Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: Primary human monocytes isolated from PBMCs from healthy donors were pretreated with water solvent (H 2 O) or 15 μM P7-Pen for 30 min, followed by stimulation with CL075, R837 (1 μg/ml) or K12 LPS (100 ng/ml) for indicated time, followed by collection of supernatants and cell lysis. ( A ) Quantification of IFNβ , TNF and IL-1β mRNA expression by qRT-PCR in primary human monocytes. Data presented as relative fold change when compared to unstimulated sample pretreated by water (H 2 O), mean ± SEM (n = 6–11). ( B, C ) Cytokine secretion was addressed by ELISA for IFNβ and by BioPlex for other cytokines and graphed as mean ± SEM (n = 5–8). ( C ). P7-Pen effect on CL075- or LPS-mediated secretion of all tested cytokines shown as Volcano plots generated using multiple paired t-test analysis, controlled for a False Discovery Rate (FDR) of 1%. ( D ) Cell viability was addressed by LDH content in supernatants using LDH cytotoxicity assay and presented as % of dead cells, mean ± SD. ( A, B, D ) Statistical testing was done by two-way ANOVA or mixed effects model on log-transformed data (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Isolation, Solvent, Lysis, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Generated, LDH Cytotoxicity Assay, Transformation Assay

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: ( A ) Whole-blood assay for samples of healthy donors with lepirudin as an anticoagulation reagent. Blood samples were pretreated with water solvent or 20 μM P7-Pen peptide for 30 min, followed by addition of CL075, R837 (2 μg/ml), K12 LPS (100 ng/ml), E. coli particles (2x10 6 /ml), or S. aureus particles (4x10 6 /ml) for 5 h before the collection of plasma samples. Plasma samples were probed for IFNβ, TNF and IL-6 secretion by ELISA. Data presented as mean ± SEM, statistical significance evaluated using Wilcoxon matched-pairs signed-rank test. ( B, C ) Quantification of phagocytosis based on flow cytometry for primary human monocytes pretreated by a solvent (water, H 2 O) or 15 μM P7-Pen, or 3 μM CytoD for 30 min and incubated with E. coli ( B ) or S. aureus ( C ) pHrodo particles for indicated time. Median pHrodo fluorescence intensity MFI and percentage of pHrodo-positive cells shown on graphs ( B, C ). Data presented as mean ± SEM, statistical significance evaluated by 2way ANOVA or mixed effect analysis. Representative images of flow cytometry results for one out of 3-5 donors are shown on the right. ( D ) Results of live bacteria phagocytosis assays for DH5α E. coli and S. aureus Wood strain by PMA-differentiated THP-1 monocytic cells. Data presented as mean ± SD, statistical significance evaluated by unpaired t-test ( A-D ) Significance levels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, nonsignificant if not shown otherwise; ND, not detected.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Whole Blood Assay, Solvent, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Fluorescence, Bacteria

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: BMDMs ( A ) and iDCs ( B ) were generated from BM cells from C57BL/6 mice. ( A-C ) M-CSF-differentiated BMDMs ( A ), bone marrow derived iDCs (GM-CSF and IL-4-differentiated) ( B ) or immortalized murine macrophages ( C ) were pretreated with water solvent (H 2 O) or 10 μM P7-Pen for 30 min and stimulated with R848 (1 μg/ml) for indicated time, followed by cell lysis, RNA isolation and RT-qPCR analysis of Ifnβ, Tnf and Il-1β mRNA expression. Data presented as relative fold change when compared to unstimulated sample pretreated by water (H 2 O), mean ± SEM (n = 3). ( D ) Immortalized murine macrophages were kept untreated or stimulated with B4 LPS (100 ng/ml) or R848 (1 μg/ml) for indicated time, followed by cell lysis and IP assays. Endogenous TIRAP was immunoprecipitated from WCL of stimulated cells (1 mg protein/IP). LPS stimulation was applied as a positive control for TIRAP recruitment to MyD88. Cellular lysates were analyzed in parallel as input control (5%), with WB performed for total levels of MyD88 and TIRAP in WCLs and IPs, and for levels of phosphorylated p38 MAPK and IKKα/β in WCLs as a positive control for cell activation by TLR ligands. A representative experiment is shown from a total of three consecutive experiments.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Generated, Derivative Assay, Solvent, Lysis, Isolation, Quantitative RT-PCR, Expressing, Immunoprecipitation, Positive Control, Control, Activation Assay

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: Recruitment of TIRAP to the TLR8-Myddosome complex 45–60 minutes after TLR8 activation (late complex) promotes Akt activation, IRF5 dimerization and nuclear translocation, and the expression and secretion of IFNβ and IL-12. The P7-Pen peptide disrupts TIRAP recruitment to the TLR8-MyD88 complex, thereby inhibiting phosphorylation of Akt and IKKα/β, IRF5 dimerization, and nuclear translocation, ultimately leading to reduced expression of IRF5-regulated genes.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Activation Assay, Translocation Assay, Expressing