Cytokine secretion and pyroptosis of cholesteatoma keratinocytes mediated by AIM2 inflammasomes in response to cytoplasmic DNA

Abstract

Cholesteatoma constitutes an acquired benign epidermal non‑permanent bone lesion that is locally destructive and patients often relapse. Inflammasomes, which mediate the maturation and production of IL‑18 and IL‑1β, resulting in pyroptosis, have been documented to serve a core function in multiple inflammatory conditions. Absent in melanoma 2 (AIM2) is an inflammasome that identifies cytoplasmic DNA and has previously been reported as a pivotal modulator of inflammatory responses. Therefore, the present study aimed to determine the expression levels of AIM2 in human cholesteatoma tissues, and elucidate its function in modulating cytokine production. The expression levels of IL‑18, apoptosis‑associated speck‑like protein containing a CARD (ASC), IL‑1β, AIM2 and caspase‑1 were markedly elevated in cholesteatoma tissues. Protein expression levels of AIM2, caspase‑1 and ASC were localized in the cellular cytoplasm, primarily in the granular and prickle‑cell layers in the cholesteatoma epithelium. Induction using IFN‑γ, as well as cytoplasmic DNA markedly activated the AIM2 inflammasome and elevated the release of IL‑18 and IL‑1β in human cholesteatoma keratinocytes. IFN‑γ was found to enhance poly(dA:dT)‑induced pyroptosis of cells and cytokine production. The results of the present study revealed that AIM2 expressed in human cholesteatoma serves a vital function in the inflammatory response by initiating the inflammasome signaling cascade in cholesteatoma.

Keywords: cholesteatoma; absent in melanoma 2; inflammasome; cytoplasmic DNA; cytokine; pyroptosis.

Figure 1.

Immunofluorescence and western blotting analyses of inflammasome components in human congenital and acquired cholesteatoma tissues. (A) Localization and expression of AIM2 in cholesteatoma tissues and healthy skin tissues. Immunofluorescence analysis revealed that AIM2 was localized in the cytoplasm of cells, primarily in granular and prickle-cell sheets. The nucleus was labelled using DAPI (magnification, ×200). (B and C) Western blotting images of AIM2 and caspase-1. (D) Corresponding semi-quantitative analysis of the AIM2 protein content in congenital and acquired cholesteatoma tissues. Localization of ASC, caspase-1, IL-1β and IL-18 in (E) congenital cholesteatoma tissues and (F) acquired cholesteatoma tissues. The immunofluorescence assay revealed that ASC and caspase-1 were mainly localized in the cytoplasm of cells, primarily in granular and prickle-cell layers. The nucleus was labelled using DAPI (magnification, ×200). (G and H) Western blotting images and (I) corresponding semi-quantitative analysis of the ASC, caspase-1, IL-1β and IL-18 protein expression levels in congenital and acquired cholesteatoma tissues. The relative protein expression content was standardized to β-actin. The columns indicate the mean ± standard deviation values (n=3 per group). *P<0.05. ASC, apoptosis-associated speck-like protein containing a CARD; AIM2, absent in melanoma 2.

Figure 2.

IFN-γ induces the expression of AIM2 in human cholesteatoma keratinocytes. (A) Cholesteatoma keratinocytes were treated with different titers of IFN-γ for 24 h. The expression levels of AIM2 were assessed using western blotting. (B) The panels indicate the ratios of the band densities of AIM2 to β-actin. The results are indicated as the mean ± standard deviation of three different experiments (n=3 per group). *P<0.05 vs. control group. AIM2, absent in melanoma 2.

Figure 3.

Expression of inflammasome components in human cholesteatoma keratinocytes following stimulation by cytoplasmic DNA after priming with or without IFN-γ (25 ng/ml). (A) Changes in AIM2, ASC, caspase-1, cleaved-caspase-1, IL-1β and IL-18 protein expression levels were determined by western blotting after stimulation with 25 ng/ml IFN-γ or 25 ng/ml IFN-γ + 2 µg/ml poly(dA:dT) for 24 h. (B) Semi-quantification of western blotting results. The columns represent the mean ± standard deviation values (n=3 per group). Data were collected from three independent assays. *P<0.05 vs. control group; ^#P<0.05 vs. IFN-γ group. AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a CARD.

Figure 4.

IFN-γ stimulation promotes poly(dA:dT)-triggered cholesteatoma keratinocyte pyroptosis. (A and B) TUNEL assay demonstrated that poly(dA:dT) promoted apoptosis in the presence of IFN-γ. (C and D) Protein expression of GSDMD-F and GSDMD-N in cholesteatoma keratinocytes following stimulation with poly(dA:dT) alone, and IFN-γ and poly(dA:dT) combined. Relative protein expression content was standardized to β-actin expression. Poly(dA:dT) enhanced the cleavage of GSDMD-N, but did not affect the expression of GSDMD-F. The presence of IFN-γ resulted in greater protein expression levels of GSDMD-N. The columns indicate the mean ± standard deviation values (n=3 per group). Data were acquired from three independent assays. *P<0.05. GSDMD-F, full-length gasdermin D; GSDMD-N, terminal cleavage product of gasdermin D.