Virginia Research Day 2021

Graduate Student Research Biomedical

08 Inflammasome-Mediated Inflammatory Cell Death Protects Against the Zoonotic Disease Brucellosis

Juselyn D Tupik 1 ; Sheryl L. Coutermarsh-Ott 1 ; Angela H Benton 1 ; Kellie A. King 1 ; Hanna D. Kiryluk 1 ; Clayton C. Caswell 1 ; Irving C. Allen 1,2 Corresponding author: jdtupik@vt.edu

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 2 Department of Basic Science Education, Virginia Tech Carilion School of Medicine

Brucellosis is a zoonotic disease caused by the bacteria genus Brucella that leads to inflammatory pathogenesis. Once transmitted to humans, often from agricultural animals through unpasteurized dairy products, brucellosis symptoms include inflammatory or flu-like characteristics such as recurring fevers, arthritis, and neurological symptoms. Currently, there is no human vaccine for brucellosis, and other therapeutics such as antibiotic regiments require lengthy treatment durations. This is because Brucella’s unique lipopolysaccharide (LPS) outer membrane layer and location primarily within immune cells called macrophages can often lead to immune evasion and further persistence of brucellosis. However, components of the innate immune system known as pattern recognition receptors are able to sense Brucella pathogen-associated molecular patterns (PAMPS). Upon sensing PAMPS such as bacterial DNA, NOD-like receptors (NLRs) can form a multi-protein signaling complex known as the inflammasome to attenuate Brucella pathogenesis.

This occurs through inflammasome activation of the pro-inflammatory cytokines IL-1ß and IL-18 to drive immune cell recruitment and subsequent inflammation. Alternatively, inflammasome activation can also initiate inflammatory cell death called pyroptosis, which through the cleavage of the protein gasdermin D leads to cell lysis to enhance bacteria clearance. In this study, we assessed inflammasome activation following Brucella abortus infection. We conducted in vivo studies infecting inflammasome ( Asc -/- ) knockout mice with B. abortus and found that in comparison with wildtype mice, inflammasome knockout mice exhibited increased mortality, decreased immune cell recruitment, and increased bacteria load. This suggests that the inflammasome plays a protective role in regulating brucellosis. To further define this mechanism, we conducted studies in vitro using inflammasome (Asc-/-) knockout macrophages. We found that the protective role of the inflammasome may result from the initiation

of pyroptosis through the cleavage of gasdermin D. Furthermore, looking at immune recognition of Brucella PAMPs, our results showed that only genomic DNA instigated a high inflammatory response that did not occur in inflammasome knockout cells. This suggests that inflammasome activation occurs through sensing gDNA instead of the unique Brucella LPS layer. Ultimately, these in vivo and in vitro results further define our understanding of inflammasome activation and subsequent initiation of pyroptosis during brucellosis. Additionally, these results support the creation of novel treatments designed to augment inflammasome activation in order to protect against brucellosis.

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