Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic: polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NF κ B) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV pro-phylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections

Decreasing IRF7 expression enhances HIV-1 and RelA transcription.

<p>(A) IRF7, (C and F) HIV-1, (B, D and G) IFNα and (E and H) RelA transcription in HIV-1-infected cervical tissues treated with random and IRF7 targeting siRNA were quantified by RT-PCR before (A) and (B) and on days 1 (C, D and E) and 3 (F, G and H) after HIV-1 infection. All data was normalized to GAPDH. Results were consistent among four donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for tissues treated with random and IRF7 targeting siRNA.</p

Poly (I:C) decreases HIV-1 replication in PBMCs.

<p>HIV-1 p24 levels (ng/ml) in HIV-1 infected PBMCs left untreated or treated with poly (I:C) at 20 μg/ml were measured after washing the residual input virus (day 0), and again on days 3, 5 and 7 after infection. Results are shown as the mean ± STDEV from three experiments with each condition tested in triplicate. * p = 0.008, p = 0.01 and p = 0.009 for days 3, 5 and 7 after infection between untreated and poly (I:C) treated PBMCs.</p

Poly (I:C) decreases HIV-1 transcription in cervical tissues by enhancing IRF7 mediated antiviral responses and reducing RelA expression.

<p>(A) Levels of HIV-1, (B) RelA, (C) IRF7, (D) IRF3, (E) RIG-1, (F) TLR3, (G) IFNα and (H) IFNβ transcription in HIV-1-infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 3 and 5 after infection. All data was normalized to glyceraldehyde 3-phophate (GAPDH). For each gene, day 3 values in untreated control tissues were set to 1. Day 5 values in untreated control tissues or days 3 and 5 values in poly (I:C) treated tissues were normalized to 1. Results were consistent among three donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for untreated and poly (I:C) treated tissues.</p

Poly (I:C) decreases HIV-1 transcription by increasing IRF7 expression in PBMCs.

<p>(A) Levels of HIV-1, (B) RelA, (C) IRF7, (D) IRF3, (E) RIG-1, and (F) TLR3 transcription in HIV-1 infected PBMCs left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 3 and 5 after infection. All data was normalized to GAPDH. For each gene, day 3 values in untreated control tissues were set to 1. Day 5 values in untreated control tissues or days 3 and 5 values in poly (I:C) treated tissues were normalized to 1. Results were consistent among three donors and are shown as the mean ± STDEV from one representative experiments with each condition tested in triplicate. * p<0.05 for untreated and poly (I:C) treated cells.</p

Poly (I:C) decreases HIV-1 replication in cervical tissues.

<p>(A) HIV-1 p24 levels (ng/ml) in HIV-1 infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were measured after washing the residual input virus (day 0), and again on days 11 and 21 after infection. Results are shown as the geometric mean ± STDEV from triplicate values of 12 individual donors. *p = 0.00005 and p = 0.07 between untreated and poly (I:C) treated tissues at days 11 and 21 after infection, respectively. (B) Levels of HIV-1 reverse transcription and (C) integration in donor matched HIV-1 infected cervical tissues left untreated or treated with poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 11 and 21 after infection. All data was normalized to human β actin. For HIV-1 reverse transcription and integration, day 11 values in untreated control tissues were set to 1. Day 11 values in poly (I:C) treated tissues or days 21 values in untreated or poly (I:C) treated tissues were normalized to 1. Results are shown as the relative geometric mean ± STDEV from triplicate values of 8 and 12 individual donors on days 11 and day 21 respectively. *p = 0.004 and p = 0.0009 between untreated and poly (I:C) treated tissues at days 11 and 21 after infection, respectively.</p

Poly (I:C) improves the efficacy of TFV in cervical tissues.

<p>(A) HIV-1 p24 levels (ng/ml) in HIV-1 infected cervical tissues left untreated or treated with TFV at 10 μg/ml alone or in combination with poly (I:C) at 20 μg/ml were evaluated after washing the residual input virus (day 0), and again on days 11 and 21 after infection. (B) Levels of HIV-1 reverse transcription and (C) viral integration in donor matched HIV-1 infected cervical tissues left untreated or treated with TFV at 10 μg/ml alone or in combination poly (I:C) at 20 μg/ml were quantified by RT-PCR on days 11 and 21 after infection. All data was normalized to human β-actin. For HIV-1 reverse transcription and integration, day 11 values in untreated control tissues were set to 1. Day 11 values in TFV or TFV/Poly (I:C) treated tissues or days 21 values in untreated; TFV or TFV/Poly (I:C) treated tissues were normalized to 1. Results were consistent among 4 donors and are shown as the mean ± STDEV from one representative experiment with each condition tested in triplicate. * p<0.05 for untreated, poly (I:C) and TFV treated tissues. (D) FACS analysis of single cell suspensions from HIV-1 infected ectocervical tissues left untreated or treated with TFV at 10 μg/ml and stained for CD3 and CD8 on day 11 after infection. CD4⁺ T cells were defined as CD3⁺ and CD8^-. For each panel the percentage of CD4⁺ T cells from the total cell number is depicted in the upper left corner. Results were consistent among 4 donors.</p