Interleukin-8 Production by Human Airway Epithelial Cells in Response to Pseudomonas aeruginosa Clinical Isolates Expressing Type a or Type b Flagellins

Abstract

Pseudomonas aeruginosa lung infection is a major cause of morbidity and mortality worldwide. P. aeruginosa flagellin, the main structural protein of the flagellar filament, is a virulence factor with proinflammatory activity on respiratory epithelial cells. P. aeruginosa bacteria express one of two isoforms of flagellin (type a or b) that differ in their primary amino acid sequences as well as in posttranslational glycosylation. In this study, the distribution of type a and b flagellins among 3 P. aeruginosa laboratory strains and 14 clinical isolates (1 ulcerative keratitis, 3 cystic fibrosis, and 10 acute pneumonia isolates) was determined, and their abilities to stimulate interleukin-8 (IL-8) production by human airway epithelial cells was compared. By comparison with the PAK (type a) and PAO1 (type b) prototype laboratory strains, 10/14 (71.4%) of clinical isolates expressed type a and 4/14 (28.6%) expressed type b flagellins. Among four cell lines surveyed, BEAS-2B cells were found to give the greatest difference between constitutive and flagellin-stimulated IL-8 production. All 17 flagellins stimulated IL-8 production by BEAS-2B cells (range, 700 to 4,000 pg/ml). However, no discernible differences in IL-8 production were evident when comparing type a versus type b flagellins or flagellins from laboratory versus clinical strains or among the clinical strains.

NEU1 Sialidase Regulates Membrane-tethered Mucin (MUC1) Ectodomain Adhesiveness for Pseudomonas aeruginosa and Decoy Receptor Release

“…Enzyme-linked Immunosorbent Assays (ELISAs)-A549 cells were incubated for 6 h with 10 ng/ml flagellin or medium alone, and culture supernatants were processed for IL-8 ELISA as described (28). For MUC1-ED ELISA, airway EC supernatants and BALFs were added to ELISA plates, and wells were blocked with PBS, pH 7.0, containing 10 mg/ml BSA, and washed with PBS containing 0.05% Tween 20 (PBS-T).…”

“…2F, lane 2 versus 1) (34) and IL-8 synthesis (Fig. 2G) (28). Infection of airway ECs with Ad-NEU1 increased the binding of Alexa Fluor 594-labeled flagellin compared with Ad-GFP-infected controls (Fig.…”

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition

“…Screening was performed in vitro using A549-Gluc cells and interluleukin-8 (IL-8) as the readout for immune stimulation. IL-8 production by human epithelial cells upon P. aeruginosa recognition is well characterized and readily observable (25,40). We tested approximately 5,800 mutants.…”

“…For instance, Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis have been demonstrated to be capable of producing altered flagellin that cannot be recognized by TLR5 (23). Modification of flagellin by glycosylation also has been observed in bacteria, although its implications in the context of host immune recognition are less clear (24,25). In P. aeruginosa, in addition to the above-mentioned flagellin degradation by AprA, a more widely known mechanism involves the repression of flagellar biosynthesis in response to environmental cues as part of a global response observed in chronic infections (26)(27)(28).…”

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

“…We report a strong induction of numerous pro-inflammatory cytokines, chemokines and AMPs such as CXCL1, CXCL2, CXCL5, CXCL8, CCL20, TNFα, TSLP, IL-23α, IL-32, IL-6, hBD2 and S100A7 during the infection of monolayered primary keratinocytes and reconstructed epidermis with the wild-type PAK strain. Some of these mediators have previously been described during lung [18,24,26–29] or corneal infections by P. aeruginosa [30], extending the potent pro-inflammatory role of this pathogen to skin infections. IL-32 is a cytokine involved in the induction of IL-6, TNFα and chemokines [31,32].…”