Key genes and immune infiltration in chronic spontaneous urticaria: a study of bioinformatics and systems biology.

Background: Chronic spontaneous urticaria (CSU) is defined by the spontaneous occurrence of wheals and/or angioedema for >6 weeks. The pathogenesis involves skin mast cells, but the complex causes of their activation remain to be characterized in detail. Objectives: To explore disease-driving genes and biological pathways in CSU. Methods: Two microarray data sets, e.g., GSE57178 and GSE72540, with mRNA information of skin from CSU patients, were downloaded from the Gene Expression Omnibus (GEO) database. An integrated bioinformatics pipeline including identification of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction (PPI) network analysis, co-expression and drug prediction analysis, and immune and stromal cells deconvolution analyses were applied to identify hub genes and key drivers of CSU pathogenesis. Results: In total, we identified 92 up-regulated and 7 down-regulated genes in CSU lesions. These were significantly enriched in CSU-related pathways such as TNF, NF-κB, and JAK-STAT signaling. Based on PPI network modeling, four genes, i.e., IL-6, TLR-4, ICAM-1, and PTGS-2, were computationally identified as key pathogenic players in CSU. Immune infiltration analyses indicated that dendritic cells, Th2 cells, mast cells, megakaryocyte-erythroid progenitor, preadipocytes, and M1 macrophages were increased in lesional CSU skin. Conclusion: Our results offer new insights on the pathogenesis of CSU and suggest that TNF, NF-κB, JAK-STAT, IL-6, TLR-4, ICAM-1, and PTGS-2 may be candidate targets for novel CSU treatments.

Involvement of CCL2 and CH25H Genes and TNF signaling pathways in mast cell activation and pathogenesis of chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU), a mast cell-driven disease, substantially affects the quality of life. While genetics affect CSU susceptibility and severity, the specific genetic factors associated with mast cell activation in CSU remain elusive. We aimed to identify key genetic factors and investigate their roles in CSU pathogenesis. Two gene expression datasets from the Gene Expression Omnibus were merged and validated using principal component analysis and boxplots. The merged dataset was subjected to limma and weighted gene co-expression network analyses. Genes whose expression correlated highly with CSU were identified and analyzed using Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. As GSEA, GO, and KEGG analyses highlighted the importance of chemokine (C-C motif) ligand 2 (CCL2) and cholesterol 25-hydroxylase (CH25H) gene and tumor necrosis factor (TNF) signaling pathways in CSU; the three corresponding genes were knocked down in human mast cell line-1 (HMC-1), followed by incubation with thrombin to mimic CSU pathogenesis. CCL2, CH25H, and TNF knockdown reduced excitability and cytokine production in HMC-1. Our findings suggest that genes involved in the CCL2, CH25H, and TNF pathways play crucial roles in CSU pathogenesis, providing insights into potential therapeutic targets for CSU treatment.

A genome-wide association study of chronic spontaneous urticaria risk and heterogeneity.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a dermatologic condition characterized by spontaneous, pruritic hives and/or angioedema that persists for 6 weeks or longer with no identifiable trigger. Antihistamines and second-line therapies such as omalizumab are effective for some CSU patients, but others remain symptomatic, with significant impact on quality of life. This variable response to treatment and autoantibody levels across patients highlight clinically heterogeneous subgroups. OBJECTIVE: We aimed to highlight pathways involved in CSU by investigating the genetics of CSU risk and subgroups. METHODS: We performed a genome-wide association study (GWAS) of 679 CSU patients and 4446 controls and a GWAS of chronic urticaria (CU)-index, which measures IgG autoantibodies levels, by comparing 447 CU index-low to 183 CU index-high patients. We also tested whether polygenic scores for autoimmune-related disorders were associated with CSU risk and CU index. RESULTS: We identified 2 loci significantly associated with disease risk. The strongest association mapped to position 56 of HLA-DQA1 (P = 1.69 × 10-9), where the arginine residue was associated with increased risk (odds ratio = 1.64). The second association signal colocalized with expression-quantitative trait loci for ITPKB in whole blood (Pcolocalization = .997). The arginine residue at position 56 of HLA-DQA1 was also associated with increased risk of CU index-high (P = 6.15 × 10-5, odds ratio = 1.86), while the ITKPB association was not (P = .64). Polygenic scores for 3 autoimmune-related disorders (hypothyroidism, type 1 diabetes, and vitiligo) were associated with CSU risk and CU index (P < 2.34 × 10-3, odds ratio > 1.72). CONCLUSION: A GWAS of CSU identified 2 genome-wide significant loci, highlighting the shared genetics between CU index and autoimmune disorders.

