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Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network
Published on: February 16, 2016
Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to
M J Lappin1, V Brown2, S S Zaric2
1Centre for Dentistry, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, UK.
Objective:
To investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge.
Design:
mRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot.
Results:
Human gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts.
Conclusion:
Our data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production.
Insights
Interferon-alpha (IFN-α) primes human gingival fibroblasts by increasing CD14 expression, enhancing their inflammatory response to bacterial lipopolysaccharides (LPS). This priming effect boosts cytokine production, indicating a heightened sensitivity to bacterial challenges.
Area of Science:
- Immunology
- Cell Biology
- Oral Microbiology
Background:
- Human gingival fibroblasts play a role in periodontal disease pathogenesis.
- Toll-like receptors (TLRs) and CD14 are key mediators of innate immune responses to bacterial components like lipopolysaccharides (LPS).
- Interferons (IFNs) are known modulators of immune cell function.
Purpose of the Study:
- To investigate the effect of Interferon-alpha (IFN-α) on the expression of CD14, TLR2, and TLR4 in human gingival fibroblasts.
- To determine if IFN-α influences the responsiveness of gingival fibroblasts to bacterial LPS stimulation.
- To elucidate the role of CD14 in IFN-α-mediated priming of gingival fibroblasts.
Main Methods:
- Quantitative polymerase chain reaction (Q-PCR) and flow cytometry were used to measure mRNA and protein expression of CD14, TLR2, and TLR4.
- Enzyme-linked immunosorbent assay (ELISA) assessed the production of IL-6 and IL-8 following LPS stimulation after IFN-α pre-incubation.
- Western blot analysis was employed to investigate LPS-induced IκBα degradation.
Main Results:
- Human gingival fibroblasts express CD14, TLR2, and TLR4.
- IFN-α, but not IL-10β, upregulated the mRNA expression of CD14, TLR2, and TLR4, and induced membrane-bound CD14 protein expression.
- Pre-incubation with IFN-α led to increased IL-6 and IL-8 production upon stimulation with Escherichia coli or Porphyromonas gingivalis LPS.
- A blocking antibody against CD14 abrogated the increased pro-inflammatory cytokine production, and both LPS types induced IκBα degradation.
Conclusions:
- IFN-α primes human gingival fibroblasts by upregulating CD14 expression.
- This priming results in an enhanced responsiveness to bacterial LPS challenge, evidenced by increased pro-inflammatory cytokine production.
- The findings highlight a novel mechanism by which IFN-α may modulate the innate immune response in the gingival tissue.
