Enhancement of antiviral immunity by small molecule antagonist of suppressor of cytokine signaling

Abstract

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

FIGURE 1

pJAK2(1001–1013) can rescue mice from an intranasal infection with vaccinia virus. Mice (C57BL/6, n = 5) were pretreated on days −2, −1, and 0 with 200 μg (□), 50 μg (▼), or 10 μg (○) lipo-pJAK2 (1001–1013) peptide or 200 μg (△) control peptide, lipo-JAK2(1001–1013) 2A. On day 0, vaccinia virus (2 ×10⁶ PFU) was given intranasally. Survival of mice was followed over a period of 30 d. The significance of difference between different treatments was measured by log-rank survival method, which gave p values of 0.002, 0.002, and 0.02 for the administration of 200, 50, and 10 μg lipo-pJAK2(1001–1013) versus the control peptide, respectively.

FIGURE 2

Lipo-pJAK2(1001–1013) is internalized by mouse peritoneal cells in vivo and by L929 fibroblast cells in culture. Lipo-pJAK2(1001–1013) was coupled to FITC as per Materials and Methods. A, Peritoneal cell uptake. Mice were injected i.p. with 15 μg FITC–lipo-pJAK2(1001–1013) or an equivalent amount of FITC alone. Peritoneal cells were harvested after 2 h, and the cells were examined by confocal fluorescent and contrast microscopy for FITC labeling. B, L929 cells uptake. Cells were incubated with 5 μM FITC–lipo-pJAK2(1001–1013) or an equivalent amount of FITC alone for 2 h, after which they were examined as above for uptake of FITC. Magnification ×20.

FIGURE 3

pJAK2(1001–1013) and IFN-γ(95–132) act synergistically. A, Mice were pretreated on days −2, −1, and 0 with 10 μg pJAK2 (○), 2 μg IFN-γ mimetic, indicated as mim (◇), 5 μg IFN-γ mimetic (▲), a control peptide JAK2(1001–1013)2A (■), or treated with a combination of 10 μg pJAK2 and 2 μg IFN-γ mimetic (▼) or 10 μg pJAK2 and 5 μg IFN-γ mimetic (□). On day 0, mice were infected intranasally with 1 × 10⁶ PFU vaccinia virus. Survival of mice was followed over a period of 30 d. The significance of difference between different treatments was measured by log-rank survival method, which gave p values of 0.002, 0.04, 0.012, 0.017, and 0.017 for the administration of 10 μg pJAK2 and 5 μg IFN mimetic, 10 μg pJAK2 and 2 μg IFN mimetic, 5 μg of IFN mimetic, 2 μg IFN mimetic, and 10 μg pJAK2 versus the control peptide, respectively. B, IFN-γ mimetic and pJAK2(1001–1013) peptides activate GAS promoter element synergistically. WISH cells were cotransfected with plasmids expressing a GAS promoter element linked to a firefly luciferase and another plasmid constitutively expressing Renilla luciferase as an internal control, followed by addition of the peptides indicated. After overnight incubation, relative luciferase activity was measured and is expressed as average ± SD. C, pJAK2(1001–1013)-treated cells had increased levels of phosphorylated STAT1α. L929 cells were seeded onto six-well plates at 1 × 10⁶ cells/well, grown overnight, and treated with pJAK2(1001–1013) (2, 10, 25 μM) or JAK2(1001–1013)2A (25 μM) for 1 h at 37°C. The cells were washed and lysed, and whole-cell extracts were resolved on 12% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with Abs to pSTAT1α or STAT1α. Similar results were obtained in three different experiments. Relative intensities of the pSTAT1α band are shown under the blot. As determined by the unpaired t test on nonnormalized readings, the intensity in the pJAK2-treated band versus the untreated had a p value of 0.0002.