GWAS of Chronic Spontaneous Urticaria Reveals Genetic Overlap with Autoimmune Diseases, Not Atopic Diseases.

Although chronic spontaneous urticaria (CSU) is a common disease, GWASs of CSU are lacking. We aimed to identify susceptibility SNPs by performing a GWAS in Chinese Han adults with CSU. The discovery cohort included 430 CSU cases and 482 healthy controls. The GWAS findings were validated in 800 CSU cases and 900 healthy controls. Genetic, functional enrichment, and bioinformatic analyses of genome-wide significant SNPs were performed to assess the association between CSU and autoimmunity or atopy. Five genome-wide significant SNPs were identified: rs434124/LILRA3, rs61986182/IGHG1/2, rs73075571/TDGF1, rs9378141/HLA-G, and rs3789612/PTPN22. The first four SNPs were in linkage disequilibrium with autoimmune-related diseases‒associated SNPs and were cis-expression quantitative trait loci in immune cells. The five SNPs-annotated genes were significantly enriched in immune processes. Higher polygenic risk scores and allele frequencies of rs3789612∗T, rs9378141∗C, and rs73075571∗G were significantly associated with autoimmune-related CSU phenotypes, including positive antithyroglobulin IgG, positive anti-FcεRIα IgG, total IgE <40 IU/ml, and positive antithyroid peroxidase IgG but not with atopic or allergic sensitized CSU phenotypes. This GWAS of CSU identifies five risk loci and reveals that CSU shares genetic overlap with autoimmune diseases and that genetic factors predisposing to CSU mainly manifest through associations with autoimmune traits.

STAT3 gain-of-function is not responsible for low total IgE levels in patients with autoimmune chronic spontaneous urticaria.

Background: The pathogenesis of chronic spontaneous urticaria (CSU) has not been clarified entirely. Type IIb autoimmune chronic spontaneous urticaria (CSUaiTIIb) is a distinct subtype of CSU that is often difficult to treat and is connected to low levels of total IgE. Previous findings indicate that an enhanced signal transducer and activator of transcription 3 (STAT3) may be responsible for reduced IgE serum levels. Objective: Our aim was to investigate a possible underlying gain-of-function mutation or activating polymorphism in STAT3 that could be responsible for the low levels of IgE in patients with CSUaiTIIb. Methods: We included 10 patients with CSUaiTIIb and low levels of IgE and sequenced selected single nucleotide polymorphisms (SNP) in STAT3 associated with common autoimmune diseases. Exon sequencing was performed for the most relevant exons of STAT3. To test for a gain-of-function of STAT3, we performed a phospho-specific flow cytometry analysis of STAT3 in peripheral blood mononuclear cells before and after stimulation with interleukin-6. Results: No differences were found in the prevalence of the tested SNPs between our patients and a control population. Moreover, we could not find any mutations or variants on the tested exons of STAT3. The function of STAT3 was also not altered in our patients. Conclusion: In total, we could not find any evidence for our hypothesis that low IgE in patients with CSUaiTIIb is linked to mutations in STAT3 or altered activity of STAT3. Thus, it remains to be discovered what causes the low serum levels of IgE in patients with CSUaiTIIb.

Association of PTPN22 single nucleotide polymorphisms with chronic spontaneous urticaria.

