Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome-dependent secretion of IL-1β and the danger signals ASC and HMGB1

Abstract

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger-associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro-inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF-κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase-1 and stimulates secretion of mature IL-1β. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high-mobility group box 1 protein and apoptosis-associated speck-like protein, with a similar time-course as caspase-1 activation. Thus, F. nucleatum expresses the pathogen-associated molecular patterns necessary to activate NF-κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.

Fig. 1

Intracellular localization of Fusobacterium nucleatum in GECs. Immunofluorescence confocal micrograph GECs infected with F. nucleatum (MOI of 100) for 1 h. The single optical section through the middle of the host cell confirms the intracellular localization of the bacteria. Fixed GECs were stained with phalloidin–tetramethylrhodamine B isothiocyanate (red) to show actin filaments, and anti-F. nucleatum antibody (green). Bar represents 20 μm.

Fig. 2

F. nucleatum infection activates caspase-1 in GECs Western blot analysis of supernatants from GECs infected with F. nucleatum A. at MOI of 25, 50 and 100 for 8 h or B. MOI of 100 for 2, 4, 6 or 8 h. Caspase-1 (22 kDa) activation was detected by Western blot, showing the small (22 kDa) fragment of activated caspase-1 in the supernatant. Uninfected samples were collected and processed after 8 h incubation. Results represent an average of three independent experiments, quantified by densitometry. Error bars represent mean ± standard deviations (SD). Asterisks designate statistically significant differences compared with control (**p < 0.01 and ***p < 0.001, Student’s t-test).

Fig. 3

NF-κB translocation to the nucleus during F. nucleatum infection. Cells infected with F. nucleatum (MOI of 100) were analysed by confocal microscopy at multiple time points. A. Image shows cells at 1 h post-infection stained with anti-NF-κB antibodies (red) and DAPI (blue) to visualize the nucleus. B. Quantification of NF-κB because of fluorescence intensity of staining relative to control at 30 min, 1, 2 and 6 h. Uninfected controls were collected at after 6 h of incubation. Data are representative of two independent experiments ran in duplicates with at least 100 cells counted per time point. Bar represents 20 μm. Asterisks designate statistically significant difference compared with control (**p < 0.01 and ***p < 0.001, Student’s t-test).

Fig. 4

F. nucleatum infection stimulates IL1B gene transcription and processing of IL-1β in GECs. Secretion of IL-1β (17 kDa) was measured in cell supernatants by SDS-PAGE Western blot assay. GECs were infected with F. nucleatum A. at an MOI of 25, 50 and 100 for 8 h, or B. at an MOI of 100 for 2, 4, 6 and 8 h. Western blots were quantified by densitometry. C. IL-1β protein secretion in GECs was confirmed by ELISA after 2, 4 and 8 h of F. nucleatum infection. D. Relative IL-1β mRNA expression compared with control was evaluated by real-time PCR of GECs infected with F. nucleatum (MOI of 100) for 1, 2, 4 and 6 h. E. Immunofluorescence staining images to evaluate IL-1β protein expression during F. nucleatum infection (MOI of 100) for 2, 6 and 8 h. Immunofluorescence intensity levels were measured using NIH-ImageJ and the values of at least 2 standard deviations above the average intensity values of the control group were considered significant. GECs were stained with FITC-conjugated antibody against IL-1β and the nuclear stain DAPI, as indicated in the Materials and methods section. Bar represents 10 μm. F. Quantification of immunofluorescence intensity measured relative to controls at 2, 6 and 8 h. Results represent an average of three independent experiments performed in duplicates. Error bars represent the mean ± SD. Asterisks designate statistically significant difference compared with control (*p < 0.05, **p < 0.01 and ***p < 0.001, Student’s t-test).

Fig. 5

ASC and HMGB1 release following F. nucleatum infection. Release of ASC protein (22 kDa) in cell supernatants was detected using Western blot assay. F. nucleatum infected GECs were infected A. at MOI of 25, 50 and 100 for 8 h, and B. at MOI of 100 for 2, 4, 6, and 8 h. HMGB1 protein (29 kDa) was measured using Western blot of supernatant from cells infected with F. nucleatum C. at an MOI of 25, 50 and 100 for 8 h an D. at MOI 100 for 2, 4, 6 and 8 h, or for 8 h in GECs pretreated with 50 mM z-YVAD-fmk for 30 min prior to infection. Western blots were quantified by densitometry. E. Immunofluorescence images of HMGB1 were captured at 3 and 8 h for infected cells (MOI of 100). Bar represents 10 μm. F. Quantification of immunofluorescence measured relative to control at 3 and 8 h with or without z-YVAD-fmk pretreatment. Results represent an average of three independent experiments performed in duplicates. Error bars represent the mean ± SD. Asterisks designate statistically significant difference compared with control (**p < 0.01, ***p < 0.001, Student’s t-test).

Fig. 6

Depletion of NLRP3 inhibits caspase-1 activation and IL-1β secretion. A, B. Wildtype and NLRP3-depleted (knockdown, KD) GECs were infected with F. nucleatum (MOI of 100) for 8 h. Supernatants were collected and analyzed by Western blot assay for A. caspase-1 activation and B. IL-1β secretion. Results represent three independent experiments. Error bars represent the mean + SD. Asterisks designate statistically significant difference compared with control (**p < 0.01).

Fig. 7

Model of pro-inflammatory cytokine expression and the inflammasome activation cascade during F. nucleatum infection F. nucleatum infection activates NF-κB, which translocates to the nucleus, where it stimulates expression of pro-inflammatory genes, including genes encoding pro-IL-1β. In addition, infection activates the NLRP3 inflammasome, which in turns activates caspase-1, resulting in processing and release of IL-1β. Inflammation is magnified further by release of the danger signal ASC, and caspase-1 secretion of HMGB1.