To examine whether systemic administration of immunostimulatory and immunosuppressive oligodeoxynucleotides (ODNs) alter host susceptibility to inflammatory arthritis.
To examine whether systemic administration of immunostimulatory and immunosuppressive oligodeoxynucleotides (ODNs) alter host susceptibility to inflammatory arthritis.
Normal BALB/c mice were treated systemically with CpG ODNs or suppressive ODNs, and then challenged intraarticularly with CpG DNA. The onset and magnitude of the resulting inflammatory response was monitored.
Systemic delivery of CpG ODNs significantly increased susceptibility to local inflammation, whereas systemic treatment with suppressive ODNs reduced this susceptibility. CD11c+ cells played a key role in mediating host sensitivity to arthritis. These cells were the dominant source of tumor necrosis factor α production in CpG-stimulated animals and transferred resistance to arthritis from mice treated with suppressive ODNs.
Systemic exposure to immunostimulatory and immunosuppressive DNA influences host susceptibility to local inflammatory challenge. Current findings raise the possibility that suppressive ODNs may be useful in the prevention/treatment of proinflammatory diseases.
Reactive arthritis presents as an asymmetric oligoarticular inflammatory condition that typically appears 1–3 weeks after microbial infection of the gastrointestinal or genitourinary tract (1, 2). Bacterial DNA, but not viable microorganisms, can be found in the affected joints, suggesting that DNA may trigger or contribute to this inflammatory process (1, 3, 4). This possibility is supported by evidence that histologic changes analogous to reactive arthritis in humans develop when purified bacterial DNA is injected into the joints of normal mice (4). Unmethylated “CpG motifs” present at high frequency in bacterial, but not mammalian, DNA are responsible for this effect, since synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs induce a similar inflammatory response (4, 5).
Numerous studies have shown that CpG DNA has immunostimulatory properties, triggering immune cells to proliferate, mature, and secrete a variety of proinflammatory chemokines and cytokines (6–9). This immune activation can be inhibited by “suppressive” ODNs, which block the production of Th1 and proinflammatory cytokines (10–13). Suppressive motifs are rich in poly G or GC sequences, tend to be methylated, and are present in the DNA of mammals and certain viruses (10, 11, 14). Recent studies show that CpG-induced arthritis can be prevented by injecting suppressive ODNs into the affected joint (5). Since reactive arthritis commonly involves multiple joints, we examined whether administering CpG or suppressive ODNs systemically could influence global susceptibility to arthritis.
Mice treated systemically with CpG ODNs showed increased susceptibility to inflammatory stimuli. In contrast, systemic administration of suppressive ODNs increased the host's resistance to arthritis. CD11c+ cells played a central role in mediating these shifts in disease susceptibility. These findings clarify the capability of immunomodulatory DNA to influence the development of autoimmunity and provide evidence that suppressive ODNs may be of therapeutic benefit.
Female BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were 8–20 weeks old when used in the experiments. Mice were housed under specific pathogen–free conditions. All experiments were approved by the Animal Care and Use Committee of the Center for Biologics Evaluation and Research (CBER).
Phosphorothioate ODNs were synthesized by the CBER Core Facility. They contained <0.1 endotoxin unit (EU) of endotoxin/mg of ODN, as assessed by a Limulus amebocyte cell lysate assay (QCL-1000; BioWhittaker, Walkersville, MD). The sequences of the ODNs we used were as follows: GCTAGACGTTAGCGT for stimulatory CpG ODNs, CCTCAAGCTTGAGGGG for suppressive ODNs, and GCTAGATGTTAGCGT for control (non-CpG) ODNs.
Inflammatory arthritis was induced as previously described (5). Briefly, 1–25 μg of stimulatory CpG ODNs in 6 μl of phosphate buffered saline (PBS) was injected with a 30-gauge needle into the knee joint of normal BALB/c mice. Joint swelling was measured daily in the coronal plane using micrometer calipers (General Tools, New York, NY). Swelling was defined as an increase in joint diameter after injection compared with the diameter of the same joint before injection. Arthritis was verified histologically. Mice were euthanized on day 4, and the knees were fixed, decalcified, sectioned, and stained with hematoxylin and eosin. Histologic sections were scored by an investigator (DV) who had no knowledge of the experimental group. A scale of 0–3 was used, where 0 = absence of inflammation, 1 = sparse, localized perivascular infiltrate, 2 = moderate infiltrate, and 3 = moderate-to-dense infiltrate, with synovial hyperplasia.
