Activation of the Stimulator of Interferon Genes (STING) adaptor attenuates experimental autoimmune encephalitis

Abstract

Cytosolic DNA sensing activates the Stimulator of Interferon Genes (STING) adaptor to induce interferon type I (IFNαβ) production. Constitutive DNA sensing to induce sustained STING activation incites tolerance breakdown leading to autoimmunity. Here 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 central nervous system (CNS) and suppressed innate and adaptive immune responses to MOG 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 IFNαβ receptor genes, but not IFNγ receptor genes, were essential for therapeutic responses to DNPs to manifest. Treatments with cyclic diguanylate monophosphate (c-diGMP) to activate STING also delayed EAE onset and reduced disease severity. Therapeutic responses to DNPs were critically dependent on indoleamine 2,3 dioxygenase (IDO) enzyme activity in hematopoietic cells. Thus DNPs and c-diGMP attenuate EAE by inducing dominant T cell regulatory responses via the STING-IFNαβ-IDO pathway that suppress CNS-specific autoimmunity. These findings reveal dichotomous roles for the STING-IFNαβ pathway in either stimulating or suppressing autoimmunity and identify STING activating reagents as a novel class of immune modulatory drugs.

Introduction

Self-tolerance is an active and constitutive process that prevents autoimmunity. Recent studies on mice with defective DNA repair enzyme expression emphasize the potential for DNA in incite lethal autoimmunity by stimulating cytosolic DNA sensors that activate STING to induce IFNαβ production (1, 2). Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease in which neuronal tissues are progressively targeted (3) due to loss of tolerance to central nervous system (CNS) antigens such as myelin basic protein and oligodendrocyte glycoprotein (MOG).

Indoleamine 2,3 dioxygenase (IDO) is a natural immunomodulatory enzyme that attenuates autoimmunity in murine disease models, including experimental autoimmune encephalitis (EAE), a model of MS, autoimmune rheumatoid arthritis (RA), type I diabetes (T1D), systemic lupus erythematosus (SLE) and inflammatory bowel disease (4). In these syndromes IDO ablation accelerated disease onset and enhanced disease severity. Moreover, the immunomodulatory properties of soluble forms of CTLA4 (5, 6) and CD83 (7) depend, in part on their ability to induce IDO-dependent regulatory phenotypes in DCs, which promote de novo Treg generation, activate resting Tregs and suppress effector T cell responses (8, 9). Moreover, some immune stimulatory reagents (adjuvants) co-induce IDO, and this property of TLR9 ligands (CpGs) inhibited type I diabetes progression in NOD female mice (10-12). Diametric responses to immune adjuvants underscore the need to evaluate innate immune responses to inflammatory stimuli to discern underlying pathways that induce dominant stimulatory or regulatory responses by T cells (13). Sustained IFNαβ production is a key feature of chronic immune activation at sites of persistent infections such as HIV-1 (14). Sustained IFNαβ release, especially by plasmacytoid dendritic cells (DCs), correlates strongly with risk of autoimmune syndromes such as SLE (15). IFNαβ and IFN type II (IFNγ) have well documented immune stimulatory properties but IFNs are also potent IDO inducers, providing a rationale for increased IDO-mediated T cell regulation, particularly at sites of chronic inflammation.

Previously, we reported that small populations of DCs co-expressing the B cell marker CD19 up-regulated IDO selectively (amongst DCs) in response to systemic treatments with soluble CTLA4 (CTLA4Ig), TLR9 ligands (CpGs) and DNA nanoparticles (DNPs) containing the cationic polymer polyethylenimine (PEI) and cargo DNA (12, 16, 17). IDO induction in CD19⁺ DCs was mediated by IFNαβ not IFNγ, and CD19⁺ DCs expressing IDO stimulated Foxp3-lineage CD4 T cells (Tregs) to acquire regulatory phenotypes that suppressed Th1 responses (8, 17). Tolerogenic responses to DNPs were dependent on cargo DNA sensing by small populations of myeloid DCs to activate STING and induced selective IFNαβ release that stimulated CD19⁺ DCs to express IDO (17, 18). In the current study, we tested the hypothesis that STING activation to induce IDO via IFNαβ signaling following systemic DNP treatment inhibits EAE progression and reduces disease severity.

