Apoptosis and Expression of Antiviral Response Genes during Ocular HSV-1 Infection in TNFR1 or TNFR2 Knockout Mice

Abstract

Purpose: : TNF-α plays a role in ocular damage during HSV-1 infection. The cellular response to TNF-α is mediated via two receptors, TNFR1(p55) and TNFR2(p75). TNFR1 can signal through a caspase 3-dependent pathway leading to apoptosis whereas TNFR2 engagement does not usually lead to apoptosis. These studies investigated the apoptotic and inflammatory/antiviral responses during HSV-1 infection.

Methods: : TNFR1 and TNFR2 knockout (KO) and wild type control C57BL/6 mice were injected with 2 × 10⁴ pfu of HSV-1 H129 via the anterior chamber route. Inoculated eyes were collected at days 2, 3, 4, 5, and 7 post inoculation (p.i.). Virus titers were measured by plaque assay, and expression of caspase 3 was determined by western blot. Expression of early immune response genes was determined at day 2 p.i. using a mouse antiviral response PCR Array (SABiosciences). NK, CD4, CD8 and Mac-1 cells were assessed by flow cytometry of single cell suspension at day 5 p.i.

Results: : Virus titers in the inoculated eyes of TNFR1 KO mice were significantly higher (Avg: 8.91×10⁵±1.05×10⁰ - day 5, 2.57×10⁶±5.53×10⁰ - day 7) compared to wild type (2.63×10⁴±3.40 ×10⁰ - day 5, 6.46×10³±2.45×10⁰ - day 7) and to TNFR2 KO mice (5.50×10⁴±3.48×10⁰ - day 5, 6.46×10² ± 1.28x10¹ - day 7), P<0.05. Caspase 3 was expressed in inoculated eyes beginning at day 3 p.i. and was increased in both TNFR1 and TNFR2 KO mice compared with wild type mice. Expression of antiviral response genes in inoculated eyes was increased in TNFR1 and TNFR2 KO mice compared with wild type control, including CD80(5.4X in TNFR1 KO mice, 4.0X in TNFR2 KO mice respectively), CD86(11.8X , 10.8X), IL-1β(13.8X, 12.6X), IL-6(8.0X, 8.1X) and TLRs(2.1-6.4X, 2.0-6.2X for TLR3, 7, 8 and 9). Among all cell types, only the percentage of Mac-1 positive cells in TNFR1 KO mice was markedly reduced compared with TNFR2 KO and wild type mice.

Conclusions: : Since both TNFR1 and TNFR2 appear to play a role in caspase 3 dependent apoptosis, it is therefore likely that non-TNF-α inducers of apoptosis produced during HSV-1 ocular infection, such as NO, may induce apoptosis in TNFR1 KO mice. The lack of TNFR1 or TNFR2 leads to a compensatory increase in the innate immune response.

Keywords: herpes simplex virus • retinitis • receptors