Splenic suppressor of cytokine signaling 3 transgene expression affects T cell responses and prevents development of collagen-induced arthritis

Authors


Abstract

Objective

Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3.

Methods

DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry.

Results

Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor α, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3–transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-γ (−43%), IL-4 (−41%), and IL-17 (−70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction.

Conclusion

This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.

Cytokines play an essential pathogenic role in rheumatoid arthritis (RA). Many cytokines involved in RA, such as interleukin-18 (IL-18), IL-1, IL-6, and IL-17, exert their biologic effect through the JAK/STAT signaling pathway. Upon binding of these cytokines to their receptors, JAKs become activated and phosphorylate tyrosine residues in the cytoplasmic domains of the receptor, which serve as binding sites for STAT molecules. Subsequently, STAT proteins become activated by JAKs and dimerize to each other, after which translocation to the nucleus takes place. This can lead to transcription of genes involved in the pathogenesis of RA (1).

Key negative intracellular regulators of cytokine responses are members of the suppressor of cytokine signaling (SOCS) family. Currently, the following 8 members of the SOCS family are known: SOCS-1 through SOCS-7 and cytokine-inducible SH2-containing protein (CIS) (2–4). Production of SOCS proteins may be induced by a wide range of stimuli via activated STATs; once expressed, these proteins provide negative feedback by inhibiting the JAK/STAT pathway. All homologs share an SH2 domain similar to that of STAT proteins, which enables binding to the phosphorylated tyrosine residues of JAKs, thereby preventing binding of STAT proteins. Furthermore, SOCS proteins can stimulate degradation of the cytokine receptor by a proteosome-mediated process via their SOCS box. SOCS-1 and SOCS-3 also have an extra kinase inhibitory region involved in the inhibition of JAK tyrosine kinase activity (4–6).

Signal inhibition by SOCS members may have an important effect on the balance of cytokines that determine Th1 and Th2 cell–mediated immune responses. For example, SOCS-1 expression in Th1 cells prevents STAT-6 signaling and thereby negatively regulates the differentiation of Th2 cells induced by IL-4. In contrast, SOCS-3 expression in Th2 cells leads to inhibition of IL- 12–dependent STAT-4 activation and, as a result, inhibits Th1 cell differentiation (7, 8). A recent study showed that enhanced SOCS-3 expression in dendritic cells (DCs) ex vivo may effectively generate tolerogenic DCs capable of directing an immune response toward Th2 cells (9). Therefore, preferential expression of SOCS proteins in the different T cell lineages and antigen-presenting cells (APCs) might have a significant role in directing Th1 and Th2 cell–mediated autoimmune diseases.

Several in vivo animal studies suggest an important regulatory role for SOCS proteins in the development and progression of experimental arthritis. For example, SOCS-1 deficiency results in aggravation of methylated bovine serum albumin (mBSA)/IL-1–induced arthritis in interferon-γ (IFNγ)–knockout mice, characterized by increased synovial inflammation and joint destruction (10). Mice that lack SOCS-3 in the hematopoietic and endothelial cell compartments also develop exacerbated mBSA/IL-1–induced arthritis with severe joint inflammation, and, in contrast to SOCS1−/− mice, IFNγ−/− mice also develop peripheral neutrophilia and splenomegaly (11). Furthermore, a point mutation of tyrosine-759 in the signal transducer gp130 receptor chain causes augmentation of arthritis in mice, probably due to the inability of this mutated form to bind SOCS-3. Mice with this point mutation develop an RA-like joint disease accompanied by autoantibody production and an increased number of activated T cells (12, 13). Periarticular injection of Ad5SOCS-3 into the ankle joints of mice with antigen-induced arthritis or collagen-induced arthritis (CIA) results in reduced disease severity and joint swelling. These in vivo effects of Ad5SOCS-3 on inflammation and bone destruction are thought to be a result of direct inhibition of synoviocytes and osteoclast activity (14).

Because SOCS-3 proteins regulate macrophage and DC activation and are essential for T cell development and differentiation, we examined the contribution of SOCS-3 in regulating cellular immune responses during experimental arthritis. We induced SOCS-3 overexpression by intravenous delivery of recombinant adenoviruses, 1 day after a booster injection of type II collagen (before the clinical onset of CIA), in order to manipulate the adaptive immune response. We showed that this route of delivery led to enhanced SOCS-3 expression in splenic APCs, which caused an immune-modulating response in the spleen. These altered splenic cellular responses were accompanied by a profound protective effect against the development of CIA.

