Previous studies have implicated PCD in the pathogenesis of immune disorders in HT. The existing reports on PCD have focused on TFCs. In this study, we used integrated bioinformatic analysis to identify six PCD-related genes that are expressed in the DCs found within the thyroid tissues of HT patients. Analysis of mRNA sequencing, microarray, and scRNA-seq data revealed that TNFAIP3, CYBB, PTPN6, STAT1, TGFB1, and NLRP3 were significantly upregulated in the thyroid tissues of HT patients. Gene overlapping analysis showed that TNFAIP3 was associated with necroptosis, ferroptosis, and PANoptosis, CYBB with necroptosis and ferroptosis, STAT1 with necroptosis, NLRP3 with necroptosis, PANoptosis, and pyroptosis, PTPN6 with ferroptosis, and TGFB1 with ferroptosis and autophagy. The correlation heatmap illustrated the interaction among these PCDDEGs. CIBERSORT analysis confirmed the positive correlation between PCDDEGs and the infiltration of TFCs. These findings were further validated using an external dataset (GSE29315) and qRT–PCR experiments. We also demonstrated that the PCDDEGs were correlated with TPOAb and TGAb, suggesting that they play a role in thyroid tissue damage. This is the first report on genes associated with PCD in HT and provides valuable insights into the identification of markers relevant to HT pathogenesis as well as the choice of immunotherapy.
DCs are essential members of the immune system and are widely recognized as the primary antigen presenters and immune regulators (Banchereau and Steinman 1998). In autoimmune diseases, DCs may capture and deliver antigens from their own tissues, leading to an aggressive immune response by TCs against these tissues. In addition, they can release proinflammatory cytokines and chemokines that attract other immune cells to the lesion and contribute to the development of tissue inflammation and injury (Sarkar and Fox 2005; Bates and Diehl 2014; Kaewraemruaen et al. 2020). DC infiltration is a common hallmark of HT. The involvement of DCs in the immunopathology of the thyroid gland has been further substantiated through various models of experimental autoimmune thyroiditis (EAT). The transfer of syngeneic splenic DCs, pulsed in vitro with TG or necrotic thyrocytes, into healthy animals resulted in the development of EAT (Watanabe et al. 1999; Verginis et al. 2005; Li et al. 2006). Similarly, the adoptive transfer of DCs isolated from animals with EAT (induced by active TG immunization) into healthy animals led to the establishment of thyroid-specific immune responses (Knight et al. 1988). These findings emphasize the significant role of DCs in autoimmune thyroiditis.
TNFAIP3, also known as A20, is a crucial negative regulator of the nuclear factor-kappa B and tumor necrosis factor signaling pathways (Lee et al. 2000; Heyninck and Beyaert 2005). It has been associated with autoimmune thyroid diseases, such as Grave’s disease (Song et al. 2014) and HT (Hori et al. 2019). The deletion of A20 caused DCs in the liver to be activated along with the infiltration of various immune cells, including T helper 1, T helper 17, regulatory T cells, and follicular T helper cells (Das et al. 2019). Additionally, in myeloid-specific A20-deficient mice, A20-deficient macrophages were susceptible to RIPK1/RIPK3/MLKL-dependent necroptosis, which activated inflammasome signaling and promoted arthritis (Polykratis et al. 2019). Furthermore, the miR17-92–TNFAIP3/A20–ACSL4 axis has been found to protect endothelial cells from ferroptosis (Xiao et al. 2019; Ajoolabady et al. 2021). This study showed that TNFAIP3 was significantly expressed in DCs within the thyroid tissues of HT patients compared with normal subjects and was correlated with disease severity, likely by influencing necroptosis or ferroptosis.
NLRP3 encodes an inflammasome sensor protein that participates in necroptosis and pyroptosis. Factors like poly(deoxyadenylic-deoxythymidylic) acid and excessive iodine intake were shown to trigger pyroptosis in TFCs, leading to the elevated expression of gasdermin D and interleukin-18 (Guo 2018; Liu et al. 2019). This study confirmed the importance of NLRP3 in the development of pyroptosis in HT. Interestingly, the pyroptosis gene signature was mainly found in the DCs rather than the TFCs of HT patients in this study. Mammalian DCs were shown to release interleukin-1β through NLRP3-mediated pyroptosis (Zhivaki and Kagan 2021). The NLRP3 inflammasome was activated when mice containing caspase-8-deficient DCs were treated with bacterial endotoxin, eventually activating other molecules and proteins, including RIPK1, RIPK3, and MLKL (Kang et al. 2013). This suggests that the NLRP3 inflammasome may activate necroptosis in DCs, the role of which should be thoroughly investigated in the pathogenesis of HT.