Methods

Mice and induction of EAE

Mice were bred under SPF conditions at GRU or purchased from Taconic Farms and procedures were approved by the IACUC. IDO1-KO, IFNAR-KO, IFNγR-KO and STING-KO, mice were described (19-21). To induce EAE, mice aged 8-12 weeks were immunized (s/c) at 2 sites on the rear flank with 100μg myelin oligodendrocyte glycoprotein peptide (MOG35–55, MEVGWYRSPFSRVVHLYRNGK, Bio Basic Canada Inc.) emulsified in CFA (Difco Laboratories, Detroit, MI) containing 4mg/ml Mycobacterium tuberculosis H37Ra (Difco Laboratories). 200ng of pertussis toxin (Sigma) was given (i/p) on days 0 and 2 post-immunization. Clinical symptoms of EAE were scored on a scale from 0 to 5 as described by Das Sarma et al. (22) with slight modification as follows; 0, no clinical signs; 0.5, partial limp tail; 1, full limp tail or waddling gait; 1.5, limp tail and waddling gait/lumbar weakness; 2, partial paralysis of one hind limb; 2.5, paralysis of one hind limb or partial paralysis of both hind limbs; 3, paralysis of one hind limb and partial paralysis of the other hind limb; 3.5, paralysis of both hind limbs or partial paralysis of both hind limbs and weakness of the upper limb; 4, ascending paralysis, i.e., complete paralysis of both hind limbs and weakness of the upper limb; 4.5, three paralyzed limbs; 5, four paralyzed limbs/moribund or dead. Some mice were given drinking water containing the IDO inhibitor (1-methyl-[D]-tryptophan, D-1MT, 2mg/ml) and Nutrasweet (to increase palatability) as described (23).

DNP and c-diGMP treatments

DNPs were prepared by mixing 7μl of polyethylenimine (PEI, 150mM) with 21μg of CpG^free LacZ pDNA (Invivogen, San Diego, CA) in 200μl of 5% glucose solution (N/P=16.7) as described (17). Chemically synthesized c-diGMP was dissolved in PBS at 500μg/ml. DNPs and c-diGMP (100μg/dose) were injected intravenously.

Immunohistochemistry

After sacrifice, mice were perfused (PBS then paraformaldehyde, PFA, 4%,) and lumbar spinal cord tissues were harvested, left in PFA (3 days) and embedded in paraffin. 5μm sections were deparaffinized, rehydrated, submerged in DAKO–TARGET solution (cat. S-1699, 20min) and incubated in 3% H₂O₂ (5min). Non-specific biotin/avidin (Vectorlabs, Burlingame, CA) and Fc/Fab staining was blocked using kits (cat. 015.000.008 Jackson ImmunoResearch). M.O.M kits (Vectorlabs, Burlingame, CA) were used to dilute primary mouse anti-mouse/human IDO mAb (E7, 1:50; Santa Cruz Biotechnology, cat. 365086), neurons (NeuN rabbit polyclonal, 1:500; Millipore cat. ABN78), microglia (IBA rabbit, 1:500; WAKO, cat. 019-19741) and astrocytes (GFAP rabbit monoclonal, 1:500; Cell Signaling, cat. 123895). After 60 min, sections were incubated with biotinylated anti-mouse IgG antibody from the M.O.M kit and Alexa fluor 488-conjugated AffiniPure F(ab′)2 fragment donkey anti-rabbit IgG (H+L) (cat. 711-546-152, Jackson ImmunoResearch) for 45 min. After washing, streptavidin conjugated with Alexa fluor 555 (1:400, Invitrogen Molecular probes, cat. S32355) was applied for 15 min. Slides were mounted in Fluor Save Reagent (Calbiochem, cat. 345789) and analyzed by confocal microscopy (Zeiss LSM 510).

Cytokines

Splenocytes (10⁶) were cultured in RPMI in triplicate in 96-well U-bottom plates in the presence or absence of 20 mg/ml MOG peptide for 72hrs. Cytokine levels in culture supernatants were analyzed using multiplex kits from Bio-Rad or eBioscience.

IDO enzyme activity

Cell-free spleen homogenates were added to IDO enzyme cocktails and kynurenine generated after 2hrs was measured by HPLC as described (24).

Flow cytometry

Single-cell suspensions of spleen and CNS (pooled brain and spinal cord) prepared as described (25) were stimulated in medium containing 20mg/ml MOG peptide (37°C, 5% CO₂, 18hr); 1μl/ml brefeldin(BD) was added for the last 4hrs of culture. After staining the surface marker CD4 (BD Pharmingen, clone RM4-5), cells were fixed and permeabilized (Foxp3/Transcription Factor Fixation/Permeabilization buffer; eBioscience), followed by staining with mAbs to mouse Foxp3 (eBioscience clone FJK-16S), IL-17A (eBioscience, clone eBio17B7) and IFN-γ (BD Pharmingen, clone XMG1.2). Flow cytometric analyses were performed using a LSRII cytometer (BD) and data generated were analyzed using FACS DIVA (BD Bioscience) or FlowJo (Tree Star, Ashland, OR) software.

Radiation chimeric mice

Mice were irradiated (900 Rads) and after 24 hours mice received ∼10⁷ nucleated bone marrow cells (i/v) harvested from the long bones of donor mice and were allowed to recover for 10 weeks before use in EAE induction experiments.