In some experiments, mice were injected intraperitoneally (IP) with 300 μg of ODNs in PBS. Then, 1–3 days later, a single-cell suspension was prepared from the spleens of these mice, and 5–200 × 105 cells were transferred intravenously to naive littermate controls (Figure 5 and Table 3). These spleen cells were enriched or depleted of various subpopulations by separation with magnetic beads (Vario-Macs System; Miltenyi, Auburn, CA), as recommended by the manufacturer. Typically, the purity of the resultant cell populations was >90%. Recipients were challenged by intraarticular injection of CpG ODNs 1 hour after cell transfer. The contralateral knee was injected with PBS as a control.
BALB/c mice were injected IP with 300 μg of CpG ODNs. A single-cell suspension was prepared from the spleens of these animals 1–3 days later. Then, 2 × 106 cells/ml were incubated for 6 hours with 1 μM CpG ODNs plus 1 μg/ml of brefeldin A (Sigma, St. Louis, MO). Cells were then fixed with paraformaldehyde and stained with fluorescein isothiocyanate–labeled anti-TNFα plus Cy3-labeled anti-CD11c, anti-B220, anti–T cell receptor, or anti-CD11b, as recommended by the manufacturer (PharMingen, San Diego, CA). Fluorescence-activated cell sorting analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson).
Differences between 2 groups were determined by Wilcoxon's rank sum test. Differences between multiple groups were evaluated using a 2-tailed analysis of variance with Tukey's posttest analysis. P values less than 0.05 were considered significant.
Intraarticular injection of high-dose (25 μg) CpG ODNs induced normal BALB/c mice to develop inflammatory arthritis (Table 1). Both the dose and type of DNA administered were critical: intraarticular delivery of low-dose (1 μg) CpG ODNs did not induce disease, nor did 25 μg of control (non-CpG) ODNs.
|Treatment||Outcome measure, day 4|
|Systemic, day 0||Local, day 3||Histologic score||Swelling, mm ×10−2|
|PBS||PBS||0.6 ± 0.1||1.7 ± 1.7|
|None||Control (non-CpG) ODNs, 25 μg||0.3 ± 0.2||0.9 ± 0.2|
|None||CpG ODNs, 25 μg||1.9 ± 0.3†||9.1 ± 2.4†|
|PBS||CpG ODNs, 25 μg||2.3 ± 0.3†||23 ± 2.5†|
|None or PBS||CpG ODNs, 1 μg||0.3 ± 0.2||2.4 ± 1.3|
|CpG ODNs, 300 μg||CpG ODNs, 1 μg||1.7 ± 0.2†||10 ± 2.2†|
In vivo and ex vivo studies have shown that systemic delivery of CpG DNA stimulates immune cells to secrete a variety of proinflammatory chemokines and cytokines (7, 15, 16). To determine whether such treatment increases host susceptibility to arthritis, BALB/c mice were injected IP with 300 μg of CpG DNA. None of these animals spontaneously developed arthritis. However, significant inflammation and joint swelling developed when these mice were subsequently injected intraarticularly with low-dose (1 μg) CpG ODNs, a dose that had no effect on naive mice (P < 0.03) (Table 1).
The time course over which systemically administered CpG DNA increased host susceptibility to arthritis was examined. Animals were injected IP with 300 μg of CpG ODNs. These animals developed significant joint swelling when challenged intraarticularly with low-dose (1 μg) CpG ODNs 3–5 days later (P < 0.05) (Figure 1). In contrast, no inflammation was observed in mice challenged immediately after systemic CpG ODN treatment or after a delay of 8 days. These findings suggest that several days are required for systemic exposure to CpG DNA to increase host susceptibility to arthritis and that this susceptibility persists for only a limited period.
To determine whether arthritis susceptibility was associated with the generation of cells capable of mediating CpG-induced inflammation, 20 × 106 spleen cells from CpG-treated donors were transferred to naive recipients. The recipients were challenged 6 hours later with intraarticular injection of low-dose (1 μg) CpG ODNs. As expected, this subarthritogenic dose of DNA had no effect on naive controls, nor did it have any appreciable effect on recipients of cells from donors treated 1 day earlier with CpG ODNs (Figure 2). In contrast, recipients of cells from donors treated 3 days earlier with CpG ODNs developed significant joint swelling and inflammation (P < 0.03) (Figure 2). Disease was limited to the CpG-injected joint; the contralateral PBS-injected knee was uninvolved.