CYBB, also known as nicotinamide adenine dinucleotide phosphate hydrogen oxidase (NOX2), plays a significant role in the immune system by generating superoxide in phagocytes. NOX2 deficiency was found to enhance the initiation and activation of the NLRP3 inflammasome (Monjarret et al. 2023). CYBB was identified as an upregulated necroptosis signature in colon, cervical, and pancreatic adenocarcinomas (Wang et al. 2022; Wu et al. 2022; Sun et al. 2022). CYBB crucially suppressed ferroptosis through the CYBB/Nrf2/SOD2 axis in mesenchymal glioblastoma multiforme (Su et al. 2023). Additionally, CYBB acts as an important chemotactic receptor for DCs. CYBB knockout in conventional DCs restricted the entry of encephalitogenic TCs into the central nervous system in experimental autoimmune encephalomyelitis (Keller et al. 2021). In the present study, CYBB was upregulated in the DCs and MaCs infiltrated into the thyroid tissues of HT patients, mainly because of necroptosis and ferroptosis. Additionally, CYBB was negatively correlated with TG, TPO, and TSHR, implicating it in the development of HT.
STAT1 is an immune response-associated signaling protein that is required for DC maturation (Darnell et al., 1994; Jackson et al., 2004). STAT1 activation in the intestinal epithelium was linked to the expression of molecules involved in apoptosis, necroptosis, and pyroptosis, highlighting its crucial role at the crossroads of key cell death pathways (Stolzer et al. 2022). The function of cytokines in HT was inhibited in STAT1-deficient mice (Kimura et al. 2009). Consistent with previous literature, we discovered STAT1 to be upregulated in DCs infiltrated in the thyroid tissues of HT patients, where it may contribute to apoptosis, necroptosis, and pyroptosis.
Within the intricate web of cellular signal transduction, PTPN6, which encodes protein tyrosine phosphatase non-receptor type 6, importantly governs the negative regulation of inflammation, necroptosis, and apoptosis (Kiratikanon et al. 2022). Null mutations in PTPN6 can give rise to the autoimmune and inflammatory phenotype observed in motheaten mice (Abram et al. 2013). PTPN6 was found to be downregulated in the DCs of type 1 diabetic patients (Ashton et al. 2019). In the present study, PTPN6 was upregulated in the DCs infiltrated in the thyroid tissues of HT patients, which was mainly associated with ferroptosis. In conjunction with previous literature, these findings suggest that PTPN6 in DCs plays a pivotal role in inhibiting immune disorders and regulating various forms of cell death.
Transforming growth factor beta is a pleiotropic cytokine that impacts all immune cells. Excessive iodine can hinder autophagy in TFCs by reducing the synthesis of TGFB1 (Xu et al. 2016). In this study, TGFB1 was upregulated in the thyroid tissues of HT patients and was negatively correlated with TFCs. Therefore, we speculate that the elevated TGFB1 levels may enhance autophagy in TFCs.
The infiltration of lymphocytes, including BCs, TCs, and PCs, into the thyroid gland is the primary pathological hallmark of HT (Antonelli et al. 2015; Zhang et al. 2022). We discovered that the predominant infiltrating cells in the thyroid tissues were TCs. Furthermore, the gene signatures of all PCD processes except cuproptosis were enriched in DCs within the thyroid tissues. DCs are recognized instigators of organ-specific autoimmune diseases, and their presence in the thyroid tissue of autoimmune thyroid disease patients and animal models is strongly suggestive of their role in triggering autoimmunity (Xu et al. 2004; Nagayama 2007). The upregulation of both the quantity and the matured functions of DCs can result in cell death, leading to inflammatory damage and the production of more autoantibodies, which may ultimately contribute to the development of autoimmune thyroid diseases.
This study has several limitations. Firstly, the sample size was relatively small, and future investigations should prioritize larger datasets for validating their results. Secondly, this study did not include detailed experiments to confirm the reliability of different cell death mechanisms in DCs. The mechanisms of these PCDDEGs in HT and their role in cell death should be explored using in vitro and in vivo HT models.