This identifies DNA-PK as a potential therapeutic target in asthma and can serve as the basis for the future development of DNA-PK inhibitors as a new treatment approach for patients with asthma. Methods Reagents NU7441 was from Tocris Bioscience (Bristol, United Kingdom). DNA-PK in DCs, attenuates the induction of allergic Rabbit Polyclonal to RBM16 sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced L-Asparagine monohydrate airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma. Introduction DNA-dependent protein kinase (DNA-PK) is a key enzyme involved in the recognition and repair of double stranded DNA breaks (DSB) by a process termed nonhomologous end-joining (NHEJ) whereby DNA ends are directly ligated1, 2, 3, 4. This represents an important mechanism of cellular repair in response to DSB induced by ionizing radiation, as well as reactive oxygen species, that prevents chromosomal translocations and genetic instability that can lead to carcinogenesis or cellular death2, 5. DNA-PK is comprised of a regulatory heterodimer of Ku proteins (Ku70 and Ku80) and a 465 kDa catalytic subunit, DNA-PKcs, which is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and functions as a serine/threonine kinase. DNA-PK also participates in additional processes that involve DSB repair, such as V(D)J recombination and class switch recombination2, 6. Mice with the mutation affecting the gene that encodes DNA-PKcs are unable to generate functional immunoglobulin and T cell receptors and are deficient in mature B and T lymphocytes7, 8, 9, 10. The missense mutation results in a premature stop codon that leads to diminished expression of the DNA-PKcs protein and specifically impairs the differentiation of stem cells into mature lymphocytes, whereas myeloid differentiation is not affected9, 11, 12. Similarly, mice with a targeted disruption of the mice stimulated with HDM (100 g/ml) for 1 h (n = 3, * P 0.01, WT + HDM vs. + HDM, one way ANOVA with Bonferroni multiple comparison test). D. CD11c+ BMDC from and WT mice were pulsed with the ovalbumin (OVA) 323C339 peptide and incubated at a 1:5 ratio with CSFE-labeled CD4+ DO11.10 T cells for 4 days. OVA-specific proliferation is presented as proliferation index (n L-Asparagine monohydrate = 8, * P = 0.0011, Mann Whitney test, pooled data from 3 independent experiments). E C G. Th2 cytokines released by co-cultures of OVA 323C329-pulsed BMDCs and CSFE-labeled DO11.10 CD3+/CD4+ T cells (n = 3, *P 0.05, WT + OVA vs. + OVA, one L-Asparagine monohydrate way ANOVA with Bonferroni multiple comparison test). H. Co-cultures of BMDCs from and WT mice incubated at a 1:5 ratio with splenic CD4+ T cells from WT mice sensitized to full-length OVA. Co-cultures were treated with PBS or OVA (1 g/ml) for 4 days and Th2 cytokines were quantified (n = 7, * P 0.05). Pooled data from 2 independent experiments. I. MFI of CD40, CD80 and CD86 cell surface expression by murine BMDCs (n = 6 mice) and human moDCs (n = 10 mice) stimulated with or without HDM (100 g/ml) for 24 h. Pooled data from 2 independent experiments (* P 0.05, Mann Whitney test). L-Asparagine monohydrate Experiments were next performed with bone marrow-derived dendritic cells (BMDCs) from mice that have a spontaneous mutation in the gene. HDM-challenged BMDCs from wild type (WT), but not mice, had increases in intracellular ROS generation (Figure 1C). Thus, in addition to being activated by ROS, DNA-PK is required for HDM-induced ROS production by BMDCs. Next, BMDCs from mice were used to investigate the role of DNA-PK in antigen-specific T cell proliferation and Th2 cytokine production. Co-culture experiments of splenic CD4+ T cells from mice expressing the MHCII-restricted DO11.10 T-cell receptor that recognizes the OVA 323C339 peptide showed that BMDCs from mice have a reduced ability to induce both T cell proliferation and Th2 cytokine production as compared to BMDCs from WT mice following stimulation with the OVA 323C339 peptide (Figures 1DCG)24. Similarly, BMDCs from mice had an impaired.
