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Transcriptional Response to Particulate Matter Is Not Dependent on the Degree of Mitochondrial Reactive Oxygen Species Generation
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A7563 - Transcriptional Response to Particulate Matter Is Not Dependent on the Degree of Mitochondrial Reactive Oxygen Species Generation
Author Block: K. Sun, A. Y. Meliton, Y. Li, J. Andrade, P. S. Woods, D. Wu, R. B. Hamanaka, G. M. Mutlu; The University of Chicago, Chicago, IL, United States.
Rationale: Particulate matter (PM) air pollution is associated with significant morbidity and mortality. We previously discovered that alveolar macrophages (AMs) and AM-derived IL-6 are required for PM-induced prothrombotic state and vascular thrombosis. We have also shown that mitochondrial reactive oxygen species (ROS) are required for PM-induced IL-6 release from alveolar macrophages. However, how varying levels of ROS production might affect the PM-induced transcriptional response is not known. We sought to determine the effect of the degree of mitochondria ROS generation on transcriptional response in macrophages. Methods: Hematopoetic cells were isolated bone marrow of C57BL/6 mice and cultured with M-CSF in vitro for 8-10 days generate bone marrow derived macrophages (BMDMs). BMDMs were then treated with either control (PBS) or PM (10 or 20 μg/cm2). After 24 hours, mitochondrial ROS levels were measured using MitoSOX-Red by flow cytometry. Cells were flow-sorted based on the degree of ROS generation (low vs. high levels). We then isolated RNA for sequencing (50bp SE). Raw data was submitted for analysis. Quality control assessment on original raw sequencing data and post-alignment mapping were done. Using a FC>2.0 and FDR adjusted p value of 0.05, we identified differential expressed gene (DEG) profiles among different groups. Results: We found that PM 20 μg/cm2 induced greater level of MitoSOX oxidation and caused higher number of DEGs than PM 10 μg/cm2. At both 10 and 20 μg/cm2, the majority (68% vs 67.2%) of the PM-induced DEGs were similar between macrophages exhibiting high or low ROS levels. Interestingly, the magnitude of change in the expression of the top 250 genes was also similar between macrophages exhibiting high ROS levels compared with those exhibiting low ROS levels. Gene ontology analysis showed that response to oxidative stress, response to stress, and inflammatory response were among the top processes induced by PM. Cells exhibiting low and high ROS levels showed 68 and 137 unique DEGs, respectively. Conclusions: These results demonstrate that gene expression to PM is dose dependent. We also found that the majority of the PM-induced transcriptional signature is not affected by the degree of mitochondrial ROS generation. These results suggest that mitochondrial ROS likely have a threshold effect on the transcriptional response.
Author Block: K. Sun, A. Y. Meliton, Y. Li, J. Andrade, P. S. Woods, D. Wu, R. B. Hamanaka, G. M. Mutlu; The University of Chicago, Chicago, IL, United States.
Rationale: Particulate matter (PM) air pollution is associated with significant morbidity and mortality. We previously discovered that alveolar macrophages (AMs) and AM-derived IL-6 are required for PM-induced prothrombotic state and vascular thrombosis. We have also shown that mitochondrial reactive oxygen species (ROS) are required for PM-induced IL-6 release from alveolar macrophages. However, how varying levels of ROS production might affect the PM-induced transcriptional response is not known. We sought to determine the effect of the degree of mitochondria ROS generation on transcriptional response in macrophages. Methods: Hematopoetic cells were isolated bone marrow of C57BL/6 mice and cultured with M-CSF in vitro for 8-10 days generate bone marrow derived macrophages (BMDMs). BMDMs were then treated with either control (PBS) or PM (10 or 20 μg/cm2). After 24 hours, mitochondrial ROS levels were measured using MitoSOX-Red by flow cytometry. Cells were flow-sorted based on the degree of ROS generation (low vs. high levels). We then isolated RNA for sequencing (50bp SE). Raw data was submitted for analysis. Quality control assessment on original raw sequencing data and post-alignment mapping were done. Using a FC>2.0 and FDR adjusted p value of 0.05, we identified differential expressed gene (DEG) profiles among different groups. Results: We found that PM 20 μg/cm2 induced greater level of MitoSOX oxidation and caused higher number of DEGs than PM 10 μg/cm2. At both 10 and 20 μg/cm2, the majority (68% vs 67.2%) of the PM-induced DEGs were similar between macrophages exhibiting high or low ROS levels. Interestingly, the magnitude of change in the expression of the top 250 genes was also similar between macrophages exhibiting high ROS levels compared with those exhibiting low ROS levels. Gene ontology analysis showed that response to oxidative stress, response to stress, and inflammatory response were among the top processes induced by PM. Cells exhibiting low and high ROS levels showed 68 and 137 unique DEGs, respectively. Conclusions: These results demonstrate that gene expression to PM is dose dependent. We also found that the majority of the PM-induced transcriptional signature is not affected by the degree of mitochondrial ROS generation. These results suggest that mitochondrial ROS likely have a threshold effect on the transcriptional response.
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