Clustering human dental pulp fibroblasts spontaneously activate NLRP3 and AIM2 inflammasomes and induce IL-1β secretion

Abstract

Objectives: The objective of the present study was to investigate whether NOD-like receptor family pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes pathways were involved in an experimental model of fibroblast activation named nemosis, which was used to mimic circumstances without bacteria stimulation.

Methods: Nemosis of human dental pulp fibroblast (DPFs) was induced by three-dimensional culture in U-shaped 96-well plates and investigated by scanning electron microscopy (SEM). DPFs monolayers were used as control. Annexin V-FITC/7-AAD apoptosis assay was performed on the DPFs spheroids by flowcytometry. Caspase-1 activity detection assay was conducted on the DPFs spheroids. Quantitative real-time polymerase chain reaction (qRT-PCR), cytokine measurements, Western blot and the effect of COX-2 inhibitor on spheroids was studied.

Results: SEM study observed human dental pulp fibroblast clusters and cell membranes damage on the surface of DPFs spheroids. The percentages of necrotic cells from DPFs spheroids gradually increased as the incubation time increased. A statistically significant increase in caspase-1 activity was observed after DPFs spheroids formation. DPFs spheroids displayed significant amounts of NLRP3, AIM2 mRNA and protein expression, caspase-1 mRNA expression and cleaved Caspase-1 protein expression and high IL-1β concentrations (P < 0.05) than DPFs monolayers. Specific COX-2 inhibitor (NS-398) decreased NLRP3 mRNA and protein expression, cleaved Caspase-1 protein expression, Caspase-1 activity and IL-1β mRNA expression and IL-1β concentrations (P < 0.05). However, Specific COX-2 inhibitor had no impact on AIM2 mRNA and protein expression, caspase-1 mRNA expression and pro-Caspase-1 protein expression.

Conclusions: In conclusion, clustering human DPFs spontaneously activated NLRP3 and AIM2 inflammasomes and induced IL-1β secretion which could be partially attenuated by COX-2 inhibitor. Thus, nemosis could become a powerful model for studying mechanisms underlying aseptic pulpitis.

Keywords: Absent in melanoma 2; Dental pulp fibroblast; Inflammasome; Nemosis; Pyrin domain-containing 3.

Fig. 1

Scanning electron microscopy images of normal monolayer of human dental pulp fibroblasts (A) and (B) and human dental pulp fibroblasts in spheroids at 24 h (C) and (D), 48 h (E) and (F), 72 h (G) and (H) and 96 h (I) and (J) after spheroid formation. (B, D, F, H and J are local magnifications of A, C, E, G and I respectively). The arrow in (F) indicates perforation of the cell membrane surface.

Fig. 2

Annexin V-FITC/7-AAD flowcytometry images of human dental pulp fibroblast (DPFs) spheroids at 0 h (A), 24 h (B), 48 h (C), 72 h (D) and 96 h (E). The necrotic cells cells are AnnexinV⁻ 7-AAD⁺(Q1). The late apoptotic cells are AnnexinV⁺ 7-AAD⁺(Q2). The early apoptotic cells are AnnexinV⁺ 7-AAD^-(Q3). The viable cells are AnnexinV⁻ 7-AAD^-(Q4). (F) indicated the necrosis rate of DPFs spheroids at 0 h, 24 h, 48 h, 72 h and 96 h ∗p < 0.05 24 h, 48 h, 72 h and 96 h vs. 0 h.

Fig. 3

Caspase-1 enzymatic activity was measured by caspase-1 activity assays. ∗p < 0.05 vs. 0 h.

Fig. 4

mRNA expression at different time points (0, 24, 48, 72 and 96 h) in human dental pulp fibroblast (DPFs) monolayers and DPFs spheroids. (A) NLRP3, (B) AIM2, (C) caspase-1, (D) IL-1β. ∗p < 0.05 spheroid vs. monolayer.

Fig. 5

Protein expression at different time points (0, 24, 48, 72 and 96 h) in human dental pulp fibroblast (DPFs) monolayers and DPFs spheroids. (A) Western blotting analysis of Protein of AIM2, NLRP3, pro-Caspase-1, and cleaved Caspase-1. (B), (C), (D), and (E) indicated relative protein expressions of AIM2, NLRP3, pro-Caspase-1, and cleaved Caspase-1 in the monolayer DPFs and DPFs spheroids, respectively. ∗p < 0.05 spheroid vs. monolayer.

Fig. 6

Concentrations of IL-1β secreted by human dental pulp fibroblasts cultured in monolayer and spheroid. ∗p < 0.05 spheroid vs. monolayer.

Fig. 7

Selective COX-2 inhibitor effect on NLRP3 and AIM2 inflammasomes gene and protein expression, Caspase-1 activity and IL-1β secretion. The mRNA expressions and protein expressions of NLRP3 and AIM2 inflammasomes and secretion of IL-1β in human dental pulp fibroblast spheroids after 72 h without or with NS-398 treatment. (A) represents NLRP3, AIM2, caspase-1, and IL-1β mRNA expression. (B) represents IL-1β secretion amount. (C) represents Caspase-1 enzymatic activity. (D) and (E) represent AIM2, NLRP3, pro-caspase-1, and cleaved caspase-1 protein expression. ∗p < 0.05 with NS-398 (NS-398) vs. without NS-398 (control). NS, not significant. mon, monolayer. sph, spheroid.