Chronic spontaneous urticaria (CSU) is one of chronic inflammatory dermatosis, which is delineated as, for recognized or unrecognized causes, angioedema, wheal, or both occurring spontaneously for more than 6 weeks (1). The CSU’s prevalence is approximately 1% of the population (lifetime prevalence = 1.4%; point prevalence = 0.7%) (2). Moreover, CSU will get the increase of risk for comorbid autoimmune diseases like autoimmune thyroid disease (3). The frequently recurring symptoms, pruritus, urticaria, and angioedema, severely affect patients’ performance at school and work and impair their quality of life, which brings much encumbrance to both their households and society (4). Unlike acute urticaria, which is usually caused by an identifiable agent like an allergic reaction to a drug or other, the cause and pathogenesis of CSU are complex and remain largely unclear (5). Consequently, it is of great significance for individualized and effective treatment to reveal the pathogenesis and recognize key biomarkers of CSU.

The CSU’s etiopathogenesis has not been totally uncovered, but the existing studies suggest that maladjustment of inflammatory cells (such as mast cells and basophils) is the potentially core contributor (6). It is well known that a series of intracellular signaling cascades result in mast cell activation, after IgE bonds to the high-affinity IgE receptor. The activated mast cell releases proteases, histamines, and cytokines with the generation of platelet-activating mediators and other arachidonic acid metabolites (leukotrienes C4, D4, and E4 and prostaglandin D2). These cytokines give rise to vascular permeability and increased vasodilation, ensuing interstitial edema and sensory nerve stimulation and causing the obvious itchiness, redness, and swelling (7, 8). Besides, some CSU patients could show signs of activation of the coagulation/fibrinolytic system, such as significant elevation of serum factors like D-dimer, sICAM-1, and sVCAM-1 (9, 10). In this setting, some treatment strategies were developed, such as antihistamine, biological agent, and immunosuppressant. The first-line symptomatic treatment for CSU is largely depending on modern 2nd-generation H1 antihistamines, but standard-dosed antihistamines are ineffective in about 40% of the patients (11). Omalizumab, a humanized anti-IgE antibody, is the first licensed biological treatment by the Food and Drug Administration (FDA) for patients with CSU refractory to H1 antihistamines. However, relapse rates following the withdrawal of omalizumab are high (12). Ciclosporin is recommended for combination treatment in patients with severe disease refractory. Nevertheless, it is a problem which cannot be ignored that long-term use of ciclosporin leads to serious side effects (13). It can thus be seen that complete control of symptoms in the majority of patients remains a worldwide challenge. Consequently, further and fuller exploring the inflammatory reaction pathogenesis of CSU is scientifically significant to the clinical therapy.

There has been extensive interest in the interaction between inflammatory reaction and pyroptosis. The pyroptosis is denoted as inflammasome-dependent cell death (14). It was found that it makes a critical difference in the development of numerous inflammatory skin diseases. A research reported that Mdivi-1 significantly suppressed the pyroptotic cell death of keratinocytes and inhibited NLRP3 inflammasome activation to play a protective role in atopic dermatitis (15). Deng et al. indicated that cycloastragenol could inhibit the liberation of inflammatory mediators and macrophage infiltration in psoriasis by inhibiting the pyroptosis which NLRP3 mediated (16). In addition, previous research has established that aberrant NLRP3 inflammasome activation in masts cell contributes to histamine-independent urticaria by production of IL-1β in cryopyrin-associated periodic syndromes (CAPSs) (17). However, our understanding of CSU with pyroptosis is still pretty limited.

Microarray technology has been widely utilized into biological studies in recent years, and the data generated by it like the mRNA dataset could be an advantageous instrument for discovering critical factors of etiopathogenesis of diseases, which offers valuable insulation and foundation for further novel studies (18, 19). In this study, via applying the bioinformatic method, we analyzed the data of the Gene Expression Omnibus (GEO) (20), which has its origin in microarray technology, to explore immune cell infiltration and ceRNA network, and further reveal the molecular mechanism of pyroptosis-related genes in CSU and identify key biomarkers.

CSU is a common chronic inflammatory dermatosis, which has significantly negative impacts on the quality of people’s life owing to its repeated outbreaks and protracted course (44). Although current treatments of CSU get little effectiveness, how to more effectively mitigate and avert recurrence is still a global challenge as there are still many unknowns in its genesis. In addition, it has reached a consensus in the last guidelines that further research in some areas of CSU is needed, such as identification of mast cell/basophil-activating factors, identification of serum biomarkers of urticarial activity/mast cell activation, and identification of new histological markers (1). Remarkably, pyroptosis was one of deaths associated with cell membrane rupture. The increased number of cell membranes in mast cells might lead to the liberation of intracellular β-hexosaminidase and histamine (45). Since pyroptosis showed a great research prospect in inflammatory skin diseases, this work tries to identify and verify the potential key biomarkers of CSU from the standpoint of pyroptosis-related genes through bioinformatics ways, especially in inflammatory response, to provide a new perspective for the etiopathogenesis and therapeutic approaches of CSU.

In the present research, we recognized 1,297 DEGs from the CSU RNA expression profile. Then crossing the DEGS with pyroptosis-related genes, 17 DEPRGs (containing 4 downregulated genes and 13 upregulated genes) were recognized and then performed into gene function analysis. As shown, the DEPRGs were mostly involved in inflammatory response, as well as in pro-inflammatory effects (such as positive regulation of interleukin-8 production, positive regulation of interleukin-1 beta production, positive regulation of interleukin-1 production) and biological regulation (including signaling receptor activator activity, receptor ligand activity, cytokine activity), the majority of which are generally accredited to constituent parts of the development of CSU. A research containing 153 CSU patients suggested that the IL1 gene had a significant role in the susceptibility to CSU (46). Kasperska-Zajac et al. indicated that severity of systemic inflammation of CSU was related to elevated il-8 (47). The DEPRGs mainly participated in inflammatory pathways according to KEGG, likely the NF-kappa B signaling pathway, TNF signaling pathway, and NOD-like receptor signaling pathway. In addition, the result of DO further confirms the above. The DEPRGs were majorly enriched in inflammatory diseases like ulcerative colitis and fever. This advises that, to some extent, the DEPRGs could have a function to participate in the systemic inflammation of CSU.

Through the PPI network and module analyses, we identified five hub genes, namely, IL1B, TNF, IRF1 (all upregulated genes) and HMGB1, P2RX72 (both downregulated genes). They are the common inflammatory cytokines, but most of them have not been reported to be implicated in the development of CSU, so this would be a new finding. To fully explore the maladjustment of inflammatory cells of CSU, we executed immune infiltration analysis. It was found that CSU tissue owned a higher proportion of mast cells activated, but relatively lower ones of T cells CD4 naive, plasma cells, and B cells memory. Previous studies indicated that CSU was considered to be principally a mast cell-driven disease (48). Moreover, it has also been reported that the etiopathogenesis of CSU was closely associated with the dynamical unbalance of Th1/Th2 cells of CD4+T cells (49). However, since there have been few research reports, the relationship between CSU and plasma cells or/and B cells memory might be an interesting finding. Moreover, our research further showed that each of the hub genes (IL1B, TNF, IRF1, HMGB1, and P2RX7) was statistically related to major infiltration cells. Especially, IL1B and TNF were statistically and positively associated with mast cells activated, which suggests that they are related to maladjustment of inflammatory cells of CSU and might be its possible immunomodulation pivots. In addition, to reveal a systemically interactive modulation in CSU, we constructed a ceRNA network, in which there were 8 axes, such as IL1B/miR-21-5p/SNHG3, HMGB1/miR-34a-5p/TUG1, and P2RX7/miR-588/TUG1. It is worth noting that we also found that SNHG3 was significantly and negatively correlated with mast cells resting and plasma cells, and TUG1 was negatively related to mast cells activated, which further verified that the DEmRNA–miRNA–DElncRNA ceRNA network did have a critical role in maladjustment of inflammatory cells of CSU.

We chose IL1B to do further analysis for three reasons. First, it had the biggest fold change in hub genes. Second, it was verified by the validation set GSE57178. Third, both it and its upstream factor SNHG3 were related to the activation of mast cells. These indicated that IL1B could be in a more critical position in the development of CSU. IL1B (IL-1β) is a potent pro-inflammatory cytokine and plays a role in the innate and adaptive immunity of humans (50). Under the stimulation of immune response, inflammation, and infection, IL1B is released from monocytes, macrophages, and dendritic cells, affects local cells by paracrine, and targets distant cells via endocrine, ultimately leading to a series of inflammatory cascade responses like activation of immune cells and pyroptosis (51). Abnormal IL1B-related signaling pathways have been shown to be connected with some immune inflammatory diseases like SLE and UC (52, 53). These were in agreement with the result of our research. We predicted that the protein subcellular localization of IL1B was extracellular, which is in accord with its intercellular regulation role. Moreover, it was found that IL1B was significantly correlated with familiar immune checkpoints in CSU, such as PDL1, CTLA4, TIM3, and TIGIT, which someway showed its role of immune regulation in CSU. Furthermore, IL1B was an overexpression examined in clinical CSU patients by ELISA, and the ROC curve analysis confirmed the dependability of its diagnostic value; the point was that mast cells were significantly activated in CSU and IL1B did correlate with it, which verified our bioinformatic analyses and suggests that IL1B could be a potential prognostic and diagnostic biomarker in CSU.

The NOD-like receptor signaling pathway was the major involved pathway according to the enrichment analysis of IL1B in a couple of databases. Moreover, NLRP3 is a subtype of NOD-like receptors and is famous for one of key pyroptosis cytokines; moreover, it is a well-known activator of IL1B (54). The study of Guo et al. showed that the increased expression of NLRP3 in mast cells leads to the activation of caspase-1 and ultimately to production and secretion of IL-1β in endometriosis (55). These are further supported in our work. In clinical CSU patients, NLRP3 was statistically overexpressed and related to the activation of mast cells. NLRP3 was also significantly correlated with IL1B, which might advise that the pyroptosis-related signaling pathway is activated in CSU and it might be related to the activation of mast cells. What is more, it might be via the NOD-like receptor signaling pathway, NLRP3, that IL1B participates in activation of mast cells. Moreover, we further identified 15 potential therapeutic drugs targeting IL1B, which provides a possible therapeutic strategy for CSU. Molecular docking revealed that the exact molecular binding makes this relationship more reliable.

Our study also had some limitations. We measured gene expression levels using sera from clinical CSU patients rather than tissues, which is not good enough but still clinically representative. Besides, we will perform the experiments in vivo and in vitro to further confirm our results in the future.

In sum, we identified 5 hub genes, IL1B, TNF, IRF1, HMGB1, and P2RX7, from pyroptosis-related genes, which are mainly involved in the inflammatory response and maladjustment of inflammatory cells of CSU. Particularly, ILB and its ceRNA axis might play a role in the activation of mast cells of CUS, and this might be achieved via the NOD-like receptor signaling pathway (NLRP3). Therefore, ILB and its related molecules might be potential key biomarkers in the development of CSU, and our study would provide a new perspective for the etiopathogenesis and therapeutic programs of CSU.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

Written informed consent was obtained from the individual(s)’ and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

SP and HF conceptualized the study design. SP and TZ drafted the manuscript. HF revised the manuscript. SP, SZ, QT, and YY collected data and performed the analysis. All authors contributed to the article and approved the submitted version.

This work was financed by the Hunan Provincial Health Commission scientific research projects (B2016229; B2019055; 202114051780) and Hunan Provincial Innovation Foundation for Postgraduate (CX20200547).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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