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CpG-containing immunostimulatory DNA sequences elicit TNF-alpha-dependent toxicity in rodents but not in humans.

ABSTRACT: CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

SUBMITTER: Campbell JD

PROVIDER: S-EPMC2735936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the function of microbial CpG-dideoxynucleotides containing DNA (CpG-DNA) are potent immune stimuli. The immunostimulatory activity and the species-specific activities of a CpG-ODN depend on its nucleotide sequence properties, including CpG-hexamer motif types, spacing between motifs, nucleotide sequence, and length. Toll-like receptor (TLR) 9 is the cellular receptor for CpG-ODNs in mammalian species, while TLR21 is the receptor in avian species. Mammalian cells lack TLR21, and avian cells lack TLR9; however, both TLRs are expressed in fish cells. While nucleotide sequence properties required for a CpG-ODN to strongly activate mammalian TLR9 and its species-specific activities to different mammalian TLR9s are better studied, CpG-ODN activation of TLR21 is not yet well investigated. Here we characterized chicken and duck TLR21s and investigated their activation by CpG-ODNs. Chicken and duck TLR21s contain 972 and 976 amino acid residues, respectively, and differ from TLR9s as they do not have an undefined region in their ectodomain. Cell-based TLR21 activation assays were established to investigate TLR21 activation by different CpG-ODNs. Unlike grouper TLR21, which was preferentially activated by CpG-ODN with a GTCGTT hexamer motif, chicken and duck TLR21s do not distinguish among different CpG-hexamer motifs. Additionally, these two poultry TLR21s were activated by CpG-ODNs with lengths ranging from 15 to 31 nucleotides and with different spacing between CpG-hexamer motifs. These suggested that compared to mammalian TLR9 and grouper TLR21, chicken and duck TLR21s have a broad CpG-ODN sequence recognition profile. Thus, they could also recognize a wide array of DNA-associated molecular patterns from microbes. Moreover, CpG-ODNs are being investigated as antimicrobial agents and as vaccine adjuvants for different species. This study revealed that there are more optimized CpG-ODNs that can be used in poultry farming as anti-infection agents compared to CpG-ODN choices available for other species.

Project description:BackgroundMammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.ResultsIn this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60-80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG→TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG→TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG→TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection. On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA→CpG substitution rate compared with those with SPM-LM.ConclusionsRelatively high numbers (approximately 60-80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG→TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA→CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

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CpG-containing immunostimulatory DNA sequences elicit TNF-alpha-dependent toxicity in rodents but not in humans.

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