CpG-ODN from Streptococcus thermophilus as a tool for inducing extrinsic apoptosis via caspase-8 activation in mouse splenocytes.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the lacZ gene of Streptococcus thermophilus ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.