Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin activates TLR signaling in cardiac fibroblasts (A) Cardiac fibroblasts were incubated with Hb (5 mg/mL) for the indicated times. Protein extracts were analyzed by immunoblotting for p-NFκB and p-MAPK. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB and p-MAPK band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). (B) Cardiac fibroblasts were incubated either vehicle or with varying concentrations of a TLR4 pharmacological inhibitor for 3 h. Hb (5 mg/mL) was then added. After 1 h, protein extracts were isolated and subsequently analyzed by immunoblotting for p-NFκB. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗ p < 0.05; ns, not significant). (C) Cardiac fibroblasts were incubated with either LPS or Hb for 24 h, after which expression of the indicated pro-inflammatory cytokines was determined by qPCR. Expression values are shown relative to the housekeeping gene Gapdh. N = 6. ANOVA with Tukey post-hoc tests were used to determine significance (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Incubation, Western Blot, Control, Isolation, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin mediates gene repression through TLR2 and TLR4 (A and B) A study was conducted to determine the effect of hemoglobin (Hb) on (A) fibroblast-to-cardiomyocyte reprogramming and (B) fibroblast gene expression. With respect to fibroblast-to-cardiomyocyte reprogramming, cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. 24 h later, the cells were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte specific genes by qPCR. Expression values were normalized to the housekeeping gene Gapdh. N = 6–10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). t test was used to determine the significance between the miR combo groups (## p < 0.01, # p < 0.05; ns, not significant). With respect to fibroblast gene expression, cardiac fibroblasts were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Expression of the indicated fibroblast-specific genes was determined by qPCR and normalized to the housekeeping gene Gapdh. N = 10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Gene Expression, Transfection, Control, Incubation, Expressing

Journal: Frontiers in Immunology

Article Title: Adsorption-mediated modulation of lipopolysaccharide bioactivity by clinoptilolite zeolite with in vitro immunomodulatory effects and in vivo safety evaluation

doi: 10.3389/fimmu.2026.1818740

Figure Lengend Snippet: TLR4 inhibition supports zeolite-mediated reduction of LPS-induced NF-κB activation in THP-1 Dual cells. THP-1 Dual NF-κB reporter cells were pre-treated with the small-molecule TLR4 inhibitor TAK-242 (5 µM) for 1 h prior to stimulation with lipopolysaccharide (LPS) or combined clinoptilolite zeolite and LPS treatment conditions. DMSO (0.1%) was included as a vehicle control. Data are presented as mean ± SD. Statistical significance was determined using an unpaired Student’s t-test. ns, not significant.

Article Snippet: To inhibit TLR4 signaling, cells were pre-treated with the small-molecule TLR4 inhibitor TAK-242 ( ) (MedChemExpress, HY-11109) at a final concentration of 5 μM for 1 h prior to stimulation.

Techniques: Inhibition, Activation Assay, Control

Journal: Advanced Science

Article Title: Integrated Single‐Cell and TCR Profiling Reveals Protection‐Associated CD8 + T Cell Subsets Linked to Viral Control in PRRSV

doi: 10.1002/advs.76732

Figure Lengend Snippet: CD8 + T cells preferentially receive signals from macrophages /monocytes for activation via TLR4 signaling. (A) Communications between APC and CD8 + T cells within each experimental group as inferred by CellPhoneDB. (B) Analysis of the expression level of antigen presentation‐related genes in indicated cell subclusters. (C) Dot plot showing normalized mean expression level of genes involved in innate immunity in indicated subclusters of macrophages and monocytes. (D) The KEGG enrichment analysis of innate immunity‐associated pathways for differentially upregulated genes in the subclusters of macrophages and monocytes. The enrichment level is determined by the number of included genes and their expression levels. (E) Percentage of cells containing virus RNA fragments in the indicated cell clusters. (F, G) Role of TLR4 signaling in T cell activation. The spleen cell mixture from three pigs of JXA1R groups at 5dpc were prepared. The ability to secret IFN‐γ was determined using ELISpot in the presence of TAK‐242, a TLR4 inhibitor or absence of macrophage/monocytes that were depleted by mouse monoclonal antibodies to CD14. The representative ELISpot wells (F) and statistical analysis (G) were presented. (H, I) Role of TLR8 signaling in T cell activation with TLR8 inhibitor. The representative ELISpot wells (H) and statistical analysis (I) were presented. Statistical analysis was performed by two‐tailed Student's t‐test, and error bars indicate means ± standard error of mean (SEM). Asterisks (*) indicate the statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: For the TLR‐4 or TLR‐8 loss‐function assay, the CD14‐deleted cells were pre‐incubated with TAK‐242 (MCE, #243984‐11‐4) or CU‐CPT8m (MCE, #HY‐112050) at a final concentration of 5 μM for 2 h, followed by the addition of virus, and ELISpot was then performed.

