Keloids are a disfiguring fibroproliferation disorder that dramatically impairs the quality of life of the affected individual. Even though there have been many studies about keloids, they remain the most challenging skin condition to treat. The theory of tumor immune microenvironment states that the occurrence and development of tumors are the result of the joint action of various cells and molecules in the microenvironment of the tissues where tumor cells are located . Keloids have certain tumor characteristics. We speculate that the imbalance of skin immune microenvironment caused by genetic, immune, inflammatory, and other factors may be key to keloid development. Among them, abnormal expression of immune-related genes is the root cause, and immune cell regulation plays a key role in the development of keloids. These factors result in keloid tumor-like scar hyperplasia formation. Aberrant gene expression often originates from congenital risk, triggered by stimulating factors. Expression profiles of mRNAs, lncRNAs, and circRNAs were altered in keloid tissue, which may partly contribute to the etiology of keloids by affecting several signaling pathways related to keloid development . DEGs can be used as biomarkers for diagnosis or as therapeutic targets for keloids. Some biomarkers are upregulated in keloids, including fibroblast-specific protein 1 (FSP1), vimentin, TGF-β1, and Smad3 phosphorylation, which were noted in keloid tissues . Expression of miRNAs in keloid tissue was shown to be significantly different from that of normal tissue. Among the miRNAs involved in keloid pathogenesis, miRNA-21, miRNA-141-3p, miRNA-181a, and miRNA-205 were thought to upregulate fibroblast proliferation and depress apoptosis of keloid-derived fibroblasts through the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway . Drugs that indirectly target the biochemical microenvironments of keloids include growth factors (e.g., TGF-bs, bFGF, and VEGF), immunomodulators (e.g., tacrolimus, trehalose, IFN, and imiquimod), and anti-inflammatory drugs (e.g., IL-10 and IL-6) [18-24]. Based on the above research, we believe that keloid patients have abnormal gene expression compared to ordinary people.
To better understand keloids pathogenesis, we collected three groups of tissues from patients in different disease stages to describe the development of keloids. To identify abnormal gene expression, we collected normal skin tissue from keloid patients, which we described as N group. To determine the gene expression changes, we collected inflammatory lesion tissue from keloid patients, which we described as I group. Based on the tumor immunological gene study, we identified hub genes from N group to I group. These genes included CCR1, CCR2, SELL, IL10, CCR7, CD40LG, CD69, CXCL8, IL6, and CXCL9. Current hypotheses of pathogenesis classify keloids as an entity of aberrant fibrosis and tumor characters. Hyperactivation of the MCP-1/CCR2 axis reportedly causes fibrosis in the liver, cirrhosis, atherosclerosis, and lung fibrosis [25-27]. Expressions of MCP-1 and its receptor CCR2 in keloid lesions were increased in the keloid tissues . IL-10 was shown to be able to significantly inhibit the proliferation of keloid fibroblasts . Moreover, proinflammatory factors, such as interleukin (IL)-1α, IL-1Ra, IL-1β, IL-6, and tumor necrosis factor-α are upregulated in keloid tissues, which suggests that, in patients with keloids, proinflammatory genes in the skin are sensitive to trauma [1,29]. Hub genes between I group and K group included IL10, ITGAM, ITGAX, IL2, IL4, IL6, IL13, IL17A, FOXP3, and CD86. The roles of several genes in keloid development, including CCR1, SELL, CCR7, CD40LG, CD69, CXCL8, ITGAM, ITGAX, CD86, and CXCL9, need to be verified. These genes may play an important role in keloid formation after stimulation by inflammatory factors. We need to further validate these hub genes to reveal the molecular mechanism of keloid development. This may help to find a new therapeutic target.
The occurrence and development of keloids involve complex molecular mechanisms. We used GO and KEGG analysis to better understand the mechanisms of keloids. A study by Dohi T et al. identified that the soft skin surrounding keloids is exposed to high mechanical strain that correlates with increased expression of the caveolin-1/rho signaling via the rho kinase mechanotransduction pathway, which may lead to keloid progression. A study by Huang H et al. identified that upregulated mRNAs were involved in cell proliferation, cell growth, and tissue repair, and downregulated mRNAs were involved in apoptosis . In our study, GO analysis revealed that there were many variations of BP between I group and N group, including regulation of lymphocyte activation and T-cell activation. The result is similar to that found in I group and K group, which may play an important role in the initiation and formation of keloids. Variations of MF were markedly enriched in cytokine receptor binding and receptor ligand activity, which are essential for signaling pathway transmission.
