Through a novel bioinformatic approach this study provided insights on the main biological pathways disrupted by genetic defects in a small cohort of unrelated PG patients. Both in the “multilesional” and “localized” groups, VEA workflow applied to WES data showed immune and wound repair processes in PG susceptibility and severity modulation.
Interestingly, most of the pathways, including those related to extracellular matrix (ECM) remodeling, type I interferons and DAP12 (DNAX activating protein of 12 kDa) Interaction pathways herein discussed have been documented to encompass genes differentially expressed in cutaneous PG samples respective to healthy control skin, further crediting their contribution to the disease pathophysiology [15].
Among the seven Immune System pathways disrupted in the “multilesional” group exclusively, Type I interferons (IFN) (R-HSA-909733) are of particular interest, representing key cytokines for the development of innate and adaptive immune responses, with important roles in host defense against viral infections and autoimmunity. Canonical type I IFN signaling activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway and is known to be involved in pathogenesis of PG. Our results fall in line with this premise and, indeed, JAK inhibition is shaping up as a compelling treatment option for PG, although its long-term safety is still under investigation [16].
Further, the CLEC7A (Dectin-1) Induces NFAT Activation pathway (R-HSA-5607763) may also fit in PG complex pathogenesis due to its association with the extracellular release of interleukin (IL)-2, a key mediator of regulatory T cells (Treg cells) functions [17–19]. Tregs cells play a central role in preventing autoimmune and autoinflammatory processes and in fact an imbalance between Tregs and T helper (Th)17 cells has been detected in PG [20].
Acting upstream of T cell polarization, the major histocompatibility complex class I (MHC-I) antigen presentation plays a key role in alerting the immune system to cells infected; a downregulation of MHC-I molecules at the cell surface decreases the ability of CD8+ T cells to recognize peptides with a consequent impairment in immune responses [21, 22]. The Nef Mediated Downregulation of MHC- I complex cell surface expression (R-HSA-164940) and the Endosomal/Vacuolar (R-HSA-1236977) pathways are necessary for antigen processing by MHC-I, suggesting that MHC-I pathway could play a role in PG pathogenesis. Furthermore, the endosomal/vacuolar pathway (R-HSA-1236977) is related to autophagy, a process that plays a housekeeping role in maintaining cellular homeostasis and protects against genome instability [23]. Dysregulated or absent autophagy results in a wide range of conditions, including inflammatory and immune-mediated skin diseases [24]. In addition, defective autophagy in endothelial cells determines excessive transendothelial migration (TEM), with subsequent neutrophil infiltration and tissue damage [25] - both key events in PG pathophysiology.
Two additional pathways may have effect on innate immune cell function, i.e., the Maresin Conjugates in Tissue Regeneration (MCTR) (R-HSA-9026762) biosynthetic pathway and the DAP12 Interaction pathway (R-HSA-2172127).
MCTR is a family of recently identified macrophage-derived mediators promoting the resolution of inflammatory processes, through a wide variety of biological actions. Although their role in the skin has been poorly elucidated, a variety of experimental models has demonstrated their effects in alleviating neutrophil infiltration, reducing IL-17A levels [26], stimulating M2-macrophage polarization [27], hastening bacterial clearance as well as the repair and regeneration of damaged tissue. Accordingly, the disruption of their biosynthetic pathway (R-HSA-9026762) may contribute to the non-healing nature of PG ulcers [28].
DAP12 is involved in the transduction of a wide variety of activating signals in neutrophils, monocytes/macrophages, natural killer (NK) and dendritic cells (DCs) [29]. Interestingly, in a landmark study comparing gene expression between PG and healthy control skin, several differentially expressed genes were noted to be associated with the DAP12 Interactions pathway (R-has-2172127) [15]. Although the latter may be but the reflection of PG cutaneous milieu, our results together with the pathway’s pivotal role in myeloid cells - mediating the production of pro-inflammatory cytokines, chemokines, reactive oxygen species, degranulation of neutrophilic granules, and phagocytosis [30] - support the idea of its contribution to the exaggerated inflammatory response observed in PG.
As most of these pathways also influence the clearance of bacterial infections, it is tempting to speculate that their disruption may translate into greater susceptibility to superinfections, prolonging/complicating PG course and adding a further layer of complexity to their biological meaning.
Considering the five Cell Metabolism pathways, their functions were related with cell survival, proliferation, migration, and vascular permeability (R-HSA-5218921) [31]; cellular respiration and catabolism (R-HSA-5661270 and R-HSA-70895) [32]; and signal transduction (R-HSA-1489509 and R-HSA-114508) [33, 34], all of which may have major repercussions on wound healing and therefore PG pathophysiology.
Regarding the two pathways associated with Structural Functions, they were mostly related with defective assembly of the extracellular matrix (R-HSA-5083629 and R-HSA-5083633) [35]. In the ECM pathway, a key role is played by metalloproteinases (MMPs), zinc-dependent extracellular proteases synthesized by inflammatory cells such as neutrophils that break down the ECM to enable tissue remodeling and wound repair [36]. MMP-9 and to a lesser extent MMP-2 have been found in the stroma of PG lesions [37] and it has been postulated that an ongoing overexpression of MMPs by neutrophils within PG lesions, could promote prolonged ulceration.
Concerning the pathways enriched in the “localized” group exclusively, the Dectin-2 family Pathway (R-HSA-5621480) was the only one related to immune system. C-type lectin-like receptors (CLRs) of the “Dectin family” represent a family of transmembrane pattern recognition receptors (PRRs), mainly expressed by myeloid cells where they induce the production and release of cytokines like TNFα and IL-6, via nuclear factor-κB (NF-κB). Furthermore, these receptors are also involved in cutaneous wound healing by regulating neutrophil extracellular traps (NET)osis. In the context of PG, an impairment of this pathway could both promote an increased inflammatory response and affect the wound healing process of lesions through regulation of neutrophilic responses [38].
Among the three pathways related to Cell Metabolism, the Telomere Extension by Telomerase pathway (R-HSA-171319), which participates both in endothelial cell proliferation and wound healing, and the Termination of O-glycan biosynthesis pathway (R-HSA-977068) are of keen interest. O-glycosylation is a post-translational modification that occurs after the protein has been synthesised, adding several different sugars to the serine or threonine residues [39]. O-glycans play various functions including cells trafficking in the immune system, recognition of foreign material, control of cell metabolism and epithelial differentiation [40]. It has been shown that alterations in O-glycosylation are important in the development of several diseases, including immune disorders, and based on its important functions it could also be involved in the susceptibility and occurrence of PG.
Concerning pathways related to Cell Signalling, we found protein kinase A (PKA) activation (R-HSA-163615) and Signaling by NTRK1 (TRKA) (R-HSA-187037). This latter is involved in skin wound healing through activation of extracellular signal-regulated kinase (ERK) and tyrosine kinases (TRK) receptors. Indeed, in a recent study, Chakrabarti et al. [41], demonstrated that faster wound healing results from an increased re-epithelialization and granulation tissue formation due to the presence of proteins such as TrkA, p-TrkA, ERK1/2, p-ERK1/2 and NF-kβ. These results allow us to assume that an alteration of this pathway could be responsible for slower wound repair in PG cases.
In conclusion, we described altered biological pathways in PG patients, particularly involved in immune system, in neutrophils-related inflammation and in wound repair, confirming that these processes are nodal pathogenic drivers in PG pathogenesis.