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.