Oligodeoxynucleotide Analogues of Circulating DNA Inhibit dsRNA-Induced Immune Response at the Early Stages of Signal Tr
Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also i
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Anna V. Cherepanova, Zhanna K. Nazarkina, and Pavel P. Laktionov
Abstract
Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also in human primary endothelial and transformed cells (Hela and A431). ODN analogues do not effect dendritic cells activation by poly(I:C). The data obtained indicate that the early stages of the signal transduction cascade are violated by ODN analogues and the effects depend on the cell type. Keywords
Circulating DNA • Double-stranded RNA • Interleukin • Interferon • Innate immunity
Introduction DNA from different sources including free and cell-surface-bound circulating DNA inhibit the poly(I:C)-activated production of pro-
A.V. Cherepanova (*) • Z.K. Nazarkina P.P. Laktionov Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev ave., 8, Novosibirsk 630090, Russia e-mail: [email protected]
inflammatory interleukins (IL-6 and IL-8) in human primary gingival fibroblasts, the inhibiting efficacy depending on the DNA sequence (Cherepanova et al. 2013). To identify the mechanisms of the immuno-inhibiting action of DNA, we investigated the influence of specific oligodeoxynucleotide (ODN) analogues on poly(I:C)induced immuno-mediators, produced via signal transduction pathways different to those for IL-6/ IL-8 and in various cell types which may differ in the expression of pattern-recognizing receptors and peculiarities of downstream pathway functioning.
© Springer International Publishing Switzerland 2016 P.B. Gahan et al. (eds.), Circulating Nucleic Acids in Serum and Plasma – CNAPS IX, Advances in Experimental Medicine and Biology 924, DOI 10.1007/978-3-319-42044-8_20
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Materials and Methods Gingival fibroblasts (GF), endothelial cells (HUVEC) and immature dendritic cells (DC) were obtained from human gingiva, umbilical cord vein and blood monocytes, correspondingly (Mailliard et al. 2004; Cherepanova et al. 2013). Primary and transformed cells (cervical carcinoma (HeLa) and human squamous carcinoma (A431)) were incubated with 100 μg/mL poly(I:C) (Sigma) and/or 1 μM ODN 14 (pctgcatgcatttccttctgcattccagctggat). After treatment, these cells and supernatants were collected for gene expression analysis and IL-6 measurement (Vector-Best, Russia), correspondingly (Cherepanova et al. 2013). Oligo(dT)18 primer and M-MuLV Reverse Transcriptase (Fermentas, Lithuania) were used for reverse transcription of total RNA. SYBRGreen Real-time PCR was performed using specific forward (f) and reverse (r) primers (5′ → 3′): IL-6, (f) tctccacaagcgccttcg, (r) ctcagggctgagatgccg; beta-defensin 2 (HBD-2) (f) ctcctcttctcgttcctcttcata, (r) tagggcaaaagactggatgac; interferon beta (hIFN1b) (f) ccaacaagtgtctcctccaaa, (r) gcagtattcaagcctcccatt; GAPDH (f) ttgacggtgccatggaatttg, (r) acggatttggtcgtattgggc. IL-6, HBD-2 and hIFN1b real-time values were nor-
Fig. 20.1 Inhibiting effects of
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