Classification of epidermal cell constitution of uninjured skin and wounds
We retrieved and analyzed the filtered read count matrix of 1170 cells from three different conditions, i.e., normal skin (n = 391), acute wounds (AW, n = 398), and pressure ulcers (PU, n = 381) (cells that passed the quality control) from the GEO(GSE137897) [21]. These epidermal cells were separated into six clusters each by Seurat clustering (Figure 1A, B, C). The annotation based on that basal layer keratinocyte had the highest expression levels of KRT5 and KRT14; spinous keratinocyte displayed high DSG1 and DSP levels with the highest expression of KRT1 and KRT10; granular keratinocyte expressed a suite of late differentiation markers, including LOR, FLG, and SPINK5; and melanocytes had a higher expression of PMEL, TYRP1, and MLANA, whereas immune cells had higher levels of CD74. Combining canonical, novel markers in differential analysis and KEGG/GO analysis allowed us to annotate different clusters (Figure 1D, E, F, G) [22-26].
In uninjured skin, we annotated one basal (n = 106), granular (n = 74), and spinous (n = 74) keratinocyte cluster, one cluster named melanocytes (n =65), one for immune cell (n = 58) and one termed as “mitotic” (n = 14) because of the high level of well-recognized DNA synthesis and cell division transcripts, such as PCNA and KI67 (Figure 1H).
Following the same criterion, we annotated two basal (n = 88 and n =72) keratinocyte clusters, one spinous (n = 100) keratinocyte cluster, one melanocytes cluster (n = 11), one immune cell cluster (n = 85) and one “mitotic” cluster (n = 42) in AW (Figure 1I). In PU, we annotated one basal (n = 66) and one spinous (n = 66) keratinocyte cluster respectively, one melanocyte (n = 31) cluster, two immune cell clusters (n = 83 and n = 81) and a “mitotic” cluster (n = 54) (Figure 1J).
Cellular and molecular interactions of epidermal cells are enhanced in chronic wounds
We used CellChat to predict the general principles of cell-cell communication. Comparing the total number of interactions and interaction strength, we found that cell-cell communication was enhanced in chronic wound conditions (Figure 2A). Furthermore, we compared the number of interactions and interaction strength among different cell populations to identify those which showed significant changes. We found that signaling was increased between immune cells and other cells in PU compared with AW. Spinous and mitotic cell population communication was increased in PU, where mitotic cell populations were the busiest in signaling. The interaction between mitotic cells and immune cells was strongest in PU (Figure 2B, C, D).
PTN and PAR signaling pathways were significantly changed in chronic wounds
We identified the significantly altered signaling pathways by simply comparing the information flow for each, which is defined by the sum of communication probabilities among all pairs of cell groups in the inferred network (i.e., the total weights in the network). Significantly changed signaling pathways were ranked based on differences in the overall information flow within the inferred networks between the AW and PU. As shown in Figure 3A, the leading signaling pathways depicted in red are enriched in AW, whereas those in green are enriched in PU. The results show the signaling pathways with the strongest variation in increase or decrease of activity, which include those of pleiotrophin (PTN), prosaposin (PSAP), Secreted Ly-6/uPAR-related protein (SLURP), epidermal growth factor (EGF), VISVATIN, which also called visfatin, calcitonin receptor (CALCR), and the protease-activated receptor (PAR) signaling pathway.
We further analyzed the outgoing and incoming signaling associated with each cell population between AW and PU (Figure 3B, C, D). In PU, results showed a major decrease in signaling from melanocytes, such as the PTN and PSAP pathways, whereas the highest increase was registered in the PAR pathway, also from the melanocyte population. Other significantly changed signaling pathways were EGF, visfatin, and CALCR from the basal cell population. Overall, signaling pathways from the melanocyte population were the most altered between AW and PU. This indicates that melanocytes may play an important role in wound healing.
The melanocyte cell population was the dominant sender in PTN and PAR signaling pathways
To define the signaling sources most subject to variation, we identified the dominant senders, receivers, mediators, and influencers in the substantially altered pathways. The PTN signaling pathway was only present in AW, where melanocytes are the senders, and spinous, basal, immune, and mitotic cells are the receivers. The major mediators and influencers of the PTN signaling pathway are the immune cells (Figure 4A). In the PAR signaling pathway, the major senders are the melanocytes, whereas the spinous and mitotic cells are the main receivers and accept almost all the other cells’ influence (Figure 4B). Therefore, the absence of the PTN signaling pathways and the increased PAR signaling pathway from the melanocytes of chronic wounds, indicate that melanocytes may play a crucial role in the healing process.
