Journal: European Journal of Immunology

Article Title: Mast Cell Phenotypic Heterogeneity Impacts the Interplay with Pathogenic Salmonella Typhimurium Bacteria

doi: 10.1002/eji.70040

Figure Lengend Snippet: Human LUVA mast cells respond to S .Tm by a two‐step activation mechanism mediated by TLR2/6 and the TTSS‐1 invasion machinery. ( A ) Representative (from two experiments) TEM images of LUVA cells uninfected or infected for 4 h with S .Tm (white arrows) at MOI 50. ( B ) Principal component analysis (PCA) plot of RNA sequencing data, showing the uninfected group as well as 4 h p.i. with S .Tm wt or S .Tm ∆invG ( n = 3, from one experiment. Same for ( C–G ). ( C, D ) Volcano plots showing the number of differentially expressed genes (DEGs, p < 0.01) with > two‐fold (log2) changed genes in red and their respective number of up‐ and downregulated genes. ( C ) shows S .Tm wt vs. uninfected, D shows S .Tm ∆invG vs. uninfected. ( E ) Venn‐diagram‐like plot showing the (complete) overlap of > two‐fold (log2) upregulated DEGs. ( F ) Heatmap of top 20 upregulated genes with baseMean > 50 and p < 0.01 by S .Tm ∆invG , sorted descending by S .Tm wt / S .Tm ∆invG ratio (z‐score transformed). ( G ) Violine plots of length‐scaled gene expression values of different PRR receptors in uninfected LUVA MCs. ( H ) Levels of secreted TNF from LUVA MCs either untreated or treated for 4 h with 0.5 µg/mL LPS, 10 µg/mL Pam3CSK4 or its vehicle (0.5% ethanol), 0.1 µg/mL Pam2CSK4, 5 µg/mL Poly(I:C) (HMW), 0.5 µg/ml Poly(I:C) (HMW)/LyoVec, 5 µg/mL R848, 0.1 µg/mL S .Tm flagellin or 5 µg/ml M‐TriDAP as well as LUVA MCs infected with MIO 50 S .Tm wt or S .Tm ∆invG , ( n = 4 from two experiments). ( I ) Separate experiment with a similar setup as ( H ), including the untr., Pam2CSK4 and S .Tm ∆invG groups ( n = 12, from three experiments). ( H ) Graph shows mean ± SEM for pooled replicates from two experiments ( n = 4). Untreated cells were compared with other groups by one‐way ANOVA and Dunnett's post hoc test. ( I ) The Graph shows mean ± SEM for pooled replicates from three experiments ( n = 12). Untreated cells were compared with other groups by the Kruskal–Wallis test. * p < 0.05, *** p < 0.001; ns, nonsignificant.

Article Snippet: The PRR agonists E. coli LPS (TLR4, Sigma‐Aldrich, #L4516), Pam2CSK4 (TLR2/6, Tocris, #4637), Pam3CSK4 (TLR1/2, Tocris, #4633), Poly(I:C) HMW (TLR3, InvivoGen, #tlrl‐pic), Poly(I:C) (HMW)/LyoVec (RIG‐I, InvivoGen, #tlrl‐piclv), S. Tm flagellin (FliC, TLR5, Sigma‐Aldrich, #SRP8029), R848 (TLR7/8, InvivoGen, #tlrl‐r848‐1), and M‐TriDAP (NOD1/2, InvivoGen, #tlrl‐mtd) were added diluted in medium and used as indicated in the respective figure legend.

Techniques: Activation Assay, Infection, RNA Sequencing, Transformation Assay, Gene Expression

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: Rac1, pPI3K, tPI3K, pGSK-3β, tGSK-3β, NF-κB p50 and NF-κB p65, pJNK, tJNK, pJUN and tJUN expression in control cells, Hp- 1 cells and Hp- 30 cells ( A ). TLR6 and pJNK expression in control cells, Hp- 1 cells, GES-1 cells treated with 1 μM C29 for 24 h and GES-1 cells treated with H. pylori and 1 μM C29 for 24 h ( B ). TLR6 and pJNK expression in control cells, GES-1 cells treated with 0.25 μg/mL, 0. 5 μg/mL, 1 μg/mL and 2 μg/mL Pam2CSK4 for 24 h ( C ). TLR6 and pJNK expression in GES-1 cells, Hp- 1 cells, GES-1 cells treated with 1 μg/mL Pam2CSK4 for 24 h, GES-1 cells treated with 2 μg/mL SP600125 for 24 h, GES-1 cells treated with H. pylori and 2 μg/mL SP600125 for 24 h and GES-1 cells treated with 1 μg/mL Pam2CSK4 and 2 μg/mL SP600125 for 24 h ( D ). All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: Expressing, Control

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: TLR6 mRNA expression ( A ) and IL-1β mRNA expression ( B ) in Hp- 30 cells, Hp- 30 cells co-culture with H. pylori for 24 h and Hp -30 cells co-culture with H. pylori and 10 μg/ml Pam2CSK4 for 24 h. Supernatant IL-1β content ( C ) in control cells, Hp- 1 cells, Hp -30 cells, Hp -30 cells infected with H. pylori for 24 h and Hp -30 cells co-culture with H. pylori and 1 μg/ml Pam2CSK4 for 24 h. pJNK expression ( D ) in Hp- 30 cells, Hp- 30 cells co-culture with H. pylori for 24 h, Hp -30 cells co-culture with H. pylori and 1 μg/ml Pam2CSK4 for 24 h. All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: Expressing, Co-Culture Assay, Control, Infection

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: Schematic ( A ) of the in vivo treatment experimental plan. Serum TLR6 and IL-8 expression ( B, C ) and gastric mucosa TLR6 and IL-8 IHC staining ( D , E ) of the H. pylori- negative Mongolian gerbils or in the H. pylori- infected gerbils after 24 h, 48 h and 72 h intraperitoneal administration of Pam2CSK4 or DMSO. pJNK expression ( F ) in the gastric mucosa of H. pylori- negative Mongolian gerbils or in the H. pylori- infected gerbils after 72 h intraperitoneal administration of Pam2CSK4 or DMSO. The Immunofluorescence staining of CD11b + /Ly6G + cells ( G ), monocyte/macrophage ( I ), the number of CD11b + /Ly6G + cells ( H ) and the number of monocyte/macrophage ( J ) in gastric mucosa of H. pylori -negative gerbils, H. pylori -positive gerbils treated with DMSO or Pam2CSK4. The level of H. pylori 23srRNA ( K ) in gastric mucosa of H. pylori -positive Mongolian gerbils treated with DMSO or Pam2CSK4. IHC staining of TLR6 expression ( L ) in the gastric mucosa of patients. × 100. The average optical density (AOD) of TLR6 expression ( M ) in patients’ gastric mucosa. All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: In Vivo, Expressing, Immunohistochemistry, Infection, Immunofluorescence, Staining