Application of the In Vitro HoxB8 Model System to Characterize the Contributions of Neutrophil–LPS Interaction to Periodontal Disease

Abstract

<jats:p>(1) Background: Studying neutrophils in vitro is difficult since these cells are terminally differentiated and are easily activated during isolation. At the same time, most of the available model cell lines are associated with certain limitations, such as functional deficiency or a lack of expression of surface markers characteristic of neutrophils. P. gingivalis is a periodontopathogen that causes dysbiosis in subgingival bacterial biofilm. This triggers the accumulation of functional neutrophils in the periodontium. However, until now, the specific effects of P. gingivalis-derived lipopolysaccharide on neutrophil functions have not been analyzed. (2) Methods: The impact of two variants of commercially available P. gingivalis endotoxin on neutrophil functions was tested using the HoxB8 in vitro system that is well suited to analyze neutrophil response to different stimuli in a controlled manner. (3) Results: The Standard P. gingivalis lipopolysaccharide (LPS), known to activate cells through Toll-like receptor 2 (TLR2)- and Toll-like receptor 4 (TLR4)-dependent pathways, prolonged neutrophil survival and exhibited pro-inflammatory effects. In contrast, Ultrapure LPS, binding exclusively to TLR4, neither protected neutrophils from apoptosis, nor induced an inflammatory response. (4) Conclusion: Two variants of P. gingivalis-derived LPS elicited effects on neutrophils and, based on the obtained results, we concluded that the engagement of both TLR2 and TLR4 is required for the manipulation of survival and the stimulation of immune responses of HoxB8 neutrophils.</jats:p>

Porphyromonas gingivalis Peptidyl Arginine Deiminase (PPAD) in the Context of the Feed-Forward Loop of Inflammation in Periodontitis

“…Throughout the study, the HoxB8 model system was used [ 24 ] (Graphical Abstract (GA)). Mouse-derived neutrophil progenitor cell lines were grown in OPTI-MEM (Gibco) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin, 250 µM L-glutamine, 30 mM β-mercaptoethanol, 1 µM β-estradiol (all from Sigma-Aldrich, St. Louis, MO, USA), and Stem Cell Factor (SCF, collected from genetically engineered CHO cell line, [ 71 ]).…”

“…The pro-survival effect of P. gingivalis is thought to be due mainly to the presence of lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria [ 21 ]. LPS is one of the most thoroughly studied P. gingivalis -associated virulence factors, inducing the expression of pro-inflammatory cytokines and prolonging neutrophil survival [ 22 , 23 , 24 ].…”

Ursolic Acid Formulations Effectively Induce Apoptosis and Limit Inflammation in the Psoriasis Models In Vitro

“…The percentage of viable cells was determined using Annexin V-APC and/or propidium iodide (BioLegend, BioCourse.pl, Katowice, Poland) according to the manufacturer's protocol, followed by flow cytometry analysis on the BD LSRFortessa TM , (Becton Dickinson, Warsaw, Poland) as described previously [20]. The flow cytometry data were analyzed with FlowJo Software (Version X for Windows, FloJo LLC, Ashland, OR, USA).…”

“…Reactive oxygen species (ROS) production was determined as described previously [20]. Briefly, cells were harvested and 10 5 cells were stained by incubation with 20 µM 2 ′ ,7 ′dichlorofluorescein diacetate (DCF) (Merck, Pozna ń, Poland) for 30 min at 37 • C and washed with ice-cold PBS (Sigma-Aldrich).…”