Journal: Frontiers in Immunology

Article Title: Identification of novel amino acid variants in the Han Chinese population that impair toll-like receptor 4 signaling and confer hyporesponsiveness to lipopolysaccharide

doi: 10.3389/fimmu.2025.1556600

Figure Lengend Snippet: The p.(Gly715Ser), p.(Arg763Cys) and p.(Arg804Trp) failed to interact with Myd88. Transient transfections in HEK293T cells with 8 µg TLR4 WT-HA (lane 1) or five variants-HA (lane 2-6) and 8 µg Myd88-Flag plasmids followed by immunoprecipitation (IP) with HA-Trap magnetic beads or DYKDDDDK (flag)-Trap magnetic beads and analyzed by Immunoblotting (IB) with anti-HA or anti-Flag Ab. Binding Control magnetic beads (lane 7) or 293T blank cells (lane 8) were used as controls. Representative experiment of at least two independent experiments.

Article Snippet: A total of 30 μg of protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% non-fat milk in TBST containing 0.1% Tween 20, and incubated with mouse monoclonal antibody against mouse TLR4 (Proteintech, Wuhan, P.R.

Techniques: Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: Identification of novel amino acid variants in the Han Chinese population that impair toll-like receptor 4 signaling and confer hyporesponsiveness to lipopolysaccharide

doi: 10.3389/fimmu.2025.1556600

Figure Lengend Snippet: The study investigated the effect of TLR4 variants on LPS-induced NF- K B signaling and IL-8 production in HEK 293T cells. Cells were co-transfected with optimal amounts of GV219-TLR4 or its variant forms (p.(Gly715Ser), p.(Phe749Leu), p.(Arg763Cys), p.(Arg804Trp), p.(Pro823Thr)) at 300 ng/well, along with MD-2 (3 ng/well), CD14 (30 ng/well), NF- K B vectors (500 ng/well), and the pRL-TK reporter plasmid (100 ng/well). Following a 24-hour transfection period, cells were challenged with LPS at a concentration of 5 ng/mL for an additional 24 hours. (A) To assess the impact on signaling activity, cell lysates were assayed for firefly and renilla luciferase activities. The firefly luciferase activity was normalized against renilla luciferase activity. The results from cells transfected with TLR4 or its mutants were further normalized against those from cells transfected with the empty GV219 vector and are presented as fold induction. (B) For IL-8 quantification, supernatants were collected after the LPS treatment and analyzed using a Luminex kit (R&D Systems) on the Luminex 100 platform. (C) The expression of TLR4 before (Pre) and after (Post) transfection, along with that of NF-κB p65, CD14, MD-2, and β-actin, is shown in representative western blots for each genotype. The data shown represent the mean ± SD from triplicate measurements performed in three independent experiments (A, B) . * P< 0.05, compared with TLR4 WT. Ns, no significance. ANOVA and Dunnett’s test.

Article Snippet: A total of 30 μg of protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% non-fat milk in TBST containing 0.1% Tween 20, and incubated with mouse monoclonal antibody against mouse TLR4 (Proteintech, Wuhan, P.R.

Techniques: Transfection, Variant Assay, Plasmid Preparation, Concentration Assay, Activity Assay, Luciferase, Luminex, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Identification of novel amino acid variants in the Han Chinese population that impair toll-like receptor 4 signaling and confer hyporesponsiveness to lipopolysaccharide

doi: 10.3389/fimmu.2025.1556600

Figure Lengend Snippet: Species-specific comparison of a segment within the TLR4 protein sequence is illustrated. The residue at position 804, Arginine (R), which is subject to variation, is emphasized with a box.

Article Snippet: A total of 30 μg of protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% non-fat milk in TBST containing 0.1% Tween 20, and incubated with mouse monoclonal antibody against mouse TLR4 (Proteintech, Wuhan, P.R.

