Tβ4 Does Not Suppress LPS IL-8 Secretion in Gingival Fibroblasts

Objectives: Porphyromonas gingivalis is a defined periodontopathic bacterium. The objective of this study was to determine if thymosin beta 4 (Tβ4), a naturally occurring peptide with anti-inflammatory properties, could inhibit IL-8 secretion in gingival fibroblasts challenged with bacterial lipopolysaccharide (LPS). Methods: The human gingival fibroblast cell line HGF-1 (ATCC CRL-2014) was cultured in medium containing 10% serum, serum-starved, pretreated with 0.01 μg/ml Tβ4 for 1 hour, then treated with 10 μg/ml purified LPS from P. gingivalis or Escherichia coli K12 strain (InvivoGen) for 24 hour. Fibroblasts were concurrently stimulated with the pro-inflammatory molecule tumor necrosis factor alpha (TNFα) to ensure the integrity of the culture system. Culture supernatants were collected, centrifuged to remove cell debris, and assayed for interleukin (IL)-8 by ELISA (R&D Systems). Statistical analyses were completed using the unpaired Student's t-test with significance set at p ≤ 0.05. Studies were also repeated in low passage human dermal and corneal fibroblasts. Results: Tβ4 significantly suppressed TNF-α-induced IL-8 secretion in gingival fibroblasts (p = 0.0083). P. gingivalis LPS challenge resulted in increased IL-8 secretion in gingival fibroblasts however Tβ4 was not able to suppress the secretion. E. coli LPS challenge resulted in minimal IL-8 secretion in gingival fibroblasts. Results for the human dermal and corneal fibroblasts were similar to the gingival fibroblasts. Conclusions: We have previously shown that Tβ4 can suppress IL-8 secretion in cells stimulated with TNF-α. However Tβ4 does not have a similar anti-inflammatory action against LPS challenge. TNF-α and LPS elicit cell signaling pathways via different receptors. Our results suggest that in the setting of gingivitis and periodontitis, Tβ4 may not play a role in suppressing LPS-induced cellular inflammation, but likely plays a significant role in inhibiting cellular damage initiated by pro-inflammatory cytokines such as TNF-α released by bacteria and inflammatory cells.