Introduction

Cell death plays a critical role in embryonic development, cell fate determination, and maintenance of immune homeostasis and is categorized into necroptosis, pyroptosis, apoptosis, ferroptosis, and necrosis [1]. However, the distinction between these cell death modalities remains to be further investigated. Necroptosis may be the pathogenesis of inflammatory bowel disease (IBD), Crohn’s disease, skin inflammation, severe cutaneous adverse drug reactions, alcohol/ non-alcoholic steatohepatitis, and drug-induced liver injury. It may be triggered by TLR4/TLR3 stimulation, DNA-dependent activator of IFN regulatory factors (DAI), TNF-TNFR1 signaling, FASL-FAS signaling, TRAIL-TRAILR signaling, and IFN-α/β-IFNAR1 signaling, and intertwined with apoptosis [2]. Apoptosis, the most studied type of “programmed or regulated cell death” (PCD), ranges from inflammation and cancer to autoimmune diseases mediated by FASL-FAS signaling [3]. Unlike apoptosis, necrosis involves the release of intracellular contents and elicits acute exudative inflammation in surrounding tissues [4]. Ferroptosis and pyroptosis have been recently identified as forms of programmed cell death amd have become novel potential therapeutic targets for cancer therapy [5, 6].

Ferroptosis, an iron-dependent cell death modality, leads to toxic accumulation of reactive oxygen species. Dysregulation of iron metabolism and phospholipid peroxidation can lead to ferroptosis. Owing to its unique mechanism, ferroptosis might be involved in cell fate decisions, inflammatory progress, and several pathologies, such as cardiomyopathy [49]. TFRC overexpression increases the intracellular iron pool and enhances lipid peroxidation. Notably, we found that melanocytes of patients (7/10) had a lower ferroptosis suppressor gene score than that of healthy donors, indicating that these melanocytes were sensitive to ferroptosis (Fig. 3C). Healthy donor-derived melanocytes stably expressed key anti-ferroptosis genes at high levels, such as GPX4, NR4A1, FTH1, FTL, MT1G, and SLC40A1 (Fig. 4D). Our results confirmed that both pyroptosis and ferroptosis are involved in the loss of melanocytes during vitiligo development.

Fig. 4
figure 4

Ferroptosis-sensitivity was increased with decreasing expression of ferroptosis-resistant genes in brain cells of patients with MS. (A) Violin plot showing the ferroptosis suppressor geneset score in the different brain-derived cell subsets. (B) Dot plot showing the expression levels of GPX4, FTH1, MT1G, CISD, ISCU, PARK7, TF, and MTF1 within neurons of MS groups and control groups. Dot plot showing the expression levels of ferroptosis-resistant genes within oligodendrocytes (C), astrocytes (D), OPCs (E), and EC/VSM (F) derived from MS groups and control groups. (G) Schematic depiction of ferroptosis-sensitive state under MS pathological condition

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The IFN-γ signaling pathway is activated in melanocytes of patients with vitiligo [50]. Therefore, B16 cell line was used as a cell model to explore the effects of IFN-γ signaling-mediated ferroptosis and pyroptosis (Fig. 3F). IFN-γ-mediated pyroptosis depends on the IFN-γ concentration (Fig. 3G). In general, IFN-γ induces pyroptosis susceptibility based on upregulation of GSDMD, GSDME, CASP1, and CASP8. Interestingly, IFN-γ exposure did not significantly reduce the expression of GPX4, SLC7A11, or SLC3A2, implying that other factors are involved in the increasing ferroptosis-sensitive state of melanocytes in patients with vitiligo. Taken together, IFN-γ is a critical factor that drives the pyroptosis-sensitive state, but not ferroptosis.