Statistical analysis

EAE scores were evaluated with two-way ANOVA. The unpaired Student's t test was used for statistical evaluations of cytokine and cell frequency measurements. Two-tailed p-values <0.05 were considered significant. Graphpad Prism was used to perform all data analyses.

Results

DNP treatment slows EAE onset and reduces disease severity

B6 mice were immunized with MOG and treated with pertussis toxin (PTx) to induce EAE (see Methods). The effects of systemic DNP treatment on EAE progression were evaluated after administering DNPs (PEI/CpG^free pDNA) intravenously (i/v) to groups of mice starting at the time of MOG immunization (day 0) and on days 2, 5, 9 and 12 (Fig. 1A, gray arrows). Control mice were given vehicle (5% glucose) and all mice were monitored to detect EAE onset and score clinical disease severity (see Methods). As expected, EAE symptoms began to manifest in vehicle-treated mice 8-12 days post MOG immunization (partial/full limp tail, clinical score 0.5-1), peak disease severity occurred 14-24 days post immunization (mean clinical scores, mcs >2), with minor relapse thereafter (Fig. 1A, closed symbols). DNP treatment delayed disease onset by ∼4 days and reduced peak disease severity (mcs ∼1.5) significantly, relative to vehicle-treated mice (Fig. 1A, open symbols).

Figure 1.

DNA nanoparticles (DNPs) delay EAE onset and reduce disease severity. A. B6 mice were injected (i/v) with freshly prepared DNPs (21μg CpG^free pDNA, 7ul 150mM PEI in 200μl 5% glucose) or vehicle (5% glucose) on days 0, 2, 5, 9, 12 (5×, grey arrows) after MOG-immunization. B. As in A, except that DNPs were administered later (from day 11) and then every other day until day 21 (6×). C. B6 mice were treated with DNPs or PEI, cargo DNA, or vehicle (5% glucose) only from days 0-12 (5×). D. B6 mice were treated with DNPs starting at peak disease from days 14-21 (5×). Mice were monitored daily for EAE onset and scored to assess disease severity using the scheme described in Methods. Clinical scores were compiled from 1 (C), 4 (A), 2 (B, D) and experiments respectively and are expressed as mean scores ±SEM at each time point. Data were analyzed by two-way ANOVA; ****p<0.0001, ***p<0.0012.

Next, we tested if administering DNPs later, when EAE first started to manifest, attenuated EAE. DNPs were administered from day 11 (i/v) and every other day until day 21 (Fig. 1B, gray arrows). DNP treatment prevented disease onset by ∼7 days and subsequent disease severity was reduced significantly (mcs <1), relative to outcomes in vehicle-treated mice (Fig. 1B, open and closed symbols, respectively). Delayed vehicle treatment had no impact on disease progression, peak disease severity or minor relapse at late times (Fig. 1B, closed symbols), relative to mice given vehicle earlier (Fig. 1A). In contrast, treatments with PEI or cargo DNA alone induced no therapeutic responses in MOG-immunized mice, relative to mice treated with vehicle (5% glucose, Fig. 1C). DNP treatments administered to MOG-immunized mice at peak EAE disease (starting on day 14 and given every other day until day 21) also induced significant therapeutic responses (Fig. 1D), indicating that regulatory responses to DNPs overcame established CNS-specific autoimmunity.

DNP treatment inhibits IDO expression in neurons during EAE

DNPs induce rapid IDO up-regulation in mucosal and lymphoid tissues (18). To evaluate if DNPs induced IDO in CNS tissues, untreated (naïve) and MOG-immunized mice treated with vehicle or DNPs three times (days 11, 13, 15) were sacrificed on day 16, and CNS tissues were stained to detect IDO and co-stained to detect neuronal (NeuN), astrocyte (GFAP) or microglia/monocytes (Iba1) specific markers (Fig. 2A). As expected, IDO expression was not detected and few astrocytes and microglia were present in CNS tissues from naïve mice (Fig. 2A, upper panels). In contrast, most neurons (NeuN⁺) from CNS tissues of vehicle-treated, MOG-immunized mice expressed IDO and expanded cohorts of microglia/infiltrating monocytes and activated astrocytes were present but these cells did not express IDO (Fig. 2A, center panels); expanded cohorts of activated glial cells are a prominent feature of EAE, which correlates with increased neuropathology (26). However, neurons did not express IDO in CNS tissues from DNP-treated, MOG-immunized mice and numbers of microglia/infiltrating monocytes and activated astrocytes in CNS tissues were markedly reduced (Fig. 2A, lower panels), relative to MOG-immunized mice, and were comparable with naïve mice. Thus, MOG-immunization stimulated neurons to express IDO, while DNP treatment to induce IDO in non-CNS tissues (17), paradoxically suppressed MOG-induced IDO expression in neurons. These findings suggest that IDO induction in lymphoid tissues inhibits CNS infiltration of effector cells that incite neurons to express IDO.

