Journal: mBio

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.1128/mbio.00527-26

Figure Lengend Snippet: IFNβ enhances the inflammatory response of epithelial cells to C. trachomatis infection. ( A ) Primary cervical epithelial cells (left panel) or HeLa cells (right panel) were incubated with IFNβ for 24 h, or with C. trachomatis for 40 h in the absence or presence of IFNβ. The expression of inflammatory cytokines IL6 was detected by quantitative PCR. IL6 transcript was measured by real‐time RT-qPCR and normalized to actin transcript following the 2 −ΔΔCt method. The data are presented as relative mRNA levels compared to uninfected cells; five (for primary cells) or eight (for HeLa cells) independent experiments are shown, each measurement is done in triplicate, and the dots represent different individuals (left panel) or experiments (right panel). The insert with paired data displays the IL6 transcripts in infected cells with or without IFNβ treatment. One-way ANOVA and Tukey multiple comparison tests were performed (* for P -value < 0.05; ** for P -value < 0.01; and ns for not significant). ( B and C ) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h prior to adding brefeldin A for 6 h. After the treatment, the intracellular level of IL6 was measured by flow cytometry using anti-human IL6-PC7 antibody. The histograms are from one representative experiment ( B ), and the quantification of four experiments is displayed. P ‐values of Student’s paired t ‐test are shown when significant (** for P -value < 0.01) ( C ).

Article Snippet: Adhered cells (0.15 × 10 6 cells/ml) in 24-well plates were treated with the indicated concentration of recombinant IFN-I, and/or infected with C. trachomatis , or with polyinosine-polycytidylic acid (polyI:C, InvivoGen, #tlrl-picw and #tlrl-pic), as synthetic agonists of TLR3 ( ).

Techniques: Infection, Incubation, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Flow Cytometry

Journal: mBio

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.1128/mbio.00527-26

Figure Lengend Snippet: TLR3 mediates the synergy process between IFNβ and C. trachomatis . ( A ) Primary cervical epithelial cells were incubated with IFNβ and/or C. trachomatis . Twenty-four hours later, the expression of transcripts coding for pattern recognition receptors (PRRs) was measured by real‐time RT-qPCR and normalized to actin transcripts following the 2 −ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SE with individual values of three independent experiments. Each experiment was conducted in technical triplicate, and each dot represented the data obtained with cells isolated from one individual. One-way ANOVA and Tukey multiple comparison tests were conducted (* for P < 0.05, ** for P < 0.01, **** for P < 0.0001, and ns for not significant). ( B ) siRNA was incubated with HeLa cells for 24 h before treatment with IFNβ and/or C. trachomatis . Brefeldin A was added 24 hpi for 6 h. Intracellular IL6 protein was measured by flow cytometry using anti-human IL6-PC7 antibody. Data from four independent experiments are shown. The P ‐value of a Student’s paired t ‐test is shown (* for P < 0.05). ( C ) TLR3-KO cells or parental HeLa cells were infected with C. trachomatis in the presence or absence of IFNβ for 30 h before measuring intracellular IL6 levels by flow cytometry. The histograms are from one representative experiment, and data from five independent experiments are displayed (right panel). The P ‐value of a Student’s paired t ‐test is shown (* for P < 0.05). ( D ) HeLa cells were incubated with IFNβ and/or low/high molecular weight (LMW/HMW) polyI:C (25 μg/mL). Sixteen hours later, the expression of IL6 was measured by real‐time RT-qPCR as described in panel A . The data are presented as the mean ± SE with individual values of three independent experiments. Each experiment was conducted in technical triplicate. P -values of Student’s paired t -tests are shown (* for P < 0.05).

Article Snippet: Adhered cells (0.15 × 10 6 cells/ml) in 24-well plates were treated with the indicated concentration of recombinant IFN-I, and/or infected with C. trachomatis , or with polyinosine-polycytidylic acid (polyI:C, InvivoGen, #tlrl-picw and #tlrl-pic), as synthetic agonists of TLR3 ( ).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Isolation, Comparison, Flow Cytometry, Infection, High Molecular Weight

Journal: mBio

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.1128/mbio.00527-26

Figure Lengend Snippet: PI3K/AKT but not STAT1 signaling pathways downstream of IFN-I are implicated in the synergy between IFN-I and C. trachomatis . ( A ) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h followed by the detection of activation and expression of STAT1 via immunoblot. The results are representative of three independent experiments. ( B ) siRNA against STAT1 or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ alone (left graph) or with IFNβ and C. trachomatis (right graph). The transcriptional level of STAT1 (left graph) was measured by real‐time RT-qPCR and normalized to actin transcript following the 2 −ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SE with individual values of three experiments. The P ‐value of a Student’s paired t ‐test is shown (* for P < 0.05). Intracellular IL6 protein (right graph) was determined by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments are shown. ( C and D ) HeLa cells were pre-incubated with wortmannin (5 μM) ( C ) or SB203580 (10 μM) ( D ) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The histograms are representatives of two ( C ) or four experiments ( D , upper panel), respectively. The results of the four independent experiments are displayed (D, lower panel), with a P ‐value of a Student’s paired t ‐test (* for P < 0.05). ( E ) The cells were incubated with SB203580 (10 μM) for 1 h before the addition of IFNβ for 30 min. Phosphorylation of p38 and AKT was detected by immunoblot. ( F ) The cells were stimulated with IFNβ and C. trachomatis for 24 h or with recombinant human TNFα (10 ng/mL) for 30 min as a positive control, before detection of p38 phosphorylation by immunoblot. The data are representative of three independent experiments.