INTRODUCTION AND OBJECTIVES: Chronic spontaneous urticaria (CSU) is thought to be an autoimmune disease in a subpopulation of patients. Protein tyrosine phosphatase-22 (PTPN22) polymorphisms are considered to be one of the strongest contributing factors to autoimmune diseases. In this study, we aimed to investigate the potential association of several PTPN22 single nucleotide polymorphisms (SNPs) with CSU in an Iranian population. MATERIAL AND METHODS: A total of 93 CSU patients and 100 healthy individuals were included in this study. Five SNPs within the PTPN22 gene were analyzed using TaqMan genotyping assays. The frequency of alleles, genotypes, and haplotypes of PTPN22 SNPs (rs12760457, rs2476601, rs1310182, rs1217414, and rs33996649) was investigated. RESULTS: A significantly higher prevalence of the rs1310182 T allele was observed among patients compared with controls [OR = 1.75 (95% CI: 1.17-2.63); P = 0.007]. In addition, the rs1310182 CC genotype and TT genotype were 0.47 and 2.06 times more common in patients, respectively (P = 0.03). Moreover, haplotype analysis demonstrated that CGCGC, CGTGC, and TGCGC (P < 0.001) were significantly associated with CSU. No significant differences were observed between the patients and controls in the other analyzed PTPN22 SNPs. CONCLUSIONS: Polymorphisms of the PTPN22 gene are associated with an increased susceptibility to CSU in the studied Iranian population.

Interleukin 17 receptor A haplotype analysis in chronic spontaneous urticaria: A preliminary study.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a distressing skin disease. Family clustering and heterogeneity in the onset and progression indicate that susceptibility to CSU is a complex trait. In this study, we performed haplotype analysis for one of the key player gene, IL17RA, for CSU to test the association with disease susceptibility and severity. METHODOLOGY: The study included 70 CSU patients and 30 healthy controls. The severity of the disease was evaluated by autologous serum skin test (ASST) and urticaria activity score (UAS). ASST test was done and quality of life was assessed using a questionnaire. Allelic discrimination analysis for rs4819554 and rs879577 was performed using real-time polymerase chain reaction technology. RESULTS: Carriers of rs4819554*G were more prone to develop CSU than its counterpart (P = .039), while rs4819554*A allele displayed more severe phenotype in the form of more prolonged disease duration (P = .040), concurrent angioedema (P < .001), higher level of treatment (P < .001), and higher score of quality of life (P < .001). Additionally, homozygote patients with rs879577*CC were associated with angioedema (P < .001). Haplotype analysis revealed that cohorts with both rs4819554*A and rs879577*T conferred protection against developing CSU (OR = 0.07, 95% CI = 0.01-0.32, P = .001). CONCLUSION: Our results showed that IL17RA gene polymorphisms might contribute to the increased susceptibility to CSU.

Interleukin 1 Gene Polymorphisms Presumably Participate in the Pathogenesis of Chronic Spontaneous Autoreactive Urticaria.

Recent studies underline a potential role of autoimmune and genetic disturbances in this disorder pathogenesis. Variants in genes related to inflammatory processes may possibly predispose to chronic spontaneous urticaria (CSU) occurrence. The objective of this study was to search for an association of Il1 genes polymorphisms with the pathogenesis of CSU. The examined group consisted of 153 unrelated chronic spontaneous autoreactive urticaria patients. The control group consisted of 104 unrelated healthy volunteers. In all studied subjects, IL1 rs1304037 and rs180058 polymorphisms were examined. The Urticaria Activity Score was used to assess disease intensity. The age of disease onset was also analyzed. Statistically significantly higher prevalence of Il1 rs1304037 TT genotype and T allele among CSU was proved. Similarly, the prevalence of Il1 rs1800587 GG genotype and G allele was statistically significantly higher in the CSU group. Haplotype combination rs1304037C/rs1800587G was statistically significantly more frequent in CSU, whereas rs1304037C/rs1800587A revealed statistically significantly less frequent occurrence in CSU. We did not observe any relationship between Il1 genotypes and the disease severity or age of disease onset. We are the first to suggest a significant role of IL1 gene polymorphisms in the susceptibility to CSU. This observation may lead to a better pathogenesis understanding and more effective treatment. We recommend further studies on other polymorphisms in chronic urticaria to analyze the role of the genetic mechanisms in the pathogenesis of this disorder.