Figure 2.

DNP treatment blocks IDO induction in neurons and reduces effector cell infiltration in CNS tissues. A. Spinal cord tissue sections were prepared from mice 16 days post MOG immunization (or from untreated controls) with or without DNP treatment on days 11, 13 and 15 (3×). Tissues were stained to detect cells expressing IDO and specific markers expressed by neurons (NeuN), astrocytes (GFAP) or microglial cells (Iba1). Scale bar, 20μm (lower right). B-E. Total cell numbers (B) and numbers of Th17 (IL-17), Th1 (IFNγ) and Tregs (Foxp3) in gated CD4⁺ T cells from CNS tissues (C-E) were assessed by flow cytometric analyses of pooled spinal cord and brains from individual mice stained to detect CD4 T cells and intracellular IL-17, IFNγ and Foxp3 expression after stimulation ex vivo with MOG-peptide (18hrs) and added Brefeldin A for the final 4 hours. F. HPLC analyses to detect kynurenine in serum samples from MOG-immunized mice (day 16) with or without DNP treatment as above. Data are representative of 2 experiments (A) or were compiled from 3 experiments (B-F). Data are expressed as means ±SEM and were analyzed using the unpaired, two-tailed Student's t-test; *p< 0.05; ns, not significant.

DNP treatment reduces Teff:Treg ratios in CNS tissues during EAE

Absolute numbers of cells in CNS tissues were reduced significantly (∼2-fold) in MOG-immunized mice treated with DNPs (3×, days 11, 13, 15 and harvested on day 16), relative to vehicle-treated controls (Fig. 2B). Consistent with this finding, numbers of CD4 T cells expressing intracellular IFNγ or IL-17 (Th1, Th17) after stimulating cells from CNS tissues with MOG peptide ex vivo (18hrs) were reduced by DNP treatment (Fig. 2CD, ∼2-3-fold) but the relative proportions of Tregs and MOG-specific Th1 and Th17 T cells amongst infiltrating CD4 T cells were unaffected by DNP treatment (Suppl. Fig. 1A). Since DNPs suppressed IDO expression in CNS tissues from MOG-immunized mice (Fig. 2A) we evaluated if DNPs induced IDO activity elsewhere in MOG-immunized mice by measuring levels of serum kynurenine, a tryptophan catabolite secreted by cells expressing IDO. Serum kynurenine increased significantly (Fig. 2F, ∼2-fold) in DNP-treated, MOG-immunized mice, relative to levels in vehicle-treated, MOG-immunized mice, indicating that systemic DNP treatment stimulated IDO activity in non-CNS tissues of MOG-immunized mice.

DNP treatment suppresses pro-inflammatory responses to MOG immunization in spleen

Systemic DNP treatment during MOG-immunization (3×, days 11, 13, 15) increased spleen cellularity (at day 16) relative to vehicle-treated MOG-immunized mice with EAE (Fig. 3A). Numbers (Fig. 3A) of Tregs and MOG-specific CD4+ T cells expressing intracellular IFNγ and IL-17 also increased significantly (>2-3 fold) as did the relative proportions of Tregs, and MOG-specific Th1 and Th17 cells amongst CD4 T cells in spleens of DNP-treated mice (Suppl. Fig. 1B). However splenocyte proliferation, measured by thymidine incorporation ex vivo (72hrs) in response to MOG peptide, was reduced substantially in DNP-treated mice (Fig. 3B). Consistent with this finding, MOG-peptide induced proinflammatory cytokine production by splenocytes from MOG-immunized mice (IL-17A/F, IL-6, IL-22, IFNγ) was reduced significantly (60-95%) by DNP treatment (Fig. 3C). Thus DNP treatment enhanced MOG-specific Th1 and Th17 T cell and Treg responses in spleen but the overall affects of DNP treatment were to attenuate innate and adaptive immunity at this site.

Figure 3.

DNP treatment attenuates Th1/Th17 responses to MOG immunization elicited in spleen. A. B6 mice were immunized with MOG and treated with DNPs or vehicle (i/v, 3×, days 11, 13, 15). Mice were sacrificed on day 16 and spleen cellularity and numbers of splenic CD4⁺ T cells expressing intracellular IL-17, IFNγ and Foxp3 were assessed by flow cytometric analyses after ex vivo culturing with MOG peptide (18hrs) and Brefeldin A (last 4hrs). B. Splenocytes from DNP-treated (i/v, 5×, days 0, 2, 5, 9, 12) or vehicle-treated MOG-immunized mice were harvested (day 13) and stimulated ex vivo with MOG peptide (20μg/ml, 72hrs), and IL-17A/F, IL-6, IL-22 and IFNγ in culture supernatants were determined by multiplex analyses. Data are representative of two experiments (n=4) and are expressed as mean ±SEM. Data were analyzed using the unpaired, two-tailed Student's t-test; ****p<0.0001, ***p<0.001, **p< 0.01, *p<0.05.