Ferroptosis participated in the pathology of multiple sclerosis (MS)

To investigate further the involvement of ferroptosis and pyroptosis in the pathology of autoimmune diseases, we compared the single-nucleus transcriptional profile of brain cells derived from MS patients and control individuals [51]. Although the two groups didn not show obvious differences in ferroptosis driver/suppressor gene scores and pyroptosis gene score (Additional file 4: Fig. S4), neurons had a lower ferroptosis suppressor gene score than other cell types (oligodendrocytes, astrocytes, immune cells, EC/VSM, and OPCs) (Fig. 4A). In addition, the neurons of MS patients expressed some important anti-ferroptosis genes (such as GPX4, FTH1, MT1G, and MTF1) at low levels (Fig. 4B). The same phenomenon was observed in some cell types, such as oligodendrocytes (GPX4, NFE2L2, FTH1, MT1G, SLC16A1, MTF1 etc.) (Fig. 4C), astrocytes (GPX4, NFE2L2, FTH1, MT1G, SLC40A1, MTF1 etc.) (Fig. 4D), OPCs (GPX4, SQSTM1, and FTH1) (Fig. 4E), and EC/VSM (GPX4, FTH1, MT1G, SQSTM1, SLC40A1, SLC16A1, and MTF1) (Fig. 4F). It has been observed that GPX4, as the most robust anti-ferrotposis functional gene, was commonly downregulated in MS patient-derived cell types, which convincingly demonstrated that ferroptosis is involved in the progression of MS. The downregulation of ferroptosis-resistant genes in MS brain cells leads to a tendency towards ferroptosis.

Pyroptosis and ferroptosis were involved in the systemic sclerosis-associated interstitial lung disease

Systemic sclerosis (SSc) is an autoimmune disorder that can lead to interstitial lung disease (ILD). To explore the role of pyroptosis and ferroptosis in SSc-ILD, we analyzed the scRNA-seq dataset from GSE128169, which included eight SSc-ILD samples and five control samples. Analysis of pyroptosis-related driver genes in several major cell types revealed many significant changes between normal lungs and SSc-ILD samples. CASP1, CASP6, CASP8, GSDMB, GSDMC, and GSDMA were markedly upregulated in alveolar type 1&2 cells derived from SSc-ILD samples (Fig. 5A). Notably, group A Streptococcus (GAS) cysteine protease SpeB virulence factor can cleave GSDMA after Gln246, which triggers pyroptosis [41]. Upregulated GSDMA levels in patient with SSc may lead to pneumonia caused by Streptococcus pyogenes or other toxins. In addition, the club/gobelet/basal cells tended to upregulate the expression of CASP8, CASP1, GSDMB, and GSDMC in the SSc-ILD samples (Fig. 5B). Smooth muscle cells and pericytes in SSc-ILD lungs showed increased expression of GSDMD, DHX9, CASP4, CASP1, CASP6, and CASP8 compared to healthy cells (Fig. 5C). SSc-ILD lung-derived ECs exhibited high expression of DHX9, GSDMB, and GSDMC (Fig. 5D). Thus, increased GSDMB expression in alveolar type 1&2 cells, club/gobelet/basal cells, and ECs derived from SSc-ILD might increase the risk of pyroptosis mediated by the GZMA of autoactive CTLs.

Fig. 5
figure 5

Pyroptosis involved in the pathological process of SSc-ILD. Expression levels of pyroptosis-related genes in different cell types, such as alveolar type 1&2 cells (A), club/gobelet/basal cells (B), smooth muscles/pericytes (C), and ECs (D) across matched samples. (E) Selected important genes are shown in monocle2 produced pseudotime trajectory plot in different representative samples. (F) Schematic depiction of typical GSDM gene expression patterns within different cell types under pathological SSc-ILD condition

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To evaluate the role of ferroptosis in SSc-ILD, the ferroptosis suppressor/driver gene set score was identified and found no significant difference between healthy lungs and SSc-ILD lungs in several cell types (fibrobasts, alveolar type 1&2 cells, smooth muscle/pericytes, club/gobelet/basal cells, and ECs) (Additional file 5: Fig. S5A). Several studies have shown that fibroblasts play a key role in fibrotic ILD owing to abnormalities in aberrant extracellular matrix remodeling [52]. Accordingly, we analyzed the cell trajectory of fibroblasts, and identified two different branches with various effector gene expression patterns. Branch 1 expressed IL6 at high level, and branch 2 highly expressed profibrotic features, such as CXCL12 [53], VEGFA [54], IGF1 [55], and TGFB1 [56] (Additional file 5: Fig. S5B). This profibrotic branch mostly exists in SSc-ILD lungs, but not in healthy lungs, and expresses ferroptosis-related genes (ferroptosis-resistant genes: GPX4 and NR4A1 [57]; pro-ferroptotic genes: NCOA4 and SAT1 [58]); and pyroptosis drivers (CASP4 and GSDMD) (Fig. 5E). These results revealed that SSc-ILD profibrotic fibroblasts had high activity of ferroptosis and pyroptosis. Interestingly, SSc-ILD mast cells substantially expressed CASP4, CASP6, CASP8, DHX9, and GSDMD (Additional file 5: Fig. S5C), suggesting relationship between pyroptosis and mast cell degranulation.

Ferroptosis was involved in the imbalance of intestinal microenvironment homeostasis in Crohn’s Disease

Crohn’s disease (CD), a common form of inflammatory bowel disease (IBD), is characterized by irreversible aberrant immune responses [59]. Public scRNA-seq dataset of the terminal ileum of patients with CD and control donors was obtained from https://www.gutcellatlas.org/ (Additional file 6: Fig. S6A). First, we investigated the ferroptosis state of fibroblasts (Additional file 6: Fig. S6B), and confirmed that some proferroptosis genes, such as ACSL4, RPL8, SAT1, and CS [60], were upregulated (Fig. 6A). In addition, CD fibroblasts highly expressed some ferroptosis-resistant genes (such as FTH1, GPX4, NR4A1, NFE2L2, and FTL), but not others (such as MT1G and SLC40A1), indicating that pathological changes in fibroblast heterogeneity existed to some extent (Additional file 6: Fig. S6C). Consequently, the analysis of the cell trajectory of fibroblasts (Additional file 6: Fig. S6D) and revealed a unique differentiated cell state with ASCL4, CTSB, SAT1, and NFE2L2 expression at high levels (Fig. 6B). This differentiated cell state was consistent with SSc-ILD profibrotic fibroblast branch 2. Interestingly, this branch expressed SLC40A1 at low levels. These results highlighted that ferrotposis affected the balance of fibroblast differentiation. However there were no obvious differences in the pyroptosis-related genes between CD patient-derived fibroblasts and normal cells (Additional file 6: Fig. S6E, F).

Fig. 6
figure 6

Aberrant ferroptosis and pyroptosis involved in the pathological process of CD. (A) Dot plot showing the expression levels of pro-ferroptosis genes within fibroblasts of CD samples and control samples. (B) Selected representative genes are shown in monocle2 produced pseudotime trajectory plots of fibroblasts under different conditions. Violin plots showing the expression levels of ferroptosis suppressor genes (C), ferroptosis driver genes (D), and pyroptosis-related genes (E) within epithelial cells of CD samples and control samples. (F) The expression levels of NCOA4, GPX4, NFE2L2, FTH1, FTL, P4HB, PRDX1, and SLC40A1 within myeloid cells of CD samples and control samples. (G, H) Selected important genes were shown in monocle2 produced pseudotime trajectory plot under different conditions. (I) i. The schematic of bulk RNA-seq of THP-1-derived cells. ii. Heatmap depicted gene expression levels of macrophage-related genes (M1: IL18, IL1B, and TNF; M2: IL10 and TGFB1). iii. Heatmap of expression levels of pyroptosis-related genes. iv. Heatmap shows expression levels of ferroptosis-related genes

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The expression of multiple ferroptosis suppressors and driver genes in the epithelial cells (Fig. 6C, D) showed that epithelial cells derived from patients with CD expressed anti-ferroptosis genes (ZFP36 [61], HSPA5 [62], AKR1C3 [63], PLIN2 [64], FTL, FTH1, GPX4, and MT1G [65]) at lower levels than those of control cells, while pro-ferroptosis gene levels (RPL8 and MTDH [66]) were higher than those control cells. However, no clear evidence of upregulating pyroptosis drivers was identified (Additional file 6: Fig. S6G and Fig. 6E).

