Synthetic oligonucleotides as modulators of inflammation

Abstract

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN directly stimulate human B cells and plasmacytoid dendritic cells, promote the production of Th1 and proinflammatory cytokines, and trigger the maturation/activation of professional APC. CpG ODN are finding use in the treatment of cancer, allergy, and infection. In contrast, ODN containing multiple TTAGGG motifs mimic the immunosuppressive activity of self-DNA, down-regulating the production of proinflammatory and Th1 cytokines. Preclinical studies suggest that “suppressive” ODN may slow or prevent diseases characterized by pathologic immune stimulation, including autoimmunity and septic shock. Extensive studies in animal models suggest that the therapeutic value of CpG and TTAGGG ODN may be optimized by early administration.

INTRODUCTION

DNA has multiple and complex effects on the immune system. Although bacterial DNA elicits an immunoprotective, inflammatory response, it can also exacerbate immune-mediated tissue damage, promote the development of autoimmune disease, and increase sensitivity to toxic shock [1,2,3,4,5,6]. In contrast, self-DNA released by injured host cells can down-regulate overexuberant inflammatory reactions. The latter effect is mediated, at least in part, by repetitive TTAGGG motifs present in mammalian telomeres [7,8,9]. Synthetic, single-stranded phosphorothioate oligodeoxynucleotides (ODN) expressing CpG or TTAGGG repeats mimic the immunomodulatory activity of bacterial and self-DNA, inducing or blocking inflammatory responses [10,11,12,13,14,15,16,17]. This work provides an overview of a lecture entitled “Synthetic oligonucleotides as modulators of inflammation,” delivered at a National Cancer Institute-sponsored symposium about “Cancer and Inflammation.” It is intended to highlight examples where CpG ODN can be used for the prevention/treatment of diseases caused by abnormalities in the inflammatory immune milieu.

Immunomodulatory properties of CpG and suppressive ODN

The recognition of CpG DNA by TLR9 expressed by human B cells and plasmacytoid dendritic cells (pDC) initiates an immunostimulatory cascade that culminates in the maturation, differentiation, and/or proliferation of multiple cell types, including NK cells, T cells, monocytes, and macrophages [13, 18,19,20,21,22,23]. Together, these secrete cytokines and chemokines that create a proinflammatory (IL-1, IL-6, IL-18, and TNF) and Th1-biased (IFN-γ and IL-12) immune milieu [12, 13, 20, 21, 24,25,26].

Reflecting the evolutionary divergence among TLR9 molecules expressed by different species, the sequence motif (unmethylated CpG dinucleotide plus flanking regions) that optimally stimulates cells from one species may be ineffective in another species [27]. For example, the sequences of murine and human TLR9 differ by 24% at the amino acid level [20]. Whereas the optimal sequence motif in mice consists of two 5′ purines, unmethylated CpG, and then two 3′ pyrimidines [12, 13, 25, 28], the optimal motif in humans is TCGTT and/or TCGTA [24, 27, 29,30,31,32]. In addition, the cell populations that express TLR9 differ among species. In mice, immune cells of the myeloid lineage (including monocytes, macrophages, and myeloid DC) express TLR9 and respond to CpG stimulation, whereas in humans, these cell types do not express TLR9 and are not directly activated by CpG ODN [33,34,35].

At least three structurally distinct classes of synthetic CpG ODN have been described that are capable of stimulating cells that express human TLR9 [30, 31, 36, 37]. “K”-type ODN (also referred to as “B” type) encode multiple CpG motifs on a phosphorothioate backbone. K ODN trigger pDC to differentiate and produce TNF-α and B cells to proliferate and secrete Ig [30, 31, 31, 38] (see Table 1). Stimulation of these cells is initiated by the binding of K ODN to TLR9 in lysosomal vesicles [39] and proceeds through an IFN regulatory factor 5 (IRF5)-mediated pathway. “D”-type ODN (also referred to as “A” type) are constructed of a mixed phosphodiester/phosphorothioate backbone and contain a single hexameric purine/pyrimidine/CG/purine/pyrimidine motif flanked by self-complementary bases that form a stem-loop structure capped at the 3′ end by a poly-G tail [30], which interacts with CXCL16 expressed on the surface of pDC, which increases their uptake and directs them into early endosomes [39, 40]. TLR9-mediated CpG stimulation proceeds through IRF7 in these lysosomes, culminating in the production of IFN-α [40]. Thus, the poly-G tail of D ODN accounts for their ability to trigger pDC to secrete IFN-α rather than TNF-α and their lack of activity on B cells that do not express CXCL16 [30, 31, 40].

TABLE 1.

