Identification of key genes and molecular mechanisms of chronic urticaria based on bioinformatics

Abstract

Chronic urticaria (CU) is characterized by persistent skin hives, redness, and itching, enhanced by immune dysregulation and inflammation. Our main objective is identifying key genes and molecular mechanisms of chronic urticaria based on bioinformatics. We used the Gene Expression Omnibus (GEO) database and retrieved two GEO datasets, GSE57178 and GSE72540. The raw data were extracted, pre‐processed, and analyzed using the GEO2R tool to identify the differentially expressed genes (DEGs). The samples were divided into two groups: healthy samples and CU samples. We defined cut‐off values of log₂ fold change ≥1 and p < .05. Analyses were performed in the Kyoto Encyclopaedia of Genes and Genomes (KEGG), the Database for Annotation, Visualization and Integrated Discovery (DAVID), Metascape, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and CIBERSOFT databases. We obtained 1613 differentially expressed genes. There were 114 overlapping genes in both datasets, out of which 102 genes were up‐regulated while 12 were down‐regulated. The biological processes included activation of myeloid leukocytes, response to inflammations, and response to organic substances. Moreover, the KEGG pathways of CU were enriched in the Nuclear Factor‐Kappa B (NF‐kB) signaling pathway, Tumor Necrosis Factor (TNF) signaling pathway, and Janus kinase/signal transducers and activators of transcription (JAK‐STAT) signaling pathway. We identified 27 hub genes that were implicated in the pathogenesis of CU, such as interleukin‐6 (IL‐6), Prostaglandin‐endoperoxide synthase 2 (PTGS2), and intercellular adhesion molecule‐1 (ICAM1). The complex interplay between immune responses, inflammatory pathways, cytokine networks, and specific genes enhances CU. Understanding these mechanisms paves the way for potential interventions to mitigate symptoms and improve the quality of life of CU patients.

1. INTRODUCTION

Chronic urticaria (CU) is a dermatological condition that manifests as an angioedema or wheal for more than 42 days. ¹ It is a chronic pruritus condition with a global prevalence of about 1.8%, steadily rising during the coronavirus 2019 (COVID‐19) pandemic period. ² , ³ CU affects the quality of life of individuals and leads to financial burdens on families seeking medical care and treatment. CU is recurrent and makes it difficult to perform daily activities with constant interruptions from recurrent pruritus, angioedema, and urticarial. ⁴

CU increases the prevalence of auto‐immune infections such as thyroid diseases and has a complex pathophysiology. ⁵ According to Peng et al., ⁶ chronic urticaria is caused by allergic reactions to drugs or foreign substances. Therefore, personalized treatment and medication have greater significance in identifying key biomarkers and pathogenesis of CU. Giménez‐Arnau et al. ⁷ postulated that the etiopathogenesis of CU is based on the maladjustment of mast cells and basophils that regulate inflammatory responses and are significant contributors in controlling intracellular signaling cascades that activate mast cells and basophils. These inflammatory cells are activated when they release cytokines, proteases, and histamines and produce platelet‐activating agents and metabolites of arachidonic acid. According to Maurer et al. ⁸ and He et al., ⁹ these inflammatory cytokines and mediators increase the permeability and vasodilation of the vascular system, therefore, exacerbating interstitial oedema and stimulation of sensory nerves that leads to swelling and redness.

According to Asero, ¹⁰ patients with CU often exhibit signs and symptoms of activated coagulation and fibrinolytic systems, as evidenced by increased levels of serum components such as D‐dimers. However, these signs and symptoms have been treated using immunosuppressors or antihistamines. ¹¹ Altrichter et al. ¹² suggested that the first line of treatment in CU is administering 2nd generation H1 antihistamines that are highly productive and safe compared to standard doses. For instance, the Food and Drug Administration (FDA) has recommended omalizumab, a human anti‐IgE antibody, for CU patients with refractory cases of anti‐H1 histamines.

