J Periodontal Implant Sci.  2014 Dec;44(6):266-273. 10.5051/jpis.2014.44.6.266.

N-acetylcysteine and the human serum components that inhibit bacterial invasion of gingival epithelial cells prevent experimental periodontitis in mice

• Alam J
• Baek KJ
• Choi YS
• Kim YC
• Choi Y

• KMID: 1820398
• DOI: http://doi.org/10.5051/jpis.2014.44.6.266

Abstract

PURPOSE
We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice.
METHODS
Immortalized human gingival epithelial (HOK-16B) cells were infected with various 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester-labeled oral bacteria, including Fusobacterium nucleatum, Provetella intermedia, Porphyromonas gingivalis, and Treponiema denticola, in the absence or presence of three major serum components (human serum albumin [HSA], pooled human IgG [phIgG] and alpha1-antitrypsin). Bacterial adhesion and invasion were determined by flow cytometry. The levels of intracellular reactive oxygen species (ROS) and activation of small GTPases were examined. Experimental periodontitis was induced by oral inoculation of P. gingivalis and T. denticola in Balb/c mice.
RESULTS
HSA and phIgG, but not alpha1-antitrypsin, efficiently inhibited the invasion of various oral bacterial species into HOK-16B cells. HSA but not phIgG decreased the adhesion of F. nucleatum onto host cells and the levels of intracellular ROS in HOK-16B cells. N-acetylcysteine (NAC), a ROS scavenger, decreased both the levels of intracellular ROS and invasion of F. nucleatum into HOK-16B cells, confirming the role of ROS in bacterial invasion. Infection with F. nucleatum activated Rac1, a regulator of actin cytoskeleton dynamics. Not only HSA and NAC but also phIgG decreased the F. nucleatum-induced activation of Rac1. Furthermore, both HSA plus phIgG and NAC significantly reduced the alveolar bone loss in the experimental periodontitis induced by P. gingivalis and T. denticola in mice.
CONCLUSIONS
NAC and the serum components HSA and phIgG, which inhibit bacterial invasion of oral epithelial cells in vitro, can successfully prevent experimental periodontitis.

Keyword

Albumins; Bacteria; Epithelial cells; Immunoglobulin G; Periodontitis

MeSH Terms

Acetylcysteine*
Actin Cytoskeleton
Albumins
Alveolar Bone Loss
Animals
Bacteria
Bacterial Adhesion
Epithelial Cells*
Flow Cytometry
Fusobacterium nucleatum
Humans
Immunoglobulin G
Mice*
Monomeric GTP-Binding Proteins
Periodontitis*
Porphyromonas gingivalis
Reactive Oxygen Species
Serum Albumin
Acetylcysteine
Albumins
Immunoglobulin G
Monomeric GTP-Binding Proteins
Reactive Oxygen Species
Serum Albumin

Figure

• 

  Figure 1 Human serum albumin (HAS) and pooled human IgG (phIgG) but not α1-antitrypsin inhibited the invasion of oral bacteria into HOK-16B cells. (A) HOK-16B cells were infected with carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled Fusobacterium nucleatum for 24 hours in the absence or presence of human serum, HSA, phIgG or α-1 antitrypsin at the indicated concentrations. After quenching the fluorescence of the bacteria bound on the surface, the cells were analyzed by flow cytometry. The FL-1 intensities of the infected cells in the presence of human serum or serum components are overlaid on those of the control infected cells in the absence of an additive (left panel). Bacterial invasion was calculated by subtracting the mean fluorescence intensity of the fixed cell control from that of the infected cells and is expressed as the percentage of the control condition without serum additives (right panel). (B) HOK-16B cells were infected with various CFSE-labeled oral bacterial species in the absence or presence of human serum, HSA (4.4 mg/mL) or phIgG (1 mg/mL) for 24 hours, and the bacterial invasion was analyzed by flow cytometry. Bacterial invasion is expressed as the percentage of the control condition without serum additives. The mean±standard error of the mean of two experiments in triplicate is presented. P. intermedia: Provetella intermedia, P. gingivalis: Porphyromonas gingivalis, T. denticola: Treponiema denticola. *P<0.05 and **P<0.0001 compared with control.

• 

  Figure 2 Human serum albumin (HAS) but not pooled human IgG (phIgG) reduced bacterial adhesion and the levels of intracellular reactive oxygen species (ROS) in HOK-16B cells. (A) HOK-16B cells were incubated with carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled Fusobacterium nucleatum on ice for 1 hour in the absence or presence of human serum, HAS, or phIgG. After washing, the cells were analyzed by flow cytometry without quenching. Bacterial adhesion was calculated by subtracting the mean fluorescence intensity of the HOK-16B cells alone from that of the bacteria-infected cells and is expressed as the percentage of the control adhesion. (B) HOK-16B cells prestained with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were incubated with various concentrations of HSA and phIgG in the absence or presence of F. nucleatum infection for 24 hours. The levels of intracellular ROS were measured by DCF fluorescence. (C) HOK-16B cells prestained with carboxy-H2DCFDA were incubated with various concentrations of N-acetylcysteine (NAC) in the absence or presence of F. nucleatum infection for 24 hours. (D) HOK-16B cells were infected with CFSE-labeled F. nucleatum in the absence or presence of various concentrations of NAC, and the invasion of F. nucleatum was analyzed by flow cytometry. The mean±standard error of the mean of two experiments in triplicate is presented. *P<0.05 and **P<0.0001 compared with control.

• 

  Figure 3 Human serum albumin (HAS), pooled human IgG (phIgG), and N-acetylcysteine (NAC) inhibited the Fusobacterium nucleatum-induced activation of Rac1. (A) The lysates of the HOK-16B cells infected with F. nucleatum (FN) for 0.5, 1, or 2 hours were subjected to pull-down assays for RhoA, Rac1, and Cdc42. As a positive and a negative control, the lysates of the HOK-16B cells alone were incubated with GTPγ and GDPγ, respectively, prior to being subjected to the pull-down assays. (B) The lysates of the HOK-16B cells infected with F. nucleatum for 2 hours in the absence or presence of HSA (4.4 mg/mL), phIgG (1 mg/mL), or NAC (5mM) were subjected to a pull-down assay for Rac1. The band intensities in the gel images from three experiments were measured and are expressed as the mean±standard error of the mean. *P<0.05 compared with control.

• 

  Figure 4 Human serum albumin (HAS)+pooled human IgG (phIgG) and N-acetylcysteine (NAC) prevented experimental periodontitis in mice. Experimental periodontitis (PD) was induced via the oral inoculation of Porphyromonas gingivalis and Treponiema denticola. During the inoculation period of two weeks, the PD_HSA+phIgG and PD_NAC groups were fed with 6.6-mg/mL HSA plus 1-mg/mL phIgG and 10mM NAC in their drinking water, respectively. Distances between the cementoenamel junction to the alveolar bone crest (ABC-CEJ) at six lingual sites per mouse were measured blindly (A), and the alveolar bone loss in experimental periodontitis was calculated (B). *P<0.01 compared with sham. #P<0.05 and ##P<0.01 compared with PD.

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