MATERIALS AND METHODS

Mice.

Male 10–12-week-old DBA/1 mice (Janvier-Elevage, Le Genest St. Isle, France) were bred and housed in filtertop cages and fed a standard diet, with water and food ad libitum. Mice injected with adenoviral vectors were housed in low-pressure isolator cages. All in vivo studies were in compliance with national legislation and were approved by local authorities for the care and use of animals, with related codes of practice.

Construction of adenoviral vectors.

Viral vectors were E1A, B, and E3 deleted and were produced according to the method described by Chartier et al (15). Briefly, SOCS-3 complementary DNA (cDNA) obtained from the mouse hybridoma B9 cell line by polymerase chain reaction (PCR) was cloned into the E1-deleted region of the serotype 5 adenoviral pShuttle-CMV transfer vector, as described previously (16). The purified recombinant adenoviral vector DNA was transfected into N52E6 viral packaging cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Ad5SOCS-3 and the control vector Ad5 luciferase were purified using 2× CsCl gradient purification and stored in small aliquots at −80°C. The viral titer of the purified viral vectors was determined in human embryonic retinoblastoma 911 indicator cells by immunohistochemical detection of viral capsid protein, 20 hours after transfection.

Induction of CIA.

Bovine type II collagen was dissolved in 0.05M acetic acid to a concentration of 2 mg/ml and was emulsified in equal volumes of Freund's complete adjuvant (2 mg/ml of Mycobacterium tuberculosis strain H37Ra) (Difco, Detroit, MI). Mice were immunized intradermally at the base of the tail with 100 μl of emulsion (100 μg of bovine type II collagen). Subsequently, mice were given an intraperitoneal booster injection of 100 μg of type II collagen dissolved in phosphate buffered saline (PBS) on day 21. One day after the booster injection, immunized mice without clinical signs of CIA were injected intravenously with 3 × 108 focus-forming units (FFU) or intraarticularly into both knees with 1 × 107 FFU Ad5SOCS-3 or Ad5 luciferase. Two independent observers monitored clinical signs of arthritis in paws and ankle joints, macroscopically and in a blinded manner, until the end of the experiment. Cumulative scoring based on redness, swelling, and, in later stages, ankylosis was as follows: 0 = no changes; 0.25 = 1–2 toes red or swollen; 0.5 = 3–5 toes red or swollen; 0.5 = swollen ankle; 0.5 = swollen footpad; 0.5 = severe swelling and ankylosis, with a maximal score of 2 per paw.

Histologic analysis.

Whole knee joints (n = 63 for the Ad5 luciferase–treated group and n = 53 for the Ad5SOCS-3–treated group) were dissected and fixed in phosphate buffered 4% paraformaldehyde followed by decalcification with 5% formic acid, and embedded in paraffin wax. Serial tissue sections (7 μm) were stained with Safranin O (BDH Chemicals, Poole, UK) and counterstained with fast green (BHD Chemicals). Serial sections were scored for histopathologic changes on a 0–3-point scale, by 2 independent observers in a blinded manner. Joint inflammation was determined by the presence of synovial cell infiltrates and inflammatory cell exudates. Connective tissue destruction was determined by the depletion of cartilage proteoglycan (loss of Safranin O staining of the noncalcified upper cartilage layer) and by cartilage and bone erosion.

Stimulation assays.

Inguinal lymph nodes from mice were mashed and filtered. Spleens were mashed and filtered, and erythrocytes were removed by osmotic shock. After washing, the splenic cell fraction was incubated in RPMI 1640 (Invitrogen) at 37°C in 5% CO2 for 1 hour in order to separate adherent cells from nonadherent cells. The adherent cell fraction mainly consisting of macrophages is termed APC. Splenic APCs were stimulated for 24 hours with 10 μg/ml palmitoyl-3-cysteine (Pam3Cys-SKKKK) or 1 μg/ml lipopolysaccharide (LPS). Splenic CD3+ T cells were purified from the nonadherent cell fractions by depletion of non–T cells, using the Pan T Cell Isolation Kit (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. Purified (>97%) primary T cells were cultured in RPMI 1640 (Invitrogen) supplemented with 5% fetal calf serum (FCS), 40 μg/ml gentamycin (Centrafarm, Etten-Leur, The Netherlands), and pyruvate. For the stimulation assay, 1 × 105 lymph node cells or 3 × 104 purified splenic CD3+ T cells together with 1 × 105 irradiated (30 Gy) splenic APCs from the same mice were cultured for 4 days and stimulated with 50 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin. To measure proliferation, 1 × 105 splenic T cells were incubated in the presence of 3 × 105 irradiated splenic APCs for 3 days with 5 μg/ml or 0.5 μg/ml bovine type II collagen (heated for 10 minutes at 80°C). Cells were pulsed with 1 μCi of 3H-thymidine during the last 16–18 hours of culture, prior to harvest.

