Antiviral responses of human Fallopian tube epithelial cells to toll-like receptor 3 agonist poly(I:C) Mimi Ghosh, Ph.D., Todd M. Schaefer, Ph.D., John.

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Antiviral responses of human Fallopian tube epithelial cells to toll-like receptor 3 agonist poly(I:C) Mimi Ghosh, Ph.D., Todd M. Schaefer, Ph.D., John V. Fahey, Ph.D., Jacqueline A. Wright, B.Sc., Charles R. Wira, Ph.D. Fertility and Sterility Volume 89, Issue 5, Pages 1497-1506 (May 2008) DOI: 10.1016/j.fertnstert.2007.05.023 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Constitutive mRNA expression of TLR by FTEC. RT-PCR determined relative levels of expression of TLR2, 3, 4, 5, 7, and 9 normalized against an endogenous control β-actin. Data were further normalized against TLR7, which was the lowest expressing gene. Results are a mean of six patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Chemokine secretion by FTEC treated with TLR agonists poly(I:C), zymosan, and LPS. After 24-hour apical treatment of FTEC with TLR agonists, conditioned media were collected and analyzed for the presence of IL-8 by ELISA. (A) Poly(I:C) stimulation showed a statistically significant increase in IL-8 secretion both apically and basolaterally. (B) Treatment with zymosan and LPS showed no statistically significant increase. P values calculated using a two-tailed paired t test: ∗∗∗Statistically significantly greater (P<.001) than control; ∗∗P<.01. Results for (A) are a mean ± SE of five patient samples. Results for (B) are a mean ± SE value of four patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Cytokine secretion by FTEC treated with TLR agonists poly(I:C), zymosan, and LPS. After 24-hour apical exposure of FTEC to TLR agonists, conditioned media were collected and analyzed for the presence of TNF-α by ELISA. (A) Statistically significant increase in TNF-α secretion detected in both the apical and the basolateral conditioned media after poly(I:C) treatment. (B) No change in TNF-α secretion observed after LPS and zymosan treatments. P values calculated using a two-tailed paired t test: ∗∗∗P<.001. Results are a mean ± SE of five patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Secretion of antimicrobials by FTEC treated with poly(I:C). FTEC were exposed to poly(I:C) apically for 24 hours, after which the apical and the basolateral media were analyzed for the presence of HBD2 by ELISA. Statistically significant increased secretion of HBD2 was observed in the apical but not the basolateral conditioned media upon poly(I:C) treatment. P values calculated using a two-tailed paired t test: ∗∗∗P<.001 Results are a mean ± SE of three patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Expression of antiviral genes by FTEC treated with the TLR3 agonist poly(I:C). FTEC were exposed to poly(I:C) for 24 hours. RT-PCR was used to determine the relative levels of expression of IFN-β, 2′,5′-OAS, MxA, and PKR, normalized against an endogenous control β-actin. Data were further normalized by using values from the control for calibration. The mRNA expression levels for all four genes were found to be up-regulated upon poly(I:C) treatment in a statistically significant manner. P values calculated using a two-tailed paired t test: ∗∗∗P<.001. Results are a mean of three patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Upregulation of TLR2, TLR3, and TLR7 mRNA following exposure of FTEC to TLR3 ligand. FTEC were treated apically with poly(I:C) for 24 hours, after which RT-PCR was used to determine relative expression levels for TLR2, 3, 4, 5, 7, and 9. The data were normalized against endogenous control β-actin and further normalized by using values from the control for calibration. The mRNA expressions of TLR2, TLR3, and TLR7 were found to be up-regulated upon poly(I:C) treatment in a statistically significant manner. P values calculated using a two-tailed paired t test: ∗∗∗P<.001. Results are a mean of two patient samples. Fertility and Sterility 2008 89, 1497-1506DOI: (10.1016/j.fertnstert.2007.05.023) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions