Search

Cytosolic DNA sensing activates the stimulator of IFN genes (STING) adaptor to induce IFN type I (IFN-αβ) production. Constitutive DNA sensing to induce sustained STING activation incites tolerance breakdown, leading to autoimmunity. In this study, we show that systemic treatments with DNA nanoparticles (DNPs) induced potent immune regulatory responses via STING signaling that suppressed experimental autoimmune encephalitis (EAE) when administered to mice after immunization with myelin oligodendrocyte glycoprotein (MOG), at EAE onset, or at peak disease severity. DNP treatments attenuated infiltration of effector T cells into the CNS and suppressed innate and adaptive immune responses to myelin oligodendrocyte glycoprotein immunization in spleen. Therapeutic responses were not observed in mice treated with cargo DNA or cationic polymers alone, indicating that DNP uptake and cargo DNA sensing by cells with regulatory functions was essential for therapeutic responses to manifest. Intact STING and IFN-αβ receptor genes, but not IFN-γ receptor genes, were essential for therapeutic responses to DNPs to manifest. Treatments with cyclic diguanylate monophosphate to activate STING also delayed EAE onset and reduced disease severity. Therapeutic responses to DNPs were critically dependent on IDO enzyme activity in hematopoietic cells. Thus, DNPs and cyclic diguanylate monophosphate attenuate EAE by inducing dominant T cell regulatory responses via the STING/IFN-αβ/IDO pathway that suppress CNS-specific autoimmunity. These findings reveal dichotomous roles for the STING/IFN-αβ pathway in either stimulating or suppressing autoimmunity and identify STING-activating reagents as a novel class of immune modulatory drugs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-β, whereas CD11b(+) non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αβ) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αβ production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.

TLR ligands are effective vaccine adjuvants because they stimulate robust proinflammatory and immune effector responses and they abrogate suppression mediated by regulatory T cells (Tregs). Paradoxically, systemic administration of high doses of CpGs that bind to TLR9 ligands stimulated Tregs in mouse spleen to acquire potent suppressor activity dependent on interactions between programmed death-1 and its ligands. This response to CpG treatment manifested 8-12 h and was mediated by a rare population of plasmacytoid dendritic cells (CD19(+) pDC) induced to express the immunosuppressive enzyme IDO after TLR9 ligation. When IDO was blocked, CpG treatment did not activate Tregs, but instead stimulated pDCs to uniformly express the proinflammatory cytokine IL-6, which in turn reprogrammed Foxp3-lineage Tregs to express IL-17. Thus, CpG-induced IDO activity in pDCs acted as a pivotal molecular switch that induced Tregs to acquire a stable suppressor phenotype, while simultaneously blocking CpG-induced IL-6 expression required to reprogram Tregs to become Th17-like effector T cells. These findings support the hypothesis that IDO dominantly controls the functional status of Tregs in response to inflammatory stimuli in physiological settings.

CpG oligodeoxynucleotides (CpG-ODNs) stimulate innate and adaptive immunity by binding to TLR9 molecules. Paradoxically, expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced following i.v. CpG-ODN administration to mice. CpG-ODNs induced selective IDO expression by a minor population of splenic CD19+ dendritic cells (DCs) that did not express the plasmacytoid DC marker 120G8. Following CpG-ODN treatment, CD19+ DCs acquired potent IDO-dependent T cell suppressive functions. Signaling through IFN type I receptors was essential for IDO up-regulation, and CpG-ODNs induced selective activation of STAT-1 in CD19+ DCs. Thus, CpG-ODNs delivered systemically at relatively high doses elicited potent T cell regulatory responses by acting on a discrete, minor population of splenic DCs. The ability of CpG-ODNs to induce both stimulatory and regulatory responses offers novel opportunities for using them as immunomodulatory reagents but may complicate therapeutic use of CpG-ODNs to stimulate antitumor immunity in cancer patients.