Activation of caspase-1-mediated pathway by herpes simplex viral CpG-DNA

Objectives: Autoimmune Sjögren's Syndrome (SjS) is characterized by chronic inflammation in salivary and lacrimal glands. Our previous study indicates the activation of caspase-1-mediated pathway in macrophages in the salivary glands and increased IL-18 secretion in saliva of SjS-prone-C57BL/6.NOD-Aec1Aec2 (B6DC) mice prior to disease onset. Concomitant up-regulation of P2X7-receptor (P2X7R) detected in B6DC salivary glands prompted us to investigate if presence of chronic stimulation such as herpes simplex virus (HSV)-derived oligodeoxyribonucleotide CpG-DNA (CpG-ODN) can activate caspase-1-mediated pathway via P2X7R and a subsequent IL-18 production in mouse macrophage cell line RAW264.7.

Methods: The expression of caspase-1 and its downstream molecules were analyzed by Western Blot analysis, RT-PCR, and ELISA. RAW264.7 cells were stimulated for 24-hours in the presence of CpG-ODN (pCpG, 3µM) and negative-CpG-ODN (nCpG, 3µM) compared to non-stimulated cells and LPS-stimulated cells (1µg/ml).

Results: IL-1b; and IL-18 gene expression was up-regulated in pCpG, similar to that of LPS stimulation, a positive control for IL-18 induction. This was further supported with IL-18 levels in cell supernatants detected by ELISA, as pCpG (145±48 pg/ml, p <0.01) showed higher expression compared to non-stimulated (119±17 pg/ml) and nCpG (90±8 pg/ml). To determine which receptor viral CpG-ODN functions through, we analyzed P2X7R by western blotting. Our findings indicated up-regulated P2X7R but not TLR-9 in pCpG, which suggests the possibility that CpG-ODN stimulates caspase-1-mediated pathway via P2X7R rather than TLR-9.

Conclusions: Our findings indicate that viral CpG-DNAs are stimulatory for pro-inflammatory cytokine production via caspase-1-mediated pathway in macrophages, which implies that chronic inflammation in SjS salivary glands prior to disease onset is caused by chronic viral stimulation in the target tissue. To confirm the role of P2X7R in the pCpG and potentially in SjS, inhibition of the receptor expression by siRNA to TLR-9 and/or P2X7R is currently under active investigation (Supported by NIH/NIDCR grants U24DE016509 and R21DE016705).