Techniques: Comparison, Sequencing, Residue

Journal: Frontiers in Immunology

Article Title: Identification of novel amino acid variants in the Han Chinese population that impair toll-like receptor 4 signaling and confer hyporesponsiveness to lipopolysaccharide

doi: 10.3389/fimmu.2025.1556600

Figure Lengend Snippet: Compared to TLR4 WT mice, the SAP-modeled p.(Arg804Trp) mice exhibited reduced responsiveness to endotoxin. (A) Overview of SAP mice model induced by cerulean and LPS and design of the experiment. (B) Representative images of pancreatic morphological changes and histologic scores in the SAP models are shown. Hematoxylin and eosin-stained histological sections of the pancreas from WT and p.(Arg804Trp) mice were selected. (C) Representative images and quantitative analysis of TUNEL staining on pancreas sections from TLR4 WT or p.(Arg804Trp) mice with saline or SAP modeling. (D, E) Representative images and quantitative analysis of neutrophil(Ly6G) and macrophage markers(F4/80) on pancreas sections from TLR4 WT or p.(Arg804Trp) mice with saline or SAP modeling. The ratios of positive cells was determined by dividing the number of positive cells by the total cell count represented by DAPI staining. The images were magnified 200×. Serum amylase (F) and lipase (G) levels were measured using an automatic biochemical analyzer. Cytokine levels of IL-6 (H) , IL-10 (I) , and TNFα (J) in serum were quantified using Luminex assay kits on the Luminex 200 system. (K) The average densitometric ratios of TLR4 to β-actin for each group are illustrated. (L) Representative western blots show TLR4 and β-actin expression in each group.* denote a statistically significant difference ( P < 0.05) between the p.(Arg804Trp) group and the wild type group in SAP modeling by ANOVA and Dunnett’s test. Data represent one experiment representative of at least three independent trials.

Article Snippet: A total of 30 μg of protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% non-fat milk in TBST containing 0.1% Tween 20, and incubated with mouse monoclonal antibody against mouse TLR4 (Proteintech, Wuhan, P.R.

Techniques: Staining, TUNEL Assay, Saline, Cell Counting, Luminex, Western Blot, Expressing

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a SEAP reporter readouts from HEK-Blue TM NOD1/2 reporter cells stimulated with MDP (200 nM), M-AE (200 nM), GM (20 μM), or M (20 μM) for 18 h. Data are presented as mean values ( n = 3). b-g PGNs induce cytokine production in macrophages and monocytes and activate dendritic cells. The respective immune cells were stimulated with the indicated PGNs, followed by cytokine analysis by RT-qPCR ( c ) or ELISA ( d ). RAW264.7 cells were treated with GM (4 mM, 2 mM, 1 mM), M (4 mM), or MDP (0.4 mM) for 24 h ( n = 4). Murine bone marrow-derived macrophages (BMDMs) and THP-1 cells were treated with GM (8 mM, 4 mM, 2 mM), M (8 mM), or MDP (0.4 mM). n = 3 or 4, respectively. e Mean fluorescent intensity (MFI) of surface CD80/86 in the cDC2s population of murine bone marrow-derived dendritic cells (BMDMs) that were treated with GM (50, 100, and 200 μM) for 18 h. f-g Assays using polymyxin B (PB) confirmed that GM was not contaminated with trace endotoxin. f ELISA analysis of the TNF- α levels in BMDMs stimulated with GM (4 mM), LPS (100 ng/mL), or polymyxin B resin (PB-resin)-neutralized GM (4 mM) or LPS (100 ng/mL) for 24 h. g The TLR4 inhibitor specifically suppresses the GM-induced TNF- α production. The RAW_Dual TM cells were pre-incubated with the respective TLR inhibitors (10 μM) for 1 h followed by GM stimulation (2 mM) for 24 h. TLR2i, TLR4i, and TLR7/9i refer to CU_CPT22, TAK-242, and AT791, respectively. ( h ) SEAP reporter readouts from HEK-Blue TM mTLR4 reporter cells stimulated with GM (1 mM) or LPS (100 ng/mL) titrated with polymyxin B (0, 0.1, 1, 10 kU) for 24 h ( n = 4). Data are presented as mean values ± s.e.m. ( n = 3 unless otherwise noted) with indicated P values determined using one-way ANOVA followed by Dunnett’s multiple comparison for ( c-d, f-g ) or two-way ANOVA followed by Tukey’s multiple comparison for ( e, h ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