Techniques: Activation Assay, Expressing, Immunopeptidomics, Virus, Enzyme-linked Immunospot, Bioprocessing, Two Tailed Test

Journal: Advanced Science

Article Title: Integrated Single‐Cell and TCR Profiling Reveals Protection‐Associated CD8 + T Cell Subsets Linked to Viral Control in PRRSV

doi: 10.1002/advs.76732

Figure Lengend Snippet: Schematic model summarizing immune features associated with protection and pathogenesis. In the protected group, antigen presentation is associated with specialized subset of macrophages and monocytes characterized by high TLR4 and pro‐inflammatory signals expression. With appropriate CD4 + T cell help, these CD8 + T cells acquire robust cytotoxic effector functions and mediate efficient antiviral immunity. In contrast, in unprotected animals, antigen presentation is associated with monocyte populations expressing suppressive and inhibitory signals. CD8 + T cells in this context exhibit increased expression of exhaustion‐associated markers, especially CTLA4 , together with reduced effector functionality. Aberrant CD4 + T cell–CD8 + T cell communication further reinforces T cell exhaustion, ultimately resulting in impaired antiviral responses and disease progression.

Article Snippet: For the TLR‐4 or TLR‐8 loss‐function assay, the CD14‐deleted cells were pre‐incubated with TAK‐242 (MCE, #243984‐11‐4) or CU‐CPT8m (MCE, #HY‐112050) at a final concentration of 5 μM for 2 h, followed by the addition of virus, and ELISpot was then performed.

Techniques: Immunopeptidomics, Expressing, Biomarker Discovery

Journal: Molecular Biomedicine

Article Title: Oncolytic virus OHSV2 induces pyroptosis in bladder cancer cells via the TLR4/NLRP3/Caspase-1/GSDMD pathway

doi: 10.1186/s43556-026-00506-4

Figure Lengend Snippet: OHSV2 induces NLRP3/Caspase-1/GSDMD-dependent pyroptosis in bladder cancer cells via TLR4. a RT-qPCR analysis of the expression levels of TLR4 in bladder cancer cells treated with different concentrations (5% PBS, MOI = 1, MOI = 5) (n = 3). b Western Blot analysis of the expression levels of TLR4 in bladder cancer cells treated with different concentrations (5% PBS, MOI = 1, MOI = 5). c Immunohistochemistry analysis to detect the expression of TLR4 proteins in subcutaneous xenograft tumor tissues from nude mice. d CCK-8 assay to evaluate the effects of OHSV2 in combination with an TLR4 inhibitor (Resatorvid) on the viability of bladder cancer cells (n = 6). e Western Blot analysis to evaluate the effects of OHSV2 in combination with an TLR4 inhibitor (Resatorvid) on the expression of TLR4, NLRP3, Caspase-1, and GSDMD. f Western blot analysis of TLR4 after TLR4 knockdown. g CCK-8 assay showing partial restoration of cell viability after TLR4 knockdown (n = 6). h Western blot analysis of NLRP3, Cleaved Caspase-1, and N-GSDMD expression in TLR4-overexpressing cells after OHSV2 treatment. i Western blot analysis of NLRP3, Cleaved Caspase-1, and N-GSDMD expression in TLR4-knockdown cells after OHSV2 treatment. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student's t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The TLR4 agonist RS09 (MCE, USA) was administered intraperitoneally at a dose of 2 mg/kg per injection every day from day 0.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, CCK-8 Assay, Knockdown, Two Tailed Test