Systemic Treatment with CpG-B after Sublethal Rickettsial Infection Induces Mouse Death through Indoleamine 2,3-Dioxygenase (IDO)

Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes

Toll-like receptors differentially induce nucleosome remodelling at the IL-12p40 promoter

Toll-like receptors (TLRs) mediate recognition of microbial components. Despite activation of a shared set of signal transduction molecules, the biological effects of certain TLR agonists differ considerably. In macrophages and dendritic cells, stimulation by the prototypical stimuli CpG-DNA (TLR9), lipopolysaccharide (LPS; TLR4) and lipoteichoic acid (LTA; TLR2) resulted in striking differences in expression of IL-12. However, these stimuli induced similar amounts of the common proinflammatory cytokine TNFα. Surprisingly, an IL-12p40 promoter reporter construct was activated equally by CpG-DNA, LPS and LTA. Examinations of the chromatin structure of the endogenous IL-12p40 promoter revealed that nucleosome remodelling contributed to differential IL-12 induction. Upon stimulation, nucleosome architecture was changed to provide increased access to the IL-12p40 promoter. In dendritic cells, a differential induction of nucleosome remodelling at the IL-12p40 promoter was observed upon triggering with different TLR agonists. These results identify nucleosome remodelling as an additional restriction point in differential TLR signalling

Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance

Lipopolysaccharide (LPS) tolerance is a state of refractoriness towards a second stimulation by LPS after a preceding stimulation. LPS is recognized by Toll-like receptor-4 (TLR-4), which belongs to a group of pattern recognition receptors mediating activation of innate immunity by microbial components. To date, it is not known in detail to what extent other TLR-dependent stimuli also induce tolerance and whether preceding and challenging stimuli are interchangeable. We have examined tolerance induction in detail for lipoteichoic acid (LTA), LPS and CpG-DNA, which are recognized by TLR-2, -4 and -9, respectively. In RAW264·7 macrophages, all three stimuli induced tolerance towards a subsequent challenge with the same stimulus used for priming, as well as cross-tolerance towards subsequent challenge with other stimuli signalling via different TLRs. However, whereas LPS/LTA cross-tolerance was also functional in an in vivo model of galactosamine (GalN)-primed liver damage, pretreatment with CpG only protected against GalN/CpG challenge and failed to induce cross-tolerance for LPS and LTA. CpG-DNA pretreatment even enhanced tumour necrosis factor (TNF)-α production and liver damage upon subsequent challenge with LPS or LTA. Stimulation with CpG-DNA resulted in a peculiar sensitization for interferon (IFN)-γ secretion. The data indicate that, in contrast to in vitro macrophage desensitization, the in vivo consequences of repeated TLR stimulation greatly differ amongst different TLR ligands