Down-regulation of Gremlin1 inhibits inflammatory response and vascular permeability in chronic idiopathic urticaria through suppression of TGF-ß signaling pathway.

Chronic idiopathic urticaria (CIU) is an unfavorable skin condition which could be maintained for six weeks or longer time. Gremlin1 (GREM1) was recently applied in treatments of many diseases. However, the possible regulatory mechanism of GREM1 in CIU remained unclear. This study aimed to explore the regulatory effects of GREM1 on the inflammatory response and vascular permeability mediated by mast cells of CIU via TGF-ß signaling pathway. Initially, microarray analysis was used to identify CIU-related differentially expressed genes and the potential mechanism of this gene. A mouse model of CIU was established. To explore the functional role of GREM1 in CIU, the modeled mice were then injected with GREM1-siRNA, SRI-011381 (the activator of TGF-ß signaling pathway), or both, followed by serum test, and immunoglobulin detection. The levels of inflammatory factors and tryptase, ß-hexosaminase, histamine in the serum were detected. Besides, vascular endothelial cell permeability and the target relation between GREM1 and TGF-ß were also examined. Mice injected with SRI-011381 exhibited higher levels of tryptase, ß-hexosaminase, histamine, inflammation-related factors and increased vascular endothelial cell permeability, while GREM1-silenced mice yet expressed opposite tendency. Silencing of GREM1 was demonstrated to inhibit the TGF-ß signaling pathway. Taken together, our results demonstrated that down-regulation of GREM1 could potentially impede inflammatory response and vascular permeability by suppressing TGF-ß signaling pathway. GREM1 may promote the development of prognosis management and therapeutic treatment in CIU.

The association between chronic spontaneous urticaria and HLA class I and class II antigen

Background/aim: Chronic spontaneous urticaria (CSU) is a chronic disease with an unknown etiology. In human leukocyte antigen (HLA) system, the association of class I and class II antigens with autoimmune diseases has been identified and HLA antigens that have a tendency to or can prevent chronic urticaria have been studied. The aim of this study is to investigate the association between chronic spontaneous urticaria and HLA class I and class II antigens. Materials and methods: A total of 80 subjects, 40 patients with CSU and 40 healthy individuals were enrolled in the study. DNA sample isolation from blood was primarily done by the real-time polymerase chain reaction (RT-PCR) technique for the first time. Using HLA SSP Typing Kit (ROSE Cat. No: 800118) PCR technique, HLA-A, B, C, DRB and DQB alleles from DNA samples were analyzed. Results: The mean age was 36.80 ± 9.48 years and the duration of the disease was 4.26 ± 5.18 years. Among the HLA class I and class II antigens, HLA-A was detected significantly more often in the control group (P = 0.039). HLA-DRB1 was more often detected in the CSU group but no statistical difference (P > 0.05). Conclusion: It can be considered that HLA-DRB1 may have a tendency to CSU, while HLA-A might prevent the disease.

Somatic Mosaic NLRP3 Mutations and Inflammasome Activation in Late-Onset Chronic Urticaria.

Chronic urticaria is a common skin disorder with heterogeneous causes. In the absence of physical triggers, chronic urticarial rash is called idiopathic or spontaneous. The objective of this study was to identify the molecular and cellular bases of a disease condition displayed by two unrelated patients aged over 60 years who presented for two decades with a chronic urticaria resistant to standard therapy that occurred in the context of systemic inflammation not triggered by cold. In both patients, a targeted sequencing approach using a next generation technology identified somatic mosaic mutations in NLRP3, a gene encoding a key inflammasome component. The study of several of both patients' cell types showed that, despite the late onset of the disease, NLRP3 mutations were not found to be restricted to myelomonocytic cells. Rather, the data obtained strongly suggested that the mutational event occurred very early, during embryonic development. As shown by functional studies, the identified mutations-an in-frame deletion and a recurrent NLRP3 missense mutation-have a gain-of-function effect on NLRP3-inflammasome activation. Consistently, a complete remission was obtained in both patients with anti-IL-1 receptor antagonists. This study unveils that in late-onset chronic urticaria, the search for autoinflammatory markers and somatic mosaic NLRP3 mutations may have important diagnostic and therapeutic consequences.