Deng and colleagues (4, 17) established that the proinflammatory cytokine TNFα is essential to the development of CpG-mediated arthritis. We therefore evaluated the effect of CpG ODNs on the production of TNFα. Systemic treatment with CpG DNA did not alter the number of cells spontaneously secreting TNFα in vivo (Figure 3). However, when spleen cells from these mice were reexposed to CpG ODNs in vitro, significantly more were stimulated to produce TNFα compared with spleen cells from naive mice (P < 0.001) (Figure 3).
The phenotype of these TNFα-secreting cells was determined by staining for intracytoplasmic cytokine plus cell surface markers. As shown in Table 2, >80% of the cells activated to produce TNFα were CD11c+ cells. The number of CD11c+ cells producing TNFα rose 38-fold following CpG stimulation.
|Cell surface marker||Fold increase in no. of TNFα-producing cells||% of TNFα-producing cells|
|CD11c||38.0 ± 6.6||80.3 ± 4.3|
|B220||6.0 ± 2.7||8.1 ± 3.2|
|TCR||2.7 ± 1.4||1.1 ± 0.6|
|CD11b||8.0 ± 2.0||5.1 ± 0.8|
Based on the observation that systemically administered CpG DNA increases host susceptibility to disease, the possibility that suppressive ODNs might reduce susceptibility to inflammation was examined. Mice were injected IP with 300 μg of suppressive ODNs and then challenged intraarticularly with high-dose (25 μg) CpG DNA. Arthritis was significantly reduced in animals treated 3 days earlier with suppressive ODNs (P < 0.03) (Figure 4). By comparison, animals injected with control ODNs or with PBS consistently developed disease.
To explore the cellular basis underlying this resistance to arthritis, 20 × 106 splenocytes from suppressive ODN–treated mice were transferred to naive recipients. Naive mice, as well as recipients of spleen cells from control donors, developed arthritis when challenged intraarticularly with 25 μg of CpG DNA (Figure 5). In contrast, joint swelling and inflammation were significantly reduced among recipients of spleen cells from donors treated with suppressive ODNs (P < 0.01 for joint swelling and P < 0.02 for histologic score).
To gain insight into the cell type(s) responsible for transferring this resistance to joint inflammation, spleen cells from mice treated 3 days earlier with suppressive ODNs were depleted or enriched in various subpopulations and transferred to naive recipients. As shown in Table 3, resistance to CpG-induced arthritis was transferred by 20 × 106 spleen cells. Depletion of CD19+ B cells or DX5+ natural killer cells had no effect on arthritis resistance, indicating that these cell types were not responsible for reducing disease susceptibility. When CD11c+ cells were removed from the donor pool, however, suppressive activity was completely abrogated (P < 0.05) (Table 3). Conversely, transfer of only 5 × 105 CD11c+ enriched spleen cells conferred resistance to CpG-induced arthritis.
|Donor treatment||Cells transferred||No. of cells, ×105||Outcome measure|
|Swelling, mm ×10−2||Histologic score|
|None||Total spleen cells||200||14 ± 5||2.3 ± 0.8|
|Suppressive ODNs||Total spleen cells||200||2 ± 1||0.5 ± 0.1|
|Suppressive ODNs||B cell–depleted spleen cells||200||0 ± 0||ND|
|Suppressive ODNs||NK cell–depleted spleen cells||200||6 ± 4||0.3 ± 0.3|
|Suppressive ODNs||CD11c-depleted spleen cells||200||18 ± 3†||1.4 ± 0.4†|
|Suppressive ODNs||CD11c-enriched spleen cells||5||1 ± 0||0.5 ± 0.1|
DNA has multiple and complex effects on the immune system. Unmethylated CpG motifs in bacterial DNA trigger an immune response characterized by the production of proinflammatory and Th1 cytokines (6–9). This evolutionarily conserved response can protect the host from infectious pathogens (18–20), but it also increases the risk of developing autoimmune and inflammatory diseases (4, 21–24). Mammalian DNA contains “suppressive” motifs that inhibit CpG-induced immune activation and may thus limit the pathologic changes caused by CpG-driven immune responses (10, 13, 25).