IFN type I (IFNαβ) not type II (IFNγ) signaling mediates therapeutic responses to DNPs

To evaluate IFN signaling requirements for therapeutic responses to DNPs to manifest, MOG immunized mice with defective IFN type I (IFNAR-KO) or type II (IFNγR-KO) receptor genes were treated with DNPs (or vehicle) during EAE induction (days 0-12) or later, at the time of EAE onset (days 11-21). DNP-treated and vehicle-treated B6 (WT) mice (Fig. 4AB), and vehicle-treated IFNAR-KO (Fig. 4CE) and IFNγR-KO (Fig. 4DF) were used as controls. DNPs had no significant therapeutic effects on EAE onset or peak disease severity when administered early (Fig. 4C) or later (Fig. 4E) to IFNAR-KO mice. Clinical scores at late stages of EAE progression (>20 days) were slightly worse in DNP-treated IFNAR-KO mice (>3) than for vehicle-treated controls (<3), suggesting that weak immune stimulatory effects of DNPs are masked by dominant regulatory responses mediated by IFNαβ receptors. In vehicle-treated IFNAR-KO mice (Fig. 4C), the time of EAE onset (days 8-12), attainment of peak EAE severity (days 14-16), and clinical scores at late stages of EAE (∼2.5) were unaffected by loss of IFNAR function, relative to B6 controls (Fig. 4A). Thus IFNαβ signaling is essential for therapeutic responses to DNPs to manifest. In contrast, DNP treatment in IFNγR-KO mice slowed EAE onset and reduced EAE severity significantly when DNPs were administered early (Fig. 4D), or late (Fig. 4F), relative to vehicle-treated controls. Consistent with previous studies on IFNγR-KO mice (27), EAE onset (∼days 12-14) and attainment of peak EAE severity (>day 20) were delayed and peak clinical scores were higher (4-5) in vehicle-treated IFNγR-KO mice (Fig. 4D), relative to vehicle-treated B6 controls (Fig. 4B), especially when vehicle was given early (Fig. 4D). Thus IFNγ signaling is not required for therapeutic responses to DNPs, even though IFNγ signaling regulates EAE progression in MOG-immunized mice (27), possibly by inducing IDO at specific phases of EAE progression (28).

Figure 4.

Therapeutic responses to DNPs are dependent on IFN type I but not IFN type II signaling. A-D. MOG-immunized B6 (AB, WT), IFNAR-KO (C) or IFNγR-KO (D) mice were treated with DNPs (days 0-12, 5×) or vehicle (5% glucose). E-F. As in C and D, except that treatments were administered to mice later (days 11-21, 6×). Mice were monitored daily for EAE onset and severity. Data were compiled from two or more experiments and are expressed as mean clinical scores ±SEM at each time point. Data were analyzed by two-way ANOVA; ****p< 0.0001, ***p<0.001, **p<0.01, NS, not significant.

STING mediates therapeutic responses to DNPs

DNPs regulate T cell responses via a STING dependent pathway (18). To test if STING was required to alleviate EAE, MOG-immunized, B6 and STING-deficient (STING-KO) mice were treated with DNPs or vehicle (Fig. 5AB). STING-KO mice succumbed uniformly to EAE (Fig. 5B, closed symbols), though EAE onset was slower (days 14-16) and disease scores were lower (<2) than in B6 mice (Fig. 5A, closed symbols). Early DNP treatment (days 0-12) accelerated EAE onset and increased disease severity (>2), relative to vehicle-treated STING-KO mice (Fig. 5B, open symbols). Administering DNPs later (days 11-21) induced no therapeutic responses in STING-KO mice, relative to vehicle-treatments (Fig. 5C). Analyses of serum kynurenine (Kyn) levels in MOG-immunized mice revealed that DNP treatments before EAE onset (days 0-12) or at the time of EAE onset (day 11) did not induce IDO activity in STING-KO mice, though MOG-immunization alone led to elevated IDO activity in STING-KO mice relative to control B6 mice (Table I). Thus therapeutic responses to DNPs via IDO were STING dependent and, as in mice lacking IFNγ receptors, loss of STING signaling revealed cryptic immune stimulatory properties of DNPs normally masked by dominant regulatory responses in mice with intact STING genes.

Figure 5.