Next, it was necessary to investigatewhether pyroptosis and ferroptosis are involved in pathological myeloid differentiation. Myeloid cells in CD patients highly express anti-ferroptosis genes, such as GPX4, NFE2L2, FTH1, FTL, P4HB [67], and PRDX1 [68]. Moreover, compared to healthy myeloid cells, these myeloid cells expressed NCOA4 at low levels. Interestingly, SLC40A1 was highly expressed in healthy donor-derived myeloid cells (Fig. 6F). Cell trajectory analysis showed that macrophage M1 and M2 polarizations both existed in myeloid cells (Fig. 6G and Additional file 6: Fig. S6J). In addition, myeloid cells of patients with CD trended to polarize into the ferroptosis-resistant M1 state (Fig. 6H). SLC40A1 may also play a critical role in the maintaining of M2 state. SLC40A1 is highly expressed in tumor-associated macrophages, which suppresses the production of IL-1β [69], and is consistent with low IL-1β expression in control myeloid cells.

To identify the ferroptosis/pyroptosis patterns of macrophages under inflammatory conditions, as described CD inflammatory microenvironment, PMA, IFN-γ, and LPS were added to induce the differentiation of active macrophages and M1 macrophages (Fig. 6I(i)). Upon the PMA stimulation, THP-1 cells were induced into an activated state by secreting both M1 cytokines (IL18, IL1B, and TNF) and M2 cytokines (IL10 and TGFB1) (Fig. 6I(ii)). The cells were then differentiated into strong M1 states under IFN-γ and/or LPS culture conditions. Interestingly, with terminal M1 differentiation, THP-1-derived cells were more susceptible to pyroptosis owing to the upregulated expression of GSDMD, CASP4, CASP9, GSDMA, CASP5, GSDMC, and CASP8, but not GSDMB (Fig. 6I(iii)). The ferroptosis-related gene expression patterns of THP-1-derived M1-like cells were consistent with those of the above patterns of CD-related macrophages at scRNA-seq levels, such as SLC40A1 (Fig. 6I(iv)). THP-1-derived M1-like cells showed increased anti-ferroptotic activity (GPX4, NR4A1, FTH1, MT1G, SLC7A11, SLC3A2, and FTL) and decreased expression of NCOA4 and ALOXE3. Together, these results suggest that inflammatory conditions could drive the reversal of the sensitivity to ferroptosis and pyroptosis, and these properties could be targeted for treatment.

The prominent role of ferroptosis in experimental autoimmune orchitis

We elucidated the roles of ferroptosis and pyroptosis in six human autoimmune diseases. Next scRNA-seq dataset analysis of experimental autoimmune orchitis (EAO) (Additional file 7: Fig. S7A, B), which is a widely used as the mouse model of testicular inflammation [70], was conducted. Compared with other cell types, spermatids had a low ferroptosis suppressor gene expression score, which indicated that spermatids were sensitive to ferrotposis (Additional file 7: Fig. S7C). Except for spermatogonia/sertoli cell cluster, control sample-derived spermatids, spermatocytes, and Leyding cells/immune cells showed a high ferroptosis suppressor geneset score (Additional file 7: Fig. S7D). There was no significant difference in the ferroptosis driver gene score and pyroptosis gene score between EAO and control samples or different clusters (Additional file 7: Fig. S7E–H). In addition, GPX4 was downregulated in EAO cells (Additional file 7: Fig. S7I). Specifically, spermatids, Leydig cells/immune cells, spermatocytes, and spermatigonia/sortoli cells in EAO trended to have reduced Gpx4 expression (Additional file 7: Fig. S7J). These results confirmed that testicular cells were sensitive to ferroptosis under EAO conditions.

