VCOM Louisiana Research Day Program

Biomedical Research

Stephanie Torres, OMS-III; Rebekah L. Morrow, PhD; Olivia Bee, OMS-II; Sarah Voth, PhD; K. Adam Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana 11 AMYLOID PRECURSOR PROTEIN MODULATES INFLAMMATORY RESPONSE TO PSEUDOMONAS AERUGINOSA INFECTION

Context: Nosocomial Pseudomonas infections amount to a great deal of distress within the healthcare system. Bacterial pneumonia has been shown to cause short- and long-term cognitive dysfunction as sequelae in a high proportion of patients. The mechanism(s) responsible for this effect are still largely unknown. It is theorized that the secondary proteinopathy following a nosocomial infection could propagate, leading to known gross hallmarks of Alzheimer’s disease. Here we attempt to elucidate the relationship between Pseudomonas infection, amyloid production, and downstream inflammatory agents such as ApoE and cytokine MCP-1. Objective: The goal of this study was to determine whether amyloid precursor protein knock out (APPKO) enhanced sensitivity of pulmonary microvascular endothelial cells to Pseudomonas aeruginosa infection and contribute to a pro-inflammatory phenotype. Methods: We utilized RTPCR on RNA that was isolated from 5 independent cell lines to stratify our experimental cell lines based on amyloid measurements. The top phenotypes after amyloid screen were subjected to Western Blot for further characterization of amyloid expression, utilizing densitometry for

confirmation. Cellular inoculation with ∆PcrV and ExoY bacterial strains was conducted in 6 well plates with our gRNA and APPKO cell types, to determine if there were differences between them to monolayer damage following inoculation with either pseudomonas strain. Supernatant collected 6 hours post-infection from cell wells were also characterized via western blot for amyloid and ApoE expression with Densitometry for confirmation. Quantification of MCP-1 from the same infectious supernatant was completed via ELISA. Results: We confirmed APPKO via RT-PCR and western blot. At baseline, APPKO cells had 2 fold higher levels of ApoE and 4 fold higher MCP-1 compared to control cells. P. aeruginosa infection caused greater barrier disruption in APPKO cells compared to control. In control cells, P. aeruginosa exoenzyme Y abrogated MCP-1 increase seen with the control strain (∆PcrV), while lack of APP correlated with higher MCP-1 levels in response to infection with both bacterial strains. Conclusions: We concluded that lack of APP contributes to a pro-inflammatory phenotype in pulmonary microvascular endothelial cells, and APP is required, at least in part, to protect against Pseudomonas aeruginosa infection.

23 2023 Via Research Recognition Day