Next, we identified the upregulated and downregulated signaling ligand-receptor pairs in PU and determined the communication probabilities mediated by ligand-receptor pairs from melanocytes to other cell groups (Figure 4C). This showed that the highest communication probabilities mediated by ligand-receptor pairs are substantially changed from melanocytes compared to other cell groups, such as CTSG-F2RL1, CTSG-PARD3, CTSG-F2R, which belong to the PAR signaling pathway, and PTN-SDC1, PTN-SDC3, and PTN-NCL, which are part of the PTN signaling pathway.
PTN-SDC1 pairs in the PTN signaling pathway may regulate epithelial cell proliferation and migration during wound healing
To determine what type of effects the substantially changed signaling pathways may bring to the receiver cell populations, we calculated the most contributing ligand-receptor pairs in cell-cell communication in the most altered signaling pathway. We narrowed down the analysis to the most highly contributing pairs to analyze the information between sender and receiver cells.
The most contributing ligand-receptor pair of the PTN signaling pathway, which is only present in AW, was PTN-SDC1 (Figure 5A, B). PTN is only expressed in the melanocyte population and is a secreted growth factor that mediates signaling via cell-surface proteoglycan and non-proteoglycan receptors via their chondroitin sulfate (CS) groups [27-31]. Syndecan-1 (SDC1) is a cell surface proteoglycan that participates in cell-cell and cell-matrix interactions, a growth factor coreceptor involved in wound healing [32-35]. SDC1 expression was the highest in almost all cells, indicating that melanocytes can affect other cell populations through PTN-SDC1 binding (Figure 5C, D).
PTN gene expression is upregulated in brain injury [36], and is involved in bone repair [37], angiogenesis [38-40], regulation of epithelial cell migration, and keratinocyte proliferation [41, 42]. In previous studies, PTN and SDC1 expression was found to be upregulated in brain injury and promote epithelial cell proliferation and migration during wound healing. However, the PTN-SDC1 ligand-receptor pair, in other words, the PTN pathway, is absent in PU, so we speculate that melanocytes may play a key role in skin regeneration, and that the absence of the PTN pathway may disrupt healing in chronic wounds.
CTSG-F2RL1 pairs in the PAR signaling pathway may lead to chronic inflammation in chronic wounds
PAR signaling pathways are only present in PU. PARs are a family of G-protein-coupled receptors that are irreversibly activated by proteolytic cleavage of their N-terminus. This exposes a peptidic ligand that binds and activates the transmembrane receptor domain, provoking a cellular cascade in response to inflammatory signals and other stimuli. PARs are involved in a wide variety of diseases, such as cancer and inflammation [43, 44].
The ligand-receptor pair in the PAR pathway that contributes most to cell-cell communication is CTSG-F2RL1 (Figure 6A, B). CTSG, also known as cathepsin G, has a serine protease with trypsin- and chymotrypsin-like specificity. CTSG is synthesized and secreted by neutrophils, mast cells, and antigen-presenting cells [45-48]. CTSG is also found in some non-immune cells, such as endothelial and smooth muscle cells [49], brain astrocytes [50], fibroblasts [51], and in Paneth cells – specialized epithelial cells underneath the crypts of Lieberkühn [52, 53]. We also found that CTSG was expressed in the melanocyte population of PU. F2RL1 is a receptor for trypsin and trypsin-like enzymes coupled to G proteins and is activated through proteolytic cleavage of its extracellular amino terminus [43]. F2RL1 is expressed in almost all cells except melanocytes, and we can deduce that melanocytes can send their signal to other cell populations through CTSG-F2RL1 binding (Figure 6C, D).
Previous studies have found that CTSG is elevated in chronic wounds and inhibits regeneration [54-56] and promotes inflammation [57]. F2RL1 can reduce cell migration through actin cabling and can prevent the internalization of E-cadherin [58]. Its stimulation can promote inflammation and inflammatory cell infiltration [59-61]. Therefore, CTSG binding to F2RL1 may promote inflammation in and reduce migration of the epithelium cells during wound healing. However, CTSG-F2RL1 ligand-receptor pair, i.e., the PAR pathway, is increased in PU, adding evidence that melanocytes may play a key role in wound healing. The increase in the PAR pathway may contribute to chronic inflammation and inhibition of cell migration in the epithelium of chronic wounds.