Success of CpG ODN Monotherapy in Cancer Trials

Tumor type	Trial phase	# Responders/total
NHL	1	2/23
Melanoma	2	6/25
Cutaneous T cell lymphoma	1, 2	9/28
Renal cell	1	2/35
Recurrent glioblastoma	1	2/24
Total:		21/135

Data in this table are excerpted from a recent review by Kreig [57]. 

“C”-type ODN resemble K type in being composed entirely of phosphorothioate nucleotides. C-type ODN were originally described as expressing a TCGTCG at the 5′ end and commonly contain an internal K-type motif (such as GTCGTT) imbedded in a palindromic sequence [41]. This class of ODN is capable of stimulating B cells to secrete IL-6 and pDC to produce IFN-α (thus, combining some of the stimulatory properties of D- and K-type ODN) [36, 37]. Of note, most of the clinical studies involving CpG ODN have been performed using K class molecules. Thus, although of potential value, little is known about the activity of D or C class ODN in humans.

Even less is known about the mechanism underlying the immunoinhibitory properties of suppressive ODN. The literature describes several structurally distinct types of immunoinhibitory ODN. Most of these contain a run of three to four poly-G but otherwise differ in nucleotide sequence, length, and activity. This report focuses on phosphorothioate TTAGGG multimers (referred to as “suppressive ODN”), patterned after the repetitive TTAGGG motifs present in murine and human telomeres that have themselves been shown to have immunosuppressive activity [7]. The poly-G motifs present in these suppressive ODN can form G-tetrads via Hoogsten bonds. As preventing G-tetrad formation reduces suppressive activity, this structural motif is considered critical to function [7]. Yet, other groups report that multimer formation is not needed for an ODN to exhibit immunoinhibitory activity [42,43,44]. Moreover, distinct types of ODN differ in their suppressive activity: The TTAGGG-based suppressive ODN inhibit the immune activation elicited by a large number of TLR ligands, as well as antigen-specific and polyclonal activators of the immune system [45]. In contrast, other inhibitory ODN containing a single poly-G run appear to selectively block the activation mediated by TLR7 and/or TLR9 [42, 43, 46]. The mechanism(s) underlying the activity of this latter type of ODN have not been established [43, 44]. Although the mechanism by which TTAGGG ODN influence immune activation is under investigation, evidence indicates that suppressive TTAGGG ODN bind to and prevent the phosphorylation of specific STATs, thereby inhibiting the signal transduction cascade needed to maintain inflammatory responses [47].

THERAPEUTIC UTILITY OF CpG ODN

CpG ODN in the treatment of cancer

CpG ODN were shown to have anti-tumor activity in a number of murine models. Multiple mechanisms by which CpG ODN facilitate the elimination of tumor cells have been identified, including their ability to induce CD8⁺ CTL capable of eliminating tumors, up-regulate the expression of MHC by tumor cells and APCs (enhancing tumor antigen recognition and presentation), contribute to the generation of NK cells that target tumors, interfere with the tumor’s ability to establish an immunosuppressive microenvironment, and block the ability of T regulatory cells to protect the tumor from being rejected [35, 48,49,50,51,52,53].

Although CpG ODN alone can successfully eliminate small and/or highly immunogenic tumors, they generally need to be combined with other treatment modalities (such as radiation therapy, chemotherapy, or surgery) to successfully eradicate larger tumors [52, 54,55,56,57]. As an example, combining CpG ODN with a tumor-specific toxin induces the complete regression of squamous cell carcinoma in 83% of mice, whereas no regression was observed in animals treated with CpG or toxin alone [58].

Despite favorable results in murine tumor studies, results from phase I–III clinical trials of CpG ODN have been less encouraging. In general, CpG ODN increase the frequency of complete or partial remission in ∼15% of patients participating in trials involving melanoma, non-Hodgkin’s lymphoma (NHL), cutaneous lymphoma, renal cell carcinoma antigen, and/or glioblastoma, although there was considerable variation in outcome among various studies (Table 1) [52, 53, 57]. What accounts for these less-impressive results when compared with much higher rates of cure in murine models? One possibility is that CpG ODN therapy is generally administered to humans late in the disease process. We postulate that success would increase if CpG ODN therapy for cancer were initiated earlier. This possibility is supported by findings in other model systems (reviewed below), indicating that the efficacy of CpG ODN treatment is optimized by early administration. Alternatively, use of a different class of CpG ODN (D or C rather than K) might provide more effective anti-tumor therapy.

Early administration of CpG ODN enhances host survival in infectious challenge models

The evolutionary conservation of CpG recognition by TLR9 suggests that the immune response elicited by this interaction contributes to host survival. Data indicate that CpG ODN reduce host susceptibility to infection by many different bacteria, viruses, fungi, and parasites (reviewed in ref. [59]). Less well appreciated is that this protective activity is typically optimized by early administering of the CpG ODN. For example, when normal mice are treated with CpG ODN prior to exposure to Listeria monocytogenes, they are fully protected against a 10,000 LD₅₀ challenge of that pathogen (Table 2). By comparison, delaying treatment for as little as 2 days after exposure significantly reduces the ability of CpG-treated mice to survive high-dose listeria challenge (Table 2).

TABLE 2.

CpG ODN Modulate Host Susceptibility to Infection

Challenge dose (LD50)	Mean time to death (days)
	Untreated	CpG Pre-Rx	CpG Post-Rx
10	8	Survive	Survive
100	6	Survive	12
1000	4	Survive	4
10,000	4	Survive	4

BALB/c mice were treated with 50 μg CpG ODN 1 day before (Pre-Rx) or 2 days after (Post-Rx) challenge with L. monocytogenes. Data reflect the mean time to death in days of all challenged animals, as excerpted from refs. [60, 61]. 

Early administration of CpG ODN reduces host susceptibility to allergy

Allergic asthma is an IgE-mediated, inflammatory disease of the airways, characterized by the overproduction of Th2 cytokines [62]. As CpG ODN down-regulate Th2 responses (in the process of up-regulating Th1 immunity), they were predicted to reduce susceptibility to allergy. Murine models of asthma commonly include a “sensitization” phase, during which mice are exposed to the allergen, followed by a “challenge” phase, in which the animals are re-exposed to the same allergen [63]. In studies of ragweed, we observed that CpG ODN significantly reduced the systemic and pulmonary manifestations of allergy {IgE levels in serum and bronchi-alveolar lavage (BAL) fluid were reduced, as was airway resistance [64]}. These beneficial effects were optimized by administering the ODN prior to sensitization (Table 3). When the ODN were delivered prior to challenge, the success of this therapy was reduced. Delaying treatment until after allergen challenge yielded little clinical benefit (Table 3). These findings support the conclusion that early intervention maximizes the therapeutic use of CpG ODN. Only one clinical trial involving the use of CpG ODN for the treatment of allergic asthma in humans has been described. That study found that CpG ODN coupled to ragweed allergen reduced the responsiveness of immune cells in the lung to allergen stimulation and reduced disease severity for two seasons [65, 66].

TABLE 3.

CpG ODN Modulate Host Susceptibility to Allergic Asthma

Treatment	Serum IgEa	BAL IgEb	BAL eosinophilsc	Airway resistanced
Normal mice	1	60	1	2.8
Allergic mice	23	6000	85	8.2
CpG treatment:
Presensitize	3	130	4	3.3
Prechallenge	8	400	15	5.1
Postchallenge	16	2900	36	6.3

BALB/c mice were sensitized and challenged with 150 μg of ragweed allergen. Animals were treated with 35 μg CpG ODN 1 day before sensitization, 2 days before challenge, or 2 days post-challenge. Responses were analyzed 3 days post-challenge. Data are largely excerpted from Ref. [64]. 

^aFold increase over naive mice; 

^bng/ml; 

^cPercent eosinophils in BAL; 

^dEnhanced pause index in mice treated with 50 mg/ml methacholine. 

CpG ODN as vaccine adjuvants

The ability of CpG ODN to promote Th1 responses and induce the maturation/activation of professional APC suggests they might be useful as vaccine adjuvants [67,68,69,70]. We studied A/J mice vaccinated with anthrax vaccine adsorbed (AVA; the licensed human vaccine) to explore this issue [71]. As seen in Table 4, the inclusion of CpG ODN with AVA increased the resultant antibody-mediated toxin-neutralizing activity (TNA) by greater than tenfold. Survival following anthrax spore challenge was also improved significantly by immunizing with CpG-adjuvanted AVA. In contrast, delaying the administration of CpG ODN until after AVA immunization yielded almost no booster effect, consistent with adjuvant activity requiring codelivery with antigen.

TABLE 4.

CpG ODN As Vaccine Adjuvants

Treatment	% Survival	TNA
None	0	0
AVA alone	30	350 + 48
AVA plus CpG ODN (simultaneous)	80a	25 + 7a
AVA plus CpG ODN (delayed)	30	37 + 6

A/J mice were immunized once with 8 μl AVA + 50 μg CpG ODN at the same time (simultaneous) or 1 day after (delayed) AVA delivery. TNA was measured 3 weeks later. Mice were challenged with 60 LD₅₀ Sterne strain anthrax at 4 weeks. Data are excerpted from refs. [71, 72]. 