Cytokine measurement.

The levels of murine IL-4, IL-6, IL-10, IL-17, IFNγ, and tumor necrosis factor α (TNFα) were determined in serum or culture supernatant using Luminex multi-analyte technology (sensitivity ∼1 pg/ml). IL-23 levels in serum or culture supernatant of stimulated adherent cells were determined by enzyme-linked immunosorbent assay (ELISA; eBioscience, San Diego, CA), according to the manufacturer's instructions. Briefly, Nunc MaxiSorp 96-well plates (eBioscience) were coated with 100 μl/well 1× capture antibody in coating buffer overnight at 4°C. On the following day, coating antibody was removed, and the wells were blocked for 1 hour with 200 μl/well 1× assay diluent. After washing, 100 μl/well of sample and standard (stock 1 μg/ml, 2-fold serial dilutions starting from 4,000 pg/ml) was added to the wells. After 2 hours of incubation and an extensive washing procedure, the wells were incubated for 1 hour with 100 μl/well 1× detection antibody, followed by incubation for 30 minutes with 100 μl/well 1× avidin–horseradish peroxidase. After washing, 100 μl/well SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was added, followed by luminometric detection using a POLARstar luminometer (POLARstar Galaxy; BMG Lab Technologies, Offenburg, Germany). The sensitivity was ∼3 pg/ml.

Flow cytometric analysis.

For the analysis of T cell subsets in spleen and inguinal lymph nodes, nonadherent lymphocyte populations and total lymph node cells, respectively, were used. All antibodies were from BD PharMingen (Hamburg, Germany). Cells were stimulated for 5 hours with 50 ng/ml PMA and 1,000 ng/ml ionomycin in the presence of GolgiPlug (BD PharMingen). Cells were stained with allophycocyanin-labeled anti-mouse CD4 (1:100 in PBS plus 1% BSA) for 30 minutes at 4°C, fixed, and permeabilized with Cytofix/Cytoperm solution (BD PharMingen) and then labeled with phycoerythrin (PE)–labeled anti-mouse IL-17 (1:500), fluorescein isothiocyanate–labeled anti-mouse IFNγ (1:500), PE-labeled anti-mouse IL-4 (1:200), or appropriate isotype controls, for 30 minutes at 4°C in PBS containing 1% BSA, 2% FCS, 0.1% saponin. Stained cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson).

Determination of specific IgG titers against type II collagen.

The IgG1 and IgG2a antibody titers against bovine type II collagen in serum were determined by ELISA. Briefly, 96-well plates were coated with 10 μg of bovine type II collagen, and nonspecific binding sites were blocked. Subsequently, serial dilutions of mouse sera were added, followed by incubation with isotype-specific goat anti-mouse peroxidase diluted 1:2,000 in PBS (Southern Biotechnology, Birmingham, AL). The conversion of the substrate 5-aminosalicylic acid (Sigma, Poole, UK) was measured by spectrophotometric analysis at an optical density of 492 nm.

RNA isolation.

The spleen and inguinal lymph node samples were ground using a Micro-Dismembrator II (Braun, Melsungen, Germany). Total RNA was extracted from these tissue homogenates and from cells using TRIzol reagent, as described by Chomczynski and Sacchi (17). Isolated RNA was treated with DNase followed by reverse transcription of 1 μg of RNA into cDNA using Moloney murine leukemia virus reverse transcriptase, 0.5 μg/μl oligo(dT) primers, and 12.5 mM dNTPs (Invitrogen).

Quantitative PCR analysis.