FIGURE 4

Time course of inhibition of vaccinia virus replication by pJAK2(1001–1013) by one-step growth curve. BSC-40 cells grown to confluency were left untreated, treated with lipo-pJAK2(1001–1013), or its alanine-substituted mutant JAK2(1001–1013)2A at 50 μM for 1 h. Cells were then infected with vaccinia virus at an moi of 5 for 1 h. After 1 h, the cells were washed and incubated in the presence of the same concentrations of peptides for the indicated times. Cell extracts (A) and supernatants (B) obtained from these were titrated for the amount of intracellular and extracellular virus, respectively. Note the difference of the scale on y-axis, indicating that there is less of extracellular virus than intracellular.

FIGURE 5

pJAK2(1001–1013) inhibits vaccinia virus replication in a dose-dependent manner as determined by a one-step growth curve. BSC-40 cells were grown to confluency and left untreated or treated with the indicated amounts of lipo-pJAK2(1001–1013) or the alanine-substituted control peptide for 1 h. Cells were next infected with vaccinia virus at an moi of 5. After 1 h, the cells were washed and incubated in the presence of the same concentrations of peptides for 1 d. Supernatant and cell extracts obtained were titrated for the amount of intracellular (A) and extracellular (B) virus, respectively.

FIGURE 6

pJAK2(1001–1013)-treated cells had increased levels of endogenous IFN-β. pJAK2(1001–1013) increases levels of endogenous IFN-β. L929 cells were seeded onto six-well plates at 1 × 10⁶ cells/well, grown to confluency, and treated with peptides at varying concentrations for 30 or 60 min at 37°C. The cells were washed and lysed, and whole-cell extracts were resolved on 12% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with Abs to IFN-β (A), IFN-α (C), or SOCS1-KIR (D). The relative intensities were tested by a nonparametric test (Wilcoxon-Mann-Whitney) and found to be statistically significant with a p value of <0.05 for antagonist versus control for A and C. B, Intracellular IFN-β levels were determined for the cell lysates with an IFN-β ELISA kit, following manufacturer’s instructions. Values represent the means (± SEM) of triplicate wells from three independent experiments. Asterisks (*) indicate statistically significant differences when compared with untreated cells as determined by two-way ANOVA with Bonferroni posttests.

FIGURE 7

pJAK2(1001–1013) exerts an adjuvant effect at both cellular and humoral levels. A, Splenocyte stimulation. Mice (n = 5) were pretreated i.p. on day −2, −1, and 0 with pJAK2(1001–1013), control peptide JAK2(1001–1013)2A, or PBS. On day 0, 50 μg BSA was injected in mice in all groups, except the naive group. Four weeks later, isolated splenocytes (5 × 10⁶/well) were seeded in quadruplicate and incubated with 0.5 μg BSA for 3 d with the addition of 1 μCi/well [³H]thymidine for the last 6 h, and its incorporation was measured. Data are representative of three individual experiments. B, IgG production. Mice (n = 5) were treated as in A. Sera obtained in the weeks indicated were diluted (1:1000) and added to microtiter plates. IgG Abs were measured in an ELISA assay. C, SOCS antagonist enhances T cell-independent Ab production. Mice (C57BL6, n = 3) were injected i.p. with T cell-independent Ag, LPS (50 μg each), or the T cell-dependent Ag BSA (50 μg). Some of the mice received SOCS antagonist (200 μg), the control peptide (JAK2A) (200 μg), or a combination of SEA/SEB (SAg, 25 μg each). A set of mice was also injected with BSA (50 μg) and SAg. Two weeks later, mice were bled. Sera were tested for IgG to LPS or BSA by ELISA. The secondary Ab used was anti-mouse IgG conjugated to HRP. After washing, substrate was added, and color was allowed to develop before reading absorbance at 490 nm. Comparison of LPS versus LPS and SOCS antagonist by Student t test resulted in p < 0.01 at 1/100 dilution. D, LPS stimulation. RAW264.7 cells (5 × 10⁶/well) were seeded in triplicate and incubated overnight. The indicated amounts of pJAK2(1001–1013) or control peptide were added to the cells and incubated for 4 h, after which 2 μg/ml LPS was added, and the cells were incubated for 3 d. NO was measured by Griess reagent, and absorbance was read. E, Poly I:C stimulation. Murine macrophages (RAW264.7) were incubated with lipophilic pJAK2(1001–1013), or control peptide for 2 h, followed by stimulation with poly I:C at 0.1 μg/ml for 72 h. Culture supernatants were collected and nitrite concentration determined using Griess reagent. *p < 0.001.