Therapeutic responses to DNPs are mediated by STING. AB. MOG-immunized B6 (A, WT) or STING-KO mice (B) mice were treated with DNPs (days 0-12, 5×) or vehicle (5% glucose). C. As in B, except that STING-KO mice were treated later (days 11-21, 6×). D-F. B6 mice were treated with c-diGMP (i/v, 100μg) or vehicle (PBS) during MOG immunization (D, days 0, 2), at initial EAE onset (E, days 11, 13) or both phases (F, days 0, 2, 9, 12). Mice were monitored daily for EAE onset and severity. Data were compiled from 1-2 experiments and are shown as mean clinical scores ±SEM at each time point. Data were analyzed by two-way ANOVA; ****p<0.0001, ***p<0.001.

Table I. IDO activity in MOG-immunized mice.

Mice	MOG (n)	DNP treatment	Serum Kyn (μM)
B6	+ (4)	-	1.54 ± 0.19
	+ (8)	+ days 0-12	2.97 ± 0.32*
	+ (5)	+ day 11	3.47 ± 0.42*
STING-KO	+ (4)	-	2.75 ± 0.91
	+ (3)	+ days 0-12	3.19 ± 0.33 (ns)
	+ (5)	+ day 11	3.76 ± 0.27 (ns)

Notes: Harvested on day 13;

^*p<0.05; ns, not significant

Cyclic dinucleotides mediate therapeutic responses to EAE via STING

Based on studies using DNP-treated STING-KO mice we hypothesized that systemic treatment with the cyclic dinucleotide, diguanylate monophosphate (c-diGMP), which activates STING (29) also attenuates EAE. To test this hypothesis, MOG-immunized B6 mice were treated with chemically synthesized c-diGMP (100μg, i/p on days 0, 2). c-diGMP treatment delayed EAE onset by ∼6 days, but did not reduce peak EAE severity relative to vehicle-treated (PBS) mice (Fig. 5D, open and closed symbols, respectively). Similarly, c-diGMP treatment later (days 11, 13) also slowed EAE progression (∼4 days) but did not reduce peak disease severity relative to vehicle-treatments (Fig 5E, open and closed symbols). In contrast, combined early and late phase c-diGMP treatments (days 0, 2, 9 & 12) delayed EAE onset by ∼15 days and reduced peak disease severity (∼1.5) significantly relative to vehicle-treated mice (Fig. 5E, open and closed symbols). Thus STING activation after c-diGMP or cargo DNA sensing or attenuates EAE progression and onset.

Therapeutic responses to STING activating reagents are IDO dependent

To test if IDO is required for therapeutic responses to DNPs B6 mice were treated with DNPs (5×, days 11-19). One group of DNP-treated mice was given the IDO-specific inhibitor 1-methyl-[D]-tryptophan (1MT) on day 11 (1mg, i/p) and then given drinking water containing 1MT (2mg/ml) ad libitum until experimental endpoints. As expected, DNPs reduced EAE clinical symptoms significantly relative to vehicle treatments (Fig. 6A, open versus closed circles). 1MT treatment reduced therapeutic responses to DNPs significantly but not completely (Fig. 6A, open squares). To further test requirements for IDO function, IDO1-deficient (IDO1-KO) mice with pure B6 backgrounds were treated with DNPs (6×, days 11-21) or c-diGMP (2×, days 11, 13). As shown in Fig. 6B, neither treatment induced therapeutic responses in IDO1-KO mice (open symbols). Rather, EAE severity was slightly higher at days 20-22 in mice treated with c-diGMP relative to vehicle-treated mice (Fig. 6B, open squares versus closed circles). Thus functional IDO expression was essential for therapeutic responses to manifest following systemic treatments with DNPs or c-diGMP.

Figure 6.

Therapeutic responses to STING activating reagents are dependent on IDO function in hematopoietic cells. A. MOG-immunized B6 mice were treated with DNPs (days 11-19, 5×) vehicle (5% glucose) or DNPs plus 1-methyl-D-tryptophan (1MT) injected (i/p, 500μl at 2mg/ml) on day 11 and then provided in drinking water (2mg/ml) from day 11 until experimental endpoints (day 27). B. MOG-immunized IDO1-KO mice were treated with DNPs, vehicle or c-diGMP as indicated. CD. Radiation chimeric mice were generated from B6 (WT) and IDO-KO (KO) donors and recipients as described in Methods, and were treated with DNPs or vehicle (days 0-12, 5×) 10 weeks after bone marrow reconstitution. Mice were monitored and scored daily for EAE as before. Data were compiled from two pooled (A) or single experiments (B-D) and are expressed as mean clinical scores ±SEM at each time point. Data were analyzed by two-way ANOVA; ****p<0.0001; NS, not significant.