Quantitative real-time PCR was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR protocol was as follows: 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. PCRs were performed in a total reaction volume of 25 μl, consisting of cDNA, 5 μM of forward and reverse primers, and SYBR Green PCR Master Mix (Applied Biosystems). Quantification of the PCR signals was achieved by calculating the difference between the threshold cycle (Ct) value of the gene of interest and the Ct value of the reference gene GAPDH for each sample (▵Ct). The primer sets used were as follows: for GAPDH, forward 5′-GGCAAATTCAACGGCACA-3′ and reverse 5′-GTTAGTGGGGTCTCGCTCCTG-3′; for SOCS-3, forward 5′-CTGGTACTGAGCCGACCTCTC-3′ and reverse 5′-CCGTTGACAGTCTTCCGACAA-3′; for IL-23 p19, forward 5′-CCAGCGGGACATATGAATCTACT-3′ and reverse 5′-CTTGTGGGTCACAACCATCTTC-3′.

Statistical analysis.

The significance of differences was determined using Student's t-test, unless stated otherwise. P values less than or equal to 0.05 were considered significant.

RESULTS

Altered cytokine production by splenic APCs after intravenous administration of adenovirus encoding SOCS-3.

SOCS-3 transgene was delivered by intravenous injection of recombinant adenoviruses, 1 day after the type II collagen booster injection but before the clinical onset of CIA. Markedly enhanced SOCS-3 transgene expression was achieved in the spleen 1 day (∼20-fold increase) and 3 days (∼12-fold increase) after virus injection, whereas no expression was observed in the inguinal lymph nodes (Figure 1A). Increased SOCS-3 expression was observed especially in the APC fraction of the spleen, whereas minimal transgene expression was detected in the purified (>97%) CD3+ lymphocyte population (Figure 1B).

Figure 1.

Cytokine profile of suppressor of cytokine signaling 3 (SOCS-3)–transduced splenic antigen-presenting cells (APCs). A, DBA/1 mice were systemically injected with 3 × 108 focus- forming units of Ad5SOCS-3 or Ad5 luciferase 1 day after the administration of a booster injection of type II collagen. Spleens and inguinal lymph nodes (LNs) were isolated 3 days after injection. Systemic injection of adenovirus encoding SOCS-3 led to enhanced expression of mRNA for the transgene only in spleens. Values are the mean and SEM results from 3 mice. B, SOCS-3 expression in CD3+ T cells and the APC-containing adherent cell fraction from the pooled spleens of 3 mice per group was determined by quantitative polymerase chain reaction, 3 days after adenovirus injection. SOCS-3 expression was shown especially in the APC fraction. C and D, Primary splenic APCs (1 × 105) obtained 1 day (C) or 3 days (D) after virus injection from mice developing collagen-induced arthritis were stimulated for 24 hours with 10 μg/ml palmitoyl-3-cysteine. Cytokine production was determined in the supernatant of these cultures by Luminex multi-analyte technology or enzyme-linked immunosorbent assay. A reduction in the levels of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-23 was observed, whereas IL-10 levels were significantly increased. The broken horizontal lines in A and B represent the control group (set at 1). Values are the mean and SEM results from triplicate cultures (n = 4 mice). ∗ = P < 0.05 versus control.

We studied the effect of enhanced SOCS-3 expression on the cytokine profile of these APCs. Figure 1C shows that ex vivo stimulation of APCs (isolated 1 day after virus injection) with a Toll-like receptor 2 (TLR-2) agonist (Pam3Cys-KKKK) induced synthesis of TNFα, IL-6, and IL-23, which was markedly reduced in the SOCS-3–transduced cells (−76%, −80%, and −83%, respectively). In contrast, TLR-induced IL-10 expression was significantly enhanced (+121%), suggesting that these cells exhibit a tolerogenic phenotype. Similar results were obtained after TLR-4 stimulation with 1 μg/ml LPS (−44% for TNFα, −78% for IL-6, −91% for IL-23, +50% for IL-10). Three days after virus injection, splenic SOCS-3–transduced APCs still exhibited a tolerogenic phenotype characterized by significantly reduced production of TNFα (−35%) and IL-6 (−29%) and enhanced production of IL-10 (+93%) after TLR-2 stimulation. Levels of IL-23 in supernatant showed a trend similar to that observed 1 day after injection, but the trend did not reach significance 3 days after virus injection (Figure 1D). These results correspond with the observed decline in viral SOCS-3 expression 3 days after injection.

Effect of SOCS-3 treatment on splenic Th cell subsets.

