Influence of Immune Cells and Circulating Inflammatory Cytokines on Pathological Scars: A Mendelian Randomization Study

Introduction

Pathological scars are the products of abnormal repair during the wound healing process. Abnormal scars, namely hypertrophic scars or keloids, may form during the healing process of some injuries, which are collectively referred to as pathological scars.¹ Pathological scars can occur in any part of the body, affecting approximately 30–90% of the population.² Furthermore, pathological scars often cause problems such as pain, itching, and cosmetic defects, bringing severe physical and psychological distress to patients.^3,4 Since there are still problems such as unsatisfactory treatment effects and high recurrence rates in the treatment of pathological scars, there is an urgent need to develop more effective treatment methods.⁵

The specific pathogenesis of pathological changes remains unclear at present. It may be related to genetic factors, immune factors, altered cytokine expression, and environmental factors.^6–8 Both hypertrophic scars and keloids demonstrate genetic susceptibility and familial recurrence. Genetic factors play a significant role in the pathogenesis of pathological scars.^9,10 The pathological scars are closely associated with the immune cells and inflammatory cytokines.^11,12 Compared with normal tissues, the levels of macrophages, T cells, and mast cells are all elevated in scar tissues. In addition, cytokines such as IL - 6, IL - 8, chemokine like factor-1, and prostaglandins produced by cyclooxygenase (COX - 1) are significantly elevated in scar tissue.^13,14 Nishiguchi et al demonstrated that the chemokine CXC chemokine ligand-12 (CXCL12) promotes scar formation in mice, whereas the knockout of CXCL12 inhibits this process.¹⁵ A study demonstrates that IL-10 can mitigate scar formation by decreasing the deposition of extracellular matrix (ECM) proteins and inhibiting the transformation of fibroblasts into myofibroblasts.¹⁶ Mice with a double deficiency of IL - 10 and IL - 4 exhibited an inflammatory response, with significantly aggravated scar formation. In contrast, mice that received recombinant human IL - 10 showed reduced inflammation and scarring.¹⁷ Cytokines serve as crucial mediators through which immune cells perform their functions, and an imbalance in cytokine levels is closely linked to abnormal scar formation. However, the causality between various immune cells, inflammatory cytokines and pathological scars are currently unclear.

We often utilize genetic data to infer causality through Mendelian randomization (MR) analysis. MR analysis can circumvent potential confounding factors and demonstrate a significant causal association. This study is the first to explore the causality among 731 immune cells, 91 inflammatory cytokines, and pathological scars through MR analysis, providing novel directions into the inflammatory immune mechanisms and treatment of pathological scars.

Methods

Study Design

The data were derived from the summary statistics of genome-wide association studies (GWAS). This analysis necessitated the fulfillment of three primary assumptions: (1) Exposure factors are strongly correlated with instrumental variables (IVs); (2) There are no known confounding factors associated with either exposure or outcome; and (3) IVs only affect outcomes when they are associated with exposure factors (Figure 1).

Figure 1 Key assumptions diagram for bidirectional MR analysis.

Date Sources

The data concerning immune cell phenotypes were sourced from the GWAS database.¹⁸ The data on 91 circulating cytokines can be found in a GWAS involving 14,824 subjects of European population.¹⁹ The GWAS data of hypertrophic scars were sourced from the latest FinnGen database, including 2068 cases and 465673 controls. The GWAS data of keloids were derived from a previously conducted meta-analysis, including 668 cases and 481,244 controls.²⁰

Selection of IVs

In this study, a significance threshold of P < 1.0×10⁻⁵ was set to screen out single - nucleotide polymorphisms (SNPs) that were associated with immune cells.²¹ We selected a P - value of 5×10⁻⁶ as the threshold to screen out SNPs associated with inflammatory cytokines.²² Furthermore, the analysis of linkage disequilibrium was conducted using the following threshold (r² = 0.001, kb = 10,000).²³ The palindromic SNPs were excluded, and the IVs was assessed using the F-statistic (F > 10) to ensure that the exposure factors were strongly related to the obtained IVs.²⁴ We employed a threshold of (5×10⁻⁶) to screen out SNPs associated with pathological scars (r² = 0.001 and kb = 10,000).²⁵