Discussion

The present study showed that in psoriasis, AD, vitiligo, MS, SSc-ILD, CD, and EAO, ferroptosis and pyroptosis act as disrupters with aberrant expression patterns. S.C cells of patients with psoriasis presented a cell death (ferroptosis/apoptosis/pyroptosis)-resistant pattern at the mRNA level. Meanwhile, S.G&S.S cells of patients with psoriasis possessed intensified resistance to apoptosis and pyroptosis, but were susceptible to ferroptosis. Our findings were consistent with those of a recent study [40], however, the roles of ferroptosis and pyroptosis in certain cell types of patients with psoriasis at the single cell level were highlighted in this work. Thus, our results provide valuable information to illustrate that some cell death-related drugs should be treated with great caution due to differences in cell death and changes in cell death sensitivity. Under AD pathological conditions, the keratinocyte lineage exhibits a unique and susceptible pyroptosis pattern. These keratinocytes tend to express both GSDMC and GSDMD, which might increase the sensitivity of pyroptosis. The high expression of GSDMC, suggests that inhibition of the TNFα-Caspase8 pathway may reduce the harmful effect of GSDMC-triggered pyroptosis [43]. Similarly, it may be helpful to reduce the risk of exposure to pathogens or other triggers of caspase-1 [71]. The pathogenesis of vitiligo is very clear [50, 72]. Epidermal melanocytes of patients with vitiligo have a remarkable features of pro-pyroptosis and pro-ferroptosis. A recent report has shown that ferroptosis is involved in the pathogenesis of melanocyte destruction in vitiligo [73], which was consistent with our results, and confirmed the reliability of our analysis methods. Some researchers have made an assumption without direct evidence that pyroptosis is involved in the pathogenesis of melanocyte destruction [74]. However, our results provide tangible evidence of pyroptosis-driven melanocyte destruction. The upregulation of CASP1/4 and GSDMD may drive canonical and non-canonical GSDMD-dependent pyroptosis of melanocytes. These findings collectively indicate that ferroptosis and pyroptosis were the important inducement of autoimmune skin diseases. What’s more, intestinal epithelial cells are ferroptosis-sensitive.

Compared with the known pyroptosis-driven melanocyte destruction in patients with vitiligo, pyroptosis-driven brain cell destruction was weak in MS. However, a pyroptosis-sensitive trend was detected (Additional file 4: Fig. S4). Indeed, GSDMD-mediated pyroptosis is involved in neuroinflammation of experimental autoimmune encephalomyelitis (EAE) model, which is initiated by peripheral myeloid cells [75]. Previous research has shown that GPX4 expression is decreased in the brains of patients with MS [76], which is consistent with our findings. Furthermore, this current study clarified which cell types were ferroptosis-sensitive at the single cell level, providing guidance on the selection of MS therapeutic strategies. Some researchers have noticed that pyroptosis may take part in the pathological alteration of pulmonary ECs in SSc [77]. Our results indicated that upregulation of GSDMB and GSDMC in SSc pulmonary ECs increased pyroptosis sensitivity. Different SSc pulmonary cell types upregulate different GSDM genes, such as GSDMA/B/C in AT1&2 cells, GSDMB/C in club/gobelet/basal cells, and GSDMD in smooth muscles/pericytes. Abnormal pyroptosis is linked to pathological transition of SSc-ILD and can be treated as a useful target. Macrophage-initiated pyroptosis is involved in the inflammatory response to CD. Pyroptosis-related features were not obvious in this work, while ferroptosis profoundly affected the balance of physiological and pathological fibroblasts, or M1 and M2 macrophages [78]. Thus, ferroptosis is involved in the pathological progression of EAO.