Next, the influence of SOCS-3 on Th cell differentiation was evaluated. Figure 2A shows that in vivo SOCS-3 delivery led to increased numbers of splenic CD4+ T cells 3 days after virus injection, during the preclinical phase of CIA. Because no enhanced SOCS-3 expression was observed in the inguinal lymph nodes, the numbers of CD4+ T cells in this secondary lymphoid tissue were determined as a control. No differences between groups were observed in the percent of CD4+ T cells (Figure 2A). Subsequently, intracellular IFNγ, IL-4, and IL-17 production in splenic CD4+ T cells as representative of the different Th cell subsets (Th1, Th2, and Th17, respectively) was determined. No differences were found between the control group and the Ad5SOCS-3–treated group (Figure 2B), suggesting that SOCS-3 treatment had no major effect on the composition of T cell subsets at this stage of the disease.

Figure 2.

Modulation of T cell activity after SOCS-3 overexpression. Spleen, inguinal lymph nodes, and/or serum from bovine type II collagen–immunized DBA/1 mice were isolated 3 days after systemic administration of adenovirus. A, SOCS-3 treatment led to increased amounts of total CD4+ T cells in the spleen, whereas no differences were observed in the inguinal lymph nodes. B, No differences were observed in the percent of splenic CD4+ T cells that expressed interferon-γ (IFNγ), IL-4, or IL-17, as measured by flow cytometry. Values are the mean and SEM results from 5 mice per group. C, Cytokine production by lymph node cells or splenic CD3+ T cells cultured with splenic APCs from the same mice was determined after stimulation with phorbol myristate acetate and ionomycin. Production of IFNγ, IL-4, and IL-17 by splenic CD3+ T cells was significantly reduced after SOCS-3 treatment. Values are the mean and SEM results from triplicate cultures (≥3 mice per group). D, Enhanced SOCS-3 expression in the spleen led to decreased splenic IL-23 mRNA expression. At the same time, levels of murine IL-23 determined in serum were significantly reduced in mice treated with Ad5SOCS-3. Values are the mean and SEM results from 3 mice per group. ∗ = P < 0.05 versus control. FSC = forward scatter (see Figure 1 for other definitions).

To examine the consequences of the altered cytokine profile of SOCS-3–modulated APCs on T cell activity, purified CD3+ T cells were cocultured for 4 days with SOCS-3–transduced APCs from the same mice and activated with PMA and ionomycin. Interestingly, this led to a significant reduction in IFNγ, IL-4, and IL-17 production (−43%, −41%, and −70%, respectively), whereas no differences were found in IFNγ, IL-4, IL-10, and IL-17 production by cells isolated from inguinal lymph nodes (Figure 2C). The effect on IL-17 production by splenic CD3+ T cells was striking; therefore, we investigated the expression of IL-23, a Th17 cell–supporting cytokine (18). The expression of IL-23 messenger RNA in spleen was reduced by SOCS-3 transduction, and even circulating levels of IL-23 in the blood of these mice were strongly reduced (Figure 2D). These results suggest that the antiinflammatory response of SOCS-3–transduced APCs can lead to the suppression of cytokine production by Th1, Th2, and especially, Th17 cells.

Impaired antigen-specific proliferative response of splenic T cells after SOCS-3 overexpression.

We examined whether SOCS-3 overexpression affects antigen-specific T cell activity. Splenic CD3+ T cells and APCs isolated 3 days after virus injection were mixed at a ratio of 1:3 and stimulated for 3 days with the immunization antigen bovine type II collagen. Figure 3A shows a significant reduction in antigen-specific proliferation of splenic CD3+ T cells from Ad5SOCS-3–treated mice compared with CD3+ T cells from control mice, after treatment with type II collagen at the lower dose. At the same time, the antibody titers against type II collagen were not affected, and titers of both IgG1 and IgG2a were elevated. Figure 3B shows the values obtained 9 days after virus injection; similar results were observed 3 days and 7 days after virus injection (data not shown).

Figure 3.

Impaired antigen-specific proliferation of splenic T cells after SOCS-3 treatment. A, Splenic CD3+ T cells and APCs from bovine type II collagen–immunized DBA/1 mice were isolated 3 days after systemic administration of adenovirus. Proliferation of splenic CD3+ T cells was determined by 3H-thymidine incorporation after stimulation with bovine type II collagen in the presence of APCs. Less antigen-specific proliferation of CD3+ T cells was observed after SOCS-3 treatment. Values are the mean and SEM results from triplicate cultures (n = 3 mice). ∗ = P < 0.05 versus control. B, Titers of bovine anti–type II collagen IgG1 and IgG2a antibody were measured. No differences were observed in anti–type II collagen IgG1 and IgG2a antibody levels in serum from Ad5SOCS-3–treated mice and control mice. Values are the mean ± SEM results from 8 individual mice per group, determined on day 31 of collagen-induced arthritis. SI = stimulation index; OD = optical density (see Figure 1 for other definitions).