Hematopoietic cells expressing IDO mediate therapeutic responses to DNPs

We hypothesized that IDO induced by DNPs in lymphoid tissues mediated therapeutic responses because MOG-immunization stimulated neurons to express IDO in CNS tissues (Fig. 2A), and IDO activity in CNS tissues generates neurotoxic tryptophan catabolites (30, 31). To test if therapeutic responses to DNPs were dependent on IDO function in hematopoietic cells B6 (WT) or IDO1-KO (KO) mice were lethally irradiated and reconstituted with bone marrow from WT or KO donors to create chimeric mice. This approach permits segregation of potential toxic effects of IDO in non-hematopoietic neurons from potential beneficial immune regulatory effects of IDO in hematopoietic cells such as DCs.

First, we monitored EAE induction and severity in irradiated B6 mice reconstituted with B6 bone marrow (WT→WT). As shown in Fig. 6C (closed symbols) EAE manifested uniformly in MOG-immunized WT→WT mice but EAE onset was delayed slightly (∼12-14 days) and peak disease severity was higher in WT→WT mice (5) relative to non-chimeric B6 mice (Fig. 1), suggesting that residual inflammation in radiation chimeras may exacerbate EAE susceptibility. Despite elevated EAE susceptibility, DNPs administered during MOG immunization (5×, days 0-12) delayed EAE onset (days 16-18) and reduced disease severity (∼2) significantly in WT→WT chimeric mice (Fig. 6C, open symbols). Thus therapeutic responses to DNPs were unaffected by residual inflammation in chimeric mice.

Next, we evaluated EAE progression in B6 mice reconstituted with bone marrow from IDO1-KO (KO→WT) mice. EAE onset (days 12-14) and disease severity (5) in vehicle-treated KO→WT and WT→WT mice were comparable, except for a slight delay in attaining peak disease severity in KO→WT mice (Fig. 6D, closed circles). DNP treatment during EAE induction (5×, days 0-12) had no significant effect on EAE progression (days 12-14) or peak disease severity (5) in MOG-immunized KO→WT chimeric mice (Fig. 6D, open circles), indicating that intact IDO1 gene function in hematopoietic cells was essential for therapeutic responses to DNPs to manifest. While some hematopoietic cells from WT donors may remain viable in KO→WT chimeric mice, these residual WT cells did not promote robust therapeutic responses to DNPs. In contrast, DNPs delayed EAE onset (days 18-20) and reduced peak disease severity (∼1.5) significantly in WT→KO chimeric mice with intact IDO1 genes in hematopoietic cells but not in non-hematopoietic cells (Fig. 6D, open squares). As therapeutic responses to DNPs were comparable in WT→KO (Fig. 6D) and WT→WT (Fig. 6C) chimeric mice, these findings indicated that IDO1 gene function was required only in hematopoietic cells, not non-hematopoietic cells, for therapeutic responses to manifest.

Discussion

In this study we show that selective STING activation attenuates lethal EAE by stimulating IFNαβ release to induce IDO-dependent T cell regulatory responses. The cyclic dinucleotide c-diGMP and DNPs containing CpG^free cargo DNA triggered the STING-IFNαβ-IDO regulatory pathway to delay EAE onset and reduce disease severity substantially, even when treatments were given at the time of EAE onset or to mice with established EAE. Therapeutic responses were not induced by DNA or PEI alone and were dependent on cargo DNA sensing after DNP uptake by hematopoietic cells in peripheral lymphoid tissues to activate STING and induce regulatory responses via IFNαβ and IDO. Thus selective STING activation is an effective strategy to induce potent regulatory responses that suppress CNS-specific autoimmunity.

Our findings appear contradictory to recent studies showing that constitutive DNA sensing to activate STING incited lethal autoimmunity in mice lacking DNA degrading enzymes (1, 2). Thus, increased access to DNA triggered sustained STING activation and constitutive IFNαβ release by stromal cells to incite progressive tolerance breakdown. Dominant regulatory responses induced by DNP cargo DNA and c-diGMP described in the current study may mimic physiologic responses to cellular DNA (chromatin) from dying (apoptotic) cells, which also promote potent regulatory responses via STING and IDO (17, 18, 21). Sustained STING activation in non-hematopoietic (stromal) tissues (1) or selective STING activation in hematopoietic cells may incite diametric immune outcomes because IDO is induced only in the second setting. Thus the STING-IFNαβ pathway activates immune cells while other microenvironmental factors, such as the balance of pro/anti-inflammatory cytokines and metabolic effects of increased tryptophan catabolism by cells expressing IDO, determine immune outcomes (32).

