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: 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

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A to D ) Monocyte-derived dendritic cells (MoDCs) were treated with NET supernatants formed by PBS, ionomycin high dose (1 μM, Iono-High), native S. Parasanguinis (SP) lysates with low dose of ionomycin (0.01 μM), or SPI (native S. parasanguinis lysates with low-dose ionomycin) with PAD inhibitor (PADi) or intact citrullinated S. parasanguinis lysates, or SPI NETs in the presence or absence of TLR4 inhibitor TAK-242, or IRAK4 inhibitor zimlovisertib for 12 hr, and visualized to detect phagocytosed citrullinated proteins (A), or the level of TLR4 and MyD88 (B) in MoDCs by confocal microscopy. Relative mean intensity of cit-H3 (A, right , n = 4), TLR4, or MyD88 (B, n = 4) was determined with image J. Scale bars, 10 μm. The expression level of TLR4 (C, n = 6) or IRAK4 (D, n = 6) in CD11c + MoDCs was assessed by flow cytometry. ( E and F ) HEK-Blue-hTLR4 cells were treated with each NET supernatant (E, n = 9) or native (Nat) or citrullinated (Cit) human proteins in different concentrations (F, n = 3) for 24 hr. The level of secreted embryonic alkaline phosphatase (SEAP) activity was measured in the supernatant. SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Article Snippet: B cells were pre-treated with pharmacologic inhibitors, including 5 μM TLR4 inhibitor, 5 μM E6446 TLR7 inhibitor (Sellekchem), 5 μM IRKA4 inhibitor, or 1 μM Evobrutinib BTK inhibitor (MedChemExpress) 30 min before stimulation of B cells.

Techniques: Derivative Assay, Confocal Microscopy, Expressing, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A ) Experimental schematic of coculture assay of in vitro MoDCs and CD8 + T cells from ACPA + RA PBMCs in presence of NETs or human citrullinated proteins with or without TLR4 or IRAK4 inhibitor, or anti-HLA class I antibody. Figure created with BioRender. ( B ) Quantification of GZMB + IFNγ + ( left , n = 7), or CD107a + ( right , n = 7) CD8 + T cells in the coculture assay measured by flow cytometry. ( C ) Representative flow cytometry histograms ( left ), and frequency ( right ) of proliferating CD8 + T cells gating with cell proliferation dye eF450 ( n = 6). ( D ) Percentage of IL-6-expressing CD11c + MoDCs in the coculture assay ( n = 5). ( E and F ) Human native- or citrullinated proteins were incubated with MoDCs in the presence or absence of inhibitors and cocultured with CFSE-stained CD8 + T cells. LPS and anti-CD40 antibody were used as a positive control to activate MoDCs. Proportion of GZMB + IFNγ + (E), or proliferating (F) CD8 + T cells was measured using flow cytometry. * indicates versus Cit Ag (E). n = 8. ( G ) Representative histogram showing TLR4 shRNA knockdown efficiency in MoDCs ( left ) or quantification of GZMB + IFNγ + CD8 + T cells ( right ) in the coculture of control ( red ) or TLR4 ( black ) shRNA-treated MoDCs with autologous CD8 + T cells. Each dot represents an independent sample ( n = 3). SPI; S. parasanguinis lysates with low-dose ionomycin. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (B-F), or two-way ANOVA with Fisher’s LSD test (G). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Article Snippet: B cells were pre-treated with pharmacologic inhibitors, including 5 μM TLR4 inhibitor, 5 μM E6446 TLR7 inhibitor (Sellekchem), 5 μM IRKA4 inhibitor, or 1 μM Evobrutinib BTK inhibitor (MedChemExpress) 30 min before stimulation of B cells.

Techniques: Co-culture Assay, In Vitro, Flow Cytometry, Expressing, Incubation, Staining, Positive Control, shRNA, Knockdown, Control

Journal: bioRxiv

Article Title: Cross-presentation of citrullinated antigens drives cytotoxic CD8 + T cell responses in rheumatoid arthritis

doi: 10.64898/2026.06.03.729882

Figure Lengend Snippet: ( A ) Pan CD19 + B cells from ACPA + RA PBMCs were treated with anti-IgM antibody, human native or citrullinated proteins, with or without the pretreatment of TLR4, TLR7, or IRAK4 inhibitor for 16 hr. Quantification of CD80 ( left ) or CD86 ( right ) in CD19 + B cells was measured using flow cytometry. n = 6. ( B ) Frequencies of CD80 ( left ), and CD86 ( right ) in ACPA-tetramer + or - tetramer − B cells. n = 8. ( C ) The secreted level of ACPA in the supernatants of pan B cells in response to each stimulation was quantified using ACPA ELISA. n = 5. ( D and E ) Percentage of ACPA + B cells in pan B cells stimulated with anti-IgM antibody, LPS, or human native or citrullinated proteins in the presence or absence of each inhibitor. D; n = 5, E; n = 9. ( F ) Quantification of GZMB + IFNγ + ( left, n = 10) or proliferating ( right, n = 7) CD8 + T cells in the coculture of ACPA + or ACPA − B cells with cell proliferation dye-stained CD8 + T cells with human native- or citrullinated proteins. ( G ) Frequencies of CD86 + ( left ) or IL-6 + ( right ) CD19 + B cells in the coculture of FACS-sorted ACPA + or ACPA − B cells with CD8 + T cells. n = 10. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test (A, C-G), or Student’s unpaired t-test (B). Data are plotted as means ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001.

Article Snippet: B cells were pre-treated with pharmacologic inhibitors, including 5 μM TLR4 inhibitor, 5 μM E6446 TLR7 inhibitor (Sellekchem), 5 μM IRKA4 inhibitor, or 1 μM Evobrutinib BTK inhibitor (MedChemExpress) 30 min before stimulation of B cells.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining