DNA DAMAGE RESPONSES IN AEROALLERGEN-INDUCED ASTHMA

Ph.DDOCTOR OF PHILOSOPH

Metabolomics Investigation Reveals Metabolite Mediators Associated with Acute Lung Injury and Repair in a Murine Model of Influenza Pneumonia

Influenza virus infection (IVI) can cause primary viral pneumonia, which may progress to acute lung injury (ALI) and respiratory failure with a potentially fatal outcome. At present, the interactions between host and influenza virus at molecular levels and the underlying mechanisms that give rise to IVI-induced ALI are poorly understood. We conducted a comprehensive mass spectrometry-based metabolic profiling of serum, lung tissue and bronchoalveolar lavage fluid (BALF) from a non-lethal mouse model with influenza A virus at 0, 6, 10, 14, 21 and 28 days post infection (dpi), representing the major stages of IVI. Distinct metabolite signatures were observed in mice sera, lung tissues and BALF, indicating the molecular differences between systematic and localized host responses to IVI. More than 100 differential metabolites were captured in mice sera, lung tissues and BALF, including purines, pyrimidines, acylcarnitines, fatty acids, amino acids, glucocorticoids, sphingolipids, phospholipids, etc. Many of these metabolites belonged to pulmonary surfactants, indicating IVI-induced aberrations of the pulmonary surfactant system might play an important role in the etiology of respiratory failure and repair. Our findings revealed dynamic host responses to IVI and various metabolic pathways linked to disease progression, and provided mechanistic insights into IVI-induced ALI and repair process.National Science Foundation (U.S.)Singapore-MIT Alliance for Research and Technology (SMART

Vitamin E isoform gamma-tocotrienol protects against emphysema in cigarette smoke-induced COPD

10.1016/j.freeradbiomed.2017.06.023FREE RADICAL BIOLOGY AND MEDICINE110332-34

House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs

Background Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. Objective We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) in vivo and in vitro. Methods We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. Results HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX) foci in bronchial epithelium. BEAS-2B cells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. Conclusion This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology

Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage

Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. Conclusions: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS

Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage

10.3389/fimmu.2018.00891FRONTIERS IN IMMUNOLOGY9MA

Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of 2 weeks in vivo. We show that influenza induces DNA damage to both, when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67-positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.Singapore National Research Foundatio

Table_2_Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage.docx

<p>Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. Conclusions: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS.</p

Table_1_Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage.docx

<p>Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. Conclusions: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS.</p