Statistical Analysis

The inverse-variance weighted (IVW) method was used as a primary analytical method.²⁶ To control potential false positives, we applied the false discovery rate (FDR) method to perform multiple testing on the results. A p - value < 0.05 suggested a suggestive causality, while an FDR - adjusted p - value < 0.05 indicated a significant causality.²⁷

Sensitivity Analysis

The Cochran’s Q test was used to identify heterogeneity.²⁸ Furthermore, horizontal pleiotropy was assessed using MR-Egger regression (P < 0.05 considered to indicate horizontal pleiotropy.²⁹ The leave - one - out method is employed to assess the stability of the research data.³⁰

Reverse MR Analysis

Hypertrophic scars and keloids were used as exposure factors, and immune cells and inflammatory cytokines were used as outcome variables. We used the IVW method as the main analytical method to analyze the reverse causality of pathological scars acting on immune cells and inflammatory cytokines.

Results

Causal Effect of Immune Cells and Inflammatory Cytokines on Hypertrophic Scars

Our analysis showed that 15 of these immune cells were suggestively associated with hypertrophic scars, including 10 risk factors and 5 protective factors. Our research findings revealed that Resting CD4 regulatory T cell %CD4 regulatory T cell, CD25++ CD4+ T cell %T cell, CD25 on CD45RA- CD4 not regulatory T cell, HLA DR on CD14+ CD16+ monocyte, CD45RA on naive CD4+ T cell, T cell Absolute Count, CD25 on transitional B cell, CD27 on IgD+ CD38- unswitched memory B cell, SSC-A on Natural Killer, and CD16-CD56 on Natural Killer show a positive correlation with the risk of hypertrophic scars, while CD20 on IgD+ CD38+ B cell, HLA DR on CD14+ CD16- monocyte, HLA DR++ monocyte %monocyte, CD8+ T cell %T cell, and HLA DR on CD14+ monocyte show a negative correlation with the risk of hypertrophic scars (Figure 2) (Table S1).

Figure 2 Association of immune cells with the risk of hypertrophic scars in MR analyses. Abbreviations: CI, confidence intervals; OR, odds ratio.

Our analysis showed that 5 inflammatory cytokines were suggestively associated with hypertrophic scars, including 3 risk factors and 2 protective factors. Programmed cell death 1 ligand 1 (PD-L1) levels, Stem cell factor (SCF) levels and Glial cell line-derived neurotrophic factor (GDNF) levels may increase the risk of hypertrophic scars, while TNF-related apoptosis-inducing ligand (TRAIL) levels and Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) levels may reduce the risk of hypertrophic scars (Figure 3) (Table S2).

Figure 3 Association of inflammatory cytokines with the risk of hypertrophic scars in MR analyses. Abbreviations: CI, confidence intervals; OR, odds ratio.

Causal Effect of Immune Cells and Inflammatory Cytokines on Keloids

Our analysis showed that 9 of these immune cells were suggestively associated with keloids, including 5 risk factor and 4 protective factors. Our research findings revealed that CD19 on naive-mature B cell, CD86 on CD62L+ myeloid Dendritic Cell, CD45 on Natural Killer T, CD25 on CD39+ CD4 regulatory T cell, and CD45RA on resting CD4 regulatory T cell show a positive correlation with the risk of keloids, while Plasmacytoid Dendritic Cell Absolute Count, Activated & secreting CD4 regulatory T cell Absolute Count, CD25 on unswitched memory B cell, CD3 on Central Memory CD8+ T cell show a negative correlation with the risk of keloids (Figure 4) (Table S3).

Figure 4 Association of immune cells with the risk of keloids in MR analyses. Abbreviations: CI, confidence intervals; OR, odds ratio.

Our analysis showed that 2 inflammatory cytokines were suggestively associated with keloids, including 1 risk factor and 1 protective factor. Our research findings revealed that Urokinase-type plasminogen activator (uPA) levels may increase the risk of keloids, while Osteoprotegerin (OPG) levels may reduce the risk of keloids (Figure 5) (Table S4).

