Th1-Like Cytokine Induction by Heat-Killed Brucella abortus Is Dependent on Triggering of TLR9

Abstract

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9−/− mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9−/−, but absent in MyD88−/− mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-α and IFN-γ, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.

TLR2 signaling subpathways regulate TLR9 signaling for the effective induction of IL-12 upon stimulation by heat-killed Brucella abortus

“…The CyD-and CQ-induced inhibition of IL-12p40 was not secondary to cell death, as the viability of the untreated and CyD-or CQ-pre-treated DCs was 96.8%61.3% and 95.4%61.6% versus 93.9%62.7% and 92.8%63%, respectively. These data confirm the indispensable role of HKBA phagocytosis and digestion in IL-12p40 induction by DCs and further supports previous data 4 suggesting that IL-12p40 secretion by HKBA-stimulated DCs is important for the activation of TLR9 localization to the endosomes.…”

“…Here, we investigated the effect on heat-killed Brucella abortus (HKBA) through the costimulation of two TLRs, particularly because it is known to activate both TLR2 and TLR9 in mouse dentritic cells (DCs). 4,5 We paid particular attention to the possibility that the cross-talk between two signal transduction pathways might require regulated integration for the effective induction of proinflammatory cytokines.…”

“…5 Importantly, it has been demonstrated that TLR2 is required for HKBA-stimulated TNF production, whereas HKBA induced the production of IL-12 in a TLR9-dependent manner in DCs. 4 TLR9 functions in the endosomes of innate immune cells and recognizes distinct patterns of nucleic acids in late-endosomal compartments; [10][11][12] its activation requires HKBA phagocytosis. 4 TLR2 and TLR4 localize to the plasma membrane, and during phagocytosis, they are recruited to the contact site with the pathogen and are highly enriched on phagosomes.…”

“…4 TLR9 functions in the endosomes of innate immune cells and recognizes distinct patterns of nucleic acids in late-endosomal compartments; [10][11][12] its activation requires HKBA phagocytosis. 4 TLR2 and TLR4 localize to the plasma membrane, and during phagocytosis, they are recruited to the contact site with the pathogen and are highly enriched on phagosomes. The recruitment of TLRs to phagosomes provides a mechanism for the association of phagocytosis with inflammatory responses.…”

Early Treatment with Chloroquine Inhibits the Immune Response against<i> Plasmodium yoelii</i> Infection in Mice

“…Thus, the decreased levels of both DCs may directly cause the negative regulation of IgG-secreting B cells in the post-6h group. Several reports have indicated that CQ can regulate innate immune functions in a TLR9-dependent manner (He et al 2004;Hong et al 2004;Coban et al 2005;Huang et al 2005). In addition, early studies in TLR9-deficient mice revealed that TLR9 was not only required for the production of pro-inflammatory cytokines, but also for the induction of Th1-based immune responses and the proliferation of B cells (Liu et al 2003;He et al 2004;Iwasaki and Medzhitov 2004).…”