Bacteria-derived hydrogen sulfide promotes IL-8 production from epithelial cells

Abstract

Hydrogen sulfide (H(2)S), a volatile sulfur compound, is implicated as a cause of inflammation, especially when it is produced by bacteria colonizing gastrointestinal organs. However, it is unclear if H(2)S produced by periodontal pathogens affects the inflammatory responses mediated by oral/gingival epithelial cells. Therefore, the aims of this study were (1) to compare the in vitro production of H(2)S among 14 strains of oral bacteria and (2) to evaluate the effects of H(2)S on inflammatory response induced in host oral/gingival epithelial cells. Porphyromonas gingivalis (Pg) produced the most H(2)S in culture, which, in turn, resulted in the promotion of proinflammatory cytokine IL-8 from both gingival and oral epithelial cells. The up-regulation of IL-8 expression was reproduced by the exogenously applied H(2)S. Furthermore, the mutant strains of Pg that do not produce major soluble virulent factors, i.e. gingipains, still showed the production of H(2)S, as well as the promotion of epithelial IL-8 production, which was abrogated by H(2)S scavenging reagents. These results demonstrated that Pg produces a concentration of H(2)S capable of up-regulating IL-8 expression induced in gingival and oral epithelial cells, revealing a possible mechanism that may promote the inflammation in periodontal disease.

Figure 1. Measurement of H₂Sreleased from oral bacteria

Fourteen bacterial strains that belong to different pathogenesis clustering complexes were cultured in anaerobic conditions until they reached the late log growth phase. The concentration of all strains was adjusted to 0.9 as measured by OD 590 nm. The concentrations of H₂S and H₂ were measured in the bacterial culture broth using a needle-type H₂S or H₂ sensor, as described in the Materials & Methods section. Data are shown as the mean ± SD of three different cultures.

Figure 2. Effect of exogenously applied H₂S on IL-8 production in OBA9 and OKF6 cells

Confluent cultures of gingival epithelial cell line (OBA9) or oral epithelial cell line (OKF6) were exposed to serial dilutions of the H₂S donor, Na₂S, for 24 hr with or without PMA (1 μmol/L) stimulation. The concentrations of IL-8 in the culture supernatant produced from OBA9 (A) or OKF6 (B) were measured by ELISA. Data indicate the mean ± SD of three cultures. *p < 0.05, **p < 0.01: Values differ significantly (t-test).

Figure 3. Effect of soluble factors, including H₂S, released from PgW83 on IL-8 production in OBA9 and OKF6

The transwell assay system is illustrated (A). The epithelial cells, OBA9 or OKF6, were cultured in the lower chamber until they reached confluent coverage of surface of tissue culture well. Subsequently, live or fixed PgW83 were applied in the upper chamber with or without H₂S scavenger. They were co-cultured for 24 hr in KSF medium containing inflammatory stimulant PMA (1 μmol/L). The concentrations of IL-8 in the culture supernatant of OBA9 (B and D) or OKF6 (C and E) were measured by ELISA. Data indicate the mean ± SD of three cultures. *p < 0.05, **p < 0.01: Values differ significantly (t-test).

Figure 4. Effects of RGP and KGP deletion mutant strains of Pg on IL-8 production in the culture of gingival epithelial cells

(A) The culture broth of wild-type Pg 33277 or Pg KDP137 (RGP-KGP-HagA triple deficient mutant strain) growing in log growth phase was tested for H₂S concentration using a needle-type H₂S sensor. Data represent the average ± SD of three cultures. (B) OBA9 cells were cultured in the lower chamber, and Pg KDP137 was applied to the upper chamber of the transwell co-culture system in the presence of PMA (1mmol/L) with or without Morpholine-resin. After co-culture for 48 hr, the concentrations of IL-8 in the culture supernatant were measured by ELISA. Data are the mean ± SD of three cultures. *p < 0.05, **p < 0.01: Values differ significantly (t-test).