^aP < .05. 

These findings were confirmed in studies of rhesus macaques, where co-administering CpG ODN with AVA induced a six-fold higher antibody response than AVA alone [73]. Serum from the primates vaccinated with AVA plus CpG ODN transferred significantly greater protection against anthrax spore challenge to murine recipients [73]. A clinical trial examining the response of 69 normal, healthy volunteers to 0.5 ml AVA ± 1 mg CpG ODN was conducted. Results from that study demonstrated that in humans, the inclusion of CpG ODN significantly accelerated the induction of protective immunity and increased serum IgG antiprotective antigen titers by ninefold when compared with AVA alone (P<0.05) [74].

THERAPEUTIC UTILITY OF SUPPRESSIVE ODN

Early administration of suppressive ODN reduces reactive arthritis

Reactive arthritis presents as an asymmetric, oligoarticular, inflammatory condition [75, 76]. This disease can be modeled by injecting CpG DNA into the knee joints of mice, which causes significant swelling and inflammation [6, 8]. As seen in Table 5, this arthritogenic response was reduced significantly by treating mice with suppressive ODN prior to injection of CpG DNA (P<.03). This effect was sequence-specific, as disease severity was unaffected by the administration of control ODN or PBS. The therapeutic benefit of suppressive ODN required early intervention; however, no reduction in inflammation was observed when treatment was delayed until 1 day after arthritiogenic challenge.

TABLE 5.

Suppressive ODN Modulate Host Susceptibility to Inflammatory Arthritis

Treatment	Arthritis induced	Histologya	Swellingb
Saline	−	0.4 + 0.2	0 + 0.1
Saline	+	1.9 + 0.3	4.8 + 1.2
Control ODN	+	1.7 + 0.4	5.9 + 1.4
Sup ODN (pre)	+	0.7 + 0.2c	0.8 + 0.3c
Sup ODN (post)	+	1.7 + 0.5	4.2 + 0.9

Arthritis was induced by injecting 25 μg CpG ODN into the knee joint of female BALB/c mice. Animals were treated systemically 3 days before or 1 day after the arthritogenic challenge with 300 μg suppressive ODN (Sup ODN). Disease severity was monitored on Day 4. 

^aInflammation scale; 0/none, 1/sparse, 2/moderate, 3/dense (as excerpted from ref. [8]). 

^bmm × 10–2; 

^cP < .05 compared with control ODN or saline. 

Early administration of suppressive ODN reduces collagen-induced arthritis (CIA)

CIA is a well-established murine model of rheumatoid arthritis (RA). This model has been used to clarify the pathogenesis of RA and evaluate potential treatments [7, 77]. CIA is elicited by injecting DBA/1 mice intradermally with type II bovine collagen (CII) in CFA, followed 3 weeks later by CII in IFA [7, 77]. Arthritis typically develops shortly after the second CII injection, manifest by swelling and inflammation of the joints that persists for many weeks.

The ability of suppressive ODN to block the induction of CIA was examined. Suppressive ODN (300 ug) were delivered twice, starting before or after the initial injection of CII. As seen in Table 6, the earliest treatment reduced the frequency and severity of disease (P<.05). These benefits were clearly mediated by the suppressive ODN, as control ODN had no effect on CIA (Table 6).

TABLE 6.

Suppressive ODN Reduce the Frequency and Severity of CIA

Treatment	Timing	Disease incidence (%)	Severity
PBS	Before CIA	100	6.9 + 0.8
Control ODN	Before CIA	95	6.2 + 1.1
Suppressive ODN	Before CIA	60a	3.2 + 0.7b
	After CIA	90	5.0 + 0.9

DBA/1 mice were injected intradermally with CII emulsified in CFA (Day 0) and then IFA (Day 21). The effect of administering 300 μg ODN on the incidence and severity of disease is shown. Animals were treated on Days −3 and 3 or Days 0 and 18. n = 10–20 mice/group. Disease severity was evaluated on a scale of 0–12. 

^aP < .05, Kaplan Meir (including onset and frequency of disease); 

^bP < .05, t-test. 

The effect of suppressive ODN on the immunologic abnormalities that accompany CIA was examined. Treatment with suppressive ODN reduced serum IgG anti-CII autoantibody levels and the number of T cells that responded to CII exposure by secreting IFN-γ by threefold (P<.05) [78].