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

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a-b WT and Tlr4 -/- RAW_Dual TM reporter cells were used to evaluate GM-induced NF-κB and IRF signaling. Cells were treated with respective PGNs at indicated concentrations for 24 h for quanti-blue analysis (top, n = 3) or quanti-luciferase analysis (bottom). LPS (100 ng/mL), MDP (0.4 mM), or Poly(I: C) (0.1 mg/mL) were used as controls. Concentrations of GM, MG, and M tested were indicated in the figures. c-e The RNAseq analysis and validation confirm that the GM-induced immune responses in BMDM require TLR4. c Heatmap plot of RNAseq results showing the mean values of differentially expressed genes ( p < 0.01, and logFC > 1) in WT and Tlr4 -/- BMDMs treated with GM (8 mM) or MDP (0.4 mM) for 24 h ( n = 3). d-e Validation of the RNAseq results by RT-qPCR ( d ) and ELISA ( e ). WT and Tlr4 -/- BMDMs were treated with GM (4 mM) or MDP (0.4 mM) for 24 h. Data are presented as mean values ± s.e.m. ( n = 4 unless otherwise noted) with indicated P values determined using one-way ANOVA followed by Dunnett’s multiple comparison for ( d-e ) or two-way ANOVA followed by Tukey’s multiple comparison for ( b ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Luciferase, Biomarker Discovery, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a-b In vitro enrichment of recombinant mTLR4 protein by immobilized GM. a Scheme of the pulldown assay, with GM- or G-immobilized agarose beads. b Coomassie blue-stained SDS-PAGE analysis of mTLR4 proteins bound or unbound to the resin. Excess GM was added to compete with mTLR4 bound to GM-immobilized beads. c Surface plasmon resonance (SPR) demonstrates direct binding of GM and mTLR4. Representative SPR sensorgrams of mTLR4 binding to MDP (left) and GM (middle) are shown. Corresponding plots and fitted data of steady-state binding for MDP (blue) and GM (black), with the estimated respective K D values, are shown (right). ND stands for not detected. The data shown are the representative results from 3 independent experiments. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: In Vitro, Recombinant, Staining, SDS Page, SPR Assay, Binding Assay

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a Analysis of specific residues in TLR4/MD-2 required for GM-induced NF-κB activity. The various mTLR4/mMD2 variants were transiently expressed in HEK-Blue TM reporter cells and treated with GM (0.4 mM, 0.2 mM) or LPS (100 ng/mL) for 24 h. The activation of NF-κB was measured based on the SEAP reporter signals. For each transfected group, the readouts were normalized to the H 2 O-treated samples. Data are presented as mean values ± s.e.m. ( n = 3). b-c Structural-activity relationship (SAR) analysis of disaccharide analogs using HEK-Blue TM mTLR4 reporter cells. Cells were treated with respective disaccharides at 0.5 or 1 mM for 24 h. LPS (20 ng/mL and 100 ng/mL) was used as controls. Data are presented as mean values ( n = 2). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Activity Assay, Activation Assay, Transfection

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a Timeline of the study, where GM was intraperitoneally administered at 10 mg/kg to mice (WT and Tlr4 -/- ) that were provided with 3% DSS or normal drinking water. I.p. injections of PBS were given to the negative control group. b-c Measurements of mice body weight ( b ) ( n = 8, 6, 10, 10, 9, 8, 10, 10, respectively) and colon length ( c ) ( n = 8, 6, 14, 14, 8, 8, 15, 14, respectively) at the endpoint of the timeline. d-e Representative images of colons ( d ) and H&E-stained colon sections ( e ) from each group ( n = 5). f Flow cytometry analysis of immune cell infiltration in the colons of each treatment group. The percentage of the CD45 + population ( n = 8, 6, 12, 13, 7, 8, 12, 12, respectively) was calculated within pre-gated live cells. The percentage of Ly6C + MHCII - ( n = 8, 6, 10, 10, 7, 8, 10, 8, respectively) and Ly6C + Ly6G + ( n = 8, 6, 8, 8, 7, 8, 11, 9, respectively) populations was calculated within the CD45 + population. g RT-qPCR analysis of colonic cytokines demonstrates the significantly reduced inflammation in the GM-supplemented DSS-induced colitis in WT mice but not Tlr4 -/- mice. Tnfα , n = 10, 10, 9, 9, 5, 8, 8, 8, respectively. Il1β , n = 12, 12, 10, 13, 5, 8, 8, 7, respectively. Ccl2 , n = 10, 10, 9, 9, 5, 8, 6, 6, respectively. Data are presented as mean values ± s.e.m. ( n as indicated) with indicated P values determined using two-way ANOVA followed by Tukey’s multiple comparison for ( c, f-g ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Negative Control, Staining, Flow Cytometry, Quantitative RT-PCR, Comparison