Prevention of CIA development by intravenous adenoviral transfer of SOCS-3.

There is emerging evidence that pathogenic Th17 cells play a dominant role in the development of CIA (19, 20). Therefore, we anticipated that the involvement of SOCS-3 in modulated IL-17 production would exert a clinical effect on CIA. Indeed, overexpression of SOCS-3 before onset of the clinical manifestations of arthritis clearly prevented the development of CIA. Table 1 shows that the incidence of arthritis in the paws of mice that received the SOCS-3 gene was considerably reduced, from 78% to 34%. Among mice in the Ad5SOCS-3–treated group in which arthritis developed, it did so within the same time frame after administration of the antigen booster and reached a macroscopic score similar to that in the control virus–treated mice. However, histopathologic examination of the arthritic joints on day 31 of the experiment showed moderately, but significantly, reduced joint inflammation and bone erosion in mice that received Ad5SOCS-3 (Figures 4A and B).

Table 1. Prevention of CIA by systemic adenoviral SOCS-3 gene transfer*
Route of administration/experimental groupNo. of arthritic mice/no. of immunized mice (%)No. of arthritic pawsArthritis score, mean ± SEMDay of CIA onset, range
  • *

    Mice immunized with type II collagen received an intraarticular (1 × 107 focus-forming units [FFU]) or intravenous (3 × 108 FFU) injection of Ad5SOCS-3 or Ad5 luciferase 1 day after the booster injection, before the onset of arthritis. In mice treated intravenously, all paws were scored for the development of arthritis; in mice treated intraarticularly, the hind paws and ankle joints were scored. The mean arthritis score was calculated by adding the total macroscopic score for each joint and dividing by the total number of arthritic mice at the indicated time points. CIA = collagen-induced arthritis; SOCS-3 = suppressor of cytokine signaling 3.

Intravenous    
 Exp. no. 1 (day 32)    
  Luciferase8/10 (80)131.6 ± 0.425–32
  SOCS-34/10 (40)81.8 ± 0.625–28
 Exp. no. 2 (day 31)    
  Luciferase8/10 (80)202.9 ± 0.623–29
  SOCS-33/10 (30)62.3 ± 1.223–27
 Exp. no. 3 (day 31)    
  Luciferase9/12 (75)182.6 ± 0.425–30
  SOCS-34/12 (33)72.8 ± 0.724–28
 Total    
  Luciferase25/32 (78)51  
  SOCS-311/32 (34)21  
Intraarticular    
 Exp. no. 1 (day 32)    
  Luciferase8/8 (100)193.0 ± 0.325–28
  SOCS-39/9 (100)203.2 ± 0.425–29
 Exp. no. 2 (day 31)    
  Luciferase7/10 (70)141.9 ± 0.423–31
  SOCS-36/10 (60)121.6 ± 0.523–29
 Total    
  Luciferase15/18 (83)33  
  SOCS-315/19 (79)32  
Figure 4.

Suppressor of cytokine signaling 3 (SOCS-3) treatment affects the histopathology of the joints and circulating tumor necrosis factor α (TNFα) levels. The knees of DBA/1 mice were dissected at the end of 3 separate experiments and processed for histologic assessment (n = 63 knee joints in the control group and 53 knee joints in the Ad5SOCS-3–treated group). A, Joint inflammation was determined by examination of synovial cell infiltrates and inflammatory cell exudates (scored 0–3). Systemic SOCS-3 gene transfer significantly reduced cell infiltrates and exudates in the knees of mice with collagen-induced arthritis. B, Connective tissue destruction was determined by assessment of cartilage proteoglycan depletion, cartilage erosion, and bone erosion (scored 0–3). Bone erosion was significantly reduced in mice treated with SOCS-3. C, On day 29, serum samples were obtained from 5 mice per group and analyzed for cytokine levels. The level of TNFα in the Ad5SOCS-3–treated group was significantly lower as compared with the control group 7 days after virus injection. Serum levels of interleukin-6 followed a similar but not significant trend. Values are the mean and SEM. ∗ = P < 0.05 versus control, by Mann-Whitney U test.

