Repetitive extragenic palindromic DNA sequences from Brucella melitensis stimulate Toll-like receptor 9 signaling in macrophages

Abstract

Brucella DNA activates the host innate immune system via the intracellular Toll-like receptor 9 (TLR9). However, the Brucella DNA sequences which are responsible for these immunostimulatory effects remain to be elucidated. The present study demonstrated that repetitive extragenic palindromic (REPs) sequences present in Brucella DNA were able to stimulate macrophages through TLR9. The induction of interferon-α (IFN-α) production by Brucella REPs was detected in cultured RAW264.7 mouse macrophages as well as in Wistar rats. Knockdown of TLR9 expression by siRNA in macrophages led to a reduction in IFN-α production following REPs stimulation. In addition, it was confirmed that the activating capacity of Brucella REPs is CpG dependent. Induction of IFN-α by Brucella REPs was completely abrogated when REP sequences were transformed into non-CpG sequences or by C-methylated modifications. Furthermore, it was observed that REPs-initiated TLR9/NF-κB and TLR9/MAPK signaling pathways contributed to the production of IFN-α. The identification of Brucella REPs as natural TLR9 agonists may be useful for the development of novel therapeutic applications.