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

Maja Sochalska,Tomasz Kantyka,Natalia Zubrzycka,Jan Potempa,Maria Użarowska,Magdalena B Stańczyk,Susanne Kirschnek,Aleksander M Grabiec

doi:10.3390/pathogens9070530

Abstract

(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.

Highlights

Studying neutrophils, called polymorphonuclear leukocytes (PMNs), in vitro is difficult since these cells are activated during isolation, die rapidly in culture and only a limited amount of neutrophils can be isolated from mice
By employing the HoxB8 system, we showed that the engagement of both Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) is required for the manipulation of neutrophil survival since Standard lipopolysaccharide, but not Ultrapure LPS from periodontal pathogen P. gingivalis, protects neutrophils from apoptosis
Neutrophils represent the overwhelming majority of the leukocytes recruited to the gingival crevice in PD patients as well as in chamber models of experimental periodontitis in mice [21,35]

Summary

Introduction

Called polymorphonuclear leukocytes (PMNs), in vitro is difficult since these cells are activated during isolation, die rapidly in culture and only a limited amount of neutrophils can be isolated from mice. Many in vitro myeloid models devoted to study. Pathogens 2020, 9, 530 the process of neutrophil differentiation and functionality have been established. Those include factor-dependent immortalized human myeloid cell lines, i.e., promyelocytic NB-4 and myeloblastic. It was reported that these cell lines fail to terminally differentiate into mature neutrophils, as they are lacking some secondary granule proteins that are expressed at the late stages of the neutrophil maturation process [2]. Many murine-inducible model cell lines are available that express the full range of neutrophil maturation markers. These include (i) Stem Cell Factor (SCF)- and Granulocyte-Macrophage

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