Journal: Nature Communications

Article Title: DPEP2 suppresses hyperinflammation via metabolic reprogramming of macrophages in sepsis

doi: 10.1038/s41467-026-70466-4

Figure Lengend Snippet: a mRNA levels of Dpep2 in RAW264.7 cells were examined after stimulation with Pam3Csk4 (TLR1/2 ligand), LPS (TLR4 ligand), R848 (TLR7/8 ligand) and poly (I:C) (TLR3 ligand) for 12 h ( n = 3 biological replicates). b , c mRNA expressions of DPEP2 in human monocytes ( b ) or bone marrow-derived macrophages (BMDM) ( c ) were examined after LPS stimulation for 12 h with different doses ( n = 3 biological replicates). d mRNA expressions of Dpep2 in RAW264.7 cells were examined after LPS stimulation for 6 or 12 h with different doses ( n = 3 biological replicates). e Protein levels of DPEP2 in RAW264.7 cells were examined after LPS stimulation for 24 h with different doses ( n = 3 biological replicates). f – g RAW264.7 cells with Dpep2 deficiency (sh Dpep2 RAW264.7 cells, n = 4 biological replicates) and control RAW264.7 cells (shNT RAW264.7 cells, n = 3 biological replicates) were obtained for RNA-seq analysis; f Volcano plot showing results from differential expression analysis. Genes with P-adjusted value < 0.05 are highlighted in red or blue; g Bar plot showing the GSEA results in sh Dpep2 vs shNT RAW264.7 cells. h – k Levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF) in sh Dpep2 vs shNT BMDMs and Dpep2 knockout ( Dpep2 KO) vs wild type (WT) mice derived BMDMs were analyzed ( n = 3 biological replicates). l, m IL-1β, IL-6 and TNF secretion of sh Dpep2 vs shNT RAW264.7 cells and Dpep2 KO vs WT mice derived BMDMs were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). n - o RAW264.7 cells and BMDMs overexpressing Dpep2 (DPEP2) and relatively control vector (Vector) cells were constructed. Levels of IL-1β, IL-6, and TNF secreted by DPEP2 cells vs Vector cells were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). Data represent the mean ± standard deviation. ns, not significant. One-way ANOVA followed by the Bonferroni multiple comparisons was used in ( a – c, e ), two-way repeated measures ANOVA with Tukey’s multiple comparisons test was used in ( d ), two-sided Wilcoxon rank-sum test was used in ( f ) and two-sided unpaired Student’s t test was used in ( h–o ). NES, n o rmalized enrichment score.

Article Snippet: RAW264.7 cells were treated with TLR1/2 ligands (1 μg/mL Pam3Csk4; HY-P1180A, MedChemExpress, Monmouth Junction, NJ, USA), TLR7/8 ligands (1 μg/mL R848; HY-13740, MedChemExpress) and TLR3 ligands (1 μg/mL poly(I:C); HY-107202, MedChemExpress) for 12 h. RAW264.7 cells, human monocytes, and BMDMs were stimulated with LPS (L4391, Sigma) to establish an in vitro inflammatory model. RAW264.7 cells were treated with LTD4 (1 μM; 20310, Cayman, Ann Arbor, MI, USA), LTE4 (1 μM; 20410, Cayman) or PGE2 (1 μM; HY-101952, MedChemExpress) in vitro.

Techniques: Derivative Assay, Control, RNA Sequencing, Quantitative Proteomics, Knock-Out, Plasmid Preparation, Construct, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Porcine B Cell Subset Responses to Toll-like Receptor Ligands

