± SEM). Paired two-tailed t -test of three biological replicates has been used to evaluate statistical significance of differences between RORAko and CD5L/RORA DKO cell lines. Both RORAko and CD5L/RORA DKO cells produce similarly higher levels of TNF than the CD5Lko cells at 3 and 6 hours after LPS stimulation (C) . CD5Lko cells produce significantly lower amounts of IL-6 at both the 3- and 6-hour timepoints, while RORAko and CD5L/RORA DKO secrete similar levels of this cytokine (D) (n = 6, ± SEM, representative of two independent experiments * p<0.05, ** p<0.01).

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Gene Expression, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: RNA-seq analysis of differentiated CD5L deletion cells. (A) Volcano plot of ANOVA results. Vertical cutoff line is at FDR-adjusted p = 0.05. Vertical cutoff lines are at fold difference = 2. A total of 1,165 annotated genes were found to be differentially expressed between CD5L deletion and control cell lines using these criteria. The majority of differentially expressed genes (879) were downregulated (blue). (B) Gene Ontology terms enriched by the differentially expressed dataset include regulation of intracellular processes as well as cell-to-cell signaling characteristic of immune cells. (C) All five most significantly enriched KEGG pathways mediate immune signaling. (D) The magnitude of changes in expression levels of genes that contribute to the top five enriched KEGG pathways. For genes that are not part of a specific pathway, the values are shown in gray. Colors represent fold difference change in gene expression when compared to controls. FDR, false discovery rate.

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: RNA Sequencing, Control, Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: Expression of genes in the TNF signaling pathway is impacted by deletion of CD5L . Deletion of CD5L results in downregulation (green) of transcription of nearly half of the genes comprising the TNF signaling pathway. Expression of signal propagating kinases is also affected by p38, and JNK is downregulated at the transcriptional level (adapted from KEGG TNF signaling pathway, hsa04668 6/25/18).

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: Lipid remodeling function of CD5L. (A) Total levels of lipid classes analyzed in mutant and control cells. (B) Proportion of side acyl chains classified according to the extent of their saturation. MUFA: one or two double bonds in a 3-acyl chain lipid or one double bond in a 2-acyl chain lipid. MUFA1: number of double bonds and acyl chains is equal. PUFA2, PUFA3, and PUFA4: number of double bonds is two-, three-, and fourfold higher compared to number of acyl chains in the lipid, respectively. (C) Deletion of CD5L significantly (*** p < 0.001, Mann–Whitney test) increases levels of highly polyunsaturated [PUFA(3,4,5)] fatty acids. (D) CD5L deletion results in an increase of free cholesterol in mutant cells. PUFA, polyunsaturated fatty acid. (E) Corrected Total Cell Fluorescence (CTCF) values calculated for 10 randomly selected cells for each cell line (* p = 0.014 unpaired two-tailed t -test).

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Mutagenesis, Control, MANN-WHITNEY, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: FASN activity in knockout and control cells. Deletion of CD5L does not significantly change the overall FASN activity of human macrophages. Lysates from 10 6 cells were used to measure FASN activity by monitoring NADPH oxidation over 15 minutes. Activity is presented as an average of replicates from three independent experiments. FASN, fatty acid synthase.

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Activity Assay, Knock-Out, Control

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: Differences in gene expression in CD5L and RORA deletion cells. (A) Self-organizing map (SOM) clustering identified six distinct patterns of changes in gene expression between respective mutant and control cells. (B) GO term analysis of genes in highlighted divergently regulated clusters identifies terms characteristic of immune response. (C) Eight out of 10 KEGG pathways enriched by genes from highlighted clusters are central to shaping the inflammatory signaling of immune cells. Genes contributing to the TNF (D) and NF-κB (E) signaling pathways are downregulated in CD5L mutant cells and upregulated in RORA mutants. GO, Gene Ontology.

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Gene Expression, Mutagenesis, Control, Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: CD5L deletion induces transcriptional changes in undifferentiated monocytes. (A) Deletion of CD5L in undifferentiated monocytic THP-1 cells significantly changed expression of 52 genes more than twofold up and 95 genes twofold down (in color). (B) Expression of atherogenesis-related genes involved in interaction of monocytes with ECM is changed in CD5Lko cells. (C) CD52 expression is significantly increased in undifferentiated (FDR = 3e−14) and differentiated (FDR = 2.9e−35) CD5Lko cells. (D) Cellular CD52 protein levels are increased in undifferentiated CD5Lko THP-1 monocytes. ECM, extracellular matrix; FDR, false discovery rate.

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Endogenous CD5L controls the metabolic and inflammatory state of human macrophages

doi: 10.3389/fimmu.2025.1677948

Figure Lengend Snippet: Model for the role of CD5L in mediating inflammatory state of human macrophages. (A) CD5L induces changes in macrophages’ lipid content, reducing the availability of RORα ligands and subsequent RORα inactivation. Thus, no inhibitory effect of RORα is applied on inflammatory signaling. (B) In the absence of CD5L, there are sufficient levels of RORα ligands driving its activation and downregulation of inflammatory signaling.

Article Snippet: The primary antibodies against CD5L (mouse monoclonal F1 clone, 1/1,000, sc-514283, Santa Cruz Biotechnology), GAPDH (1/2000, sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA USA), RORA (1/1,000 PP-H3910-00, R&D Systems), and the HRP-conjugated goat anti-mouse secondary antibody (1/5000, #P0447, Dako) were diluted in 0.5× blocking buffer/PBS.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Schematic of experimental approach for RNA sequencing of islets treated with vehicle or pro-inflammatory cytokines (PIC). (B) RNA sequencing data showing relative expression of Gpr31b in WT and KO islets treated with PIC (n=3 mice per condition). (C) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with vehicle (n=3 mice per condition). (D) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with vehicle. Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (E) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). (F) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (G) Analysis of CD5L concentration by enzyme-linked immunosorbent assay (ELISA) in the supernatant of islets treated with vehicle or PIC. (H) Gene set enrichment analysis (GSEA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition) using hallmark gene sets. Both suppressed ( top panel ) and activated gene sets ( bottom panel ) are shown. Normalized enrichment score (bars) and log-transformed P-value (blue dots). All pathways meeting significance criteria of adjusted P-value <0.05 are shown. (I) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for differentially expressed genes meeting more stringent criteria of absolute fold change ≥2 with adjusted P-value <0.05. (J) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for genes in the leading edge for the ROS hallmark gene set. Data presented as mean with SEM. Statistical significance determined by Student’s t-test or paired one-way analysis of variance (Friedman ANOVA).

Article Snippet: CD5L protein levels in islet supernatants were quantified using a mouse CD5L enzyme-linked immunosorbent assay (ELISA) kit (orb565622; Biorbyt, Cambridge, UK) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay