CpG-ODN and Poly (I: C)LL induce increased expression of IL-6 and IL-8.

<p>(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).</p

TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells <i>In Vitro</i>

<div><p>The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC's) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC's can alter immune responses of macrophages and dendritic cells (DC's) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-β dependent. In summary, the results of our study indicate that EEC's play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.</p></div

Epithelial cell derived TGF-β mediates anti-inflammatory effects on MDMs.

<p>(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.</p

Secretions of End1/E6E7 cells modulate cytokine levels by MDMs in response to LPS.

<p>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 (<b>A</b>), IL-10 (<b>B</b>), GM-CSF (<b>C</b>), IFN-γ (<b>D</b>) and IL-2 (<b>E</b>) 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.</p

Polarized endocervical cells respond to apical stimulation with CpG-ODN and poly (I: C) LL.

<p>(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.</p

Secretions of ligand stimulated End1/E6E7 cells enhance chemotaxis of U937 cells.

<p>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.</p

Stimulation with CpG-ODN and Poly (I: C)LL activates NF-kB pathway.

<p>(<b>A</b>). 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>B</b>). Confocal images of phosphorylated p65-NF-<i>κ</i>B 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).</p

Secretions of End1/E6E7 cells modulate cytokine levels by DC: T cell-co-cultures in response to LPS.

<p>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<b>⁺</b> T cells for 24 hrs and spent media were analyzed for TNF-α (<b>A</b>) and IL-12 (<b>B</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.</p

qPCR analysis of <i>Hb-α</i> and <i>Hb-β</i> expression in hPVECs and VK2/E6E7 cells before and after induction with LPS.

<p>Cells seeded at a density of 10⁶/well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of <i>Hb-α</i> and <i>Hb-β</i> was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p< 0.001 Vs Untreated).</p

Immunofluorescence localization of cytokeratin-13.

<p>Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).</p