doi: 10.3389/fimmu.2017.01044

Figure Lengend Snippet: Toll-like receptor (TLR) mRNA expression in B cells, monocytes, and plasmacytoid dendritic cells (pDCs). (A) CD21 + B cells and CD14 + monocytes were sorted by magnetic cells sorting (purity > 98%); CD172a + CD4 high pDCs were purified by fluorescence activated cell sorting (FACS) (purity > 99%). RNA was isolated for RTqPCR analysis of TLR expression. Data are from three biological replicates generated using cells from three different pigs. In B cells, TLR7 expression was significantly higher than all other TLR (*** P < 0.0001). In pDC, TLR3, TLR7, and TLR9 were found to be significantly higher when compared to the other TLRs (** P < 0.001). (B) Expression of TLR2 on CD21 + cells after stimulation of peripheral blood mononuclear cells with Pam3Cys-SK4 for 24 h. A representative experiment from three independent experiments is shown. (C) TLR mRNA expression of B cell subsets P1–P4 sorted by FACS according to the gates defined in Figure . Mean values calculated from three different animals with standard deviations are shown. P4 was statistically significant from P1 and P2 for TLR3 expression (* P < 0.05).

Article Snippet: The TLR7 ligands loxoribine and gardiquimod, as well as the TLR7/8 ligand resiquimod were acquired from Chemdea (Ridgewood, NJ, USA), and the TLR7 ligand imiquimod was purchased from Invivogen.

Techniques: Expressing, Purification, Fluorescence, FACS, Isolation, Generated

Journal: Cancer treatment reviews

Article Title: In vivo cancer vaccination: which dendritic cells to target and how?

doi: 10.1016/j.ctrv.2018.10.012

Figure Lengend Snippet: Use of TLR7 agonists as adjuvant in active clinical trials (* www.clinicaltrials.gov ).

Article Snippet: 5 , NCT02556463 A Study of MEDI9197 in Subjects With Solid Tumors or CTC Landin Combination With Durvalumab and/ or Palliative Radiation in Subjects With Solid Tumors , Solid Tumors and early-stage cutaneous T Cell lymphoma [CTCL] (135) , • TLR7/8 agonist-MEDI9197 • Durvalumab (Fc optimized anti programmed cell death-ligand 1 [PD-L1] monoclonal antibody) • Radiation , • Phase I, non- randomized and open- labeled , • Recruiting. • Study Start date: 4th Nov 2015 • Completion date: 19th Aug 2020 , • MedImmune LLC.

Techniques: Adjuvant, Clinical Proteomics, Labeling

Journal: Cancer treatment reviews

Article Title: In vivo cancer vaccination: which dendritic cells to target and how?

doi: 10.1016/j.ctrv.2018.10.012

Figure Lengend Snippet: Use of TLR3 agonists as adjuvant in a selection of active clinical trials (* www.clinicaltrials.gov ).

Article Snippet: 5 , NCT02556463 A Study of MEDI9197 in Subjects With Solid Tumors or CTC Landin Combination With Durvalumab and/ or Palliative Radiation in Subjects With Solid Tumors , Solid Tumors and early-stage cutaneous T Cell lymphoma [CTCL] (135) , • TLR7/8 agonist-MEDI9197 • Durvalumab (Fc optimized anti programmed cell death-ligand 1 [PD-L1] monoclonal antibody) • Radiation , • Phase I, non- randomized and open- labeled , • Recruiting. • Study Start date: 4th Nov 2015 • Completion date: 19th Aug 2020 , • MedImmune LLC.

Techniques: Adjuvant, Selection, Clinical Proteomics, In Situ, Activity Assay, Immunofluorescence, Recombinant, Immunopeptidomics, Labeling

Journal: PLoS ONE

Article Title: Cis Association of Galectin-9 with Tim-3 Differentially Regulates IL-12/IL-23 Expressions in Monocytes via TLR Signaling