Elevated IDO activity in CNS tissues may contribute to EAE pathogenesis due to local catabolism of tryptophan (Trp), the substrate for serotonin synthesis and release of neurotoxic Trp catabolites (30, 31, 33). Another apparent paradox from the current study is that treatment with DNPs attenuated IDO expression in neurons of MOG-immunized mice with EAE, even though DNPs stimulate IDO activity in many tissues (17). Glial cells in inflamed CNS lesions expressed IDO in neuro-inflammatory syndromes including EAE, though neuronal IDO expression was not reported (30). In the current study, MOG immunization stimulated neurons but not glial cells to express IDO, and DNP treatments attenuated neuronal IDO expression induced by MOG immunization. Loss of neuronal IDO expression following DNP treatments may reflect reduced CNS infiltration by inflammatory cells expressing IFNγ, a potent IDO inducer (30). Thus DNP and c-diGMP treatments may inhibit neuronal IDO expression indirectly, by suppressing MOG-specific responses and thereby reducing immune effector cell infiltration into CNS tissues. Similarly, vitamin D treatments also alleviated EAE by inducing IDO-dependent tolerogenic phenotypes in DCs that enhanced Treg functions (34).

DNPs enhanced spleen cellularity in MOG-immunized mice but reduced pro-inflammatory cytokine production substantially, even though more MOG-specific Th17 and Th1 effector cells were present in DNP-treated mice. Thus regulatory responses to DNPs did not prevent T cell priming but were functionally dominant in the EAE model. In a previous study, systemic DNP treatments stimulated antitumor immunity and enhanced serum IFNγ and IL-12 levels (35). It is unclear how to reconcile these diametric effects of systemic DNP treatments in these disease models but distinct features of inflammatory responses to local tumor growth and MOG-immunization to induce EAE may be key factors as well as different DNP formulations. Our findings suggest that optimizing T cell regulatory responses induced by DNPs may improve therapeutic responses that attenuate EAE and other autoimmune syndromes, while anti-tumor effects of DNPs may be attenuated by their ability to induce IDO, a potent regulator of anti-tumor immunity (36).

CpG^free plasmid DNA was used as DNP cargo DNA in the current study to avoid IFNγ production by TLR9-activated NK cells (17). As for other nanoparticle formulations used to deliver antigen-specific therapies to treat EAE (37), DNPs can be engineered to deliver defined autoantigens and cargo DNA can transduce genes encoding autoantigens. DNPs containing biodegradable poly-β-amino ester derivatives as cationic polymers (38) instead of non-degradable PEI also induced potent and dominant regulatory responses in naïve mice (H.L., unpublished data), suggesting that biodegradable DNPs necessary for metronomic treatment regimens in clinical settings may possess immunomodulatory properties comparable with DNPs containing PEI.

Like DNPs, synthetic cyclic dinucleotides offer a versatile platform to develop STING activating reagents as immunomodulatory drugs. Limited c-diGMP treatments slowed EAE onset and reduced disease severity substantially, suggesting that more aggressive treatments may be even more effective. Cyclic dinucleotides are produced by some microbial organisms such as Listeria and are sensed via STING at sites of infection to stimulate host defense (29). Mammalian cells synthesize cyclic guanosine-adenosine monophosphate (c-GAMP) when cytosolic DNA is sensed by the nucleotidyltransferase c-GAMP synthase (cGAS), a key pathogen sensor that drives host immunity (39-41). Thus cyclic dinucleotides from external or internal sources activate STING in mammalian cells. Subcutaneous c-diGMP administration enhanced antigen-specific immunity in mice, indicating that c-diGMP has immune stimulatory (adjuvant) properties in this setting (42). Thus the route of c-diGMP administration is a critical determinant of immune responses, even though STING activation to induce IFNαβ may be the common pathway that incites or inhibits immunity. Genetic studies revealed that polymorphisms in human STING genes abrogate responsiveness to the vascular disrupting reagent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), suggesting that STING signaling is defective in some humans (43). However a recent study revealed that responsiveness to cyclic dinucleotides is widespread amongst humans carrying an array of single nucleotide polymorphisms (SNPs) in human STING genes, though some SNPs correlated with defective IFNβ release following treatment with specific cyclic dinucleotides (44). Clearly, more research is needed to discern optimal reagents to manipulate immune responses in humans.

IFNβ is used to treat MS patients though the mode of action is not fully understood. IFNβ may recapitulate some downstream regulatory responses elicited by systemic DNP or c-diGMP treatments to activate STING and induce endogenous IFNβ release. However IFNβ elicits multiple side effects that can be severe, causing some MS patients to discontinue treatment (30). Systemic administration of STING activating reagents may offer improved ways to target immune regulatory pathways responsive to IFNβ, while avoiding some detrimental responses to IFNβ, including immune stimulation.

In summary, DNPs and cyclic dinucleotides are two classes of STING activating reagents that can be developed as immunomodulatory drugs to treat patients with hyper-immune syndromes such as autoimmune diseases. Diametric responses to STING activators highlight pivotal roles for the STING-IFNβ pathway in activating immune cells but emphasize the importance of rigorous evaluation of innate and adaptive immune responses to these treatments to discern dominant immune outcomes in particular settings of inflammatory diseases and treatments.