CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation

Abstract

Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40-CD40 ligand-independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG-DNA induces phosphorylation of Jun N-terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein-1 (AP-1) via phosphorylation of its constituents c-Jun and ATF2. Moreover, stress kinase activation is essential for CpG-DNA-induced cytokine release of tumor necrosis factor α (TNFα) and interleukin-12 (IL-12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non-CpG-DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG-ODN-triggered activation of antigen-presenting cells.

The Toll‐like receptor 7 (TLR7)‐specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily

“…TLR9 is the only TLR family member known to be located in endosomal/lysosomal compartments but not at the cell surface [30]. Furthermore, TLR9 signaling requires CpG-ODN uptake and endosomal maturation, which can be blocked by bafilomycin A1 or dominant negative Rab5 GTPase [31]. Upon TLR9 activation MyD88 is recruited to LAMP-1-positive [43] endosomal/ lysosomal vesicles where signaling is initiated [30].…”

“…Among the TLR characterized so far, only CpG-DNAdriven signaling via TLR9 requires acidification and therefore maturation of endosomes [30,31]. CpG-DNA signaling is efficiently blocked by a dominant negative version of the Rab5 GTPase, which interferes with early steps in endosomal fusion, as well as by bafilomycin A1, a specific inhibitor of the V-type ATPase responsible for acidification of endosomes and lysosomes [30][31][32][33].…”

“…CpG-DNA signaling is efficiently blocked by a dominant negative version of the Rab5 GTPase, which interferes with early steps in endosomal fusion, as well as by bafilomycin A1, a specific inhibitor of the V-type ATPase responsible for acidification of endosomes and lysosomes [30][31][32][33].…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Bafilomycin A1 acts as an endosomal TLR inhibitor by selectively blocking the vacuolar H + -ATPase. As a result, bafilomycin increases the acidic endosomal pH, which is thought to be essential for the activation of TLR mediated signal transduction [92][93][94]. As for bafilomycin A1, the inhibitory acitivity of chloroquine has been generally ascribed to the inhibition of endosomal acidification.…”

Adaptive and innate immune responses to gene transfer vectors: role of cytokines and chemokines in vector function

“…25 CpG DNA also induces the activation of c-Jun Nterminal kinase and p38 MAPK, which lead to activation of the transcription factor AP-1. 26,27 The actions of CpG motifs stimulate the secretion of cytokines such as TNFa and IL-6, monocyte activation, and NK cell activity. 23,28,29 The induction of type I interferons, IFNg, IL-12, and IL-18, stimulates Th1 responses and enhance the development of adaptive immunity.…”