doi: 10.1371/journal.pone.0072488

Figure Lengend Snippet: A) RT-PCR detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 mRNAs in THP-1 cells transfected with Gal-9 plasmid in the absence or presence of TLR stimulations. THP-1 cells were transfected with either pBKCMV3-Gal-9 plasmid (Gal-9) or pBKCMV3 empty vector (Con) for 24 h, stimulated with or without LPS and R848 for 6 h, followed by RT-PCR measuring Tim-3, IL-12p35, IL-23p19, IL-12/IL-23p40 mRNA expressions. β-actin served as loading control to normalize target gene levels. Data are shown as representative imaging (left) and mean ± SE of corrected optimal densitometry (O.D) values from three independent experiments (right). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. B) RT-PCR detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 mRNAs in THP-1 cells transfected with Gal-9 siRNAs in the absence or presence of TLR stimulations. THP-1 cells were transfected with either Gal-9 or control siRNAs for 48 h, stimulated with or without LPS/R848 for 6 h, followed by RT-PCR measuring Tim-3, IL-12p35, IL-23p19, IL-12/IL-23p40 mRNA expressions. β-actin served as loading control. Data are shown as representative imaging (left) and mean ± SE of corrected optimal densitometry (O.D) values from three independent experiments (right). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. C) Enhanced Gal-9 expression and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities. pBKCMV3-Gal-9 or control vector was transiently transfected into THP-1 cells, along with either Tim-3, or IL-12p35, or IL-23p19, or IL-12/IL-23p40 promoter luciferase reporter vectors. 24 h after transfection, the cells were stimulated with or without LPS/R848 for 6 h, followed by luciferase assays for reporter gene transcriptional activities as described in Methods. Data are shown as mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. D) Silenced Gal-9 expression and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities. Gal-9 or control siRNAs was transiently transfected into THP-1 cells, along with either Tim-3, or IL-12p35, or IL-23p19, or IL-12/IL-23p40 promoter reporter vectors. 48 h after transfections, the cells were stimulated with or without LPS/R848 for 6 h, followed by luciferase assays. Data are shown as mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software.

Article Snippet: Following 24~48 h of transfection or co-transfections, the cells were stimulated with or without 1 μg/ml of TLR 4 ligand - lipopolysaccharide (LPS, Santa Cruz) and 2.5 μg/ml of TLR 7/8 ligand - R848 (Santa Cruz) for 6 h, followed by detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 gene transcriptions with reverse transcription-polymerase chain reaction (RT-PCR) and luciferase assay as described below.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control, Imaging, Software, Expressing, Luciferase

Journal: PLoS ONE

Article Title: Cis Association of Galectin-9 with Tim-3 Differentially Regulates IL-12/IL-23 Expressions in Monocytes via TLR Signaling

doi: 10.1371/journal.pone.0072488

Figure Lengend Snippet: A) Western blot detection of Gal-9 and phosphorylation of STAT-1 proteins in THP-1 cells transfected with Gal-9 plasmid or siRNA. THP-1 cells were transfected with either pBKCMV3-Gal-9 plasmid (Gal-9) or pBKCMV3 empty vector (Con), or Gal-9 silencing siRNA or control siRNA. After 24~48 h transfection, the cells were subjected to Western blot analysis of Gal-9 and pSTAT-1 proteins. β-actin or total STAT-1 served as loading control to normalize target gene levels. Data are shown as representative imaging (left) and corrected optimal densitometry (O.D.) values from three independent experiments for STAT-1 (right). NS = no significance. B) Western blot detection of pSTAT-3 protein in THP-1 cells transfected with Gal-9 plasmid in the presence or absence of TLR stimulations. THP-1 cells were transfected with either Gal-9 or Control plasmid for 24 h, stimulated with or without LPS/R848 for 6 h, followed by Western blot analysis of pSTAT-3 protein. Total STAT-3 served as loading control to normalize target gene levels. Representative imaging and corrected O.D. values from three independent experiments are shown. *P<0.05. C) Western blot detection of pSTAT-3 protein in THP-1 cells transfected with Gal-9 siRNAs with or without TLR stimulations. THP-1 cells were transfected with either Gal-9 or control siRNAs for 48 h, stimulated with or without LPS/R848 for 6 h, followed by Western blot analysis of pSTAT-3 protein. Total STAT-3 served as loading control. Representative results and corrected O.D. values from three experiments are shown. *P<0.05, NS = no significance. D) Luciferase assay for Tim-3, IL-12, IL-23 promoter activities in Gal-9-transfected THP-1 cells in the presence of STAT-3 inhibitor. pBKCMV3-Gal-9 or control plasmid was transfected into THP-1 cells, along with Tim-3, IL-12p35, IL-23p19, or IL-12/IL-23p40 promoter reporter vectors, in the presence of STAT3-specific inhibitor or DMSO for 24 h, followed by luciferase assays. Data are mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance. E) Intracellular Gal-9 and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities in THP-1 cells treated with STAT-3 inhibitor and TLR stimulation. pBKCMV3-Gal-9 or control vector was transfected into THP-1 cells, along with Tim-3, IL-12p35, IL-23p19, or IL-12/IL-23p40 promoter reporter vectors, in the presence of STAT3-specific inhibitor for 24 h. The cells were stimulated without or with LPS/R848 for 6 h, followed by luciferase assays as described in Methods. Data are mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance.

