Figure 3 . Significantly enriched cell processes identified in all three tested groups (subjects with allergy, subjects with allergy without asthma and AD, and subjects with allergic asthma and AD) in comparison to control group using SNEA. Individual cells present a p value of selected pathways.
Discussion
As the type I hypersensitivity can manifest as atopic dermatitis, conjunctivitis, rhinitis, rhinoconjuctivitis, or allergic asthma, we compared gene transcripts common for participants with these conditions (between allergy group, allergy without asthma and AD group, and allergic asthma and AD group) and confirmed that there are shared genes among these groups. There were 61 altered transcripts identified commonly for all three tested groups (Figure 1, Supplementary Material 1: Figure S2). Among them, 7 genes were found to be involved in immune-related pathways in GO database (Figure 1 and 2). These seven genes have established roles in immunity; however, it is important to emphasize that these genes often have multiple functions beyond immunity, and their roles can be complex and context-dependent. With respect to immune processes, this cluster of genes is associated with T cell activation, differentiation, and migration (PRNP, IL27RA, RIPOR2)27 ,28 ,29, immune cell regulation (L3MBTL3, IL27RA)30, 31, and is linked to the severity of immune mediated diseases such as asthma and dermatitis (IL27RA) and anaphylactic response (PTAFR)28, 32. When assessing gene function by Gene Ontology (GO)33, the top 15 biological processes (FDR <0.05) indicate pro-inflammatory machinery as the biological processes include T cell migration, leukocyte activation and production of pro-inflammatory signals IL-6 and IL-17.
By employing both GSEA and SNEA on a whole genome profile, we identified cell processes and other functional entities enriched by estimated gene profiles related with type I hypersensitivity. During both the sensitization phase and effector phase, many signaling molecules are employed, e.g., interleukins, immunoglobulins, or CD molecules, whose signaling pathways were revealed by GSEA analysis (Table 3). Cell processes identified by SNEA were shared among at least two of the three groups. These shared pathways were involved in APC function, Th cell signaling, including Th2 cell’s function leading to IgE production. These processes are crucial in the sensitization phase of the type I hypersensitivity. Further, mast cells, basophils, and eosinophils, whose signaling pathways were also enriched in our analyses, are important for the process of atopy, namely for the effector phase.
SNEA identified various cell processes belonging to cell groups such as APCs, Th cells, and B cells (Table 4), that are involved in sensitization phase. The sensitization phase starts by reaction of naive T cells to an antigen (allergen) by APCs8. At this stage, APCs, namely dendritic cells capture and process the antigen and, through major histocompatibility complex (MHC) trigger T-cell receptors (TCRs) presented on naive T cells34. Engagement of MHCs class II with TCRs activates the proliferation of naive T cells and promotes inflammatory signaling cascades, resulting in differentiation of T cells into Th cells and regulatory T cells (Tregs)35.
GSEA revealed roles for many immune-related molecules, including CD80, CD86, IL-3 and IL-4. Specifically, CD80 and CD86 are membrane proteins expressed by APCs during sensitization/regulation. Engaging with CD28 receptor, CD80 and CD86 act as co-stimulators in APC:T cell interaction and enhance TCR signaling36, 37. TCRs and CD28 downstream signaling is associated with activation of NF-κB transcription factor, one of crucial regulators of IL-4 expression, that plays an essential role in Th2 cell differentiation38. Beside IL-4, IL-25, IL-33, and Thymic Stromal Lymphopoietin (TSLP) cytokines, IL-3 has also been found to be involved in Th2 cell differentiation. While IL-4 directly drives Th2 cell differentiation, IL-3 regulates the process indirectly by enhancing the production of IL-439, 40.
In addition to ACPs, Th cells, and B cells, SNEA identified enriched cell processes related with Th2 cells and germinal centers (GC). B cells interact with Th cells. Th2 cells play a crucial role in the entire inflammatory process. Th2 cells stimulate activation of B cells via production of IL-4 and IL-13. The interaction induces B cell differentiation and isotype switching of Ig heavy chain, and therefore, differentiated B cells migrate into secondary lymphoid organs where they induce GC formations. In germinal centers, B cells undergo somatic hypermutation and proliferation, and start to produce IgE antibodies41, 42, 43, 44. Consequently, IgE antibodies bind to IgE receptors presented on mast cells and basophils whereby trigger the effector phase8, 45, 46.
Other enriched entities provided by GSEA include receptor tyrosine kinases, specifically VEGFA, VEGFR, EREG, TGFA and HBEGF, in conjunction with the forkhead box O3a (FOXO3A). In addition to these expression targets, enriched entities related to IL-17 were observed in our study. Both FOXO3A and IL-17 are involved in the IgE production network. Moreover, Tgfb expression has an impact on many cell processes related with T cells and B cells (for more details of the genes regulating these pathways, see Overlapping genes in Supplementary Material 4). Production of IgE antibodies is indirectly controlled by Tregs. Tregs, together with regulatory B cells (Bregs), act as an inhibitor of production of IgE antibodies through regulation of transforming growth factor β (TGFβ) and IL-10. Therefore, this cytokine (TGFβ) plays a dominant role in IgE regulation during inflammation. TGFβ is known as an inhibitor of B cell differentiation and proliferation and can induce Ig isotype switching into IgA antibodies47.
Simultaneously, TGFβ induces conversion of CD4+ T cells to Tregs under regulation of FOXO3A protein48. FOXO3A protein was examined as a suppressor of both pro-inflammatory cytokines, such as IL-4 and IL-13, and anti-inflammatory cytokines, namely IL-1048, 49, 50. Another member of the FOXO family, the FOXO1 transcription factor, negatively regulates the generation of Th17 cells and, consequently, the secretion of IL-17, which also promotes IgE production48, 51. Although, the regulatory network involving FOXO transcription factors underscores its role in regulating cytokine expression, which in turn affects IgE production and contributes to immune homeostasis, the impact of the observed tyrosine kinases on the FOXO3A transcription factor remains understudied.
Lastly, both GSEA and SNEA identified enriched processes related to mast cells that are involved in effector phase of type I hypersensitivity reaction. The effector phase starts by degranulation of activated basophils and mast cells8, 45. Activated mast cells secrete their mediators, namely cytokines (IL-4, IL-5, IL-13, and TNFs), leukotrienes, and during degranulation, histamine, heparins, tryptases or chymases. The degranulated mediators provoke eosinophil adhesion, accumulation, and migration, and neutrophil accumulation52. The cytokines produced by mast cells, especially TNFs, promote the mast cell development and migration that support the recruitment of eosinophils, neutrophils, and mast cells (themselves), and overall allergic reaction53, 54. Moreover, IL-3 stimulates TNF production that is crucial for mast cell development55.
In conclusion, expression profiling blood in participants with allergic manifestations including allergic asthma, atopic dermatitis and other symptoms provided insights into immune-related pathways. Crucial processes involved antigen presentation, Th2 cell signalling leading to IgE production, and mast cell, basophil, and eosinophil involvement in the effector phase. Moreover, the 7 observed common genes may play an important role in immune processes related to type I hypersensitivity and may be candidates for new potential biomarkers. However, these genes should be confirmed by further clinical or focused in vivostudies. Understanding these mechanisms is vital for targeted therapies and improved management of atopic diseases.