J Bacteriol Virol.  2011 Mar;41(1):9-18. 10.4167/jbv.2011.41.1.9.

Effect of Weissella cibaria on Fusobacterium nucleatum-induced Interleukin-6 and Interleukin-8 Production in KB Cells

• Kang MS
• Lim HS
• Kim SM
• Lee HC
• Oh JS

• KMID: 1449858
• DOI: http://doi.org/10.4167/jbv.2011.41.1.9

Abstract

Oral microorganisms, including pathogens together with commensals, interact with oral epithelial cells, which can lead to the activation and expression of a variety of inflammatory mediators in epithelial cells. Fusobacterium nucleatum is a filamentous human pathogen that is strongly associated with periodontal diseases. Our previous data suggest that Weissella cibaria, an oral commensal, inhibits the proliferation of periodontopathic bacteria including F. nucleatum. The aim of this study was to examine the effects of W. cibaria on the inflammatory mediators, interleukin (IL)-6 and IL-8, in KB cells stimulated by F. nucleatum. In a reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay, live F. nucleatum alone induced high levels of gene expression and protein release of IL-6 and IL-8, whereas W. cibaria alone did not induce IL-6 and IL-8 responses in KB cells. W. cibaria dose-dependently inhibited the increases of the IL-6 and IL-8 gene expression as well as IL-6 protein level in KB cells which was induced by F. nucleatum. Bacterial viability and its coaggregation with F. nucleatum are not essential in the inhibitory effect of W. cibaria. Visible effects of W. cibaria on the attachment and invasion of KB cells by F. nucleatum were observed. In conclusion, W. cibaria may exert immunomodulatory effects on the IL-6 and IL-8 responses to F. nucleatum-activated KB cells.

Keyword

Weissella cibaria; Fusobacterium nucleatum; Interleukin-6; Interleukin-8; KB cells

MeSH Terms

Bacteria
Enzyme-Linked Immunosorbent Assay
Epithelial Cells
Fusobacterium
Fusobacterium nucleatum
Gene Expression
Humans
Interleukin-6
Interleukin-8
Interleukins
KB Cells
Microbial Viability
Periodontal Diseases
Weissella
Interleukin-6
Interleukin-8
Interleukins

Figure

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  Figure 1. Dose responses of W. cibaria on F. nucleatum-induced IL-6 and IL-8 production in KB cells. IL-6 and IL-8 mRNA (A) and protein (B) were assayed by RT-PCR and ELISA, respectively. F. nucleatum was used at a MOI of 100. W. cibaria was used at different MOI, from 10 to 1,000 (which are relatively expressed as 0.1, 1, or 10 W. cibaria per F. nucleatum). The data is expressed with the mean ± SD of a representative experiment performed in triplicate. C, control cells without stimulation; Fn, cells stimulated with F. nucleatum; Wc, cells stimulated with W. cibaria; Fn+Wc, F. nucleatum with W. cibaria. ∗p < 0.05, compared with F. nucleatum alone.

• 

  Figure 2. Effects of live and killed W. cibaria on F. nucleatum-induced IL-6 and IL-8 production in KB cells. IL-6 and IL-8 mRNA (A) and protein (B) were assayed by RT-PCR and ELISA, respectively. KB cells were stimulated at an F. nucleatum to live, heat-killed, or formalin-killed W. cibaria ratio of 1:1, as described in Materials and Methods. The data is expressed with the mean ± SD of a representative experiment performed in triplicate. C, control cells without stimulation; Fn, F. nucleatum; L-Wc, live W. cibaria; HK-Wc, heat-killed W. cibaria; FK, formalin-killed W. cibaria; Fn+L-Wc, F. nucleatum with live W. cibaria; Fn+HK-Wc, F. nucleatum with heat-killed W. cibaria; Fn+FK-Wc, F. nucleatum with formalin-killed W. cibaria. ∗p < 0.05, compared with F. nucleatum alone.

• 

  Figure 3. Effect of the separation of W. cibaria from host cells (A) and coaggregation effect (B) on F. nucleatum-induced IL-6 and IL-8 production in KB cells. IL-6 and IL-8 protein were assayed by ELISA. The data is expressed with the mean ± SD of a representative experiment performed in triplicate. C, control cells without stimulation; Fn, F. nucleatum; Wc, W. cibaria; Wc-I, W. cibaria separated from KB cells through a tissue insert; Fn+Wc, F. nucleatum with W. cibaria; Fn+Wc-I, F. nucleatum with W. cibaria separated from KB cells through a tissue insert; C-Fn, L-canavanine pre-treated F. nucleatum; C-Fn+Wc, L-canavanine pre-treated F. nucleatum with W. cibaria. ∗p < 0.05, compared with F. nucleatum alone; #p < 0.05, compared with L-canavanine pre-treated F. nucleatum alone.

• 

  Figure 4. Effects of various lactobacilli on F. nucleatum-induced IL-6 and IL-8 production in KB cells. W. cibaria and four different Lactobacillus species were used, either alone or in combination with F. nucleatum, to infect the KB cells. IL-6 and IL-8 protein were assayed by ELISA. The data is expressed with the mean ± SD of a representative experiment performed in triplicate. C, control cells without stimulation; Fn, F. nucleatum; La, L. acidophilus; Lc, L. casei; Lf, L. fermentum; Lr, L. reuteri; Wc, W. cibaria; Fn+La, F. nucleatum with L. acidophilus; Fn+Lc, F. nucleatum with L. casei; Fn+Lf, F. nucleatum with L. fermentum; Fn+Lr, F. nucleatum with L. reuteri; Fn+Wc, F. nucleatum with W. cibaria. ∗p < 0.05, compared with F. nucleatum alone.

• 

  Figure 5. Effect of W. cibaria on the attachment (A) and invasion (B) of F. nucleatum to KB cells. KB cells were pre-treated with W. cibaria for 1 h and stimulated with F. nucleatum, as described in Materials and Methods. The levels of attachment and invasion is expressed as a percentage of bacteria retrieved following cell lysis relative to the total number of bacteria initially added. The data is expressed as the mean of a representative experiment performed in triplicate. Fn, F. nucleatum; Wc, W. cibaria. ∗p < 0.05, compared with F. nucleatum alone; #p < 0.05, compared with W. cibaria alone.

• 

  Figure 6. Cytotoxicity of W. cibaria on KB cells. (A) KB cells were stimulated with W. cibaria at various concentrations (MOI of 10, 100, 1,000) for 24 h. (B) W. cibaria and F. nucleatum were co-cultured with various concentrations (0.1:1, 1:1, 10:1). Untreated cells were used as a control. The cell viability was assessed using a MTT assay. The data is expressed at the mean of a representative experiment performed in triplicate. Fn 1, F. nucleatum was used at a MOI of 100; Wc 0.1, W. cibaria was used at a MOI of 10; Wc 1, W. cibaria was used at a MOI of 100; Wc 10, W. cibaria was used at a MOI of 1000; Fn+Wc (1:0.1), 0.1 W. cibaria per F. nucleatum; Fn+Wc (1:1), 1 W. cibaria per F. nucleatum; Fn+Wc (1:10), 10 W. cibaria per F. nucleatum.

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