6th Annual Judith Ramaley Celebration of Research and Creative Scholarship
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Poster #56 Cell Death Caused by M. Haemolytica and its Leukotoxin
Chelsea Humpal and Nate Reimnitz
Faculty Mentor: Nicole Aulik
Bovine respiratory disease (BRD) is a multifactorial disease that causes pleuropneumonia in cattle. It is characterized by intense inflammation, vast neutrophil infiltration, fibrin deposition, and consolidation of the lungs. Extracellular DNA has also been detected within the airways of cattle with BRD. The extracellular DNA build up that forms an antimicrobial-studded DNA fibrillar network, is also known as the phenomenon extracellular trap formation. Extracellular trap formation is one of several forms of leukocyte cell death. In the body, macrophages provide the first line of cellular defense when the lungs are threatened with infection. Our laboratory has demonstrated that macrophages produce a type of extracellular trap in response to Mannheimia haemolytica or leukotoxin (LKT) known as macrophage extracellular traps (ETs). Macrophages are sentinel cells that signal neutrophils to enter the lungs in high numbers. We have also shown that bovine neutrophils produce neutrophil ETs in response to M. haemolytica and its LKT in vitro. It has long been theorized that the leukotoxin induces necrosis at high concentrations and apoptosis at low concentrations. Here, we quantify necrotic, apoptotic, and ET formation of monocyte-derived bovine macrophages in response to the M. haemolytica LKT. We observed all three forms of cell death using fluorescent microscopy using a three dye system. The first dye, Hoechst, is part of a family of blue fluorescent dyes used to stain DNA, in particular apoptotic condensed chromatin. This dye is excited by ultraviolet light and also helps us observe extracellular trap formations as long strands of DNA. The Yo-Pro-1 dye is a green fluorescent dye which has the ability to permeate apoptotic cell membranes, but will not stain live, healthy cells. When combined with propidium iodide (PI), which stains necrotic phospholipids, we are able to distinguish between cells that are undergoing apoptosis and cells which are undergoing necrosis. This is because PI (red) can only enter a cell after the membrane’s integrity is lost. These data indicate that the M. haemolytica LKT causes necrosis, apoptosis and ET formation by bovine macrophages. This provides information on how the host leukocytes interact with LKT leading to three different forms of cell death, which could enhance our understanding of how the lungs are consolidated during BRD. |
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