Toll-like receptor 2 signaling is a mediator of apoptosis in herpes simplex virus-infected microglia

Abstract

Background: Information regarding the response of brain cells to infection with herpes simplex virus (HSV)-1 is needed for a complete understanding of viral neuropathogenesis. We have recently demonstrated that microglial cells respond to HSV infection by producing a number of proinflammatory cytokines and chemokines through a mechanism involving Toll-like receptor 2 (TLR2). Following this cytokine burst, microglial cells rapidly undergo cell death by apoptosis. We hypothesized that TLR2 signaling might mediate the cell death process as well.

Methods: To test this hypothesis, we investigated HSV-induced cell death of microglia obtained from both wild-type and TLR2-/- mice. Cell death was studied by oligonucleosomal ELISA and TUNEL staining, and the mechanisms of apoptosis were further analyzed using murine apoptosis-specific microarrays. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays.

Results: HSV infection induced apoptotic cell death in microglial cells from wild-type as well as TLR2 cells. However, the cell death at 24 h p.i. was markedly lower in knockout cells. Furthermore, microarray analyses clearly showed that the expression of pro-apoptotic genes was down-regulated at the time when wild-type cells were actively undergoing apoptosis, indicating a differential response to HSV in cells with or without TLR2.

Conclusion: We demonstrate here that HSV induces an apoptotic response in microglial cells which is mediated through TLR2 signaling.

Figure 1

HSV infection induces apoptosis in murine microglial cells. Wild-type and TLR2^-/- C57BL/6 microglial cells were infected with HSV at a MOI of 2. (A) The cells were examined for apoptotic DNA fragmentation using an oligonucleosomal ELISA at 8 and 24 h p.i. Data are presented as optical density (OD) per 10⁴ cells and are representative of three independent experiments using cells isolated from different brain specimens. (B) TUNEL staining of wild-type and TLR2^-/- microglia at 24 h p.i. After fixing and staining the wells, TUNEL positive cells from at least five fields were counted for each well. Data presented were representative of three independent experiments.

Figure 2

Differential expression of apoptotic genes in microglial cells obtained from wild-type and TLR2^-/- mice. Real-time PCR was performed using RNA from uninfected and HSV-infected microglia with primers specific for the apoptotic genes indicated. β-actin was used to normalize the values of apoptotic genes tested. Data presented are representative of three independent experiments.