Toll-Like Receptor 9
Toll receptors are transmembrane proteins that are evolutionarily conserved. These receptors were first recognized in Drosophila, as an essential molecule for embryogenic patterning (Anderson 2000).
Activation of these receptors in Drosophila initiates an intracellular kinase cascade that produces a translocation of transcription factors from cytoplasm to nucleus. These factors activate a variety of inflammatory mediators and cytokines, initiating an immune response (Imler and Hoffmann 2000).
Considering these facts, some researchers began a search for toll-related proteins in humans. In 1997, they identified the first human homologue, initially termed “human toll” and subsequently termed TLR4 (Medzhitov et al. 1997). After this, more toll-like receptors were discovered, as TLR1, TLR2, TLR3, TLR5, and TLR6.
In 2000, Du et al. examined the human genomic sequence database in an effort to identify novel TLRs (Du et al. 2000). He found more three TLRs, which were designated TLR7, TLR8, and TLR9. Two of these TLRs are X-linked and lie in close opposition to one another at Xp22. A third is located at human chromosome 3p21.3.
Molecular Cloning and Sequence Analysis of TLR9
All the information about structure of TLR9 was well described by Chuang et al. (Chuang and Ulevitch 2000). TLR9 exhibit features of type I transmembrane proteins. Type I proteins are single pass molecules anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to the endoplasmic reticulum lumen during synthesis.
TLR9 contains two hydrophobic regions: (1) a transmembrane domain, and (2) a signal peptide at the amino-terminus. The transmembrane regions separate TLR9 into two domains: an extracellular domain with more than 800 amino acid residues and a cytoplasmic domain with around 200 amino acid residues. External domain contains copies of leucine-rich repeats, more than found in other TLRs. Following the leucine-rich repeats-flanking region, there is a sequence of about 60 amino acid containing cysteine residues, which are conserved in all TLRs. This conserved area can be found in other proteins such as platelet glycoprotein IX and 1b–alpha (Hickey et al. 1989). The cytoplasmic domains of TLR9 share sequence homology with other members of human TLR family, although there is some variation within these regions.
Tissue Distribution of TLR9
Toll-like receptors can be found in different tissues. In contrast to all of the other TLRs, the TLR9 was initially found in immune-cell-rich tissue, including spleen, lymph node, bone marrow, and peripheral blood leukocytes. However, the TLR9 can also be found in the skin and endothelial cells (Tuon et al. 2010a, b).
Activation of TLR9
Some TLRs recognize protein and lipid structures found in microorganisms. However, toll-like receptor 3, 7, 8, and 9 recognize nucleic acid fragments from microorganisms. TLR3 respond to specific double-stranded RNA segments, TLR9 respond to DNA-specific oligonucleotides-bearing unmethylated CpG base pairs (CpG motifs), and TLR7 and TLR8 recognize specific single-stranded RNA segments (Barton et al. 2006).
The TLR9 can recognize CpG motifs which can be found in many bacterial and viral genomes (Gangloff and Gay 2008). After the recognition of the CpG motifs, several intracellular enzymatic reactions occur and the translocation of NF-kB to the nucleous induces the production of cytokines, initiating the innate immune response.
The role of TLR9 in CpG motifs detection was suggested by the observation that the activation of CpG-induced responses requires components of TLR signaling pathways, including MyD88 and TRAF6 (Hacker et al. 2000; Schnare et al. 2000). TLR9 was identified as a receptor by demonstrating that responses to CpG DNA are abrogated in mice lacking TLR9 (Hemmi et al. 2000). Furthermore, sensitivity to CpG DNA can be reconstituted by expression of TLR9, without the co-expression of CD14 (Bauer et al. 2001).
Although NF-kB is known to function as an activator of transcription, its role in the differential transcription of cytokines is less well defined. Some interactions between NF-kB and other activated transcription factors are important for determining the transcription rates of cytokine genes. NF-kB may also produce preferential binding, which could favor the production of certain cytokines. For example, cooperative binding with the transcription factor NF-IL-6 (a variance of NF-kB) is required for the transcriptional activation of IL-8 and IL-6 (Baeuerle and Baltimore 1996). In addition, NF-kB may have direct protein–protein interactions with other transcription factors, such as the glucocorticoid receptor, that alter the ability of NF-kB to bind to DNA.
The activation of TLR9 is associated with the expression of IL-1, IL-8, TNF-alpha, IFN-gamma, CD80 and CD86.
TLR9 agonists are currently in clinical trials for use in lung cancer, as antiviral therapy, as adjuvants, and as immune modulators in asthma and allergies. TLR9 antagonists, such as the antimalarial drug chloroquine, have been used since the 1950s to treat immune-mediated inflammatory disorders such as rheumatoid arthritis, systemic lupus erythematosus, and Sjögren’s syndrome. However, the use of antimalarials is limited due to the side effects or suboptimal efficacy (Sun et al. 2007).