Interestingly, circulating TNFα levels before the onset of arthritis were low and were not different between the 2 groups (mean ± SEM 33 ± 10 pg/ml versus 37 ± 6 pg/ml in the control and Ad5SOCS-3 groups, respectively). TNFα levels increased in the control group (94 ± 12 pg/ml) during arthritis development, whereas this up-regulation was absent in the SOCS-3–treated group (46 ± 12 pg/ml) (Figure 4C). The circulating levels of IL-6 followed a similar trend, but the trend did not reach significance (data not shown), and both IL-17 and IL-10 levels were below the detection level. The difference in TNFα levels and bone erosion is indicative of reduced Th17 cell involvement, as may be the result of impaired splenic T cell activation after SOCS-3 treatment.

Lack of protection after intraarticular overexpression of SOCS-3.

Because we could not exclude the possibility that distribution of systemically delivered adenoviruses to arthritic joints led to a protective effect of SOCS-3 locally, a high dose of adenovirus encoding SOCS-3 was injected intraarticularly 1 day after the booster injection and before the onset of arthritis. One day after injection, an ∼75-fold increase in SOCS-3 transgene expression was detected in synovium from mice injected with Ad5SOCS-3 compared with control mice. Interestingly, local SOCS-3 overexpression had no effect on the development or severity of CIA in the treated knees (data not shown), paws, or ankle joints (Table 1). This supports our concept that SOCS-3 transgene expression in splenic APCs can lead to modulation of autoreactive T cell responses and, thus, prevention of CIA development.

DISCUSSION

In the present study, we demonstrated that intravenous administration of recombinant adenoviruses of serotype 5 resulted in ectopic expression of SOCS-3 in splenic APCs. Applying this strategy led to the generation of tolerogenic APCs producing increased levels of IL-10 and decreased levels of IL-6, TNFα, and IL-23 after TLR-2 or TLR-4 stimulation. Hence, the antigen-specific T cell activity was impaired, and the production of IFNγ, IL-4, and IL-17 was significantly reduced. This suggests that the adaptive immune response was impaired by ectopic expression of SOCS-3 in splenic APCs and this, as a result, could lead to clear protection against the development of arthritis.

Our data show that enhanced SOCS-3 expression in splenic APCs led to an altered cytokine profile. Li and colleagues reported that forced SOCS-3 expression in DCs blocks maturation and leads to increased production of IL-10 but lower amounts of IL-12 and IFNγ (9). This change toward a tolerogenic phenotype of DCs is consistent with the reduced IL-6, TNFα, and IL-23 production and enhanced IL-10 production by SOCS-3–transduced splenic APCs, as observed in our study. NF-κB is the main transcription factor in TNFα, IL-6, and IL-23 gene expression (21–23). Alternatively, it has been reported that TLR agonists can promote IL-10 production in a MAPK- and phosphatidylinositol 3-kinase–dependent manner (24–26). It has also been reported that vaccinia virus protein A52R interacts with TNF receptor–associated factor 6 (TRAF6) and inhibits NF-κB signaling but at the same time enhances TLR-induced p38 kinase and MAPK production, thus enhancing IL-10 production (27). Interestingly, it is known that SOCS-3 can inhibit TLR signaling through its association with the TRAF6 protein, thereby blocking transforming growth factor β–activated kinase 1–mediated NF-κB activation. Therefore, it is tempting to propose a similar mechanism for SOCS-3 in the enhanced production of IL-10 by the transduced APCs in our study.

APCs such as DCs and macrophages initiate and shape the adaptive immune response by controlling the differentiation and activation of T cells. Here, we observed that splenic T cells from mice receiving adenoviruses encoding SOCS-3 showed decreased antigen-specific T cell proliferation and also produced significantly less IL-4 (−41%), IFNγ (−43%), and IL-17 (−70%) in the presence of SOCS-3–transduced APCs. Because we observed minimal transgene expression in CD3+ T cells, this altered T cell proliferation is probably caused indirectly via the affected APCs. The dramatic decline in IL-17 production can be explained by the reduction in production of IL-23, which is a Th17 cell–supporting cytokine, by splenic APCs (28). Several studies also show that SOCS-3 can down-regulate cell-surface expression of class II major histocompatibility complex (MHC) molecules (9, 29). In addition, it is well established that IL-10 is a powerful inhibitor of antigen-specific T cell proliferation through down-regulation of class II MHC molecules on APCs (30). Therefore, SOCS-3–transduced APCs might affect T cell activity by providing suboptimal stimulatory signals and via the altered cytokine profile.

