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Bacteria associated with apical periodontitis promotes in vitro the differentiation of macrophages to osteoclasts

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A. P. Torres-Monjarás
A. P. Torres-Monjarás
A. P. Torres-Monjarás
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Raquel Sanchez at Texas Tech University Health Sciences Center
Raquel Sanchez
Berenice Hernández at Universidad Autónoma de San Luis Potosí
Berenice Hernández
L. González-Baranda
L. González-Baranda
L. González-Baranda
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Abstract and Figures

Objective To analyze the possible in vitro effect of the cytokine RANKL and bacteria involved in apical periodontitis on the differentiation of macrophages into osteoclasts. Material and methods Bacteria were isolated (mainly E. faecium and E. faecalis) from the root canal of fifty patients with apical periodontitis, the possible effect of these bacteria on the phagocytic activity of the monocyte cell line THP-1 was analyzed by flow cytometry. Furthermore, the effect of these bacteria (alone or in combination with the cytokine RANKL) on the differentiation of THP-1 macrophages into osteoclasts was analyzed through the expression of the receptor RANK and the tartrate-resistant acid phosphatase TRAP. Finally, the release of different cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) by THP-1 cells induced to differentiate into osteoclasts was also analyzed. Results We observed a significant proportion of THP-1 cells were able to internalize E. faecium and E. faecalis. Furthermore, these bacteria were able to induce (alone or in combination with RANKL) a significant expression of RANK by THP-1 macrophages; accordingly, E. faecium and E. faecalis induced very significant levels of TRAP in these cells. Finally, during the differentiation of THP-1 macrophages induced by RANKL or bacteria, a significant release of the pro-inflammatory cytokines IL-6 and TNF-α was observed. Conclusions and clinical relevance Our data suggest that the causative agents of apical periodontitis can induce the differentiation of osteoclasts as well as the release of pro-inflammatory cytokines, phenomena that may have an important role in the bone damage observed in this condition.
Analysis of phagocytosis of E. faecalis by macrophages. The internalization of carboxy-fluorescein labeled E. faecalis by THP-1 macrophages was analyzed by flow cytometry, as described in Materials and Methods. A, B Dot plot images of a representative experiment. Gating of cells with characteristics of macrophages for their subsequent analysis is shown (A). A representative dot plot image of bacteria internalization at a MOI = 10 and macrophages incubated at high density (5.0 × 10⁵/well) is shown (B). Percentage of positive cells (macrophages that internalized labeled bacteria) is indicated. FITC, intensity of fluorescein isothiocyanate staining. C Quantitative analysis of the phagocytic activity of THP-1 macrophages under the indicated MOIs and at cellular densities of 5 × 10⁶ (high) and 1.25 × 10⁵ (low) cells/well. Data correspond to the arithmetic mean and SD of phagocytic cells of five independent experiments. *** p < 0.01; ns, nonsignificant
Analysis of phagocytosis of E. faecalis by macrophages. The internalization of carboxy-fluorescein labeled E. faecalis by THP-1 macrophages was analyzed by flow cytometry, as described in Materials and Methods. A, B Dot plot images of a representative experiment. Gating of cells with characteristics of macrophages for their subsequent analysis is shown (A). A representative dot plot image of bacteria internalization at a MOI = 10 and macrophages incubated at high density (5.0 × 10⁵/well) is shown (B). Percentage of positive cells (macrophages that internalized labeled bacteria) is indicated. FITC, intensity of fluorescein isothiocyanate staining. C Quantitative analysis of the phagocytic activity of THP-1 macrophages under the indicated MOIs and at cellular densities of 5 × 10⁶ (high) and 1.25 × 10⁵ (low) cells/well. Data correspond to the arithmetic mean and SD of phagocytic cells of five independent experiments. *** p < 0.01; ns, nonsignificant
… 
Analysis of the induction of differentiation of osteoclasts. THP-1 macrophages were cultured in the presence (experimental condition) or not (negative control) of human recombinant RANKL and the indicated bacteria, and then the expression of the receptor RANK was analyzed by flow cytometry, as indicated in Materials and Methods. A Flow cytometry image of a representative experiment. The percentages of RANK⁺ cells are indicated. B Quantitative analysis of the induction of expression of RANK in THP-1 macrophages by bacteria alone or in combination with RANKL. Data correspond to the arithmetic mean and SD, n = 20. *p < 0.05 compared to all other conditions tested. C Comparison of expression of RANK+ cells in THP-1 macrophages by E. faecalis and E. faecium.D Analysis of the increase in the levels of RANK⁺ cells induced by E. faecalis or E. faecium, in the presence of RANKL. Data correspond to the arithmetic mean and SD, n = 20. *p < 0.05
Analysis of the induction of differentiation of osteoclasts. THP-1 macrophages were cultured in the presence (experimental condition) or not (negative control) of human recombinant RANKL and the indicated bacteria, and then the expression of the receptor RANK was analyzed by flow cytometry, as indicated in Materials and Methods. A Flow cytometry image of a representative experiment. The percentages of RANK⁺ cells are indicated. B Quantitative analysis of the induction of expression of RANK in THP-1 macrophages by bacteria alone or in combination with RANKL. Data correspond to the arithmetic mean and SD, n = 20. *p < 0.05 compared to all other conditions tested. C Comparison of expression of RANK+ cells in THP-1 macrophages by E. faecalis and E. faecium.D Analysis of the increase in the levels of RANK⁺ cells induced by E. faecalis or E. faecium, in the presence of RANKL. Data correspond to the arithmetic mean and SD, n = 20. *p < 0.05
… 
