Biofilm and Planktonic Bacteria Differentially Express a Small Molecule (<3kD), Heat‐Stable, Protease‐Resistant Factor that Affects Bovine Neutrophil Function in a NF‐Kappa β‐Independent Mechanism

Introduction Chronic bacterial infections and the associated inflammation have a significant impact in both veterinary and human medicine. Bacterial biofilms are a major cause of the persistent inflammation observed in chronic infections and nearly 80% of all infections involve biofilms. Biofilms are typically polymicrobial in composition and many of these mixed‐infections involve anaerobic bacteria. Recent evidence demonstrates that immune cells often disregard the signals released from biofilm bacteria, and ineffectual clearance of biofilms may explain the chronicity of this sort of infection. A mixed‐species model of anaerobic biofilm growth was developed to evaluate medically relevant biofilms and their interactions with bovine neutrophils. Aims To generate mixed‐species bacterial biofilms composed of the two opportunistic pathogens Fusobacterium necrophorum and Porphyromonas levii , and to employ the in vitro system to investigate factors associated with biofilms that may limit neutrophil functional ability. Methods Mixed‐species biofilm formation was verified with SEM, CLSM and viable cell counts. Neutrophil oxidative burst was assessed with a fluorescent microplate assay for hydrogen peroxide. Chemotaxis was measured with a Transwell transmigration assay. Extracellular factors released from planktonic cultures and biofilms were assessed by size‐exclusion filtration, lipopolysaccharide (LPS) removal, protease and heat treatment. Mechanistic experiments to study neutrophil responses to these supernatants were conducted with specific inhibitors of function, such as NF‐κβ signaling (BAY‐11) and inflammasome activation (glyburide). Results Neutrophils exposed to planktonic bacterial supernatants showed significantly elevated oxidative (4‐fold increase in fluorescence) and chemotactic responses compared to neutrophils exposed to biofilm products from identical bacteria. LPS plays a significant role in the stimulation of neutrophils; in this experiment biofilms produced substantially more LPS than planktonic bacteria despite inducing less neutrophil response. Removal of LPS resulted in similar neutrophil responses to planktonic and biofilm supernatants. Size‐exclusion filtration of bacterial supernatants and subsequent exposure to neutrophils revealed that the active stimulatory molecule is below 3 kD and is released strictly from planktonic cultures. Intensive heat and protease treatment of the <3kD fractions did not alter neutrophil functional responses. Treatment with a NF‐κβ inhibitor demonstrated that neutrophils respond specifically to planktonic supernatants independently of NF‐κβ signaling. Conclusions We describe a heat‐stable, protease‐resistant, non‐LPS, small (<3kD) factor produced by in vitro planktonic cultures of F. necrophorum and P. levii that activates bovine neutrophils. Neutrophils did not exhibit the same oxidative and chemotactic responses upon exposure to in vitro generated biofilm supernatants and these differential responses are the result of a NF‐κβ independent mechanism, perhaps through an inflammasome‐mediated process. Support or Funding Information Funding provided by the University of Calgary Grant 10002461 to DW Morck.

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