In contrast to the reduced cytokine production, fluorescence-activated cell sorting analysis showed increased CD4+ T cells in the spleen after SOCS-3 treatment, with no difference in Th cell subsets between control and AdSOCS-3–treated mice on day 25 of CIA. These data show that T cells from the Ad5SOCS-3–treated group were not in an anergic state and were still responsive in the absence of the antiinflammatory milieu, suggesting that SOCS-3 influences APC-mediated T cell activation rather than affecting T cell subset composition at this time point after immunization. Wong et al (11) reported that mice lacking SOCS-3 in the hematopoietic and endothelial cell compartments have reduced percentages of CD4+ T cells in the spleens following mBSA/IL-1–induced arthritis, and CD4+ T cells isolated from untreated mice are hyperproliferative in vitro. This change in the CD4+ T cell amount and proliferation is consistent with the results we observed after SOCS-3 overexpression.

Normally, antigen-specific T cells that are activated in the spleen have undergone several changes in their cytokine and homing molecule expression, allowing them to travel to inflamed tissue and carry out effector functions locally (31). The effect of SOCS-3 on T cell activity might lead to inhibition of T cell migration, thereby explaining the increased number of CD4+ T cells in the spleen. The equal levels of CD4+ T cells in the inguinal lymph nodes and the similar cytokine production by lymph node cells from Ad5SOCS-3–treated and control mice suggest that enhanced SOCS-3 expression is necessary to modulate cellular immune responses at this stage of the disease. Overall, preventing T cell activation in the spleen and possibly also T cell migration by ectopically expressing SOCS-3 might have an inhibitory effect on the onset of CIA, as shown in this study.

The difference in proinflammatory and antiinflammatory cytokine production by splenic cells due to enhanced SOCS-3 expression has an important inhibitory effect on arthritis development. In a study using blocking of IL-10 with neutralizing antibodies it was shown that endogenous IL-10 plays a dominant role in the natural suppression of CIA (32). Although circulating levels of IL-10 were not detectable, enhanced IL-10 production by SOCS-3–transduced APCs might contribute to this protection. Furthermore, we have previously shown that the onset of arthritis in this model is dependent on TNFα (33). Interestingly, the levels of circulating TNFα were significantly lower in the Ad5SOCS-3–treated mice compared with those in the control group 7 days after virus injection. It has been reported that modulation of TNFα in CIA by periarticular injection of retrovirus encoding TNF receptor shifts the anti–type II collagen IgG2a:IgG1 ratio toward Th2-driven IgG1 and, as a result, inhibits the progression of CIA (34). Although SOCS-3 gene transfer markedly reduced TNFα levels, the anti–type II collagen IgG2a:IgG1 ratio remained unchanged, suggesting that either the antigen-specific humoral response was already established at the time of adenovirus injection, or enhanced SOCS-3 expression in APCs did not affect the humoral response.

To exclude the possibility of a local role of SOCS-3 transgene expression, we injected the adenoviruses directly into the knee joints before the onset of arthritis; however, no protection was observed. Therefore, we are convinced that the protection observed with systemic delivery of SOCS-3 was attributable to T cell modulation rather than spillover of viruses to the inflamed joints. In contrast, Shouda et al reported that periarticular injection of SOCS-3/CIS3 adenovirus drastically reduced the severity of CIA (14). It is clear that treatment via the periarticular route also affects the disease at a systemic level (35, 36), possibly affecting immunity by spillover of viruses to the draining lymph nodes.

In summary, our study shows that adenovirus-mediated systemic delivery of SOCS-3 has a major effect on the immunomodulatory capacities of splenic APCs and, as a result, has a prophylactic effect on experimental arthritis. Our findings show a critical function for SOCS-3 in regulating proinflammatory and antiinflammatory cytokine production in the spleen, which eventually affects the outcome of the adaptive immune response. Based on our data, it is tempting to suggest that agents able to influence SOCS-3 expression may offer new therapeutic applicability for APC-based immunologic intervention in autoimmune diseases.

AUTHOR CONTRIBUTIONS

Dr. Veenbergen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Veenbergen, Smeets, van de Loo.

Acquisition of data. Veenbergen, Bennink, de Hooge, Arntz, Smeets.

Analysis and interpretation of data. Veenbergen, Bennink, Smeets, van de Loo.

Manuscript preparation. Veenbergen, van den Berg, van de Loo.

Statistical analysis. Veenbergen.

Acknowledgements

We thank Dr. S. Kochanek (Center for Molecular Medicine, University of Cologne, Cologne, Germany) for kindly providing the N52E6 viral packaging cell line.

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