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. 2017 Jul 3;3(3):115-120.
doi: 10.1002/cre2.69. eCollection 2017 Jun.

Hydrogen sulfide exposure induces NLRP3 inflammasome-dependent IL-1β and IL-18 secretion in human mononuclear leukocytes in vitro

Amina Basic  1 , Sara Alizadehgharib  1 , Gunnar Dahlén  1 , Ulf Dahlgren  1
Affiliations

Hydrogen sulfide exposure induces NLRP3 inflammasome-dependent IL-1β and IL-18 secretion in human mononuclear leukocytes in vitro

Amina Basic et al. Clin Exp Dent Res. .

Abstract

The aim was to investigate if hydrogen sulfide (H2S) induces the formation of the NLRP3 inflammasome and subsequent IL-1β and IL-18 secretion in human peripheral blood mononuclear cells (PBMCs) and in the human monocyte cell line THP1. Bacterial production of H2S has been suggested to participate in the inflammatory host response in periodontitis pathogenesis. H2S is a toxic gas with pro-inflammatory properties. It is produced by bacterial degradation of sulfur-containing amino acids, for example, cysteine. We hypothesize that H2S affects the inflammatory host response by inducing formation of the NLRP3 inflammasome and thereby causes the secretion of IL-1ß and IL-18. PBMCs from eight healthy blood donors, the human monocyte cell line THP1 Null, and two variants of the THP1 cell line unable to form the NLRP3 inflammasome were cultured in the presence or absence of 1 mM sodium hydrosulfide (NaHS) in 24-well plates at 37°C for 24 hr. Supernatants were collected and the IL-1β and IL-18 concentrations were measured with DuoSet ELISA Development kit. PBMCs exposed to NaHS produced more IL-1ß and IL-18 than unexposed control cells (p = .023 and p = .008, respectively). An increase of extracellular potassium ions (K+) inhibited the secretion of IL-1ß and IL-18 (p = .008). Further, NaHS triggered the secretion of IL-1ß and IL-18 in human THP1-Null monocytes (p = .0006 and p = .002, respectively), while the NaHS-dependent secretion was reduced in the monocyte cell lines unable to form the NLRP3 inflammasome. Hence, the results suggest that NaHS induces the formation of the NLRP3 inflammasome and thus the secretion of IL-1ß and IL-18. Enhanced NLRP3 inflammasome-dependent secretion of IL-1β and IL-18 in human mononuclear leukocytes exposed to NaHS in vitro is reported. This may be a mode for H2S to contribute to the inflammatory host response and pathogenesis of periodontal disease.

Keywords: IL‐18; IL‐1ß; NLRP3 inflammasome; hydrogen sulfide; monocytes; periodontitis.

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Figures

Figure 1
Figure 1
The IL‐1ß and IL‐18 secretion of peripheral blood mononuclear cells, measured from eight donors exposed to 1 mM hydrogen sulfide ions (NaHS) for 24 hr. The cells were pre‐exposed to lipopolysaccharides prior to NaHS. A Wilcoxon Signed‐Rank Test revealed a statistically significant difference (p = .023) in IL‐1ß secretion when the cells were exposed to NaHS compared with unexposed control cells. IL‐18 secretion was also statistically significant (p = .008). A statistical difference (p = .008 for both IL‐1ß and IL‐18) was also seen between the cells exposed to NaHS and KCl compared with only NaHS. The different symbols in the figures illustrate eight different blood donors
Figure 2
Figure 2
The IL‐1ß and IL‐18 secretion of three THP1 cell lines exposed to 1 mM NaHS for 24 hr. Prior to NaHS, the cells were exposed to lipopolysaccharides. The THP1‐Null cells showed a statistically higher IL‐1ß and IL‐18 secretion when exposed to NaHS (Mann–Whitney U test, p = .0006 for IL‐1ß and p = .002 for IL‐18) compared with unexposed cells. When the other two cell lines were tested, both unable to form the NLRP3‐inflammasome, there was no difference in IL‐1ß and IL‐18 secretion when exposed to NaHS compared to control. The median of the group is shown as a vertical line
Figure 3
Figure 3
Peripheral blood mononuclear cells from 10 donors not pre‐exposed to lipopolysaccharides, but only exposed to 1 mM NaHS for 24 hr. The exposure to NaHS resulted in a statistically significant difference in IL‐1ß secretion compared with control cells (p = .020, Wilcoxon Signed‐Rank Test). A reduction in IL‐1ß secretion was seen when the cells were exposed to increased extracellular KCl concentrations (p = .004)
Figure 4
Figure 4
A schematic figure of an inflamed gingival pocket with subgingival plaque (biofilm) and monocytes/macrophages

  1. Serum exudate from blood vessels containing serum proteins, peptides, and amino acids including cysteine.

  2. The exudate (gingival crevicular fluid) continues through the thin pocket epithelium (junctional epithelium) into the subgingival pocket.

  3. The subgingival plaque, containing numerous, mainly Gram‐negative, anaerobic bacteria with proteolytic capacity, degrade proteins, peptides, and amino acids including cysteine.

  4. Growing Gram‐negative anaerobes release lipopolysaccharides that penetrate the junctional epithelium into gingival connective tissues.

  5. Growing Gram‐negative anaerobes (Fusobacterium spp., Porphyromonas gingivalis, Treponema spp., and others) produce metabolites, for example, hydrogen sulfide (H2S).

  6. The inflammatory lesion attracts monocytes that migrate into the connective tissue and differentiate to macrophages.

  7. The effect of lipopolysaccharides and H2S on macrophages and the subsequent secretion of the pro‐inflammatory cytokines IL‐1β and IL‐18

See this image and copyright information in PMC

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