Astrocyte indoleamine 2,3-dioxygenase is induced by the TLR3 ligand poly(I:C): mechanism of induction and role in antiviral response

Abstract

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism and has been implicated in neurotoxicity and suppression of the antiviral T-cell response in HIV encephalitis (HIVE). Here we show that the Toll-like receptor 3 (TLR3) ligand poly(I:C) (PIC) induces the expression of IDO in human astrocytes. PIC was less potent than gamma interferon (IFN-gamma) but more potent than IFN-beta in inducing IDO. PIC induction of IDO was mediated in part by IFN-beta but not IFN-gamma, and both NF-kappaB and interferon regulatory factor 3 (IRF3) were required. PIC also upregulated TLR3, thereby augmenting the primary (IFN-beta) and secondary (IDO and viperin) response genes upon subsequent stimulation with PIC. In HIVE, the transcripts for TLR3, IFN-beta, IDO, and viperin were increased and IDO immunoreactivity was detected in reactive astrocytes as well as macrophages and microglia. PIC caused suppression of intracellular replication of human immunodeficiency virus pseudotyped with vesicular stomatitis virus G protein and human cytomegalovirus in a manner dependent on IRF3 and IDO. The involvement of IDO was demonstrated by partial but significant reversal of the PIC-mediated antiviral effect by IDO RNA interference and/or tryptophan supplementation. Importantly, the cytokine interleukin-1 abolished IFN-gamma-induced IDO enzyme activity in a nitric oxide-dependent manner without suppressing protein expression. Our results demonstrate that IDO is an innate antiviral protein induced by double-stranded RNA and suggest a therapeutic utility for PIC in human viral infections. They also show that IDO activity can be dissociated from protein expression, indicating that the local central nervous system cytokine and nitric oxide environment determines IDO function.

FIG. 1.

IDO induction by PIC, IFN-γ, and IFN-β in primary human astrocytes. (A) Astrocyte cultures were stimulated with 10 μg/ml of PIC or 10 ng/ml of recombinant IFN-γ or recombinant IFN-β for 1 or 3 days. The Western blot shows IDO (∼45 kDa) induction by all stimuli, with IFN-γ being the most potent and IFN-β the least potent. Blots were stripped and reprobed for vinculin (117 kDa) as a control for protein loading. (B) Dose-dependent IDO induction in astrocytes. Astrocyte cultures were stimulated with 10 U (0.125 ng/ml), 100 U, or 1,000 U of recombinant IFN-β, 10 U (0.5 ng/ml), 100 U, or 1,000 U of recombinant IFN-γ, or 0.1 μg/ml, 1 μg/ml, or 10 μg/ml of PIC for 1 day. Blots were probed for IDO and then stripped and reprobed for vinculin. Astrocyte IDO is induced in a dose-dependent manner with the following ranking of potency: IFN-γ > PIC > IFN-β. Results are representative of three independent experiments.

FIG. 2.

PIC-induced expression of IDO is dependent on IFN-β but not IFN-γ. (A) Astrocytes were pretreated for 30 min with neutralizing antibodies to human IFN-β (2 × 10⁵ U/ml; PBL) or control normal rabbit serum (NRS) and then stimulated with PIC (10 μg/ml) or recombinant IFN-β (3 ng/ml) for 24 h. Immunoblotting was performed for IDO and vinculin. Neutralizing IFN-β antibody inhibited PIC-induced IDO expression as well as IFN-β-induced IDO expression in astrocytes. (B) Pooled densitometry data (means plus SEM) from three separate cases show significant inhibition of IFN-β- and PIC-induced astrocyte IDO expression by anti-IFN-β antibody compared to NRS-treated controls (*, P < 0.05, t test). (C) Astrocytes were pretreated for 30 min with a neutralizing antibody to human IFN-γ (10 μg/ml; PBL) or normal mouse IgG1 and then stimulated with PIC or IFN-γ (5 ng/ml) for additional 24 h. Immunoblotting was performed for IDO and vinculin. IFN-γ-induced IDO, but not PIC-induced IDO, was inhibited by anti-IFN-γ antibody in astrocytes. (D) Pooled IDO/vinculin densitometry data (means plus SEM) of Western blots from three different cases show complete inhibition (***, P < 0.001, t test) of IFN-γ-induced IDO by IFN-γ antibody. PIC-induced IDO was not affected by incubation with the anti-IFN-γ antibody.

FIG. 3.

PIC-induced expression of IDO is dependent on NF-κB and IRF-3. (A) Astrocytes were infected for 48 h with increasing concentrations (multiplicities of infection) of adenoviral vectors expressing a DN NF-κB (srIκBα) or EV. Cells were then stimulated with 10 μg/ml of PIC for 24 h, and total cell homogenates were prepared and immunoblotted for IDO and vinculin. PIC-induced IDO was dose dependently inhibited by the SR IκBα but not EV. Adenovirus expressing the SR IκBα or EV alone did not induce IDO. (B) The role of DN IRF3 was examined by adenoviral vectors as for panel A. The results showed that IDO expression was dose dependently inhibited by DN IRF3, whereas EV were without effect. The data shown are from one of two separate experiments with identical results.