Early administration of suppressive ODN controls murine lupus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of anti-nuclear autoantibodies, immune complex-mediated glomerulonephritis, and multifocal end-organ damage [79,80,81]. Female NZB/W mice provide a useful model for studying the pathogenesis and treatment of human SLE [82, 83]. When female NZB/W mice were treated systemically with 300 ug suppressive ODN twice monthly starting at 6 weeks of age (before the development of clinical abnormalities), the onset and magnitude of proteinuria were reduced significantly, and survival was prolonged (Table 7, P<.01). No such beneficial effects were observed in mice treated with control ODN.

TABLE 7.

Suppressive ODN Modulate Host Susceptibility to Lupus Nephritis

Timing	Treatment	Proteinuria (0–4 scale)	%
Survival
Starting at 6 weeks of age
	PBS	2.4 + 0.4	10
	Sup ODN	0.8 + 0.3a	75a
Starting at 7 months of age
	PBS	3.9 + 0.1	0
	Sup ODN	2.3 + 0.3b	40b

Female NZB/W mice were treated with 300 μg suppressive ODN. Mean proteinuria + sem at 10 months and survival at 12 months is shown for animals treated from 6 weeks of age. Proteinuria at 9 months and survival at 12 months are shown for animals with established disease treated from 7 months of age. Data are excerpted from Ref. [84]. 

^aP < .01; 

^bP < .05. 

To examine whether suppressive ODN treatment could benefit animals with established disease, treatment was initiated in a cohort of NZB/W mice with 2+ proteinuria (at ∼7 months of age). Treating these animals twice weekly for 3 months slowed (but did not halt) the progression of proteinuria and glomerulonephritis (P<.05; Table 7, P<.02) [84]. The benefit of initiating treatment late in the disease process was thus of smaller magnitude and shorter duration than that induced by initiating suppressive ODN therapy early.

Early administration of suppressive ODN reduces susceptibility to toxic shock

LPS-induced toxic shock is a major contributor to septic shock in humans [85]. LPS binds to TLR4 expressed on macrophages and monocytes, triggering a cascade of cytokine and chemokine production that culminates in the death of the host [85,86,87]. BALB/c mice challenged with 200 ug Escherichia coli LPS uniformly succumb to endotoxic shock within 2 days. Treating these mice with suppressive ODN immediately prior to challenge results in the survival of all LPS-challenged animals (Table 8, P<.001) [47]. This correlated with a concomitant reduction in the production of IFN-γ by the LPS-challenged mice (P<0.001) [47]. In contrast, delaying ODN delivery until 1 h after LPS challenge resulted in no significant benefit on mortality, although mean time to death was prolonged.

TABLE 8.

Suppressive ODN Modulate Host Susceptibility to LPS-Induced Toxic Shock

Treatment	Time of Rx	% Survival	MTD (h)
PBS	Anytime	0	26 + 4
Control ODN	Anytime	0	28 + 5
Suppressive ODN	−3 h	100a
Suppressive ODN	1 h	20	42 + 6a

BALB/c mice were treated with 300 μg suppressive or control ODN 3 h before or 1 h after challenge with 200 μg E. coli LPS. The percent of mice surviving challenge and mean time to death (MTD) are shown. Data are excerpted from ref. [84]. 

^aP < .05. 

COMMENTARY

DNA has multiple and complex effects on the immune system. CpG ODN trigger cells expressing TLR9 to initiate an immunostimulatory cascade, culminating in the broad activation of the immune system and the production of Th1 and proinflammatory cytokines and chemokines [12, 13, 20, 21, 24,25,26]. In multiple model systems, the therapeutic use of CpG ODN was optimized by early delivery. This was observed using ODN, which reduce host susceptibility to infection, and to allergic asthma and as vaccine adjuvants. We postulate that early treatment with CpG ODN may also enhance their potential as anticancer agents.

Studies involving suppressive ODN similarly indicate that early intervention is key to therapeutic success. This was consistently observed in murine models of arthritis, lupus, and toxic shock [8, 47, 78, 84]. For example, treating lupus-prone mice with suppressive ODN starting at 6 weeks of age significantly delayed the onset of renal disease and prolonged survival, while waiting until 7 months slowed the rate of progression but had only modest impact on survival [84]. Similarly, suppressive ODN reduced mortality when administered prior to but not after LPS challenge in a murine toxic shock model [47].

Based on our growing knowledge of the mechanism(s) of action of CpG and suppressive ODN, we believe it likely that these agents will prove useful in the prevention/treatment of cancer in addition to autoimmune and infectious diseases. In this context, studies are under way to examine whether ODN can be used to modulate the immune milieu of animals genetically predisposed to develop cancer prior to tumor formation. If successful, such studies might pave the way to instituting the use of CpG or suppressive ODN as prophylactic agents in populations (e.g., the elderly) at increased risk of cancer.