Multi-regulatory potency of USP1 on inflammasome components promotes pyroptosis in thyroid follicular cells and contributes to the progression of Hashimoto's thyroiditis

Abstract

Background: Inflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis.

Methods: Our study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation.

Results: Our findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram.

Conclusions: Our study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT.

Keywords: Hashimoto's thyroiditis; Inflammasome; Pyroptosis; Thyroid follicular cells; USP1.

Fig. 1

Upregulation of inflammasomes components in HT patients. A Immunohistochemical staining images of NLRP3, AIM2, NLRP1, and NLRC4 in thyroid tissue sections from patients with HT and control subjects. B Immunohistochemical staining images of ASC, caspase-1 and IL-1β in thyroid tissue sections from patients with HT and control subjects. C Quantified expression levels of NLRP3, AIM2, NLRP1, NLRC4, ASC, caspase-1, IL-1β in thyroid tissue sections from HT patients and controls using Image J (n = 5 for each group). Three views were randomly selected in each subject. *** p < 0.001, ****p < 0.0001, versus control. D The mRNA expression levels of NLRP3, AIM2, NLRP1, NLRC4, ASC, caspase-1 and IL1β in thyroid tissues from HT patients and controls (n = 7 for each group). *p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001, versus control. The relative mRNA expression levels were corrected to those of controls. E Western blot analysis of NLRP3, AIM2, Caspase-1, IL-1β in thyroid tissue from HT patients and controls (7 specimens for each group). F, G, H Comparison of NLRP3, AIM2, and IL1-β expressions between HT and normal thyroid (NT) tissues in GSE138198 dataset

Fig. 2

Simultaneous screening of upregulated USPs and activated inflammasome components in TFC cell line upon stimulation with inflammatory cytokines. A GSEA showing hallmark gene sets of DEGs in HT versus normal thyroid tissues in GSE138198 dataset. B GSEA of TNF-α response pathway. C GSEA of IFN-γ response pathway. D The relative mRNA levels of NLRP3, AIM2, NLRP1, NLRC4, ASC, caspase-1, IL-1β, IL-18, GSDMD and GSDME in Nthy-roi 3-1 cells upon stimulation with TNF-α or IFN-γ. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus TNF-α 0 ng/ml; ^##p < 0.01, ^####p < 0.0001, versus TNF-α 5 ng/ml; ^&p < 0.05, ^&&p < 0.01, ^&&&&p < 0.0001, versus IFN-γ 0 ng/ml; ^▲▲p < 0.01, ^▲▲▲▲p < 0.0001, versus IFN-γ 12.5 ng/ml. Data were obtained from three independent experiments. E, F Real-time PCR assessment of mRNA expression of USPs in Nthy-roi 3-1 cells after TNF-α or IFN-γ stimulation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data were obtained from three independent experiments. G Immunohistochemical staining images of USP1 in thyroid tissue sections from HT patients and controls. H Quantified expression levels of USP1 in thyroid tissue sections from HT patients and controls using Image J (n = 5 for each group). Three views were randomly selected in each subject. **** p < 0.0001, versus control. I Western blot analysis of USP1 in thyroid tissue from HT patients and controls. J Quantified expression levels of USP1 in thyroid tissue from HT patients and controls using Image J (n = 7 for each group). ****p < 0.0001, versus control