Reactive arthritis is characterized by asymmetric, self-limiting inflammation of one or a few joints (1, 2). Disease generally develops 1–3 weeks after bacterial infection of the gastrointestinal or urogenital tract (1). Since routine diagnostic measures fail to detect viable bacteria in affected joints and antibiotic treatment rarely provides effective therapy, it is widely believed that bacterial products (rather than infectious microorganisms) are responsible for the observed inflammation (26). Evidence consistent with the contribution of bacterial DNA to this inflammation include the detection of CpG DNA in the joints (27) and the ability of CpG DNA to induce inflammatory arthritis when injected into the knees of normal mice (4, 5).
Intraarticular challenge with high-dose (25 μg) CpG ODNs induces joint swelling and histopathologic changes in mice that resembles the reactive arthritis observed in humans (5). Blinded histologic evaluation of joints 4 days after injection of CpG ODNs or bacterial DNA provides rigorous evidence of pathology, which correlates with joint swelling (Pearson's correlation coefficient r = 0.59, P < 0.0001) (5). Although systemically administered CpG DNA stimulates B cells, natural killer cells, and dendritic cells to proliferate, mature, and/or secrete in vivo (6–9), no direct effect on the joints has ever been reported (Table 1) (4). The findings of the present study demonstrate that the generalized immune activation triggered by systemic CpG ODN treatment increases host susceptibility to a subsequent arthritogenic stimulus. Thus, mice injected IP with 300 μg of CpG ODNs developed arthritis when challenged intraarticularly with a dose of ODNs that does not cause disease in naive mice (1 μg).
This increased susceptibility to arthritis developed several days after CpG administration and waned by day 8. A similar time course was observed for the induction of immunoprotection by CpG ODNs in mice (18–20, 28, 29): protection generally developed several days after administration and persisted for 1–2 weeks (depending upon the pathogen). A longer delay is typical for the development of reactive arthritis in humans following bacterial infection (2, 3). The delayed kinetics may reflect differences in the exposure to DNA. In humans, infection presumably results in prolonged exposure to small amounts of CpG DNA released over the course of the infection, whereas in the murine model, a single high dose of CpG ODNs is administered IP.
There are several mechanisms by which systemically administered CpG DNA might affect the host's susceptibility to arthritis. Circulating DNA might reach the joints directly or might be taken up by immune cells, which then migrate to the joints. In previous studies, systemically administered mycobacterial DNA was found to reach the primary lymphoid organs but not the joints (30). We found that systemically administered Cy3-labeled ODN was taken up by bone marrow cells within 4 hours of injection and persisted at that site for at least 3 days (data not shown). In contrast, very few cells containing ODN were detected in joint fluid or synovium obtained from the knee over the same time period (data not shown). Rather than a local effect, we found that systemic exposure to CpG DNA increased the responsiveness of CD11c+ cells in the lymphoid organs to further stimulation. This is consistent with previous studies demonstrating that CD11c+ dendritic cells are highly responsive to CpG stimulation (31, 32). Deng et al (4, 17) showed that TNFα is a critical mediator of CpG-induced arthritis. Results of the present study indicate that large numbers of CD11c+ cells respond to CpG ODNs by producing TNFα. Of interest, this effect “matured” over several days in vivo, consistent with the time course over which increased susceptibility to arthritis developed (Figure 3).
Krieg et al (10) were the first to document that “suppressive” or “neutralizing” sequence motifs could inhibit CpG-induced immune activation. We subsequently documented that suppressive ODNs could prevent arthritis when injected directly into inflamed joints (5). The mechanism by which suppressive ODNs inhibit the activity of CpG DNA is a matter of considerable interest. Recent studies suggest that suppressive ODNs interfere with CpG-mediated cell signaling upstream of nuclear factor κB activation (11, 25). However, suppressive ODNs do not block the cellular uptake of CpG DNA or binding to Toll-like receptor 9 (13). Studies in our laboratory failed to detect the production of “inhibitory factors” or changes in the expression of surface activation markers among immune cells cultured with suppressive ODNs (data not shown). Nevertheless, we found that systemic treatment with suppressive ODNs significantly reduced the host's susceptibility to inflammatory arthritis. This effect peaked 3–5 days after IP administration of suppressive ODNs. Of interest, CD11c+ spleen cells transferred arthritis resistance to naive recipients. These findings raise the possibility that suppressive ODNs might be useful for the prevention/treatment of inflammatory arthritis and other rheumatic diseases, particularly those exacerbated by TNFα (33, 34).
The authors thank Drs. Paul Plotz and Fred Steinberg for their discussion of this research.