DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4+ T-cell function without causing severe combined immunodeficiency

Abstract

Background We reported that DNA-PK is critical for the expression of NF-κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. Objective To investigate the role of DNA-PK in asthma. Methods Cell culture and ovalbumin or house dust mite (HDM)-based murine asthma models were used in this study. Results DNA-PK was essential for monocyte adhesion to TNF-α–treated-endothelial cells. Administration of the DNA-PK inhibitor, NU7441, reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA-specific IgE production in mice that were pre-challenged with ovalbumin. Such effects correlated with a marked reduction in lung VCAM-1 expression and production of several cytokines, including IL-4/IL-5/IL-13/eotaxin/IL-2/IL-12 and the chemokines MCP-1/KC with negligible effect on IL-10/IFN-γ production. DNA-PK inhibition, by gene heterozygosity, also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma using HDM, a human allergen. Remarkably, such protection occurred without causing SCID. Adoptive transfer of Th2-skewed OT-II-WT CD4+ T cells reversed IgE and Th2 cytokine production but not AHR in ovalbumin-challenged DNA-PKcs+/− mice. DNA-PK inhibition reduced IL-4/IL-5/IL-13/eotaxin/IL-8/MCP-1 production without affecting IL-2/IL-12/IFN-γ/IP-10 production in CD3/CD28-stimulated human CD4+T cells potentially by blocking expression of gata-3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4+T cells, DNA-PK inhibition, in vitro, severely blocked C3/CD28-induced gata-3 and t-bet expression in CD4+T cells and prevented differentiation of Th1 and Th2 cells under respective Th1- and Th2-skewing conditions. Conclusion Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

Fuelling the mechanisms of asthma: Increased fatty acid oxidation in inflammatory immune cells may represent a novel therapeutic target

“…Although measuring lung resistance is considered a more reliable way of evaluating the lung function, Penh has been shown to correlate well with invasive measures of AHR . Moreover, our studies consistently showed an increased AHR in allergen‐exposed C57BL6 mice compared to control mice . In line with these reports, AHR to methacholine was significantly increased in OVA‐sensitized and challenged mice versus controls, while etomoxir‐treated mice had a significant reduction in AHR (Figure B).…”

“…For the OVA‐induced asthma model, mice were sensitized by intraperitoneal injection of 50 μg grade V chicken OVA (Sigma‐Aldrich) mixed with 2 mg aluminium hydroxide in saline once a week for 2 weeks. After two weeks, mice were challenged with aerosolized 3% OVA for 30 minutes . Mice were left untreated or treated with intraperitoneal injection of the CPT1 inhibitor etomoxir (Sigma‐Aldrich, St. Louis, MO) or the HADHA inhibitor ranolazine (Sigma‐Aldrich) at 50 mg/kg (both diluted in saline) 30 minutes after challenge.…”

“…Control mice were injected with 0.2 mL control (PBS) liposomes (ClodronateLiposomes.com). For the HDM‐induced asthma model, mice were anaesthetized with isoflurane and sensitized intranasally with 25 μL saline (control) or 1 mg/mL whole HDM ( Dermatophagoides pteronyssinus ) extract (Greer Labs Lenoir, NC) three times a week for 5 weeks . Mice were euthanized two days after the last HDM exposure to determine the expression of FAO mediators (Figure C).…”

“…After two weeks, mice were challenged with aerosolized 3% OVA for 30 minutes. 14,15 Mice were left untreated or treated with intraperitoneal injection of the CPT1 inhibitor etomoxir (Sigma-Aldrich, St. Louis, MO) or the HADHA inhibitor ranolazine (Sigma-Aldrich) at 50 mg/kg (both diluted in saline) 30 minutes after challenge. Mice received another intraperitoneal injection of etomoxir or ranolazine 24 hours later (30 minutes after AHR assay).…”

Sulfated non-anticoagulant heparin blocks Th2-induced asthma by modulating the IL-4/signal transducer and activator of transcription 6/Janus kinase 1 pathway

“…injection twice, once a week as previously described [16, 17]. Mice were then challenged with aerosolized 3% OVA for 30 min once for the acute model or 3 times per week for 4 weeks for the chronic model.…”