FIGURE 8

pJAK2(1001–1013) possesses antiviral activity against EMCV. A, L929 cells were treated with IFN-γ or different peptides for 2 h, after which 200 PFU/well EMCV was added. After 1 h, virus was removed, and media was added, followed by incubation for 24 h. Cells were stained with crystal violet, and plates were scanned. National Institutes of Health ImageJ software was used for analysis. B, Synergy between SOCS-1 antagonist and IFN-γ mimetic in inhibition of EMCV. pJAK2(1001–1013) at 2 μM and IFN-γ(95–132) at 5 μM together were incubated with L929 cells, after which the cells were infected as in A. C, L929 cells were incubated with the peptides in the presence or absence of 500 U/ml neutralizing Ab to IFN-β for 2 h, after which cells were infected with EMCV and processed as in A. D, pJAK2(1001–1013) protected mice from EMCV infection. Mice were injected daily i.p. beginning at day −2 with pJAK2(1001–1013) at 50, 100, and 200 μg and control peptide at 200 μg. On day 0, 50 PFU/mouse was injected i.p. Survival data are presented as Kaplan-Meier plots. The significances of difference were p < 0.005, p < 0.005, and NS for 200, 100, and 50 μg antagonist versus the control, respectively. E, Synergy in protection of mice infected with EMCV as for D above using suboptimal levels of pJAK2(1001–1013) (10 μg) and IFN-γ(95–132) (2 μg).

FIGURE 9

pJAK2(1001–1013) binding to SOCS1-KIR and SOCS3-KIR as determined by an Ab ELISA and its reversal of SOCS-1 and SOCS-3 mediated inhibition of GAS promoter activity. SOCS1-KIR (A) and SOCS3-KIR (B) bind to pJAK2(1001–1013). pJAK2 or control peptide IFN-γ(95–106) was immobilized at 3 μg/well in a 96-well plate. Following blocking, various concentrations of SOCS1-KIR or SOCS3-KIR were added, and the plates were incubated for 1 h. Following washing, 1:500 dilution of SOCS1-KIR or SOCS3-KIR Ab was added for 1 h incubation. Bound SOCS peptide was detected with a goat anti-rabbit IgG-HRP conjugate, followed by the addition of OPD substrate and 2 N H₂SO₄. Absorbance was measured in a plate reader. The statistical significance of the binding was tested by a paired t test, and p values of <0.001 and 0.0166 for the binding in A and B, respectively, were observed. C, pJAK2(1001–1013) reverses SOCS-1 and SOCS-3 inhibition of GAS promoter activity. L929 cells were transfected with a GAS promoter linked to firefly luciferase reporter gene and a control plasmid with constitutively expressed Renilla luciferase. Where indicated, SOCS-1– or SOCS-3–expressing plasmids were included in the transfection. A plasmid without a promoter attached to firefly luciferase was used as a control in the first bar indicated as empty vector. After 24 h of transfection, cell extracts were assayed for relative luciferase activities. pJAK2 peptide was used at 30 μM. D, JAK2 associates with SOCS-3 in cells. Whole cell extracts of L929 cells were subjected to IP with Abs to JAK2. Western blot analysis of the immunoprecipitates showed association of SOCS-3. IP with nonspecific IgG was run as a control and showed no SOCS-3 association. IP, immunoprecipitation.