Article Snippet: Adhered cells (0.15 × 10 6 cells/ml) in 24-well plates were treated with the indicated concentration of recombinant IFN-I, and/or infected with C. trachomatis , or with polyinosine-polycytidylic acid (polyI:C, InvivoGen, #tlrl-picw and #tlrl-pic), as synthetic agonists of TLR3 ( ).

Techniques: Protein-Protein interactions, Incubation, Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Phospho-proteomics, Recombinant, Positive Control

Journal: mBio

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.1128/mbio.00527-26

Figure Lengend Snippet: mTOR signaling is involved in the synergy between IFN-I and C. trachomatis . ( A ) HeLa cells were pre-incubated with mTOR inhibitors, rapamycin and torin1 (1 μM) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The results of five independent experiments are displayed with a P ‐value of a Student’s paired t ‐test (* for P < 0.05). ( B ) HeLa cells were incubated with SB203580 (10 μM) for 1 h before the addition of IFNβ for 30 min. Phosphorylation of mTOR was detected by immunoblot, and β-actin was used as a loading control. The results are representative of three independent experiments. ( C ) HeLa cells were treated with pharmacological inhibitors (left and middle panels) or transfected with siRNA (right panel) as described in , followed by IFNβ and/or C. trachomatis treatment for 24 h. TLR3 transcripts were measured by real-time RT-qPCR as above. Data from three independent experiments and P ‐values of Student’s paired t ‐test are shown (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Adhered cells (0.15 × 10 6 cells/ml) in 24-well plates were treated with the indicated concentration of recombinant IFN-I, and/or infected with C. trachomatis , or with polyinosine-polycytidylic acid (polyI:C, InvivoGen, #tlrl-picw and #tlrl-pic), as synthetic agonists of TLR3 ( ).

Techniques: Incubation, Expressing, Flow Cytometry, Phospho-proteomics, Western Blot, Control, Transfection, Quantitative RT-PCR

Journal: mBio

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.1128/mbio.00527-26

Figure Lengend Snippet: TLR3 modulates the host inflammatory response to the infection through ERK-ATF2. ( A ) HeLa cells were treated with IFNβ and/or C. trachomatis for 24 h. The activation of ERK and its expression were determined by immunoblot using anti-phosphorylated ERK and anti-ERK antibodies, respectively. The blots are representatives of three experiments. ( B ) The cells were pre-treated with U0126 at the indicated concentrations. One hour later, HeLa cells were infected with C. trachomatis for 24 h, still in the presence of U0126, followed by the detection of ERK phosphorylation as in panel A . The blots are representatives of three experiments. ( C ) After 1 h pre-treatment with U0126 (10 μM), HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h in the presence of U0126, prior to adding brefeldin A for 6 h. Intracellular levels of IL6 were measured by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments and the P -values of Student’s paired t ‐tests are shown (* P < 0.05 and ** P < 0.01). ( D ) HeLa cells were transfected with control siRNA or siRNA (final concentration 30 nM) against human TLR3 for 24 h before C. trachomatis infection for an additional 24 h. The activation and expression of ERK were determined as in panel A. The blots are representative of three experiments. ( E ) HeLa WT cells and TLR3-KO clone were infected with C. trachomatis for 24 h before determining ERK activation and expression. The data are representatives of three experiments. ( F ) siRNA against c-Fos (upper panel), c-Jun (middle panel), ATF2 (bottom panel), or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ and/or C. trachomatis for 24 h. Intracellular levels of IL6 were measured as in panel C . The results of three or four independent experiments and the P -values of Student’s paired t ‐tests are shown (* P < 0.05 and ** P < 0.01). ( G ) Graphical summary created by BioRender. ns, not significant.

Article Snippet: Adhered cells (0.15 × 10 6 cells/ml) in 24-well plates were treated with the indicated concentration of recombinant IFN-I, and/or infected with C. trachomatis , or with polyinosine-polycytidylic acid (polyI:C, InvivoGen, #tlrl-picw and #tlrl-pic), as synthetic agonists of TLR3 ( ).

Techniques: Infection, Activation Assay, Expressing, Western Blot, Phospho-proteomics, Incubation, Flow Cytometry, Transfection, Control, Concentration Assay

Journal: PLOS One

Article Title: Screening a library of antibacterial compounds leads to discovery of novel inhibitors for Neisseria gonorrhoeae and Chlamydia trachomatis

doi: 10.1371/journal.pone.0340486

Figure Lengend Snippet: (A) Results of screening the antibacterial compounds library (containing 1,128 bioactive antibacterial agents), at a fixed concentration of 1 μM, against N. gonorrhoeae FA1090. Drugs exhibiting ≥ 90% inhibition of bacterial growth were considered positive hits (red). (B) Schematic of the screening process in this study, and the number of test agents at each step to finally obtain two molecules that are potent against N. gonorrhoeae and C. trachomatis .

Article Snippet: N. gonorrhoeae FA1090, C. trachomatis serovar L2 (ATCC VR-902B), and McCoy cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Concentration Assay, Inhibition