Phosphoinositide 3-Kinaseγ Controls the Intracellular Localization of CpG to Limit DNA-PKcs-Dependent IL-10 Production in Macrophages

Abstract

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ−/−). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ−/− cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ−/− cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ−/− cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ−/− cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages.

The Effect of High-Mobility Group Box 1 in Rat Steatotic and Nonsteatotic Liver Transplantation From Donors After Brain Death

“…Further studies will be required to clarify whether PI3K/Akt regulation is responsible for the benefits of HMGB1 in these experimental settings. In fact, it should be taken in account that both of the drugs used to inhibit PI3K/Akt pathway, wortmannin and LY29002, also inhibit mammalian target of rapamycin and DNAPK . Moreover, it is not possible to know whether the observed effects result from the combined action of both inhibitors, inhibiting PI3K and Akt equally, or are related to the predominant action of one inhibitor on its target molecule.…”

Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages

“…Activation of different TLRs in macrophages, for instance, with LPS or CpG differentially regulates the production and subsequent secretion of IL-10 [36,37]. CpG stimulation of macrophages can lead to IL-10 production in a manner dependent on PI3K␥, but PI3K␥ does not mediate LPS-stimulated IL-10 production [36,37].…”

Pharmacological inhibition of PI3K class III enhances the production of pro- and anti-inflammatory cytokines in dendritic cells stimulated by TLR agonists

“…This includes evidenced obtained with wortmannin, the most widely used PI3K inhibitor, known to be free of the specificity problems affecting LY294002 in particular [12] . In contrast, for IL-10 upregulation, results obtained with wortmannin [16] , [17] , often clash with the evidence based on genetically modified mice [14] , [15] , [18] , [19] . However, the results generated using a specific inhibitor of the catalytic subunit p110δ do agree with the data from genetically modified mice [14] .…”