Article Snippet: Following 24~48 h of transfection or co-transfections, the cells were stimulated with or without 1 μg/ml of TLR 4 ligand - lipopolysaccharide (LPS, Santa Cruz) and 2.5 μg/ml of TLR 7/8 ligand - R848 (Santa Cruz) for 6 h, followed by detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 gene transcriptions with reverse transcription-polymerase chain reaction (RT-PCR) and luciferase assay as described below.

Techniques: Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Control, Imaging, Luciferase

Journal: PLoS ONE

Article Title: Cis Association of Galectin-9 with Tim-3 Differentially Regulates IL-12/IL-23 Expressions in Monocytes via TLR Signaling

doi: 10.1371/journal.pone.0072488

Figure Lengend Snippet: A) The kinetic presentation of Tim-3 on the surface of M/M Ø following TLR stimulation analyzed by immunofluorescent microscopy. Tim-3 on the surface of live M/M Ø was labeled with a primary Tim-3 antibody (Santa Cruz); the cells were stimulated with LPS/R848 for 0, 1, 2, 3, 6 h, followed by staining with a fluorescence-conjugated secondary antibody and analysis by AMG fluorescence phase microscope. Magnification 20x for all panels and scale bar = 100 µm. B) The presentation of Tim-3 on the surface of M/M Ø following TLR stimulation analyzed by flow cytometry. M/M Ø were stimulated with LPS/R848 for the same period of time (0, 1, 2, 3, 6 h), immunostained for Tim-3 cell surface expression using a different source of antibody (eBioscience), and analyzed by flow cytometry as described in the Methods. Isotype-matched control antibodies (eBioscience) and fluorescence minus one (FMO) controls were used to determine background levels of staining and adjust multicolor compensation as gating strategy. Representative dot plots with percentages of cells in the gated area are shown. C) The presentation of Tim-3 inside the cytoplasm of M/M Ø following TLR stimulation analyzed by flow cytometry. First, M/M Ø were incubated with 10 µg/ml of un-conjugated mAb (F38-2E2, Biolegend) to saturate surface Tim-3 epitopes; after TLR stimulation for the same period of time and fixation/permeabilization procedures, staining of intracytoplasmatic Tim-3 epitopes was performed using the same clone of conjugated Tim-3 mAb (F38-2E2), followed by flow cytometric analysis. Representative dot plots with percentages of cells gated based on the same strategy are shown on the left. The percentage of Tim-3 presentation (mean ± SD) on the surface and intracellular of M/M Ø following TLR stimulation analyzed by flow cytometry from 3 independent experiments is shown on the right. D) The amount of Tim-3 in M/M Ø following TLR stimulation analyzed by Western blot. Purified M/M Ø were stimulated with LPS/R848 for the same period of times, and total Tim-3 protein expression was detected by Western blot as described in the Methods. E) Representative dot plots for intracellular Gal-9 detection in purified resting human M/M Ø by flow cytometry.

Article Snippet: Following 24~48 h of transfection or co-transfections, the cells were stimulated with or without 1 μg/ml of TLR 4 ligand - lipopolysaccharide (LPS, Santa Cruz) and 2.5 μg/ml of TLR 7/8 ligand - R848 (Santa Cruz) for 6 h, followed by detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 gene transcriptions with reverse transcription-polymerase chain reaction (RT-PCR) and luciferase assay as described below.