According to Kristjansson et al., ¹³ mast cells and basophils have a crucial role in the pathophysiology of CU by mediating the production and release of tryptases preceded by vasodilation and infiltrating lymphocytes. The serum extracted from patients with CU with their corresponding anti‐IgE and anti‐FceRI antibodies has the potential to induce degranulation of mast cells and basophils. Mast cells are regarded as the main effector cells, with low levels of basophils being a common sign in CU. Basophils and mast cells have a modulating effect of enhancing the development of CU and can be targeted in the development of autoantibodies. ¹⁴

Altman et al. ¹⁵ suggested that effective treatment of CU includes the elimination of external stimuli and drugs involving high doses of antihistamines. The efficacy of antihistamines reduces with an increase in doses and could develop into ant‐histamine resistance. The main goal in the treatment of CU is 100% full remission. Complete remission can achieve full treatment of intractable and refractory CU using a combination of therapies involving hormones, omalizumab, anticoagulants, and immunomodulators. Recently, the pathologic mechanisms of CU include an imbalance of T‐helper 1/2 cells (Th1/Th2 cells), aberrant activation of immune cells and blood coagulation. ¹⁶ The practical and efficient control of clinical signs and symptoms of CU involves understanding the molecular mechanisms of its occurrence and pathogenesis.

Bioinformatics allows analysis of gene expression profiles and high‐throughput sequencing that are critical in understanding the pathogenesis of CU. Our study adopted the gene expression omnibus (GEO) database to identify high‐throughput gene sequencing datasets related to chronic urticaria. Bioinformatics is vital for analyzing genes in chronic urticaria. It manages and integrates diverse genetic and clinical data, detects variations associated with the condition, and elucidates disrupted pathways. ¹⁷ , ¹⁸ , ¹⁹ It studies gene expression, annotates functions, and constructs interaction networks to identify critical genes. Bioinformatics prioritizes relevant variants, compares genomes across populations, and employs machine learning for prediction. Visualization tools make data understandable. Bioinformatics uncovers genetic factors driving chronic urticaria, advancing understanding and potentially guiding personalized treatments.

Our main objective is identifying key genes and molecular mechanisms of chronic urticaria based on bioinformatics.

2. METHODS

2.1. Microarray datasets

We used the GEO database (http://www.ncbi.nlm.nih.gov/geo) generated by the National Centre for Biotechnology Information (NCBI) that contains the gene expression profiles of various sequencing and high‐throughput data of various experiments. We systematically searched the database using keywords such as “Urticaria” and “chronic urticaria” and retrieved two GEO datasets, GSE57178 and GSE72540. The GSE57178 dataset consisted of eight patients' gene expression profiles in chronic idiopathic urticaria and six healthy controls. The experiment involved 4 mm of punch biopsies of active lesions from patients with chronic idiopathic urticaria and the healthy controls that were subjected to histological examinations of ribonucleic acid (RNA) isolation and gene expression using an Affymetrix Human Gene 1.0 ST Array (www.affymetrix.com). The final sample consisted of six skin lesions, seven non‐skin lesions, and six normal skin samples.

The GSE72540 consisted of patients with moderate and severe chronic spontaneous urticaria gene expression profiles. The experiment included 20 patients with moderate to severe chronic urticaria and 10healthy controls. The urticaria scores were determined using a urticaria activity score (UAS) greater than or equal to 8, showing signs of itchiness. Furthermore, patients remained symptomatic despite using antihistamines, and the diagnosis of chronic urticaria was determined based on physical examination and clinical history. Total 4 mm of cutaneous biopsies were obtained and subjected to histological analysis, and genes were confirmed using quantitative polymerase chain reaction (Q‐PCR). The dataset was based on the GPL16699 platform (Agilent‐039494 SurePrint G3 Human GE v2 8 × 60K Microarray 039381).

2.2. Identification of differentially expressed genes (DEGs)

The raw data from the GEO datasets were extracted, pre‐processed, and analyzed using the GEO2R tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/) to identify the DEGs. In the GEO2R tool, we adjusted the Benjamini & Hochberg to regulate false discovery rates and test for multiple corrections to eliminate false positives. The samples were divided into two groups: healthy samples and CU samples. We defined cut‐off values of log₂ fold change as greater than or equal to 1 with a statistical significance of less than 0.05 (p < 0.05). Furthermore, genes that did not have symbols and had multiple probes were excluded from the analysis before obtaining volcano plots, boxplots and mean difference plots. Our analysis identified genes that were differentially expressed in pyroptosis. Additionally, we incorporated the Express Network Analyst (https://www.networkanalyst.ca/) in analyzing the gene matrix files to determine the number of up‐regulated and down‐regulated genes in chronic urticaria.