Figure 5 Association of inflammatory cytokines with the risk of keloids in MR analyses. Abbreviations: CI, confidence intervals; OR, odds ratio.

Causal Effect of Pathological Scars on Immune Cells and Inflammatory Cytokines

In the reverse causal analysis, the findings indicated that there was no significant causality between hypertrophic scars and 15 immune cells, and there was also no significant causality between hypertrophic scars and 5 inflammatory cytokines. In addition, there was no significant causality between keloids and 9 types of immune cells, nor was there a notable causality with 2 inflammatory cytokines (Tables S5–S6).

Sensitivity Analysis

The MR-Egger intercept indicated no significant horizontal pleiotropy (p> 0.05). IVW and MR-Egger results indicated the absence of heterogeneity (p> 0.05) (Table S7). The leave-one-out analysis, scatter plots and funnel plots also supported these findings (Figures S1–S12).

Discussion

Our research indicated that 15 immunophenotypes and 5 inflammatory cytokines exhibit potential causal relationships with hypertrophic scars. 10 immunophenotypes, including Resting CD4 regulatory T cell %CD4 regulatory T cell, CD25++ CD4+ T cell %T cell, etc., are identified as risk factors for hypertrophic scars. 5 immunophenotypes, including CD20 on IgD+ CD38+ B cell, HLA DR on CD14+ CD16- monocyte, etc., are identified as protective factors for hypertrophic scars. In addition, our research revealed that 9 immunophenotypes and 2 inflammatory cytokines exhibit potential causal relationships with keloids. 5 immunophenotypes, including CD19 on naive-mature B cell, CD86 on CD62L+ myeloid Dendritic Cell, etc., are identified as risk factors for keloids. 4 immunophenotypes, including Plasmacytoid Dendritic Cell Absolute Count, Activated & secreting CD4 regulatory T cell Absolute Count, etc., are identified as protective factors for keloids.

Inflammatory cytokines play a vital role in the formation of scars. We explored the effects of 91 circulating cytokines on pathological scars. The finds showed that the PD-L1 levels, SCF levels and GDNF levels were risk factors for hypertrophic scars. In contrast, TRAIL levels and 4E-BP1 levels were protective factors for hypertrophic scars. Meanwhile, uPA levels was a risk factor for the occurrence of keloids, and OPG levels was a protective factor for the occurrence of keloids. We comprehensively explore the immune cells and inflammatory cytokines associated with pathological scars, aiming to provide references for future in - depth studies on the roles of immune cells and inflammatory cytokines in pathological scars.

The proportion of T regulatory (Treg) cells in keloid tissue was considerably higher.³¹ Treg can inhibit effector T cells and M1 macrophages and impede the differentiation of M1 macrophages into M2 macrophages, thereby promoting a persistent inflammatory response.³² We revealed that CD25 on CD39+ CD4 regulatory T cell and CD45RA on resting CD4 regulatory T cell might increase the risk of keloids, while Resting CD4 regulatory T cell might increase the risk of hypertrophic scars. During the wound - healing process, the proliferation and differentiation of fibroblasts, along with the massive deposition of collagen, jointly contribute to the formation of scars.^33,34 An in vitro experiment revealed that CD4+ T cells from burn patients with scarring tendency could promote the proliferation of fibroblasts and the synthesis of collagen.³⁵ We revealed that CD25++ CD4+ T cell and CD45RA on naive CD4+ T cell might increase the risk of hypertrophic scars. The levels of CD28, CD80 and CD8+ T cell in the skin tissues of keloid patients are higher than those in healthy individuals. The binding of CD28 to its ligand CD80 further induces the activation and differentiation of T cells.³⁶ CD8+ T cells may inhibit keloid by suppressing fibroblast proliferation. It is noteworthy that we revealed that CD8+ T cell might decrease the risk of hypertrophic scars and CD3 on Central Memory CD8+ T cell might decrease the risk of keloids. CD8+ T cells may serve as a protective factor in inhibiting scar hyperplasia, but the specific mechanism of action remains to be further explored.

