hTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis

Łagosz-Ćwik, Katarzyna B.; Wielento, Aleksandra; Lipska, Weronika; Kantorowicz, Małgorzata; Darczuk, Dagmara; Kaczmarzyk, Tomasz; Gibbs, Susan; Potempa, Jan; Grabiec, Aleksander M

doi:10.1038/s41598-021-90037-5

Cited by 16 publications

(23 citation statements)

References 52 publications

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“…A Bradford assay was used to determine protein concentration in cell lysates. Equal amounts of protein were analyzed by Western-blot as described previously (25). Specific bands corresponding to a protein of interest were detected by primary antibodies against p65 (#8242), p-p65 (#3033), p-p38 (#9211) and b-actin (#4967) (all from Cell Signaling Technology) or antisera against FimA (26), and horseradish peroxidase (HRP)-conjugated anti-rabbit Ig secondary antibodies (Dako).…”

Section: Western-blotmentioning

confidence: 99%

“…First, we established a reporter system (U251 MG-hTLR2) using the U251 MG cell line co-transfected with a NF-kB-dependent reporter (Firefly luciferase) and TLR2 coding vectors. Of note, U251 MG cells express undetectable level of TLR2 (25) and have very low constitutive expression of TLR4 (Human Protein Atlas). Cells transfected with the empty vector (pcDNA) had low constitutive luciferase activity, which was unaffected by infection with P. gingivalis or stimulation with the TLR2 ligand Pam3CSK4.…”

Section: P Gingivalis Stimulates Proinflammatorymentioning

confidence: 99%

“…To verify if the results obtained using reporter cell lines can be reproduced in primary cells, we performed experiments using PHGFs, which are an important source of inflammatory mediators in gingival tissues (39) and respond to P. gingivalis infection predominantly through TLR2 (25). Since engagement of TLRs initiates the NF-kB and mitogen-activated protein kinase (MAPK) signaling pathways (40), we investigated the activation status of these pathways upon infection with various P. gingivalis strains and mutants.…”

Section: A Lack Of Ppad or Fima Reduces Inflammatory Responses By Phgfsmentioning

confidence: 99%

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TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

et al. 2022

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Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer’s. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-ĸB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection.

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Section: Western-blotmentioning

confidence: 99%

Section: P Gingivalis Stimulates Proinflammatorymentioning

confidence: 99%

Section: A Lack Of Ppad or Fima Reduces Inflammatory Responses By Phgfsmentioning

confidence: 99%

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TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

et al. 2022

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“…To mimic the oral mucosa and submucosa, we employed human primary gingival (keratinocyte and stomal) cells; specifically, stromal cells were embedded in type I collagen extracellular matrix to provide structural integrity inside the scaffold (Figure 1, B-C) , while keratinocytes were seeded on the closed porosity of the scaffold. Previous gingival studies have used immortalized cell lines that can be expanded for prolonged passages and recapitulate the histological features of the gum [23] [15] ; however, immortalized cells have been shown to fail to mimic cellular responses to P. Gingivalis , a keystone pathogen in periodontal disease [29] , suggesting their limited usability. To assess cellular viability in the construct, we labeled gingival cells with Calcein-AM indicators showing prolonged viability in the construct after six weeks in culture (Figure 2, A-B) .…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional humanized gingival tissue model to study oral microbiome

Adelfio

Martín‐Moldes

Erndt-Marino

et al. 2022

Preprint

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The oral cavity contains different microenvironments, as the non-shedding surface of the teeth and the epithelial mucosa, where oral barriers and microbial communities coexist. The interactions and balances between these two communities are responsible for oral tissue homeostasis or dysbiosis, that ultimately dictate health or disease. Disruption of this equilibrium is the first necessary step towards chronic inflammation and permanent tissue damage in the case of chronic periodontitis. There are currently no experimental models able to mimic the structural, physical, and metabolic conditions present in the oral gingival tissue to support the long-term investigation of host-pathogens unbalances. Herein, we report a 3D anatomical gingival in vitro model based on human primary culture that recapitulates the native tissue organization, and a native oxygen gradient within the gingival pocket to support human microbiome persistence with a physiologically relevant level of microbial diversity as well as native spatial organization. The modulation of inflammatory markers in the presence of oral microbiome suggested the humanized functional response of this model. The model will be used in future studies to investigate host-pathogen unbalances in gingivitis and periodontal disease.

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“…In this study, we demonstrate that PL-derived EVs increase wound closure both in gingival keratinocytes and fibroblasts, despite being aware that cell lines may not behave like primary cells [ 24 ]. Previous studies had already proven the regenerative effect of platelets, but these effects were mainly attributed to the released proteins, such as growth factors [ 4 , 5 , 6 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications

Antich-Rosselló

Munar-Bestard

Forteza-Genestra

et al. 2022

IJMS

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Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

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hTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis

Cited by 16 publications

References 52 publications

TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

Three-dimensional humanized gingival tissue model to study oral microbiome

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications

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