Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor ligands correlates with beta interferon production

Abstract

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-beta) but not production of IFN-alpha, IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-beta in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-beta in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-alpha or IFN-gamma in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-alpha(-/-) and IFN-gamma(-/-) mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-beta, mediates innate protection, mice unresponsive to type I interferons (IFN-alpha/betaR(-/-) mice) and mice lacking IFN regulatory factor-3 (IRF-3(-/-) mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-alpha/betaR(-/-) or IRF-3(-/-) mice. Local delivery of murine recombinant IFN-beta protected C57BL/6 and IRF-3(-/-) mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-beta production and innate antiviral immunity against HSV-2.

FIG. 1.

Vaginal delivery of certain TLR ligands increases resistance to IVAG HSV-2 infection in B6 mice. Six- to eight-week-old female B6 mice were treated with CpG ODN (100 μg/mouse, n = 20), poly(I:C) (100 μg/mouse, n = 20), PGN (100 μg/mouse, n = 10), FliC (80 μg/mouse, n = 10), or LPS (5 μg/mouse, n = 20) or left untreated 24 h prior to IVAG HSV-2 challenge (1 × 10⁴ PFU/mouse). Challenged mice were monitored daily for genital pathology, survival, and vaginal virus titers. (a) poly(I:C)- and CpG-treated mice showed 100% survival compared to LPS- or PGN-treated or untreated mice. FliC-treated mice showed 50% protection. (b) Vaginal HSV-2 titers were examined on days 1 to 3 following viral challenge. B6 mice treated with poly(I:C) and CpG had no viral titers, while all other groups showed significant viral titers.

FIG. 2.

Effects of TLR ligands on morphology of genital mucosa. Six- to eight-week-old female B6 mice were treated with progesterone and, 4 days later, received CpG ODN (100 μg/mouse), poly(I:C) (100 μg/mouse), PGN (100 μg/mouse), FliC (80 μg/mouse), LPS (5 μg/mouse), or PBS (control). Twenty-four hours later, vaginal tissues were isolated and processed for histological testing. Photomicrographs represent cross-sections of vaginal tissues from mice from the various treatment groups. Magnification, ×200.

FIG. 3.

Local delivery of certain TLR ligands in B6 mice induced production of TNF-α and IFN-β. Six- to eight-week-old female B6 mice were treated with CpG ODN (100 μg/mouse), poly(I:C) (100 μg/mouse), PGN (100 μg/mouse), FliC (80 μg/mouse), or LPS (5 μg/ml) or left untreated. Vaginal washes from five mice were collected at 12 and 24 h posttreatment, pooled, and used to measure levels of TNF-α, IFN-γ, IFN-β, and IFN-α by use of ELISA. No IFN-γ (a) or IFN-α (b) was detected in these washes; however, all treatments caused the production of TNF-α (a). IFN-β production (b) was seen with CpG ODN- and poly(I:C)-treated mice, while no or very little IFN-β was detected in the washes of PGN-, LPS-, and FliC-treated mice. The experiments were repeated two times with similar results.

FIG. 4.

Local delivery of poly(I:C) provides innate protection against IVAG HSV-2 challenge in the absence of IFN-γ or TNF-α. Six- to eight-week-old female IFN-γ^−/− mice were treated with poly(I:C) (100 μg/mouse, n = 10) or CpG ODN (100 μg/mouse, n = 10) or left untreated (n = 10). Six- to eight-week-old female TNF-α^−/− mice were also treated with poly(I:C) (100 μg/mouse, n = 5) or left untreated (n = 5). Twenty-four hours posttreatment, mice were challenged IVAG with their lethal doses of HSV-2. Challenged mice were monitored daily for genital pathology, survival, and vaginal virus titers. Poly(I:C)-treated IFN-γ^−/− mice and TNF-α^−/− mice showed 100% and 80% survival, respectively. All control untreated mice died within 10 days after IVAG HSV-2 infection.

FIG. 5.

Treatment of IFN-α/βR^−/− or IRF-3^−/− mice with poly(I:C) does not provide protection against vaginal HSV-2 challenge. (a) Six- to eight-week-old female IFN-α/βR^−/−, 129SVPasCrl (129SV) (congenic control for IFN-α/βR^−/− mice), or IRF-3^−/− mice were treated with poly(I:C) (100 μg/mouse) or left untreated. Vaginal washes were collected at 12 and 24 h posttreatment, pooled (n = 5), and used to measure levels of IFN-β by use of ELISA. Poly(I:C) induced significant levels of IFN-β in IFN-α/βR^−/− mice. However, IRF-3^−/− mice treated with poly(I:C) showed no detectable IFN-β in the vaginal washes. Vaginal washes from four mice were pooled for ELISA. The experiment was repeated two times with similar results. (b) Six- to eight-week-old female IFN-α/βR^−/− mice and their congenic controls were treated with poly(I:C) (100 μg/mouse, n = 10) or CpG ODN (25 μg/mouse, n = 5) or left untreated (n = 5) 24 h prior to IVAG HSV-2 challenge. Challenged mice were monitored daily for genital pathology and survival. Poly(I:C)- or CpG ODN-treated 129SVPasCrl mice showed 100% survival compared to untreated 129SVPasCrl control mice. There was no protection against IVAG HSV-2 challenge in poly(I:C)- or CpG ODN-treated IFN-α/βR^−/− mice compared to results with untreated control mice. (c) Six- to eight-week-old female IRF-3^−/− mice and their congenic control B6 mice were treated with poly(I:C) (100 μg/mouse) or CpG ODN (25 μg/mouse) or left untreated 24 h prior to IVAG HSV-2 challenge. Challenged mice were monitored daily for genital pathology and survival. Poly(I:C)- or CpG ODN-treated B6 mice showed 100% survival compared to untreated B6 control mice. There was no protection against IVAG HSV-2 challenge in poly(I:C)-treated IRF-3^−/− mice compared to results with untreated control mice. All IRF-3^−/− mice treated with CpG ODN survived against IVAG HSV-2 challenge.

FIG. 6.

Local delivery of mrIFN-β provides innate protection against vaginal HSV-2 challenge of B6 and IRF-3^−/− mice. Six- to eight-week-old female IRF-3^−/− mice (n = 8) or control B6 mice (n = 10) were treated with cell supernatants containing mrIFN-β (mrIFN-β sup) or mock supernatants (mock sup) or left untreated. Twenty-four hours posttreatment, mice were challenged with IVAG HSV-2. Challenged mice were monitored daily for genital pathology and survival. Local delivery of mrIFN-β provided B6 mice with 80% protection and IRF-3^−/− mice with 75% protection against subsequent IVAG HSV-2 challenge. Mice treated with mock supernatants showed no protection against IVAG HSV-2 challenge compared to naïve mice.