The number of monocytes increase significantly during the wound - healing process, and they further differentiate into macrophages and fibroblasts.³⁷ A study found that CD14+ monocytes promoted the proliferation of fibroblasts by releasing monocyte chemoattractant protein-1 (MCP-1), which in turn contributes to keloid formation.³⁸ HLA-DR is frequently utilized as a marker to assess the activation or maturation status of monocytes.³⁹ We revealed that HLA DR on CD14+ CD16- monocyte, HLA DR++ monocyte, and HLA DR on CD14+ monocyte might decrease the risk of hypertrophic scars. Natural killer (NK) cells can defend against viral infections and the development of malignancies by destroying target cells.⁴⁰ We revealed that SSC-A on Natural Killer and CD16-CD56 on Natural Killer might increase the risk of hypertrophic scars. However, high-quality relevant studies are still necessary to further confirm these research findings.

PD-L1 can inhibit the inflammatory and accelerate wound healing by regulating the expression of fibroblast-like cells.⁴¹ This study reveals that there is an interaction between PD-L1 and fibroblast-like cells, but the impact on scars still needs further research. SCF is important in the wound healing process. The level of SCF is notably increased in keloid tissues and fibroblasts.^42,43 We revealed that SCF can increase the risk of hypertrophic scars. SCF may serve as a potential therapeutic target for pathological scars. GDNF is a neurotrophic factor. A previous study showed that GDNF can promote hair follicle neogenesis and skin regeneration by regulating dermal fibroblasts. However, the relationship between GDNF and scar formation still needs further exploration.

uPA and its receptor play vital regulatory roles in cellular processes such as tissue remodeling and cell migration. A study found that uPA receptor is highly expressed in the extracellular matrix of keloid tissue.⁴⁴ The results of another trial conducted on patients with hypertrophic scars showed that the level of uPA in scars was notably increased.⁴⁵ We revealed that uPA can increase the risk of keloids, and uPA is closely related to scar formation. However, further relevant clinical studies are still needed to confirm the above results. A previous study suggested that the content of OPG increased during the process of culturing keloid fibroblasts.⁴⁶ OPG is associated with the formation of keloids. We revealed that OPG can decrease the risk of keloids. Conversely, there is still a lack of studies on the correlation between OPG and keloids to confirm the conclusion of this study.

PD- L1 is expressed on T cells following stimulation of the T-cell receptor.⁴⁷ Another research result indicated that following co-activation with human dendritic cells, human naive CD4+ effector T cells and CD4+ regulatory T cells exhibit elevated PD-L1 expression.⁴⁸ The expression of PD-L1 on activated CD4+ T cells facilitates its interaction with ligands such as PD-1 and CD80.^49,50 The proliferation and differentiation of CD8+ T cells into effector and memory states are energy-intensive processes that involve significant changes in cellular metabolism. The 4E-BP1 protein plays a crucial role in the proliferation of murine CD8+ T cells and in the development of antiviral effector functions.⁵¹ CD8+ T cells eliminate abnormal cells and maintain homeostasis by mediating TRAIL-induced apoptosis in target cells. uPA plays a crucial role in cell activation, adhesion, and migration, and is highly expressed in macrophages and dendritic cells.⁵² Immune cells and inflammatory cytokines exhibit complex interactions in pathological scars, further in-depth research on the underlying mechanisms is necessary.

This research investigates the causality between 731 immune cells, 91 inflammatory cytokines and pathological scars. However, this article still has a few drawbacks. The GWAS data is originated solely from European populations, the generalizability of the conclusion of this study to patients of other ethnicities is limited. After FDR correction, our research results showed no statistically significant differences. The findings presented in this paper should be interpreted with caution. The pathogenesis of pathological scars remains unclear. Therefore, large-scale, multicenter studies are necessary in the future to validate the findings of this study.

Conclusion

This study reveals the roles of immune phenotypes and cytokines in the pathogenesis of pathological scars, and emphasizes the complex interactions among the immune system, cytokines, and pathological scars. The research findings of this paper offer a novel direction for future in - depth exploration of the pathogenesis of pathological scars and offers valuable references in research areas such as early identification and intervention treatment of pathological scars.