Table 1.
List of primer sequences used for qPCR in the study.
Figure 1.
Endocervical cells (End1/E6E7) express TLR9 and RIG-I.
A. RT-PCR analysis of TLR9 and RIG-I gene expression. (a & b). End1/E6E7 cells ells were seeded at a density of 1×105 cells/well in a 24-well plate and treated for 6 hrs with TLR9 and RIG-I ligands (10 µg/ml). RT-PCR results show that expression of TLR9 and RIG-I mRNAs was significantly upregulated in End1/E6E7 cells upon stimulation with TLR9 and RIG-I ligands, CpG-ODN and poly (I: C)LL respectively compared to medium controls. (c) & (d) A quantitative densitometry analysis of TLR9 (c) and RIG-I (d) expression in End1/E6E7 cells. Values were calculated as the mean (± SD) of three separate experiments performed on different days. Level of significance (**p<0.01; ***p<0.01) was calculated by unpaired t test. B. Analysis of TLR9 and RIG-I mRNA in End1/E6E7 cells by qPCR. End1/E6E7 cells (1×105) were stimulated with ligands of TLR9 (CpG-ODN: 10 µg/ml) and RIG-I {poly (I: C) LL, 10 µg/ml} for 6 hrs. Values represent mean (±SD) of three experiments performed in duplicates on different days (p<** 0.01; ***p<0.001) were calculated by ANOVA test followed by Bonferroni analysis. C. Flow cytometry analysis of TLR9 and RIG-I expression. Flow cytometry was used to compare the percentage of permeabilized End1/E6E7 cells labeled by anti-TLR9 and anti-RIG-I antibodies incubated with media (a, c) and after stimulation with their cognate ligands, CpG-ODN and Poly (I: C)LL (b, d) respectively. Comparison of TLR9 and RIG-I positivity was made by comparing fluorescence in the M2 channel. The figures shown are the representative pictures from three independent experiments. (FLI: log fluorescence intensity). D. Confocal images showing the expression of TLR9 and RIG-I in End1/E6E7 cells. Figures (a–f) End1/E6E7 cells were treated with media or CpG-ODN for 6 hrs and subsequently stained with anti-TLR9 antibody. Figures (g–l) End1/E6E7 cells were treated with media or Poly(I:C)LL for 6 hrs and subsequently stained with anti-RIG-I antibody. The figures shown are the representative picture from three independent experiments. (Magnification X 63) (Scale 10 µm).
Figure 2.
CpG-ODN and Poly (I: C)LL induce increased expression of IL-6 and IL-8.
(A) End1/E6E7 cells were stimulated with CpG-ODN and poly (I: C)LL (10 µg/ml) for 6 hrs. Conditioned media was collected for quantification of IL-6 and IL-8 by ELISA. Each bar represents mean (± SD) of three separate experiments performed on different days. Level of significance (**p<0.01) was calculated by ANOVA test followed by Bonferroni analysis. (B) End1/E6E7 cells (1×105 cells/well) were stimulated with ligands of TLR9 (CpG-ODN: 10 µg/ml) and RIG-I {poly (I: C) LL, 10 µg/ml} for 6 hrs. Values represent mean (±SD) of three experiments performed in duplicates on different days (**p<0.001).
Figure 3.
Stimulation with CpG-ODN and Poly (I: C)LL activates NF-kB pathway.
(A). End1/E6E7 cells were seeded at a density of 1×105/well in a 24-well plates and stimulated with CpG-ODN and Poly(I:C)LL (10 µg/ml for 30 min). At the end of the treatment, cells were collected, lysates were prepared and analyzed for phospho-p65-NF-kB levels. Values were calculated as the mean (± SD) of three separate experiments performed on different days. Level of significance (**p<0. 01; n.s: not significant) was calculated by ANOVA test followed by Bonferroni analysis. (B). Confocal images of phosphorylated p65-NF-κB expression in End1/E6E7 cells before and after stimulation for 30 min with TLR9 and RIG-I ligands, CpG-ODN and Poly(I:C)LL (10 µg/ml) respectively. The images shown are the representative pictures of one of three identical experiments performed on three different days. (Magnification X 63) (Scale 10 µm).
Figure 4.
Polarized endocervical cells respond to apical stimulation with CpG-ODN and poly (I: C) LL.
(A) End1/E6E7 cell monolayers were stimulated apically with CpG-ODN or Poly (I: C)LL (10 µg/ml) for 24 hrs. Following incubation, spent media from basal chamber was collected IL-6, IL-8 and GM-CSF concentrations were determined by ELISA. Each bar is the mean (± SD) of six individual observations obtained from three independent experiments. Level of significance (*p<0.5; **<0.01; ***p<0.001) was calculated by ANOVA test followed by Bonferroni analysis. (B) End1/E6E7 cell monolayers were treated apically with CpG-ODN or Poly(I:C)LL (10 µg/ml) for 4 hrs, following RNA was extracted. Abundance of transcripts of IL6, IL8 and GM-CSF was determined by qPCR. Values represent mean (±SD) of three experiments performed in duplicates on different days (p<** 0.01)were calculated by ANOVA test followed by Bonferroni analysis.
Figure 5.
Secretions of End1/E6E7 cells modulate cytokine levels by MDMs in response to LPS.
Monocyte derived macrophages (MDMs) were incubated for 24 hrs with fresh medium (No-SM) or spent media from untreated (UT-SM), CpG-ODN (CpG-SM) or poly (I: C) LL (poly-SM) treated End1/E6E7 cells. Later MDMs were stimulated with LPS (100 ng/ml) for an additional 24 hrs and supernatants were analyzed for cytokines viz., TNF-α (A), IL-10 (B), GM-CSF (C), IFN-γ (D) and IL-2 (E) levels Values represent mean ± SD of three experiments performed on different days. Level of significance (*p<0.05; **p<0.01; ***p<0.001; n.s: not significant) was calculated by ANOVA test followed by Bonferroni analysis.
Figure 6.
Secretions of End1/E6E7 cells enhance phagocytosis.
U937 cells were incubated for 3(control) (A), untreated spent media (UT-SM) of End1/E6E7 cells (B) or spent media obtained from CpG-ODN (C) and poly(I :C)LL (D) treated End1/E6E7 cells. Quantitative analysis of U937 cells with FITC-labeled E. coli was performed by flow cytometry.
Figure 7.
Secretions of ligand stimulated End1/E6E7 cells enhance chemotaxis of U937 cells.
Spent media of End1/E6E7 cells stimulated with CpG-SM and Poly-SM were used in the lower Transwell chamber, and U937 cell were placed on the upper Transwell chamber. The data were normalized to U937 chemotaxis observed in response to 0.1 mM (N-formyl-methionyl-leucyl-phenylalanine (fMLP) in PBS-BSA, which was used as positive control (100%). Each bar is the mean ± SD from six individual observations obtained from three independent experiments. Level of significance (*p<0.05; **P<0.01) was calculated by ANOVA test followed by Bonferroni analysis.
Figure 8.
Secretions of End1/E6E7 cells modulate cytokine levels by DC: T cell-co-cultures in response to LPS.
Monocyte derived dendritic cells (MDDCs) were incubated with fresh medium (No SM) or UT-SM, CpG-SM or Poly-SM for 24 hrs. Later MDDCs were stimulated with LPS (100 ng/ml) and autologous CD3+ T cells for 24 hrs and spent media were analyzed for TNF-α (A) and IL-12 (B). Values represent mean ± SD of three experiments performed on different days. Level of significance (*p<0.05; ***p<0.001) was calculated by ANOVA test followed by Bonferroni analysis.
Figure 9.
Epithelial cell derived TGF-β mediates anti-inflammatory effects on MDMs.
(A) Spent media (UT-SM) was incubated with anti-TGF-β antibody at varying concentrations for 1 hr at 37°C and was then incubated with MDMs for 24 hrs. Post incubation, MDMs was stimulated with LPS for 24 hrs and secretion of TNF-α was quantitated by ELISA. (B) MDMs were stimulated with recombinant human TGF-β (0.5–2 µg/ml) for 24 hrs and further stimulated with LPS for 24 hrs. Secretion of TNF-α was quantitated by ELISA. Treatment with recombinant TGF-β decreased secretion of TNF-α from MDMs in a dose dependent manner. (C) Spent media of End1/E6E7cells stimulated with CpG-ODN or Poly-(I:C)LL were analyzed for secretion of TGF-β after 24 hrs of stimulation. Values represent mean ± SD of three experiments performed on different days Level. Of significance (*p<0.05; **p<0.01; *** p<0.001 was calculated by ANOVA test followed by Bonferroni analysis.