2.3. Enrichment analyses

We carried out gene ontology (GO) involving cellular components, molecular functions, and biological processes through the metascape database (https://metascape.org/gp/index.html) and identified the key processes of the differentially expressed pyroptosis genes. In metascape, the minimum enrichment was set to 2.0, an overlap of greater or equal to 3 and statistical significance was inferred at a p‐value of less than 0.01. False discovery rates were determined at a significance level of 0.05. Furthermore, we used the database for annotation, visualization, and integrated discovery (DAVID) (https://david.ncifcrf.gov/) to carry out functional annotation of all genes. In the DAVID database, Kappa scores were utilized to determine the correlations between annotated genes depending on fuzzy algorithms and co‐associated networks.

We used the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html) to determine the molecular pathways utilized by the differentially expressed pyroptosis genes. All analyses determined the pathways at 10 and batch effects were reduced using a p‐value of less than 0.05.

2.4. Protein‐protein interaction (PPI) networks

Our study determined PPI networks using the Search Tool for the Retrieval of Interacting Gene Database (STRING) (https://string‐db.org/). The STRING tool allowed us to determine the levels of interactions of all hub genes implicated in chronic urticaria. In STRING, all the genes were matched to Homo Sapiens before producing a suitable network consisting of several edges that exhibit correlations between functional genes. In the first shell, the minimum interaction score was below 10 with a moderate confidence interaction score of 0 and 400 before exporting the networks to Cytoscape for further analysis.

In Cytoscape (https://cytoscape.org/), the networks were analyzed using molecular complex detection (MCODE) in the areas of dense PPI interactions. The lowest k‐interaction score was 6, with a maximum depth of 40 and a cut‐off of 0.5. All differentially expressed genes were classified based on measures of degree and centrality using the CytoHubba extension.

2.5. Immune infiltration

We carried out immune infiltration analysis using CIBERSOFT (https://cibersortx.stanford.edu/) consisting of deconvolution algorithms. CIBERSOFT evaluated the percentages of 22 infiltrating lymphocytes in all the tissue samples, after which we carried out correlation analyses between the immune cells in healthy samples and CU samples.

3. RESULTS

3.1. Identification of DEGs in CU

We obtained and processed all the raw data from the GEO datasets to identify 1613 differentially expressed genes. The top 10up and down‐regulated genes that have been previously reported for chronic urticaria are presented in Table 1 as shown.

TABLE 1.

The top 10up‐and‐down regulated differentially expressed genes in CU.

Gene name	Regulation (up/down)	Previously reported in literature (Yes/No)
IL‐6	Up (42‐fold)	Yes
CD14	Up (13‐fold)	Yes
MYC	Up (17‐fold)	No
TLR4	Up (36‐fold)	Yes
ICAM1	Up (21‐fold)	Yes
CD53	Down (12‐fold)	No
SOD2	Down (12‐fold)	No
S100A8	Down (13‐fold)	No
NCF2	Down (14‐fold)	Yes
PTGS2	Down (21‐fold)	Yes

According to Figure 1, 342 genes were differentially expressed in the GSE57178 dataset, while 1271 genes were differentially expressed genes in the GSE72540 dataset (see Figure 1A, B). The Venn diagram revealed 114 overlapping genes in both datasets, out of which 102 genes were up‐regulated. In comparison, 12 genes were down‐regulated (see Figure 1C). The volcano plots (See Figure 1A, B) were produced based on the Benjamini & Hochberg adjusted p‐value of less than 0.05 to increase the precision of finding differentially expressed genes. The measure of log2 fold change defines the changes in gene expression levels in chronic urticaria against the healthy samples. In the blue region, down‐regulated genes show reduced expression in chronic urticaria compared to healthy samples. In contrast, the red section shows up‐regulated genes whose expression levels are increased in chronic urticaria compared to the healthy samples.

FIGURE 1.

Volcano plots of differentially expressed genes in GSE72540 and GSE57178 and overlapping genes.

3.2. Boxplots and uniform manifold approximation and projection (UMAP) plots

We normalized the datasets to adjust for batch effects and reduce the chances of false positives (see Figure 2A, B). We achieved log2 transformation of all the gene profiles as evidenced by the median values that allow for multiple comparisons between CU and healthy samples in all the datasets. These normalized boxplots represent the gene profiles of both samples in their respective datasets.

FIGURE 2.

Boxplot of normalized gene expression profile in CU and healthy GSE57178 (A) and GSE72540 (B) samples.

According to Figures 3A, B, the two main clusters in the plot correspond to the two groups of samples: chronic urticaria and healthy controls. The genes in the upper cluster are more highly expressed in patients with chronic urticaria, while the genes in the lower cluster are more highly expressed in healthy controls. Each dot on the plot represents a gene, and the distance between dots corresponds to the similarity between the genes' expression profiles.

FIGURE 3.

Uniform Manifold Approximation and Projection (UMAP) plots for GSE57178 (A) and GSE72540 (B).

3.3. Heatmaps

The heatmaps (see Figure 4A, B) show the changes in gene expression levels and intensities of CU and healthy samples. The heatmap is organized into rows and columns, with the columns showing the samples of various genes while the rows show the values of gene expressions. At the intersections, light green indicates gene expressions of lower intensities, while red indicates higher intensities. Furthermore, these heatmaps allow easy comparison of DEGs in healthy and CU samples.

FIGURE 4.

Heatmap of differentially expressed genes in GSE57178 dataset (A) and GSE72540 dataset (B).

3.4. Enrichment analysis of DEGs

Our enrichment analysis of DEGs showed that the DEGs were enriched in various biological processes, cellular components, and molecular functions. The biological processes included activation of myeloid leukocytes, response to inflammations, response to organic substances, defense, external stimulus, locomotion, and processes of immune systems (see Figure 5A).

FIGURE 5.

Enrichment analysis of differentially expressed genes. (A) Biological processes, (B) Cellular components, (C) Molecular functions, and (D) KEGG pathways.

According to Figure 5B, the DEGs were enriched in cellular components of secretory granules, vesicles, cytoplasmic vesicles, intracellular vesicles, tertiary granules, vesicles, sides of membranes, and membranes of secretory granules. Additionally, a low number of genes were observed in the extracellular matrix and external encapsulating structures. According to Figure 5C, the DEGs were enriched in molecular functions of binding of receptor signals, integrins, activities of N‐formyl peptide receptors, binding of RAGE receptors, complementing activity of receptors, and binding of scavenger receptors. Moreover, fewer genes were enriched in the binding of Toll‐like receptors and arachidonic acid.

According to Figure 5D, we observed that KEGG pathways of chronic urticaria were enriched in infections of staphylococcus, neutrophil extracellular trap formation, phagosome, lipid and atherosclerosis, Nuclear‐Factor Kappa B (NF‐kB) signaling pathway, Tumour Necrosis Factor (TNF) signaling pathway, interleukin‐17 (IL‐17) signaling pathway, Janus kinase/signal transducers and activators of transcription (JAK‐STAT) signaling pathway and legionellosis. The pathway of Staphylococcus Infections involves the interaction between the Staphylococcus bacteria and the host immune system. Staphylococcus infections can trigger immune responses and inflammation, which might contribute to the development of chronic urticaria.

The pathway of Neutrophil Extracellular Trap (NET) Formation involves neutrophils that release extracellular traps to capture and neutralize pathogens. Excessive NET formation can lead to inflammation and tissue damage, potentially contributing to chronic urticaria symptoms. Phagosomes are cellular compartments that engulf and digest foreign particles, including bacteria. Dysfunction in this pathway could lead to improper immune responses and the accumulation of immune cells, potentially contributing to chronic urticaria. Lipid metabolism pathways involve the processing of fats and cholesterol. Enrichment in this pathway might indicate a role for lipid‐related factors in chronic urticaria, possibly influencing inflammation and vascular processes.

NF‐Kappa B is a key regulator of inflammation and immune responses. Activation of this pathway can produce pro‐inflammatory cytokines, contributing to the persistence of chronic urticaria symptoms. Tumor Necrosis Factor (TNF) is a cytokine involved in inflammation and immune responses. Dysregulation of the TNF pathway can lead to chronic inflammation, potentially contributing to chronic urticaria. Interleukin‐17 (IL‐17) is a cytokine that promotes inflammation and immune responses. Aberrant IL‐17 signaling may lead to chronic inflammation and contribute to the development of chronic urticaria. Janus kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) proteins are involved in cell signaling. Dysregulation of this pathway can impact immune responses and inflammation in chronic urticaria. Legionellosis is a disease caused by the Legionella bacterium. Enrichment in this pathway suggests a potential role of bacterial infections in chronic urticaria or shared immune responses.

3.5. Protein‐protein interaction networks

According to Figure 6, we identified 27 hub genes that were implicated in CU alongside their measures of degree (deg) and centrality. Among the significantly differentially expressed genes in the study were Interleukin‐6 (IL‐6) with a 42‐fold increase, Cluster of differentiation 14 (CD14) and 163 (CD163), both showing 13‐fold upregulation, followed by Triggering receptor expressed on myeloid cells 1 (TREM1) and Tissue inhibitor of metalloproteinases 1 (TIMP1) at 12‐fold. Formyl peptide receptor 2 (FPR2), cluster of differentiation 53 (CD53), and Superoxide dismutase 2 (SOD2) also exhibited 12‐fold increases. Pentraxin 3 (PTX3), Thrombospondin 1 (THBS1), and Interferon regulatory factor 1 (IRF1) showed 10‐fold changes, while cluster of differentiation 69 (CD69) displayed similar upregulation. Notably, Myc proto‐oncogene (MYC) and Hematopoietic cell kinase (HCK) demonstrated substantial increases of 17‐fold and 16‐fold, respectively. S100 calcium‐binding protein A9 (S100A9), Fc receptor, gamma chain (FCGR2A), and Macrophage receptor 1 (MRC1) all exhibited 15‐fold upregulation. Nuclear factor of activated T cells, cytoplasmic 2 (NFAT2), and Signal transducer and activator of transcription 6 (STAT6) showed 14‐fold changes, while S100 calcium‐binding protein A8 (S100A8) mirrored the 13‐fold increase seen in CD163. Toll‐like receptor 4 (TLR4) stood out with a remarkable 36‐fold increase, followed by Tyrosine‐protein kinase receptor with immunoglobulin‐like and epidermal growth factor‐like domains, beta (TYROBP) at 22‐fold. Cytochrome b561 (CYBB), Intercellular adhesion molecule 1 (ICAM1), and Prostaglandin‐endoperoxide synthase 2 (PTGS2) all showed 21‐fold upregulation, while Integrin alpha X (ITGAX) and Formyl peptide receptor 1 (FPR1) exhibited 20‐fold changes.

FIGURE 6.

PPI networks of hub genes.

3.6. Immune infiltration analysis

According to Figure 7, the immune infiltration analysis in chronic urticaria showed that the hub genes infiltrated immune cells such as neurons, dendrites, Megakaryocytic‐erythroid progenitors (MEP), preadipocytes, endothelial cells, macrophages, T‐helper 2 (Th2) cells and Mast cells (section A). In sections B and C, the common immune cells in both samples were analyzed with consistent findings showing higher infiltrations into preadipocytes, monocytes, and endothelial cells.

FIGURE 7.

Immune infiltration analysis. (A) Immune infiltration cells, (B) Immune cells in GSE57178, and (C) Immune cells in GSE72540.

4. DISCUSSION

CU is an immune‐regulated inflammation associated with systemic response to inflammations and auto‐immune processes. The pathogenesis of CU has been associated with an imbalance in Th1/Th2 cells. Our findings through the PPI networks, enrichment analysis, immune infiltration, and KEGG pathways revealed insights into the molecular mechanisms of CU. We established that hub genes such as IL‐6, PTGS2, and ICAM1 were critical in the pathogenesis of CU coupled with signaling pathways of TNF, JAK‐STAT, and NF‐kB signaling.

We suggest that the pathway of TNF signaling plays a significant role in inflammation and immune responses. When the body encounters harmful stimuli, such as pathogens or tissue damage, immune cells release TNF‐alpha. TNF‐alpha binds to its receptors on cell surfaces, triggering a cascade of events that coordinate the immune response. ²⁰ Dysregulation of the TNF signaling pathway contributes to the development and persistence of CU due to the release of cytokines, inflammation, vascular effects, activation of immune cells, and autoimmunity. ²¹ TNF‐alpha is a potent pro‐inflammatory cytokine. Upon release, it attracts immune cells, particularly neutrophils and macrophages, to the infection, injury, or inflammation site. In CU, excessive levels of TNF‐alpha resulted in a continuous influx of immune cells to the skin, leading to chronic and inappropriate inflammation. This persistent inflammation is a key factor behind the characteristic hives, itching, and symptoms seen in CU and other skin infections. ²²

Our study suggests that in CU, the body experiences persistent triggers like allergens, infections, or auto‐immune reactions, leading to an overproduction of TNF‐alpha. This excess release can contribute to a sustained immune response, potentially increasing inflammation and skin‐related symptoms. TNF‐alpha can influence blood vessels by promoting vasodilation and increasing permeability of blood vessels. ²³ In CU, the effects of TNF‐alpha on blood vessels lead to increased permeability, allowing fluid to leak from blood vessels into the surrounding tissue. This fluid leakage contributes to edema and redness commonly observed in hives or urticarial lesions. Furthermore, TNF‐alpha activates immune cells, including neutrophils and macrophages. When activated by TNF‐alpha, these immune cells release additional inflammatory mediators such as histamines and cytokines. ²¹ In CU, prolonged activation of these immune cells could sustain the inflammatory response, contributing to the persistence of symptoms like itching, swelling, and redness.

TNF‐alpha has been implicated in cell signaling and autoimmunity. ²¹ Bracken et al. ²¹ suggested that TNF‐alpha binds to specific receptors on the surface of cells and triggers a series of intracellular signaling events. Dysregulation of these pathways from genetic factors, environmental triggers, or underlying immune system imbalances could lead to the inappropriate activation of immune responses and the continuous release of inflammatory molecules contributing to CU. In certain cases of CU, the immune system mistakenly targets the body's healthy tissues due to a dysregulation of the TNF signaling pathway. These auto‐immune responses result in urticarial symptoms, and therapies targeting TNF‐alpha might help modulate this immune response and alleviate symptoms. ²¹

Our study proposes that the JAK‐STAT Signaling Pathway was an essential molecular pathway in CU and contributes to its growth and development. The pathway is initiated by binding certain molecules, often cytokines or growth factors, to their respective receptors on the cell surface. ²⁴ According to Feng et al., ²⁴ this binding leads to activating Janus kinase (JAK) proteins associated with these receptors. Once activated, JAK proteins phosphorylate tyrosine residues on the receptors. This creates docking sites for Signal Transducer and Activator of Transcription (STAT) proteins. Phosphorylated STAT proteins are recruited to the receptors, where they are also phosphorylated by JAK. Once phosphorylated, STAT proteins form dimers and translocate to the nucleus. In the nucleus, STAT dimers bind to specific DNA sequences, thereby influencing gene expression. This can lead to the transcription of genes that drive various cellular responses, including immune responses and inflammation. ²⁴

Feng et al. ²⁴ proposed that dysregulation of the JAK‐STAT pathway leads to overactivation of immune cells, such as mast cells and basophils, which are key players in urticarial reactions. ²⁴ Increased activation of these cells results in the release of histamines and other inflammatory mediators, contributing to hives. Moreover, its dysregulation leads to the excessive production of cytokines. Therefore, enhanced chronic inflammation and sustained appearance of hives and other skin symptoms in CU.

Immunoglobulin E (IgE) antibodies play a significant role in CU. ²⁵ The JAK‐STAT pathway can influence the production of IgE, potentially contributing to allergic responses. During the initial exposure to an allergen, B cells are activated and produce IgE antibodies designed to bind specifically to the allergen. ²⁶ These IgE antibodies attach to the surfaces of mast cells and basophils. Upon re‐exposure to the same allergen, it binds to the IgE antibodies on mast cells and basophils, triggering the release of various inflammatory substances, including histamine. The release of histamine and other inflammatory mediators leads to the classic symptoms of urticaria, including itching, hives, redness, and swelling.

The JAK‐STAT pathway regulates the production of IgE antibodies by influencing the differentiation and activity of B cells and plasma cells. ²⁶ , ²⁷ Some cytokines, such as interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13), promote the production of IgE antibodies. The JAK‐STAT pathway is activated when these cytokines bind to their respective receptors on B cells and other immune cells. JAK proteins phosphorylate STAT proteins, allowing them to form dimers and translocate to the nucleus.

We established that the Nuclear Factor‐Kappa B (NF‐Kappa B) pathway is a critical signaling cascade that controls the transcription of numerous genes involved in inflammation, immune responses, cell survival, and various physiological processes. Our findings align with Abd‐Elhakim et al., ²⁸ who suggested that the pathway is initiated in response to various external stimuli, including infections, cytokines, stress, and tissue damage. In the inactive state, NF‐Kappa B is sequestered in the cytoplasm by a family of inhibitor proteins (I‐Kappa B proteins). Upon activation by various stimuli, these inhibitors are phosphorylated and degraded, freeing NF‐Kappa B. ²⁹ Once released, NF‐Kappa B proteins translocate into the nucleus, where they bind to specific deoxyribonucleic acid (DNA) sequences. In the nucleus, NF‐Kappa B binds to DNA and influences the transcription of target genes. These genes encode various molecules involved in immune responses, inflammation, and cell survival, including cytokines, chemokines, adhesion molecules, and enzymes.

NF‐Kappa B activation influences the expression of immune response‐related genes, including those involved in recruiting immune cells to sites of inflammation. ³⁰ Zhou et al. ³¹ suggested that in CU, this leads to the recruitment and activation of immune cells like mast cells, which play a central role in urticarial reactions. Moreover, it influences the expression of enzymes involved in histamine release. Histamine is a potent vasodilator and plays a crucial role in the formation of CU. The pro‐inflammatory cytokines induced by the NF‐Kappa B pathway can sensitize nerve endings, leading to heightened itch sensations.

The molecular function of Receptor signaling is a crucial aspect of cell communication. ³² , ³³ Enrichment of DEGs in binding receptor signals suggested that molecular interactions occur between cells or molecules that transmit signals. CU implies that various cell types, including immune cells, mast cells, and skin cells, are involved in abnormal receptor‐mediated interactions, potentially leading to heightened immune responses and inflammation. Additionally, the molecular function of integrins consists of cell surface receptors that play a key role in cell adhesion and signaling ³⁴ , ³⁵ ; their enrichment among DEGs points to altered cell‐matrix interactions in CU. Dysregulated integrin activity affects cell behavior, immune cell migration, and inflammation, contributing to the persistence of urticarial symptoms.

We observed that molecular functions of activities of N‐Formyl Peptide Receptors were critical in controlling immune responses, particularly in neutrophil chemotaxis. ³⁶ Enriching DEGs related to N‐formyl peptide receptor activities suggests potential immune cell recruitment and inflammation, which could contribute to the characteristic skin manifestations seen in CU. Furthermore, the Receptor for Advanced Glycation End products (RAGE) is involved in inflammation and immune responses. ³⁷ Enrichment in DEGs related to RAGE receptor binding pointed to potential interactions between RAGE and its ligands, contributing to immune dysregulation and inflammation.

Our analysis showed that DEGs involved cellular components such as secretory granules. Secretory granules are specialized cellular compartments that store and release molecules, often proteins or hormones. ³⁸ , ³⁹ Enriching DEGs in secretory granules suggests that certain cells involved in CU, such as mast cells or immune cells, are actively producing and storing various signaling molecules that might be released during immune responses or inflammation. Additionally, the enrichment of DEGs in intracellular and cytoplasmic vesicles indicated dynamic cellular processes. It suggests the involvement of vesicle‐mediated transport and communication in CU, potentially related to immune cell activation and signaling.

The lower enrichment of genes in the extracellular matrix and external encapsulating structures suggests that the focus might be more on intracellular processes and cellular compartments rather than interactions with the extracellular environment. This might imply that the internal processes of cells play a significant role in chronic urticaria, possibly linked to immune responses and inflammation. ⁴⁰

Our enrichment analysis of biological processes showed genes enriched in the activation of myeloid leukocytes. Myeloid leukocytes, including neutrophils, monocytes, and macrophages, are key players in the immune response. ⁴¹ The enrichment of DEGs in the activation of myeloid leukocytes suggests that these immune cells are being triggered and mobilized in response to factors involved in CU. Thus, this activation contributes to inflammation and immune responses in CU. When exposed to triggers like allergens or pathogens, receptors on the surface of myeloid leukocytes recognize these foreign substances or altered self‐components and trigger a cascade of intracellular signaling events. ⁴² Recognizing triggers leads to activating various signaling pathways within myeloid leukocytes, such as kinases, which act as molecular switches, transmitting signals and initiating cellular responses.