The ROS not only induece ferroptosis but also pyroptosis [79, 80], which points to the application of antioxidants in the treatment of some autoimmune diseases. Conventional antioxidant therapies have been proven to be less effective for several reasons, such as their inability to cross the blood–brain barrier, poor structural stability, low ROS and RNS scavenging activity, and low durability in vivo [81]. Nanomaterial antioxidants are good choice to solve these difficult problems, and several effective therapeutic nanomaterial antioxidants have been developed, such as two-dimensional (2D) transition-metal dichalcogenide (TMD) nanosheets [81], triapazamine-loaded hollow mesoporous bilirubin nanoparticles (HMBRN) [82], Fe3O4 @ TAn nanoflowers [83], monodispersed hydrophilic carbohydrate-derived nanoparticles (C-NP) [84], Pt-iNOS@ZIF nanoreactors [85], and ceria nanoparticles [86,87,88]. A recent study reported that an orally administered antioxidant nanoplatform based on simulated gastric fluid (SGF)-stabilized titanium carbide MXene nanosheets (Ti3C2 NSs) could be used to treat inflammatory bowel disease [89]. Nanoparticles can also be modified to load dexamethasone and antioxidants, which integrate ROS scavenging and anti-inflammatory drug delivery [90]. Some nanomaterial antioxidants, such as nanoenzyme-reinforced injectable hydrogel [91] and CIP-loaded and ceria-decorated polymer vesicles (CIP-Ceria-PVs) [92], have wide application potential in skin-related diseases. Mitochondria also participates in apoptosis via lipid peroxidation [93]. Many mitochondria-targeted ROS scavengers, such as mitoquinone (MitoQ) [94], SkQ1 [95], and melatonin [96] can prevent mitochondrial ROS formation and inhibit ferropotosis. Our results provide a theoretical foundation for the use of antioxidants based on ferropotosis and pyroptosis in autoimmune diseases.

Many studies on non-apoptotic regulated cell death (RCD) in tumor immunotherapy, including autophagy, ferroptosis, pyroptosis, and necroptosis, highlights the importance of research on non-apoptotic cell death mechanisms, and also indicate that these complex interactions can flow into several core molecular mechanisms [97]. Various microbial pathogen components and autoinflammatory factors also trigger these core molecular mechanisms, such as the CASP family/Granzyme-GSDM family axis and SLC7A11-GPX4 axis [98]. For example, Talaromyces marneffei can activate pyroptosis mediated by AIM2-caspase-1/-4-GSDMD in hepatocytes [99]. Moreover, some researchers have begun to pay attention to pyroptosis and ferroptosis in autoimmune diseases and drugs toxicity [100,101,102,103,104]. Our results provide pyroptosis/ferroptosis-associated signatures in several autoimmune diseases at the single-cell level, which provide extremely precise target information for the application of novel therapeutic approaches.

Taken together, the data presented herein strongly indicate that pyroptosis and ferroptosis are involved in autoimmune diseases (Table 2). scRNA-seq analysis was applied to understand the potential of programmed cell death in target cell types under pathological conditions. Our partial results were consistent with previous studies, demonstrating the reliability of the research methods adopted. In addition, the pattern and extent of ferroptosis and pyroptosis involvement in some autoimmune diseases determine the drugs that can be adopted to prevent uncontrolled cell death and inflammation. Notably, we found that IFN-γ is a key factor in increasing the sensitivity of pyroptosis, which provides a novel view for the role of IFN-γ-triggered pyroptosis in autoimmune diseases. Therapeutic strategies designed to inhibit the accumulation of ferroptosis/pyroptosis-sensitive target cells may improve clinical responses to autoimmune diseases. Thus, further studies based on in vivo experiments are required to screen for suitable drugs.

Table 2 Summary of ferroptosis and pyroptosis in autoimmune diseases
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