Analysis of induction of expression of TRAP. THP-1 macrophages were cultured in the presence (experimental condition) or not (negative control) of human recombinant RANKL, alone or in combination with E. faecalis or E. faecium, for 7 days, and then the differentiation into osteoclasts was assessed by the expression of the tartrate-resistant acid phosphatase (TRAP), as described in Materials and Methods. A Representative image of the detection of TRAP (brown staining) in the indicated conditions. B Quantitative analysis of induction of expression of TRAP. Data correspond to the arithmetic mean and SD of TRAP⁺ cells of five independent experiments. *p < 0.05 compared to all other conditions tested; **p < 0.05 compared to all other conditions tested
Analysis of induction of expression of TRAP. THP-1 macrophages were cultured in the presence (experimental condition) or not (negative control) of human recombinant RANKL, alone or in combination with E. faecalis or E. faecium, for 7 days, and then the differentiation into osteoclasts was assessed by the expression of the tartrate-resistant acid phosphatase (TRAP), as described in Materials and Methods. A Representative image of the detection of TRAP (brown staining) in the indicated conditions. B Quantitative analysis of induction of expression of TRAP. Data correspond to the arithmetic mean and SD of TRAP⁺ cells of five independent experiments. *p < 0.05 compared to all other conditions tested; **p < 0.05 compared to all other conditions tested
… 
Analysis of cytokine release during osteoclast differentiation. Supernatants of THP-1 macrophage cultures induced to differentiate into osteoclasts were obtained and assayed for the presence of the indicated cytokines by flow cytometry analysis, as indicated in Materials and Methods. A–D Concentrations of the indicated cytokines under the specified cell culture conditions. CTR, negative control; **p < 0.01, compared to CTR or E. faecium
Analysis of cytokine release during osteoclast differentiation. Supernatants of THP-1 macrophage cultures induced to differentiate into osteoclasts were obtained and assayed for the presence of the indicated cytokines by flow cytometry analysis, as indicated in Materials and Methods. A–D Concentrations of the indicated cytokines under the specified cell culture conditions. CTR, negative control; **p < 0.01, compared to CTR or E. faecium
… 
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Vol.:(0123456789)
1 3
Clinical Oral Investigations (2023) 27:3139–3148
https://doi.org/10.1007/s00784-023-04920-8
RESEARCH
Bacteria associated withapical periodontitis promotes invitro
thedifferentiation ofmacrophages toosteoclasts
A.P.Torres‑Monjarás1· R.Sánchez‑Gutiérrez2· B.Hernández‑Castro2,3· L.González‑Baranda2·
D.L.Alvarado‑Hernández2,3· A.Pozos‑Guillén4· A.Muñoz‑Ruiz5· V.Méndez‑González1· R.González‑Amaro2,3·
M.Vitales‑Noyola1,2
Received: 20 September 2022 / Accepted: 10 February 2023 / Published online: 17 February 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023
Abstract
Objective To analyze the possible invitro effect of the cytokine RANKL and bacteria involved in apical periodontitis on
the differentiation of macrophages into osteoclasts.
Material and methods Bacteria were isolated (mainly E. faecium and E. faecalis) from the root canal of fifty patients with
apical periodontitis, the possible effect of these bacteria on the phagocytic activity of the monocyte cell line THP-1 was
analyzed by flow cytometry. Furthermore, the effect of these bacteria (alone or in combination with the cytokine RANKL)
on the differentiation of THP-1 macrophages into osteoclasts was analyzed through the expression of the receptor RANK and
the tartrate-resistant acid phosphatase TRAP. Finally, the release of different cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10,
and IL-12p70) by THP-1 cells induced to differentiate into osteoclasts was also analyzed.
Results We observed a significant proportion of THP-1 cells were able to internalize E. faecium and E. faecalis. Furthermore,
these bacteria were able to induce (alone or in combination with RANKL) a significant expression of RANK by THP-1
macrophages; accordingly, E. faecium and E. faecalis induced very significant levels of TRAP in these cells. Finally, during
the differentiation of THP-1 macrophages induced by RANKL or bacteria, a significant release of the pro-inflammatory
cytokines IL-6 and TNF-α was observed.
Conclusions and clinical relevance Our data suggest that the causative agents of apical periodontitis can induce the differ-
entiation of osteoclasts as well as the release of pro-inflammatory cytokines, phenomena that may have an important role in
the bone damage observed in this condition.
Keywords Apical periodontitis· E. faecalis· RANK· RANKL· Osteoclasts· Bone loss
Introduction
Apical periodontitis is a common disease that is character-
ized by inflammation, pain, and damage to the alveolar bone,
periodontal ligament, and cement [1]. The pathogenesis of
this condition involves bacterial infections as well as other
factors, including toxins, chemical agents, mechanical irri-
tation, and trauma. In addition, it has been considered that
endogenous factors such as urate and cholesterol crystals,
cytokines, and other mediators of inflammation also partici-
pate in the pathogenesis of this disease [2–4].
Different bacteria are considered the main causative
agents of apical periodontitis, including Fusobacterium
sp., Prevotella sp., Porphyromonas sp., and Actinomyces
sp., as well as facultative anaerobic gram-positive coccus
and bacilli, such as Streptococcus sp., Enterococcus sp.,
* M. Vitales-Noyola
marlen.vitales@uaslp.mx
1 Endodontics Postgraduate Program, Faculty ofDentistry,
UASLP, SanLuisPotosí, SLP, México
2 Research Center forHealth Sciences andBiomedicine,
UASLP, Sierra Leona 550, SanLuisPotosí78210,SLP,
México
3 School ofMedicine UASLP, SanLuisPotosí, SLP, México
4 Basic Sciences Laboratory, Faculty ofDentistry, UASLP,
SanLuisPotosí, SLP, México
5 Postgraduate Dental Science Program, Faculty ofDentistry,
UASLP, SanLuisPotosí, SLP, México
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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