Neutrophils constitute the first line of defense against inflammation. They are recruited to the affected tissues and release enzymes and other substances to neutralize threats. ⁴³ , ⁴⁴ , ⁴⁵ However, their excessive activation leads to tissue damage and symptoms of CU. In contrast, monocytes, upon activation, differentiate into macrophages. Macrophages play a role in phagocytosis and producing cytokines that regulate immune responses. ⁴⁶ Dysregulated activation of these cells can contribute to chronic inflammations in CU. Response to organic substances implies that the cells and tissues involved in CU react to specific allergens. Therefore, this response leads to the release of inflammatory mediators and contributes to the characteristic symptoms of CU. Once activated, myeloid leukocytes release inflammatory mediators, including cytokines, chemokines, and reactive oxygen species that attract other immune cells to the site of inflammation and contribute to redness, swelling, and itching. ⁴³ , ⁴⁴

Our study established that hub genes could be targeted to develop new therapeutic techniques in the management of CU. IL‐6 is a cytokine that is central in regulating immune responses and inflammation. ⁴⁷ Its overexpression is often associated with chronic inflammatory conditions. In the context of CU, elevated levels of IL‐6 contribute to the chronic and sustained inflammation observed in the skin, leading to the development and persistence of urticarial lesions and associated symptoms. ⁴⁸ , ⁴⁹ IL‐6 can stimulate immune cells and contribute to the production of other inflammatory mediators.

PTGS2 encodes the COX‐2 enzyme, which is involved in the production of prostaglandins (molecules that mediate inflammation and various physiological processes). Overexpression of COX‐2 can lead to increased production of prostaglandins, promoting inflammation and vasodilation. ⁵⁰ , ⁵¹ In CU, up‐regulated PTGS2 contributes to the vasodilation, edema, and itching associated with urticarial reactions. ICAM1 (Intercellular Adhesion Molecule 1) is involved in cell adhesion and migration, critical for immune cell interactions and inflammatory responses. ⁵² Elevated levels of ICAM1 can lead to increased adhesion and migration of immune cells to inflammatory sites. In CU, increased expression of ICAM1 contributes to immune cell infiltration, inflammation, and the formation of urticarial lesions.

5. CONCLUSION

All the hub genes play interconnected roles in the pathogenesis of CU, contributing to immune dysregulation, chronic inflammation, and immune cell activation. Understanding the significance of these genes provides a foundation for further research and the development of potential therapeutic interventions. Enriching DEGs in these biological processes highlights chronic urticaria's immune and inflammatory nature. The immune system's response to various triggers, including organic substances, contributes to persistent inflammation, hives, and other symptoms. Understanding these biological processes offers insights into potential targets for therapeutic interventions that can modulate immune responses, reduce inflammation, and alleviate chronic urticaria symptoms.

Enriching DEGs in these cellular components reflects the intricate cellular mechanisms in chronic urticaria. It suggests that a variety of immune cells, including mast cells neutrophils, are actively participating in the release of signaling molecules, which contribute to immune responses, inflammation, and symptoms in chronic urticaria. Understanding these cellular processes guides the development of targeted therapies to manage chronic urticaria. Enriching KEGG pathways in chronic urticaria points to complex interactions involving infections, immune responses, inflammation, and cellular signaling. Investigating these pathways further could better understand the underlying mechanisms driving chronic urticaria and potentially inform targeted therapeutic approaches.

Enriching DEGs in these molecular functions highlights several interactions between immune cells, receptors, and signaling molecules in CU. These findings suggest potential targets for therapeutic interventions aimed at modulating immune responses, reducing inflammation, and alleviating the symptoms associated with CU.

AUTHOR CONTRIBUTIONS

Haichao Guo and Yanjun Wang designed the study. Lifang Guo and Li Li performed the data analysis. Na Li and Xiaoyun Lin collected background information. Haichao Guo drafted the manuscript. All authors contributed to the article and approved the submitted version.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Guo H, Guo L, Li L, Li N, Lin X, Wang Y. Identification of key genes and molecular mechanisms of chronic urticaria based on bioinformatics. Skin Res Technol. 2024;30:e13624. 10.1111/srt.13624

DATA AVAILABILITY STATEMENT

Data for this study is available from the corresponding author upon reasonable request.