Virginia Research Day 2021

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 , and Irving C. Allen 1,2 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 2 Department of Basic Science Education, Virginia Tech Carilion School of Medicine, Roanoke, VA

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-1 β 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.

Defining Phenotype of Brucella Infection The inflammasome attenuates Brucella pathogenesis and is critical for host survival

Determining Mechanism of Immune Protection

Brucella abortus gDNA provokes an inflammatory response

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Figure 3: Brucella Infected −/− mice show lower % survival Asc −/− ( n = 19) and WT ( n = 26) mice were injected intraperitoneally with 1 × 10 5 B. abortus CFUs and assessed daily for excessive weight loss (>20%) warranting euthanasia according to the Institutional Animal Care and Use Committee (IACUC). All the mice were weighed for a 24-day period, with morbidity of 5 Asc −/− mice (26.3%) warranting euthanasia only at Day 7. * p < 0.05

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Figure 7: Infected −/− BMDMs exhibit decreased IL-1 β protein in response to B. abortus gDNA Bone marrow-derived macrophages (BMDMs) (500,000 cells per well, n = 3 per group) were harvested from C57BL/6 WT and Asc −/− mice and challenged with (A) a MOI 100:1 (5 x 10 7 CFUs) of live Brucella abortus or (B) 1 μ g/mL gDNA internally into the BMDMs or externally into the supernatant . BMDMs were analyzed for IL-1 β protein in the supernatant for a 48-hour period using an ELISA. ( A ) Live Brucella infection had no significant difference between cell types. (B) Internally introduced gDNA at 24 hours provoked a significant IL-1 β protein response in WT macrophages.* p < 0.05 **** p < 0.0001

The inflammasome contributes to elevated inflammatory pathogenesis

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The inflammasome induces pyroptosis (cell death) in macrophages

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Figure 4: Brucella Infected −/− mice show a significant decrease in inflammation of the liver A total of 34 C57BL/6 WT ( n = 11 uninfected [U], 23 infected [I]) and 20 Asc −/− ( n = 6 U, 14 I) livers and spleens were evaluated by histopathology 3 d.p.i. (A) H&E stained histological slides of the liver and spleen from WT and Asc −/− mice. All spleen images were taken at 4 × power and all liver images were taken at 40 × power. Inflammation and extramedullary hematopoiesis (EMH) were the dominant features observed in the histopathology evaluation. (B) Bar graphs of WT and Asc −/− histopathology composite scores were generated based on inflammation and EMH in the liver and spleen, with significant inflammation occurring in WT spleens versus Asc -/- .* p < 0.05, ** p < 0.01.

Figure 8: −/− BMDMs exhibit decreased pyroptosis after Brucella abortus infection BMDMs were lysed at 24 hours post-challenge and used for Western blot analysis. BMDM protein (20 μ g) was probed for cleaved gasdermin D and β -actin as a control for the protein amount. Invitrogen IBright Analysis was used to determine the density ratio between the cleaved gasdermin D over β -actin. There was more cleaved gasdermin D protein in infected WT macrophages than Asc -/- macrophages.

The inflammasome attenuates bacterial load in the liver

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Inflammasome Drug Therapies

Figure 5: Brucella Infected Asc -/- mice show increased Brucella CFUs in the liver Three d.p.i., liver and spleens from infected C57BL/6 WT ( n = 23) and Asc −/− ( n = 14) mice were homogenized and counted for CFUs/gram. There was significantly elevated CFUs in the liver of Asc -/- mice. * p < 0.05

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Figure 1: Brucella Challenge in Mice C57BL/6 WT & −/− mice were injected intraperitoneally with live B. abortus (10 5 CFUs) to initiate a mild infectious response for two different protocols. First, mice were measured for % survival for a 24-day period. Secondly, mice were culled on Day 3 at the height of innate immune activation in order to harvest spleens and livers. In these organs, we measured immune cell recruitment through histopathology, number of B. abortus colony forming units (CFUs) to assess bacteria load, and pro-inflammatory protein IL-1 β with ELISA kits.

The inflammasome initiates a weak inflammatory protein response

Figure 6: Brucella Infected WT and Asc -/- mice show no significant difference in IL-1 β proteins Three d.p.i., livers and spleens of 34 C57BL/6 WT ( n = 11 U, 23 I) and 20 Asc −/− ( n = 6 U, 14 I) mice were homogenized and analyzed for their IL-1 β protein concentration through ELISA. There was only a significance difference in protein concentration between uninfected and infected WT mice in the liver. * p < 0.05.

Figure 2: Brucella Challenge in Bone Marrow-Derived Macrophages (BMDMs) Bone marrow from the tibias and femurs of C57BL/6 WT and −/− mice were harvested on Day 1 for white blood cells (WBCs). WBCs were cultured Day 2-6 in L929 conditioned media (LCM) in order to differentiate the cells into macrophages (BMDMs). On Day 7, macrophages were plated at 5 x10 5 cells/well and challenged with 3 − 6 x 10 7 CFUs of live B. abortus (MOI 100:1). Additionally, 1 μ g/mL of B. abortus genomic (g)DNA was introduced externally into media and internally through the Lipofectamine 3000 protocol. Supernatant was measured for the pro-inflammatory protein IL-1 β throughout a 48-hour period using ELISA kits. Gasdermin D cleavage was measured through Western blot at 24 hours.

All models were produced using BioRender and all data figures were generated using Prism.

For questions, please contact jdtupik@vt.edu

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