FIG. 4.

Astrocytes pretreated with PIC show upregulation of TLR3, IFN-β, and IDO mRNA. The effect of pretreatment of astrocytes with PIC or IL-1 on IDO expression was examined. Astrocytes were treated with medium (Ctrl), PIC once on day 1 (pICx1), PIC daily for 3 days (pICx3), IL-1 once on day 1 (IL-1x1), IL-1 daily for 3 days (IL-1x3), or IL-1 on day 1 and 2 and PIC on day 3 (IL-1x2-pICx1). Total RNA was harvested on day 4 and subjected to Q-PCR for IDO, viperin/cig5, IFN-β, and TLR3 mRNA expression as described in Materials and Methods. Results are means plus SEM from triplicate samples. Note that some error bars are too small to be seen. Also note that failure to induce IFN-β in some samples reflects the discordant time points for RNA harvest (24 h) and the peak IFN-β mRNA expression (3 to 6 h).

FIG. 5.

Astrocyte IDO expression in vivo in HIVE. IDO immunohistochemistry was performed on paraffin-embedded brain sections of healthy control and HIVE subjects. While no immunoreactivity was detected in controls (A), abundant IDO expression was detected in reactive astrocytes (arrows) in the white matter remote from the sites of HIV infection (B), as well as those (arrows) in the vicinity of infected macrophages and microglia forming microglial nodules (C) in HIVE. Note the IDO immunoreactivity in multinucleated giant cells and microglia in HIVE lesions (C, center). Astrocytes, macrophages, and microglia were identified by their characteristic morphology and their immunoreactivity to glial fibrillary acidic protein and CD68 in serial sections (not shown). (D) Q-PCR analysis of IDO, viperin, IFN-β, and TLR3 mRNA in brain homogenates of HIVE (n = 3) normalized to that of healthy brain (n = 3). Data are means plus SEM.

FIG. 6.

Astrocyte HIV replication is inhibited by PIC in an IRF3-dependent manner. (A) Astrocytes were first infected with adenovirus (DN IRF3 or WT IRF3). Two days later, cells were infected with VSV-G HIV with or without 10 μg/ml of PIC for an additional 3 days, as described in Materials and Methods. Cells were harvested for HIV Gag expression by Western blot analysis. Overexpression of DN (N-terminally truncated) IRF3 and WT IRF3 was confirmed by immunoblotting using an antibody against the C terminus of IRF3. A faint 55-kDa endogenous IRF3 is visible in control (Ctr) cultures. Results show that PIC suppresses HIV Gag expression, and this is partially reversed by DN IRF3 overexpression. (B) Pooled densitometric ratios (means plus SEM) of HIV Gag (total band densities including p55 Gag, p39/41 MA-CA when present, and p24 CA) to vinculin from three cases. PIC-induced inhibition of HIV expression was significant in control (Ctr) or WT IRF3-overexpressing (WT) cultures but not in DN IRF3-overexpressing (DN) cultures. Overexpression of DN IRF3 significantly reversed the antiviral effect of PIC (**, P < 0.01 by t test versus WT + PIC). (C, D) Astrocytes in 96-well plates were exposed to PIC, adenovirus, and HIV as described for panel A, and HIV Gag expression was determined by immunocytochemistry and cell-based ELISA. The number of HIV⁺ cells (C) and the ODs (D) (means plus SEM) are reduced by PIC, and this was significantly reversed by DN IRF3 (***, P < 0.001 by t test versus WT + PIC).

FIG. 7.

IDO siRNA partially reverses the anti-HIV effect of PIC. Astrocytes were transfected with IDO siRNA or control siRNA for 2 to 3 days as described in Materials and Methods. Cells were infected with VSV-G HIV with or without PIC for an additional 3 days. (A) Immunoblot showing the effect of IDO siRNA on IDO and HIV expression. (B and C) Pooled densitometric ratios of IDO and HIV Gag to vinculin from seven cases. (B) IDO siRNA suppressed PIC-induced IDO expression compared to control siRNA (*, P < 0.05, n = 7). In the same cases, IDO siRNA increased the expression of HIV Gag compared to control siRNA (***, P < 0.001, n = 7), indicating a significant reversal of PIC-induced anti-HIV activity by IDO knockdown. (C) IDO and HIV Gag expression in PIC-treated, IDO siRNA-treated samples (n = 7) are normalized to the levels in control (Ctr) siRNA samples, showing a reciprocal relationship. (D) Percent HIV (HIV Gag/vinculin) inhibition by PIC in three cases with matching IDO siRNA and control (Ctr) siRNA samples show a decrease in inhibition by IDO knockdown. (E) Tryptophan supplementation also reverses the anti-HIV effect of PIC. Astrocytes were pretreated with PIC for 3 days or cotreated with PIC simultaneously with HIV with or without l-tryptophan (Trp; 100 μg/ml). HIV Gag expression was determined by cell-based ELISA. Pretreatment with PIC shows a more potent antiviral effect than cotreatment. l-Tryptophan significantly reversed PIC effects in both treatment regimens (**, P < 0.01; *, P < 0.05). Data are means plus SEM and are representative of two experiments with similar results.