TLR9 agonists have shown activity against several neoplasms. Phase I and II clinical trials have indicated that these agents have antitumor activity as single agents and enhance the development of antitumor T-cell responses when used as therapeutic vaccine adjuvants. The efficacy of these anticancer agents is being tested in several tumor types (Krieg 2008). The activation of the innate response has direct antitumor effects and the enhanced presentation of the tumor antigen in a Th1-like cytokine and can promote an better antitumor immune response.
Synthetic oligodeoxynucleotide (ODN) agonists for TLR9 are currently in development for the treatment of cancer. Because the phosphodiester bond of native DNA is rapidly degraded by endonucleases, these investigational CpG ODNs use a nuclease-resistant phosphorothioate backbone that improves the half-life in the body. These agonists have been tested in cervical carcinoma, sarcoma, breast cancer, chronic lymphocytic leukemia, lymphomas, lung and renal cancer, and glioblastoma.
Although the results are encouraging evidence of the potential clinical benefits of TLR9 activation, the greatest improvement in patient outcomes is likely to result from the use of this approach in combination with other therapies that work in a synergistic manner. However, two phase III trials of PF-3512676 administered concurrently with chemotherapy for patients with lung cancer have reported a lack of incremental efficacy.
The exacerbation of inflammatory response by TLRs cytokine increases the production of antibodies, and several studies have been done to evaluate this overexpression of cytokines as a cause of autoimmune disorders.
Murine studies of lupus have examined whether CpG ODN would induce or flare lupus. These studies have shown increase in anti-DNA antibodies, proteinuria, and glomerulonephritis. Treatment with CpG ODN also exacerbated disease in mice, perhaps because of a more sustained immune stimulatory effect (Krieg and Vollmer 2007).
There are controversial findings of the role of TLR9 in central nervous system autoimmune disorders, including multiple sclerosis. CpG ODN accelerated the development of multiple sclerosis. Furthermore, TLR9−/− and MyD88−/− mice were resistant and partially resistant to multiple sclerosis development, respectively. These findings suggest an association of TLR9 with autoimmune disorders in central nervous system. Nevertheless, in some in vivo models, TLR9 activation has decreased the severity of multiple sclerosis, once mice injected with CpG ODN starting 4 days before multiple sclerosis model induction had a marked reduction in the subsequent disease severity, compared with mice treated with a control ODN (Krieg and Vollmer 2007).
Several investigators have examined the effects of CpG DNA on experimental models of arthritis. Because disease in these models results from Th1-mediated inflammation, TLR9 stimulation would be expected to aggravate the disease process. When injected into joints, CpG ODNs induce a transient self-limited inflammatory arthritis with a peak severity at 3 days, followed by resolution. However, systemic injection of CpG does sensitize the mouse, resulting in an exaggerated arthritis when the mice are subsequently given an intra-articular injection of a low dose of CpG DNA.
TLR9 activation on intestinal epithelial cells suppresses gut inflammation, in contrast to the immune stimulatory effects in immune cells. Mice genetically deficient in TLR9 are susceptible to acute colitis. TLR9 activation promotes the development of T regulatory cells that protect against gut inflammation. These studies suggest that in this anatomic region, TLR9 activation has a constitutive role in preventing inflammation.
Recent evidence has suggested that selective, specific antagonists for TLR9 might be beneficial in certain diseases, such as lupus. Thus, the use of suppressive ODN or novel small molecule TLR inhibitors with a larger safety window and differentiated selectivity may potentially have significant clinical utility.
Therapeutic treatment with TLR9 agonists might protect against intracellular microorganisms. Studies in mice have demonstrated that the innate immune defenses activated by CpG ODNs can protect against a wide range of viral, bacterial, and even some parasitic pathogens (Krieg 2007). The adjuvant therapy increases the pro-inflammatory cytokine synthesis, improving the antigen presentation, macrophage activation, and adequate Th1-like adaptive immune response. Animal models have been shown efficacy in chronic viral infection as hepatitis C and B, and mycobacterial infection by M. tuberculosis.
However, the major application of TLR9 agonist is related with the gold standard vaccine adjuvant, capable of inducing powerful antigen-specific antibody and Th1 cellular immune responses. This strong adjuvant activity results from synergy between TLR9 and B-cell receptor, inhibition of B-cell apoptosis, enhanced IgG class switch DNA recombination, and dendritic cell maturation and differentiation, resulting in enhanced activation of Th1 cells and strong cytotoxic T lymphocyte generation.
In humans, CpG ODNs have been used as adjuvants in controlled randomized trials for hepatitis B vaccination, anthrax vaccine, and influenza vaccine.
In summary, TLR9 exhibit structural features shared by all the other TLRs. TLR9 activate NF-kappaB through the same signal transduction pathway used by other TLRs. TLR9 activation induces powerful Th1-like innate and adaptive immune responses and is emerging as a promising therapeutic approach in several disease fields. TLR9 agonists can provide a temporary protection against diverse pathogen and have become a powerful vaccine adjuvant, inducing faster and stronger humoral and cellular immune responses. However, the cause of some autoimmune disorders has been attributed to TLR9 stimulus or, at least, worse the clinical evolution of autoimmune diseases.