Anaerobic Bacterial Biofilms Inhibit Neutrophil Responses via the Release of Stable Small‐Molecule Effectors

Abstract

Background and ObjectiveFusobacterium and Porphyromonasare two genera of opportunistic pathogens that often aggregate in complex communities known as biofilms. Host innate immune cells are frequently frustrated when they encounter biofilm bacteria, resulting in ineffectual clearance and persistent infection. Anaerobic pathogens generate or accumulate organic molecules and use them as defense against environmental pressures such as oxidative stress or attack from host immune cells. Porphyromonasspecies utilize protoporphyrin IX (PPIX) to protect against neutrophil oxidative‐killing. The objective was to develop a model of mixed‐species biofilm generation and to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that biofilm‐conditioned growth medium would contain more PPIX than planktonic medium and therefore inhibit the functional responses of peripheral neutrophils.MethodsNeutrophils were isolated from whole blood via hypotonic lysis and bacterial biofilms were generated using previously validated methods for the cultivation of mixed‐species biofilms. Spent media from biofilm and overnight planktonic cultures were collected, centrifuged, and filtered to remove viable bacteria. Supernatants were fractionated via <3kDa‐cutoff filtration and exposed directly to neutrophils. Neutrophil responses to the supernatants were assessed with a fluorescent microplate assay for reactive oxygen species (ROS) and chemotaxis was measured with a Transwell® transmigration assay. The specific mechanism of neutrophil activation was investigated by employing NF‐κB and NADPH oxidase inhibitors.ResultsNeutrophils exposed to planktonic supernatant showed significantly elevated oxidative and chemotactic responses compared to biofilm products from identical bacteria. Ultra‐filtration revealed that a <3kDa molecule is differentially expressed in the supernatants and allows biofilm‐bacteria to inhibit neutrophil responses. Intensive heat, nuclease, lipase, and protease treatment of the <3kDa fractions did not alter neutrophil functional responses. Preliminary experiments suggest that the accumulation of PPIX in the biofilm supernatant attenuates neutrophil chemotaxis and ROS production. In addition, chemical inhibition experiments indicated that neutrophils respond to planktonic supernatants independently of NF‐κB signaling.ConclusionsExperiments reveal a minimal neutrophil response to molecules released from biofilm bacteria and may explain the inadequate host response to biofilm‐mediated infections. The phenotypic changes in the biofilm mode of growth allow for the generation and accumulation of PPIX, providing protection from host immune clearance mechanisms. Understanding the mechanism of immune avoidance is a crucial step to reduce the persistent inflammation and negative clinical outcomes associated with chronic biofilm‐mediated disease.