In this study, analysis of the KEGG pathway between I group and N group revealed that all of the DEGs were primarily enriched in cytokine−cytokine receptor interaction and viral protein interaction with cytokine and cytokine receptor, reflecting that these pathways may affect keloid formation. A study by Zhong L et al. identified that target genes were associated with the MAPK signaling pathway and HIF-1 signaling pathway . Our study provides a new field for the molecular study of keloids.
The mechanistic details of keloid formation remain poorly understood. Given that the immune system is engaged in skin lesion repair, we explored cell-targeted analysis in this study. We utilized CIBERSORT to transform gene expression profiles to cell composition of complex tissues. There are immune cells, fibroblasts, endothelial cells, and an abundant collection of cytokines, chemokines, and growth factors in keloid tissue. These components and their complex interactions form a tumor-associated microenvironment, and tumor cells may use immune cells to seek benefits of growth, invasion and transfer. The main host cells recruited and activated in the tumor microenvironment are various immune cells. These immune cells have important prognostic relevance due to the dual role of the immune system in promoting and suppressing tumors. Searching for immune cells that may play a major role in keloid development will be beneficial to clinical application of cell therapy as a target. M1/M2 proportion describes the two major and opposing activities of macrophages . M1 macrophages secrete pro-inflammatory factors, chemokines, and presenting antigens full-time, participate in the positive immune response, and play a role in immune surveillance . M2 macrophages are an important immune cell in downregulating the immune response . Remarkably, the molecules primarily responsible for these “Fight” (NO) or “Fix” (Ornithine) activities both arise from arginine, and via enzymatic pathways (iNOS and arginase) that down regulate each other . The names M1 and M2 were chosen because M1 and M2 macrophages promote T1 and T2 responses, respectively. Products of T1 and T2 responses (e.g., IFN-γ and IL-4) also downregulate M2 and M1 activity, respectively . Thus, M1/M2 proportion demonstrated the importance of innate immunity, and how it is linked to adaptive immunity in a beautifully counterbalanced system [35,36].
M2 macrophages have only a weak antigen presentation function and play an important role in immune regulation by secreting congruent cytokines, such as IL-10 and TGF-β. They are an important immune cell in downregulating immune response . In this study, we identified a higher proportion of M2 macrophages in N group than in I group. M2 macrophages may lead to immunosuppressive regulation. Its high proportion in skin tissue in keloid patients may indicate the abnormal gene expression in the skin, which leads to a state of immunosuppression. This also means that the skin of keloid patients is more vulnerable to infections that cause inflammation and keloid formation. However, this difference was not significant probably due to insufficient sample size and still need to be verified by comparing with the skin from normal person.
Our research showed that there was a significant difference in M1 macrophages between I group and K group. The proportion of M1 in I group was higher than in K group. It is supposed that the state of immunosuppression could be activated by inflammatory factors. M1 macrophages play an important role in activating the immune state in keloid patients during infection and inflammation. This may indicate the presence of immune activation in the inflammation stage of keloid development. A study by Jin Q et al. identified that macrophages in keloid tissues were polarized toward the M2 subtype . In our study, the proportion of M2 in K group was higher than in I group, although the difference was not obvious. Keloid tissues presented significantly elevated infiltration by CD14+ macrophages . Myofibroblast proliferation and heterogeneity are supported by M2 macrophages during skin repair . This means that M1/M2 proportion in different disease stages may play a major role in keloid development.
In addition, the proportions of CD8+T cells varies significantly between both N group and K group.
Contrary to our findings, Jin Q et al. identified that keloid tissues presented higher infiltration by CD3+ T cells, of which the majority were CD4+ T cells. This difference may be caused by different tissue sample origination, and needs to be clarified with large sample analysis.
Despite the rigorous bioinformatics analysis in this study, there are still some limitations. First, the sample size of our study was small, which might result in some deviations in the results. Second, we only conducted bioinformatics mining and lack experimental verification, which will be carried out in the future.