Techniques: Microscopy, Labeling, Staining, Fluorescence, Flow Cytometry, Expressing, Control, Incubation, Western Blot, Purification

Journal: PLoS ONE

Article Title: Cis Association of Galectin-9 with Tim-3 Differentially Regulates IL-12/IL-23 Expressions in Monocytes via TLR Signaling

doi: 10.1371/journal.pone.0072488

Figure Lengend Snippet: Detection of Tim-3 and Gal-9 localization/co-localization in resting and TLR-stimulated M/M Ø by confocal microscopy. Purified M/M Ø were stimulated with LPS/R848 for 0, 1, 2, 3, 6 h; following fixation/permeabilization, intracytoplasmic Tim-3 and Gal-9 staining as well as DAPI nuclear staining (blue), isotype control staining, and their imaging merges observed by confocal microscopy as described in the Methods. Magnification 100x and 1.4 oil DIC (total of 140x) for all panels. A typical cell with Tim-3 (green) expression, Gal-9 (red) expression, and imaging merge as evidence of their co-localization (yellow) in M/M Ø at each time point is shown (denoted in the square) and further amplified below.

Article Snippet: Following 24~48 h of transfection or co-transfections, the cells were stimulated with or without 1 μg/ml of TLR 4 ligand - lipopolysaccharide (LPS, Santa Cruz) and 2.5 μg/ml of TLR 7/8 ligand - R848 (Santa Cruz) for 6 h, followed by detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 gene transcriptions with reverse transcription-polymerase chain reaction (RT-PCR) and luciferase assay as described below.

Techniques: Confocal Microscopy, Purification, Staining, Control, Imaging, Expressing, Amplification

Journal: PLoS ONE

Article Title: Cis Association of Galectin-9 with Tim-3 Differentially Regulates IL-12/IL-23 Expressions in Monocytes via TLR Signaling

doi: 10.1371/journal.pone.0072488

Figure Lengend Snippet: A) Detection of Tim-3/Gal-9 physical association in un-stimulated and TLR-stimulated M/M Ø by immunoprecipitation. The purified M/M Ø were stimulated with or without LPS/R848 for 6 h; the procedure for co-immunoprecipitation of Tim-3 (40 Kd) and Gal-9 (35 Kd) was described in the Methods. Samples were pulled down by anti-Gal-9 or IgG antibodies and protein A/G PLUS-agarose; then probed with anti-Tim-3 and HRP-secondary antibody. β-actin was used to probe cell lysates for equal protein inputs. Repeated co-ip experiment by M/M Ø in the presence of α-lactose in the cell lysates to inhibit random Gal-9 bound of Tim-3 are shown in the right panel. B) Activation of IL-12/IL-23 expressions by M/M Ø following TLR stimulation. Purified M/M Ø were cultured in the presence or absence of LPS/R848 for 6 h. Tim-3 cell surface expression and intracellular IL-12p35, IL-12p70, IL-23p19 productions were analyzed by flow cytometry as described in the Methods. Representative dot plots of the relationship between IL-12p35 and IL-23p19, IL-12p70 and IL-23p19, Tim-3 and IL-23p19, Tim-3 and IL-12p70, and summary data derived from multiple subjects were shown. Each line-linked symbol represents one subject’s M/M Ø before and after TLR stimulation.

Article Snippet: Following 24~48 h of transfection or co-transfections, the cells were stimulated with or without 1 μg/ml of TLR 4 ligand - lipopolysaccharide (LPS, Santa Cruz) and 2.5 μg/ml of TLR 7/8 ligand - R848 (Santa Cruz) for 6 h, followed by detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 gene transcriptions with reverse transcription-polymerase chain reaction (RT-PCR) and luciferase assay as described below.

Techniques: Immunoprecipitation, Purification, Co-Immunoprecipitation Assay, Activation Assay, Cell Culture, Expressing, Flow Cytometry, Derivative Assay