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Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice.

Ao M, Miyauchi M, Furusho H, Inubushi T, Kitagawa M, Nagasaki A, Sakamoto S, Kozai K, Takata T - PLoS ONE (2015)

Bottom Line: P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2.In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Pathobiology, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan; Department of Pediatric Dentistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.

ABSTRACT

Background: Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.

Methods: To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.

Results: Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.

Conclusions: Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

No MeSH data available.


Related in: MedlinePlus

P.g. LPS Up-regulates Expression of Inflammatory Mediators via NF-κB Signaling in Trophoblasts.(A) HTR-8 trophoblasts were seeded (5x105 cells/well) in 6-well culture plates and culture media were changed once before stimulation. Cells were treated by P.g.-LPS (1 μg/ml unless otherwise noted) and both culture medium and cells were collected. mRNA expressions of COX-2, IL-8 and TNF-α were analyzed from cell pellets. (B) HTR-8 cells were stimulated with P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) and cell lysates were examined by immunoblotting analysis using p-p65, p65. Molecular weight is labeled to the right of each band. (C,D) HTR-8 cells were pretreated with or without CAPE for 4 hrs and then stimulated with or without P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) for 24 hrs. mRNA expressions of COX-2, IL-8 and TNF-α (C); protein secretion of TNF-α were examined (D). TNF-α amount in negative controls were subtracted as basal levels when calculating the percentage of down-regulation. GAPDH or β-actin was used as internal control. MOI, multiplicity of infection. *p < 0.05, **p < 0.01. Experiments were performed at least three times with similar results.
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pone.0137249.g005: P.g. LPS Up-regulates Expression of Inflammatory Mediators via NF-κB Signaling in Trophoblasts.(A) HTR-8 trophoblasts were seeded (5x105 cells/well) in 6-well culture plates and culture media were changed once before stimulation. Cells were treated by P.g.-LPS (1 μg/ml unless otherwise noted) and both culture medium and cells were collected. mRNA expressions of COX-2, IL-8 and TNF-α were analyzed from cell pellets. (B) HTR-8 cells were stimulated with P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) and cell lysates were examined by immunoblotting analysis using p-p65, p65. Molecular weight is labeled to the right of each band. (C,D) HTR-8 cells were pretreated with or without CAPE for 4 hrs and then stimulated with or without P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) for 24 hrs. mRNA expressions of COX-2, IL-8 and TNF-α (C); protein secretion of TNF-α were examined (D). TNF-α amount in negative controls were subtracted as basal levels when calculating the percentage of down-regulation. GAPDH or β-actin was used as internal control. MOI, multiplicity of infection. *p < 0.05, **p < 0.01. Experiments were performed at least three times with similar results.

Mentions: Due to the prominent degenerative changes observed in placental tissues in the P.g. infected group (Fig 3), we examined markers of cellular damage in endothelial cells and trophoblasts. Immunostaining for CD-31, a marker of endothelial cells, revealed consistently strong expression in endothelial cells in the NC group (Fig 4I). However, much weaker and discontinuous staining was observed in the P.g.-infected group (Fig 4J), indicating endothelial cell damage. Further pathological changes in the P.g.-infected group were suggested by increased Immunostaining of both 8OHdG (an indicator of oxidative DNA damage) and cleaved caspase-3 (one of the key executioners of apoptosis)(Fig 4K–4N). PCR analysis of placental tissues confirmed detectable up-regulation of COX-2, IL-8 and TNF-α mRNA in the P.g.-infected group (Fig 4Q)


Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice.

Ao M, Miyauchi M, Furusho H, Inubushi T, Kitagawa M, Nagasaki A, Sakamoto S, Kozai K, Takata T - PLoS ONE (2015)

P.g. LPS Up-regulates Expression of Inflammatory Mediators via NF-κB Signaling in Trophoblasts.(A) HTR-8 trophoblasts were seeded (5x105 cells/well) in 6-well culture plates and culture media were changed once before stimulation. Cells were treated by P.g.-LPS (1 μg/ml unless otherwise noted) and both culture medium and cells were collected. mRNA expressions of COX-2, IL-8 and TNF-α were analyzed from cell pellets. (B) HTR-8 cells were stimulated with P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) and cell lysates were examined by immunoblotting analysis using p-p65, p65. Molecular weight is labeled to the right of each band. (C,D) HTR-8 cells were pretreated with or without CAPE for 4 hrs and then stimulated with or without P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) for 24 hrs. mRNA expressions of COX-2, IL-8 and TNF-α (C); protein secretion of TNF-α were examined (D). TNF-α amount in negative controls were subtracted as basal levels when calculating the percentage of down-regulation. GAPDH or β-actin was used as internal control. MOI, multiplicity of infection. *p < 0.05, **p < 0.01. Experiments were performed at least three times with similar results.
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pone.0137249.g005: P.g. LPS Up-regulates Expression of Inflammatory Mediators via NF-κB Signaling in Trophoblasts.(A) HTR-8 trophoblasts were seeded (5x105 cells/well) in 6-well culture plates and culture media were changed once before stimulation. Cells were treated by P.g.-LPS (1 μg/ml unless otherwise noted) and both culture medium and cells were collected. mRNA expressions of COX-2, IL-8 and TNF-α were analyzed from cell pellets. (B) HTR-8 cells were stimulated with P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) and cell lysates were examined by immunoblotting analysis using p-p65, p65. Molecular weight is labeled to the right of each band. (C,D) HTR-8 cells were pretreated with or without CAPE for 4 hrs and then stimulated with or without P.g.-LPS, Li-P.g. (live-P.g.) or D-P.g. (dead-P.g.) for 24 hrs. mRNA expressions of COX-2, IL-8 and TNF-α (C); protein secretion of TNF-α were examined (D). TNF-α amount in negative controls were subtracted as basal levels when calculating the percentage of down-regulation. GAPDH or β-actin was used as internal control. MOI, multiplicity of infection. *p < 0.05, **p < 0.01. Experiments were performed at least three times with similar results.
Mentions: Due to the prominent degenerative changes observed in placental tissues in the P.g. infected group (Fig 3), we examined markers of cellular damage in endothelial cells and trophoblasts. Immunostaining for CD-31, a marker of endothelial cells, revealed consistently strong expression in endothelial cells in the NC group (Fig 4I). However, much weaker and discontinuous staining was observed in the P.g.-infected group (Fig 4J), indicating endothelial cell damage. Further pathological changes in the P.g.-infected group were suggested by increased Immunostaining of both 8OHdG (an indicator of oxidative DNA damage) and cleaved caspase-3 (one of the key executioners of apoptosis)(Fig 4K–4N). PCR analysis of placental tissues confirmed detectable up-regulation of COX-2, IL-8 and TNF-α mRNA in the P.g.-infected group (Fig 4Q)

Bottom Line: P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2.In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Pathobiology, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan; Department of Pediatric Dentistry, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.

ABSTRACT

Background: Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.

Methods: To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.

Results: Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.

Conclusions: Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

No MeSH data available.


Related in: MedlinePlus