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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 645-647, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.F268

Perspective

Immunostimulatory DNA for Asthma

Better than Eating Dirt

Eric S. Silverman and Jeffrey M. Drazen

Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School; and Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachussetts

Address correspondence to: Eric S. Silverman, M.D., Physiology Program, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115-6021. E-mail: esilverm{at}HSPH.harvard.edu

Abbreviations: interleukin, IL • immunostimulatory sequence-ODN, ISS-ODN • natural killer, NK • oligodeoxynucleotides, ODN • pathogen-associated molecular patterns, PAMP • pattern recognition receptors, PRRs • transforming-growth factor-ß1, TGF-ß1 • T helper, TH • toll-like receptor 9, TLR9 • T regulatory, Tr


   Innate Immunity and the Hygiene Hypothesis
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 Innate Immunity and the...
Immunostimulatory Effects of CpG...
 ISS-ODN Therapy for Airway...
 ISS-ODN Therapy for Asthma:...
 References
 
The innate immune system provides an early line of defense against infection by recognizing and processing evolutionarily conserved structural motifs, so-called "pathogen-associated molecular patterns (PAMP)," on bacteria, viruses, and other invading pathogens and responding to these "danger signals" by priming the adaptive immune system for appropriate action. Macrophages, dendritic cells, and other cells of the innate immune system lack the highly-specific antigen receptors of T cells and B cells of the adaptive immune system, but can recognize these foreign motifs by a limited number of germline-encoded "pattern recognition receptors (PRRs)." The binding of PAMP to PRR can enhance phagocytosis, cell proliferation, and cytokine expression, and may have profound and long-lasting immunostimulatory effects. These effects are essential for the elimination of infectious organisms, but may also be important for immune system development and homeostasis (1).

In the absence of early life PAMP stimulation, immune balance may be upset and individuals may be susceptible to the development of disease reflecting a skewing of the immune system toward the T-helper type 2 (TH2) phenotype. This concept, known as the "hygiene hypothesis," is thought to be a major reason for the dramatic increase in asthma prevalence in Western countries over the past 40 years (2–4). The developing immune system of humans has a TH2 bias in utero and in early life that gradually diminishes over time in individuals without asthma. Exposure to PAMP during childhood infections may provide a TH1 stimulus that diminishes TH2 responses and the likelihood of developing asthma. Furthermore, exposure to PAMP may dampen both TH1 and TH2 immune responses by increasing the expression of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor-ß1 (TGF-ß1) from T regulatory (Tr) cells and antigen-presenting cells (4). The "hygiene hypothesis" predicts that asthma is increasing in highly developed regions of the world because of decreased exposure to PAMP in affluent settings and disturbing the homeostasis of a human immune system that has evolved to fight infection. Is there a way, other than exposure to childhood disease, to keep the immune system in balance?

One way of modifying immune responses is to use immunostimulatory DNA; these are specific DNA sequences that have been shown by several groups to be effective in the prevention and treatment of allergen-induced airway inflammation in animals, and may have a role in the prevention and treatment of asthma. In this issue of the AJRCMB, Ikeda and colleagues compared the ability of immunostimulatory DNA and glucocorticosteroids to treat allergen-induced airway inflammation in mice (5). Does the use of immunostimulatory DNA make sense for asthma treatment?


   Immunostimulatory Effects of CpG DNA
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 Innate Immunity and the...
 Immunostimulatory Effects of CpG...
ISS-ODN Therapy for Airway...
 ISS-ODN Therapy for Asthma:...
 References
 
For more than a century it has been known that bacterial extracts have immunostimulatory effects that can be used to fight disease. Important work over the past 10 years by a number of investigators has proven that many of these effects are mediated by unmethylated CpG dinucleotides in the DNA of bacteria (6–9). CpG dinucleotides are common in bacterial genomes, occurring at the expected frequency of ~ 1 in 16 dinucleotides, and less than 5% of the cytosines in these dinucleotides are methylated. In contrast, CpG dinucleotides are greatly underrepresented in vertebrate genomes, occurring at a frequency of ~ 1 in 125 dinucleotides, and 70–90% of the cytosines in these dinucleotides are methylated, which greatly diminishes their immunostimulatory effects (7, 10). Moreover, the sequences flanking CpG dinucleotides are not random in vertebrates or bacteria, and can dramatically impact the immunostimulatory activity of the DNA sequence. In general, strong immunostimulatory effects are elicited by unmethylated CpG sequences within a palindromic hexamer that follows the formula 5'-R1R2CGY1Y2-3', where R1 is a purine (preference for G), R2 is a purine or T, and Y1 and Y2 are pyrimidines (e.g., 5'-GACGTC-3', 5'-AGCGCT-3', and 5'-AACGTT-3') (8, 11). In vertebrates, the most common 5' flanking base is C and the most common 3' flanking base is a G, which produces a sequence that does not evoke a strong immunostimulatory effect and may actually antagonize the effects of more potent immunostimulatory sequences (7, 12). It has been hypothesized that evolutionary forces have suppressed immunostimulatory motifs in the genomic DNA of vertebrates so that PRR can more effectively distinguish endogenous DNA from the DNA of foreign invaders. It is also likely that evolutionary forces have suppressed immunostimulatory motifs in certain pathogens to avoid their detection and elimination (13).

The immunostimulatory effects of bacterial DNA can be reproduced in vivo by synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides, so-called immunostimulatory sequence-ODN (ISS-ODN). The effects of ISS-ODN on the immune system depend on the specific sequence, animal species, dose, time course, and route of delivery, so generalizations must be made with care. For example, the sequence GACGTT activates innate immune cells of the mouse much more efficiently than similar cells from humans (9). The sequence, GTCGTT confers optimal immunostimulatory effects in human cells (14). Some of the differences in response to ISS-ODN variants between species appear to be caused by genetically determined toll-like receptor 9 (TLR9) sequence-binding selectivity (15). Chemical modifications are being systematically incorporated into ISS-ODN to design second generation reagents with more potent anti-inflammatory effects and less potential for adverse reactions (16).

In general, ISS-ODN elicit TH1-biased immune responses in mammals that may prevent and reverse TH2-mediated inflammation associated with asthma and other allergic disease. CpG DNA directly activates dendritic cells and B-cells that express TLR9, a transmembrane receptor that binds CpG DNA and transduces its immunostimulatory effects (17–19), and indirectly activates other cells of the immune system such as T cells, NK cells, and neutrophils, by altering the expression of specific cytokines, receptors, costimulatory molecules, and adhesion molecules. Figure 1 is a simplified scheme of how these immunostimulatory events may block or reverse the TH2-mediated inflammation associated with asthma and other allergic disease. A detailed discussion of these complex immunomodulatory effects is beyond the scope of this Perspective. Comprehensive reviews of the effects of CpG DNA on the immune system are available (7, 19).



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  		Figure 1. Immunomodulatory effects of CpG DNA. Allergen is presented by dendritic cells to CD4+ TH0 lymphocytes and may result in their differentiation into TH2 cytokine-producing cells. Interleukin (IL)-4, IL-13, and IL-5 regulate the asthmatic response. IL-4 directs the growth and differentiation of T cells toward a TH2 cytokine profile. IL-4 and IL-13 regulate the synthesis of IgE by B cells. IL-5 regulates the differentiation and egress of eosinophils from the bone marrow into the blood. Tissue eosinophils and activated mast cells release a variety of inflammatory mediators that increase mucus production, airway edema, and smooth muscle contraction. In general, CpG DNA elicits TH1-biased immune responses that prevent or reverse TH2-mediated inflammation. CpG DNA activated pathways and critical mediator are shown in bold. CpG DNA-inhibited pathways are shown with dashed lines. ISS-ODN increase production of TH1 cytokines interleukin (IL)-12, IL-18, interferon-{gamma}, and IL-6, and decreased production of TH2 cytokines IL-4 and IL-5. T cells are inactivated by increased production of anti-inflammatory cytokines IL-10 and TGF-ß1 by dendritic and T regulatory (Tr) cells. B-cells proliferate and produce IgG2 antibody. Natural killer (NK) cells are activated by IL-12 and IL-18. Modified from Ref. 4 with permission.

 

   ISS-ODN Therapy for Airway Inflammation: Animal Models
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 Innate Immunity and the...
 Immunostimulatory Effects of CpG...
 ISS-ODN Therapy for Airway...
ISS-ODN Therapy for Asthma:...
 References
 
Ikeda and colleagues, as well as others, have shown that ISS-ODN treatment decreases eosinophilic airway inflammation, blood and bone marrow eosinophilia, IgE levels, TH2 cytokine expression (interleukin [IL]-5), and airway hyperresponsiveness, and increases the expression of TH1 cytokines (interferon-{gamma}, IL-12) and anti-inflammatory cytokines (TGF-ß1) (5, 20–25). It has also been shown that ISS-ODN attenuate allergen-induced airway remodeling in mice as manifested by decreased subepithelial collagen deposition and goblet cell metaplasia (26). ISS-ODN may be effective in the context of airway inflammation caused by infectious agents. Administration of ISS-ODN before respiratory syncytial virus infection inhibited viral replication, airway inflammation, and mucus secretion, but not airway hyperresponsiveness (27). The minor inconsistencies in some of the many studies examining the role of ISS-ODN in airway inflammation may be due to differences in the mouse strain and allergen studied, specific ISS-ODN sequence, and timing, frequency, and route of administration.

ISS-ODN have an effect in animal models similar to those observed with asthma treatments in current use (28, 29). For example, one treatment of ISS-ODN was as effective as seven daily injections of dexamethasone in protecting sensitized mice from inflammation with nebulized allergen challenge (23). Ikeda and colleagues have now shown that the combination of ISS-ODN and corticosteroid is more effective at treating established inflammation than either agent alone and discuss some of the distinct and shared anti-inflammatory pathways by which these agents mediate their effects (5). On the downside, the effects of ISS-ODN therapy appear to be transient, suggesting that optimal ISS-ODN therapy may require repeat administrations (20). The combination of ISS-ODN with allergen in the form of allergen-ISS conjugates may offer longer-lasting and allergen-specific protection (30).


   ISS-ODN Therapy for Asthma: Will It Work?
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 Innate Immunity and the...
 Immunostimulatory Effects of CpG...
 ISS-ODN Therapy for Airway...
 ISS-ODN Therapy for Asthma:...
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ISS-ODN are just beginning to be tested in human asthma. If the murine models are a true beacon, we may find that this form of immunotherapy can decrease many of the inflammatory effects characteristic of human asthma. Although ISS-ODN treatment may be better than eating dirt as a way to keep our immune system in balance (2), it makes sense to be concerned about the effects of too much immune suppression or too marked a skew toward the TH1 phenotype. Phase 1 clinical trials suggest that ISS-ODN are well tolerated in humans, but long-term effects are unknown (7, 31). In a choice between autoimmune hepatitis or diabetes and asthma, most people would choose asthma.

Received in original form April 2, 2003


   References
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 Innate Immunity and the...
 Immunostimulatory Effects of CpG...
 ISS-ODN Therapy for Airway...
 ISS-ODN Therapy for Asthma:...
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