Characterization of Campylobacter jejuni–Neutrophil Interactions

Abstract

Campylobacter jejuni is the leading cause of bacterial‐derived gastroenteritis worldwide, infecting 96 million individuals annually. During infection, inflammation and tissue pathology occur in the lower gastrointestinal tract, including the recruitment of leukocytes. Neutrophils are the most abundant leukocyte in humans, and recruitment is associated with bacterial infections and the development of various inflammatory disorders, including inflammatory bowel disease. Neutrophils possess three main antibacterial functions: phagocytosis and degradation of microbes, degranulation to release antimicrobial proteins, and extrusion of neutrophil extracellular traps (NETs). Because neutrophils are recruited to the site of C. jejuni infection and they are associated with damaging inflammation in other diseases, it is imperative to understand the immunopathology that occurs during C. jejuni infection and thoroughly study the neutrophil response to the pathogen. Detailed protocols for human and ferret neutrophil isolations, neutrophil gentamicin protection assay, neutrophil activation flow cytometry assay, NET induction and quantification, and neutrophil western blot analysis are included in this article. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human and ferret neutrophils Basic Protocol 2: Neutrophil gentamicin protection assay Basic Protocol 3: Neutrophil activation flow cytometry analyses Basic Protocol 4: Neutrophil extracellular trap induction and quantification Basic Protocol 5: Western blot detection of neutrophil‐derived antimicrobial proteins

Battle for the histones: a secreted bacterial sirtuin from Campylobacter jejuni activates neutrophils and induces inflammation during infection

“…Neutrophils were isolated as previously described (Callahan et al, 2020, 2021b). Briefly, 10 ml of blood was drawn from healthy donors into heparinized Vacutainer tubes and mixed 1:1 with sterile 1× PBS (approval UTK IRB-18-04604-XP) (Akhtar et al, 2010).…”

“…Flow cytometry detection of C. jejuni induced NETs was performed as previously described (Callahan et al, 2020, 2021b). Briefly, C. jejuni strains were resuspended in RPMI 1640 and incubated with neutrophils for 3 hours under microaerophilic conditions at an equal multiplicities of infection (MOI) of 50:1 (bacteria: neutrophil).…”

“…SYTOX NET quantification was performed as previously described (Callahan et al, 2020, 2021b). Briefly, Following the C. jejuni NET induction described above, cells were added to wells of black 96-well plates and centrifuged at 400 x g .…”

Campylobacter jejuni induces differentiation of human neutrophils to the CD16hi/CD62Llo subtype which possess cancer promoting activities

“…Isolation of primary human neutrophils: Human neutrophils were isolated from venous blood from healthy adult volunteers as described previously and in accordance with the Institutional Review Board at the University of Tennessee (UTK IRB-18-04604-XP) [45]. Venous blood was drawn into EDTA-coated vacutainers before being added to 20 mL 1 X phosphate buffered saline (PBS).…”

“…Flow cytometry analysis of neutrophil populations: Neutrophils were prepared for flow cytometry as previously described [45]. Unless specified otherwise, 10 6 neutrophils were used for each replicate.…”