Fig. 3

USP1 facilitates cytokines-induced inflammasome activation and pyroptosis in TFCs. A Real-time PCR measurement of mRNA levels of NLRP3, AIM2, ASC, caspase-1, IL-1β, IL-18, and GSDMD following different stimulations. TNF-α, 10 ng/ml; IFN-γ, 25 ng/ml; ML323, 50 μm. *p < 0.05, ***p < 0.001, ****p < 0.0001, versus control 1; ^#p < 0.05, ^###p < 0.001, ^####p < 0.0001, versus TNF-α; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p < 0.0001, versus control 2; ^▲p < 0.05, ^▲▲p < 0.01, ^▲▲▲p < 0.001, ^▲▲▲▲p < 0.0001, versus IFN-γ. Data were obtained from three independent experiments. B, C Western blotting of NLRP3, AIM2, caspase-1, caspase-1 p20 and GSDMD-N in the indicated groups. TNF-α, 10 ng/ml; IFN-γ, 25 ng/ml; ML323, 50 μm. D Calcein AM/EthD-1 double staining of Nthy-roi 3-1 cells under different conditions. TNF-α, 10 ng/ml; ML323, 50 μm. E Western blotting of caspase-3, cleaved caspase-3 and Bax in the indicated groups. TNF-α, 10 ng/ml; ML323, 50 μm. F Protein levels of NLRP3, AIM2, caspase-1, caspase-1 p20, and GSDMD-N in normal, USP1-deficient Nthy-roi 3-1 cells assessed by Western blotting. G Protein level of NLRP3, AIM2, caspase-1, caspase-1 p20 and GSDMD-N in Nthy-roi 3-1 cells transfected with USP1 overexpression plasmid and USP1 C90S mutant plasmid. H, I Quantitative analysis of the bands conducted using ImageJ. *p < 0.05, **p < 0.01. Data were obtained from three independent experiments

Fig. 4

USP1 enforces pyroptosis in TFCs by dual regulation of NLRP3 and AIM2. A Co-immunoprecipitation (Co-IP) assay showing the endogenous interaction between USP1 and NLRP3 in Nthy-roi 3-1 cells. B Partial rescue of repressed NLRP3 expression by ML323 (50 μM) through treatment with the proteasome inhibitor MG132 (10 μM). C Protein expression of p65 upon TNF-α and USP1 knockdown. D Immunofluorescence analysis of p65 upon TNF-α and USP1 knockdown. E Co-IP assay revealing the endogenous interaction between USP1 and AIM2 in Nthy-roi 3-1 cells. F Immunofluorescence analysis demonstrating colocalization of USP1 with AIM2. G Partial rescue of repressed AIM2 expression by ML323 (50 μM) through treatment with the proteasome inhibitor MG132 (10 μM). H Determination of AIM2 protein half-life in Nthy-roi 3-1 cells under different treatments using cycloheximide (CHX, 100 μg/ml) at indicated time intervals. I Increased ubiquitination level of AIM2 upon inhibition of USP1 in Nthy-roi 3-1 cells. J Enhanced protein levels of caspase-1 p20 and GSDMD-N through overexpression of AIM2 in TNF-α + ML323 treated Nthy-roi 3-1 cells. TNF-α, 10 ng/ml; ML323, 50 μM

Fig. 5

USP1 driven pyroptosis is associated with HT pathogenesis. A Experimental process of the animal model. BTg: bovine thyroglobulin. CFA: complete Freund’s adjuvant. IFA: incomplete Freund's adjuvant. B: Thyroid tissues (marked with yellow line) from mice. C H&E staining of mouse thyroid tissues. D Lymphocyte infiltration scores of mouse thyroid tissues. n = 6. E, F, G Expression of TGAb, TPOAb and TSH in plasma of animal models. *p < 0.05, **p < 0.01, ***p < 0.001. n = 6. Data were obtained from three independent experiments. H Immunohistochemical staining images of USP1, NLRP3, AIM2, caspase-1, and IL-1β in sections of mouse thyroid tissues. I Quantified expression levels of USP1, NLRP3, AIM2, caspase-1and IL-1β in thyroid tissue sections from mouse models using Image J. ** p < 0.01, ****p < 0.0001. n = 6

Fig. 6

The schematic diagram of the study. USP1 promotes inflammasome-related pyroptosis in TFCs by stabilizing NLRP3/AIM2 and transcriptional activation of NLRP3 through facilitating P65 nuclear accumulation