Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

Abstract

Ocular bacterial keratitis, often associated with Pseudomonas aeruginosa bacterial infection, commonly occurs in contact lens wearers and may lead to vision impairment. In this study, we analyzed the contribution of neutrophil extracellular traps (NETs) to the mediation of protection during ocular keratitis. Both invasive and cytotoxic P. aeruginosa clinical isolates induced NET release by neutrophils. NETs carried the characteristic histone proteins, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the invasive P. aeruginosa strains PAO1 (serogroup O5) and 6294 (serogroup O6) were trapped by NETs, the cytotoxic P. aeruginosa strains 6077, 6206 (serogroup O11), and PA14 (serogroup 010) were less sensitive to NET capture. The mechanism of escape by the cytotoxic strains from adhesion to NETs involved the shedding of outer membrane vesicles (OMVs) that outcompeted the cytotoxic P. aeruginosa strains for NET binding. When ocular infection was caused by an invasive strain in vivo, NETs were released at the ocular surface to capture bacteria, limiting their spread. Treatment with MNase I had a dose-dependent effect, with low doses of MNase speeding up bacterial clearance and high doses of MNase having toxic consequences. Cumulatively, our data suggest that NET-mediated immunity is a two-step process. Initially, pathogens attach to NET fragments; subsequently, upon nuclease activity, active serine proteases, which proteolytically degrade NET-associated proteins and promote DNase activity, are released. Therefore, a balance between NET production and NET degradation is needed to achieve maximal NET immunity. Infections caused by Pseudomonas aeruginosa are frequently associated with bacterial ocular keratitis, a condition that carries the risk of vision impairment and occurs in contact lens wearers or after ocular trauma. The majority of bacterial clinical isolates derived from corneal ulcers arising during keratitis can be divided into two functionally distinct groups: invasive and cytotoxic isolates. These two groups have distinct phenotypes; the cytotoxic strains cause rapid cell lysis, whereas the invasive strains replicate in the target cells (1, 2). These differences translate into various severities of disease; in mouse keratitis studies where infection is induced by cytotoxic strains, disease pathology is worse than that caused by invasive strains (3). Consistent with these findings, patients infected with cytotoxic strains have experienced more severe pathology and, consequently, suffered from greater visual impairment after recovery from infection (4). The immune response to both invasive and cytotoxic strains is dominated by dense neutrophil infiltrates, yet the infiltration pattern and responses of polymorphonuclear neutrophils (PMNs) differ depending on the type of strain. Due to the recently described ability of neutrophils to release DNA in the form of neutrophil extracellular traps (NETs) (5) and the presence of extracellular DNA during cytotoxic infections, we questioned whether the cyt...

TSP-1 Deficiency Alters Ocular Microbiota: Implications for Sjögren's Syndrome Pathogenesis

“…The cell number and viability were evaluated by counting trypan blue dye impermeable and permeable cells. This purification procedure yielded 99% viable bone marrow-derived neutrophils with 90% purity as described in Siemsen et al 10 and Shan et al 16 Opsonophagocytic assay…”

Cancer and inflammation

“…They also contain granules and can release anti-microbial molecules similar to the resident granulocytes and can create “neutrophil extracellular traps” (NETs). NETs are networks of DNA-rich fibers embedded with histones and granule proteins including neutrophil elastase, cathepsin G and myeloperoxidase that can trap and kill bacteria and fungi extracellularly, without phagocytosis (40, 45, 93, 107). The monocytes can differentiate into macrophages when they enter the tissue and also phagocytose cells and debris.…”

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

“…HL‐60 cell line, human promyelocytic leukemia cells, can be differentiated into granulocyte‐like cells using a wide variety of agents, such as all‐trans retinoic acid (ATRA), butyrate, hypoxanthine, dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) . Differentiated HL‐60 cells (dHL‐60) have already been applied in studies deciphering mechanisms of NETs formation . It was reported that dHL‐60 cells were able to release NETs in response to a variety of stimuli, including both synthetic (ROS‐dependent phorbol 12‐myristate 13‐acetate (PMA) and ROS‐independent calcium ionophore (CI)) and physiological NETosis inducers ( C. albicans, S. flexneri, S. aureus and interleukin‐8).…”