Wildfire smoke-induced secretions from RAW264.7 macrophages cause inflammatory activation of primary C57BL/6 mixed glia
Category: Research Poster
Author(s): Zachary Fields, Julie Moreno, Luke Montrose
Presenter(s): Zachary Fields
Mentors(s): Julie Moreno
Zach Fields 1, 3, Sean Boland 2, Luke Montrose 1, and Julie A. Moreno 1, 3 1Environmental Health and Radiological Department, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Colorado State University, Fort Collins, Co 80525 2Microbiology, Immunology, and Pathology Department, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Colorado State University, Fort Collins, CO 80525 3Brain Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80525 Wildfire smoke (WFS) in the environment is a large source of particulate matter (PM) exposure, due to increased wildfire activity. Smoke resulting from these conditions have been associated with respiratory and neurological outcomes derived from increased inflammation in the lungs causing systemic inflammation and leading to neuroinflammation in the brain, but the direct outcomes in the brain remain unclear. To understand the effects of the 2.5m PM fraction (PM2.5) on the inflammatory signaling of the brain we established an in vitro indirect glial exposure method using PM2.5 ¬generated from a 50:50 mixture of Eucalyptus: Manzanita (E/M)—a common wildfire smoke source in California and Australia. We hypothesize that increased E/M PM2.5 exposure elicits secretion of pro-inflammatory mediators in macrophage cells that will lead to increased pro-inflammatory gene transcripts in glial cells. In this experiment supernatants from murine RAW264.7 macrophages exposed to WFS PM2.5 doses ranging from 0.01-10 ug/mL media was collected 24 hours later and stored at –80C for exposure to glia cultures. Using primary C57BL/6 mixed glia, we exposed the cells to the various macrophage supernatants ¬dosed with WFS PM2.5 for 24 hours. Following exposure we collected cell lysates, isolated RNA and performed cytokine gene profiling of IL-1b, TNF-a,, C3, C1QA, CCL2, and CCL5 using quantitative real-time PCR (qPCR) with GAPDH as our loading control. We are also performing immunocytochemistry to observe the expression of the inflammatory signaling pathway transcription factor subunit, p65, in the mixed glial cultures. Our results show how exposure of the macrophage supernatant on mixed glial cultures generated upregulation of proinflammatory gene transcripts such as IL-1b, C3, CCL2, CCL5 and TNF-a at the 24-hour time point. Currently we are analyzing the p65 nuclear translocation in these glial cells. Overall, our findings show that indirect exposure to wildfire smoke PM2.5 macrophage supernatant increases inflammatory pathway activation and protein expression in mixed glia. This foundational in vitro work allows for us to develop hypotheses for potential studies investigating specific neuroinflammatory signaling molecules in vivo to identify the long-term effects of WFS PM2.5.