C-reactive protein (CRP) rs3093059C predicts poor mizolastine response in chronic spontaneous urticaria patients with elevated serum CRP level.

Chronic spontaneous urticaria (CSU) is a frequent disorder with recurrent itchy wheals and/or angioedema, and nearly 35% patients respond poorly to non-sedating H1 antihistamine treatment. CRP gene encodes the C-reactive protein, which is involved in the pathogenesis of CSU. To investigate the impacts of CRP polymorphisms on the susceptibility and therapeutic efficacy in the South Han CSU patients, we enrolled 145 CSU patients in our study. After 4-week non-sedating H1 antihistamine monotherapy treatment, more than 50% reduction of the severity score is considered as effective, or else non-effective. The CRP rs3093059T/C and rs2794521G/A genotypes of patients were determined by Sequenom MassARRAY. Functional studies including relative luciferase assay and ß-hexosaminidase assay were conducted in HEK293T cells or RBL-2H3 cells to explore the function of variants. Forty (62.50%) CSU patients were effective when treated with mizolastine, and 55 (72.4%) patients were effective in the desloratadine group. We found that the patients carried with rs3093059TT genotype were significantly associated with good response (OR = 4.20, P = 0.015), had lower serum CRP, IL-6 and TNF-α levels than the CT/CC genotypes. In vitro, the rs3093059C allele exhibited significantly higher luciferase activity than wild allele (P < 0.001). From the ß-hexosaminidase assay, we observed the inhibiting degranulation effects by mizolastine and this effect is weakened when with a higher dose CRP in RBL-2H3 cells. Our findings suggested that CSU patients carrying the rs3093059C allele may respond poorly to mizolastine with elevated serum CRP level.

Omalizumab normalizes the gene expression signature of lesional skin in patients with chronic spontaneous urticaria: A randomized, double-blind, placebo-controlled study.

BACKGROUND: Omalizumab, a humanized recombinant monoclonal anti-IgE antibody, proved to be effective in patients with chronic spontaneous urticaria (CSU), including severe and treatment-refractory CSU. Here, we report omalizumab's effect on gene expression in skin biopsies from CSU patients enrolled in a double-blind, placebo-controlled study. METHODS: Chronic spontaneous urticaria patients (18-75 years) were randomized to either 300 mg omalizumab (n = 20) or placebo (n = 10) administered s.c. every 4 weeks for 12 weeks (NCT01599637). Lesional and nonlesional skin biopsies were collected from the same area of consenting patients and assessed at baseline and on Day 85 compared with skin biopsies from the same area of 10 untreated healthy volunteers (HVs). Gene expression data were generated using Affymetrix HG-U133Plus2.0 microarrays. Statistical analyses were performed using R packages. RESULTS: At baseline, 63 transcripts were differentially expressed between lesional and nonlesional skin. Two-thirds of these lesional signatures were also differentially expressed between lesional and HV skin. Upon treatment with omalizumab, >75% of lesional signatures changed to reflect nonlesional skin expression levels (different vs placebo, P < 0.01). Transcripts upregulated in lesional skin (vs nonlesional and/or HV skin) suggested increased mast cell/leukocyte infiltration (FCER1G, C3AR1, CD93, S100A8, and S100A9), increased oxidative stress, vascularization (CYR61), and skin repair events (KRT6A, KRT16). Lesional signatures were not modulated by treatment in nonresponders (defined based on UAS7 longitudinal changes ≥16). CONCLUSION: Omalizumab, in treatment responders, reverted transcriptional signatures associated with CSU lesion phenotype to reflect nonlesional/HV expression levels; this is consistent with observed omalizumab-mediated clinical improvement observed in patients with CSU.