FIG. 8.

PIC limits CMV replication in astrocytes: the effect of tryptophan supplementation and the role of IRF3. (A) Astrocyte cultures in 96-well plates were infected with the AD169 strain of HCMV with IL-1β (10 ng/ml), PIC (50 μg/ml), and IFN-γ (10 ng/ml) with or without 100 μg/ml of l-tryptophan (Trp). Three days later, cultures were fixed and immunostained for HCMV IE1, as described in Materials and Methods. Astrocytes infected with CMV demonstrate IE1 immunoreactivity (purple) as well as typical CMV cytopathic effects such as cytomegaly, multinucleated giant cell formation (arrows), and cytolysis (empty areas devoid of cells, as evident in IL-1β-treated cultures). (B) Cell-based ELISA demonstrates that both PIC and IFN-γ reduced CMV expression (**, P < 0.01; ***, P < 0.001), while IL-1β had no significant effect. Tryptophan significantly reversed the effect of PIC and IFN-γ (*, P < 0.05; **, P < 0.01). These results support the conclusion that IDO mediates its anti-CMV effect through tryptophan starvation. (C) Effect of cytokines at low HCMV concentrations. Astrocyte cultures in four replicate wells were infected with low-dose HCMV (∼2 μl), which resulted in 1 to 3% cell infection on day 3. CMV⁺-cell counts were determined by enumerating all IE1⁺ cells in a random 100× microscopic field per well. Data are means plus SEM from four wells. IFN-γ is no longer effective at low viral doses, while PIC is effective (ANOVA with Dunnett's test). Tryptophan partially reverses the PIC effect (P < 0.01, t test). Data are representative of two experiments with similar results.

FIG. 9.

Endogenous NO inactivates IDO in cytokine-treated astrocytes. (A to C) To determine the relationship between iNOS expression and IDO expression in human astrocytes, cells were treated with cytokines (IL-1β, IFN-γ, or together) for 1 to 7 days as indicated and immunoblotted for iNOS and IDO. The results are compared with those obtained with PIC-treated astrocytes. l-NAME (NOS inhibitor) at 100 μM was added to cultures to determine the effect of NO. (D) IDO activity was also determined by measurement of KYN concentrations in day 3 culture supernatants as described in Materials and Methods. Data are means plus SEM from three cases. (E) iNOS activity was determined by measurement of nitrite in day 3 culture supernatants. Data are means plus SEM from four cases. Statistics in panels D and E are repeated-measure ANOVA with Tukey's post hoc test (**, P < 0.01; ***, P < 0.001). KYN induction by both IFN-γ and PIC was significant (P < 0.01 and P < 0.001 versus control). The results show that coinduction of iNOS by the cytokine IL-1 inactivates IDO enzyme in astrocytes without decreasing the amounts of IDO expression.

FIG. 10.

Schematic representation of the TLR3 signaling leading to the antiviral immunity in astrocytes. TLR3 located in the intracellular endosomal membrane is triggered by binding with dsRNA such as PIC. TLR3, unlike other TLRs, uses the adaptor protein TLR IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) to transduce signals to activate IRF3 kinases, such as TBK1 or IKKɛ. Both NF-κB and IRF3 are necessary to transactivate the human IFN-β gene (primary response gene). Secreted IFN-β then binds to the type I IFN receptor (IFNR) in an autocrine manner and transactivates the ISGs by formation of the ISG factor 3 (ISGF3:Stat1, Stat2, and IRF9) that simulates the IFN-stimulated response element (ISRE) of the gene promoter. One of the genes induced in this manner is the transcription factor IRF7, which synergizes with IRF3 in the activation of IFN-β gene and is necessary for the activation of all human IFN-α genes, thereby establishing a positive feedback mechanism for ISG expression (46). Another positive feedback mechanism appears to include upregulation of the receptor TLR3 itself by PIC. We have identified IDO and viperin (65) as antiviral effector molecules induced by TLR3, in addition to classical IFN-induced antiviral proteins, such as dsRNA-dependent protein kinase and 2′,5′-oligoadenylate synthetase.