Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: Cytotoxic necrotizing factor 1 (CNF1), but not CNF1-C866S, induces phenotypic maturation of immature monocyte-derived dendritic cells (moDCs). ( A – C ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with different concentrations of CNF1 or CNF1-C866S before the expression of phenotypic maturation markers was measured by flow cytometry. ( A ) Representative pseudocolor dot plot showing surface expression of CD83 and CD86. ( B ) Graphs summarizing the mean percentages of CD83+CD86+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to immature moDCs cultured without toxin using a two-way ANOVA with Sidak’s multiple comparisons test. ( C ) Histogram plots showing the surface expression of HLA-DR on moDCs cultured without toxin (green), with CNF1 (red) or with CNF1-C866S (blue). Arrows indicate increasing concentrations (0.004, 0.02 and 0.1 nM) of CNF1 and CNF1-C866S. The plots are representative of 3 independent experiments each using moDCs derived from different donors ( n = 3).

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1-induced moDC maturation is not driven by endotoxin or other putative contaminants in the toxin preparation. ( A ) Culture media alone (Ctrl) or culture media containing 0.1 nM CNF1, 50 ng/mL lipopolysaccharide (LPS) or 50 ng/mL tumor necrosis factor alpha (TNFα) was pre-incubated for 30 min at room temperature with 5 µg/mL Polymyxin B (PMB) or vehicle (endotoxin-free water). Then, immature moDCs were added to each media sample and cultured for 24 h before the expression of CD83 and CD86 was determined by flow cytometry. Shown is the mean percentage of CD83+CD86+ moDCs from 4 different donors ( n = 4) analyzed in 3 independent experiments. Error bars represent SEM and * denotes a significant difference ( p < 0.05) between vehicle- and PMB-treated cells using a paired Student’s t -test. ( B ) CNF1 diluted in moDC medium was incubated with (2) uncoated Protein A agarose beads or (3) Protein A agarose beads that had been pre-coated with a monoclonal anti-CNF1 (αCNF1) antibody or (4) an isotype control (IC) antibody. As a negative control, (1) moDC medium without CNF1 was in parallel incubated with uncoated Protein A agarose beads. After removal of the beads, the level of CNF1 in the supernatants was analyzed by western blotting. ( C ) Immature moDCs were cultured for 24 h with the supernatants (1–4) obtained in ( B ) and the expression of CD83 and CD86 analyzed by flow cytometry.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Incubation, Cell Culture, Expressing, Flow Cytometry, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 does not induce apoptosis or cell death in moDCs. ( A , B ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with 0.1 nM CNF1 before the percentages of Annexin V- and propidium iodide (PI)-positive cells were determined by flow cytometry. moDCs cultured in medium without fetal bovine serum and cytokines (Starved) were included in the analysis as a positive control for induction of apoptotic cell death. ( A ) Histogram summarizing the mean percentages of Annexin V+ and/or PI+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to Ctrl moDCs at the same timepoint using a two-tailed paired Student’s t -test. ( B ) A representative pseudocolor dot plot of an Annexin V and PI staining after 48 h of culture.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Cell Culture, Flow Cytometry, Positive Control, Derivative Assay, Two Tailed Test, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 induces functional maturation of moDCs. ( A , B ) Immature moDCs were cultured for 24 h without toxin (Ctrl), with 0.1 nM CNF1 or with 0.1 nM CNF1-C866S before the concentrations of ( A ) interleukin-6 (IL-6) and ( B ) TNFα in the cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). ( A , B ) Shown is the mean cytokine concentration + SEM in cell culture supernatants from 3 independent experiments each using moDCs derived from different donors ( n = 3). ( C ) Immature moDCs were cultured for 48 h with 0.1 nM CNF1, 0.1 nM CNF1-C866S, 50 ng/mL LPS or without toxin (Ctrl). The moDCs were subsequently washed and different numbers cultured with allogenic naïve CD4+ T cells for 4 days. Then [ 3 H]-thymidine was added, and the cells cultured for 24 h further, before determination of [ 3 H]-thymidine incorporation. Shown are the mean counts per minute (CPM) from 3 independent experiments. Each independent experiment was performed using moDCs and allogenic naïve CD4+ T cells derived from different donors (total of 6 donors). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to CD4+ T cells cultured with the same number of Ctrl moDCs using a two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Functional Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay