Genetic Evidence Supports the Therapeutic Potential of Immune Checkpoint PD-L2 in the Development of Keloid

Genetic Evidence Supports the Therapeutic Potential of Immune Checkpoint PD-L2 in the Development of Keloid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Evidence Supports the Therapeutic Potential of Immune Checkpoint PD-L2 in the Development of Keloid Ben Wang, Yiwen Deng, Zhujie Ran, Bo Pan, Haiyue Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5235659/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Keloids are pathological scars that extend beyond the original skin injury, resembling tumor-like growth, with unclear etiology and challenging treatment. This study aims to determine whether there is a causal link between the immune checkpoint PD-L2 and keloid risk, assessing immune checkpoints as potential therapeutic targets. A Mendelian randomization analysis was conducted using genome-wide association study summary statistics. PD-L2 expression levels were analyzed in Asian and European cohorts to explore their association with keloid risk. Two clinical cohorts with different ancestries were used to investigate association between PD-L2 expression and the risk of keloids. The Asian cohort included 178,726 samples (1055 cases), while the European cohort included 481,912 samples (668 cases). Our findings indicate that higher levels of PD-L2 expression in plasma proteome are associated with an increased risk of keloids. This conclusion holds true in both European and Asian large-scale cohorts, with OR (Asia) values of 1.25 (1.04-1.51), P_IVW=0.02, and OR (Europe) of 1.24 (1.07-1.43), P_IVW=0.004. This study reveals for the first time a causal relationship between the expression levels of the immune checkpoint PD-L2 and the risk of keloids. This provides a new perspective for understanding the pathogenesis of keloid and for developing new treatment and prevention strategies. Keloid PD-L2 Immune checkpoint Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The pathological characteristics of keloid are excessive collagen deposition and sustained abnormal proliferation of fibroblasts during wound healing 1 . Keloid involves numerous characteristics resembling those of tumors, including unregulated cellular proliferation. Current treatments for keloid mainly involve intralesional corticosteroid injection combined with surgical excision followed by local adjuvant chemotherapy 2,3 . Unfortunately, clinical observations indicate that approximately 50% of keloids do not exhibit a positive response to corticosteroid injections 4 , while 9-50% of initially responsive keloids eventually experience recurrence. 5 . Therefore, the treatment of keloids presents a significant challenge, primarily due to the limited understanding of their specific pathogenesis, small sample sizes in previous studies, and a lack of effective research methods. These factors are crucial limitations that hinder the development of more targeted approaches for keloid treatment. In this study, we attempt to identify treatment targets associated with keloids from two large-scale clinical cohorts to advance keloid treatment-related progress. The effectiveness of glucocorticoid therapy suggests that the abnormal immune microenvironment within keloids may be a driving factor in their persistent growth. Therefore, inhibiting abnormal immune responses has become a potential therapeutic approach for keloids. Some cytokines have been explored for their therapeutic value in keloids, such as interferon-gamma, which is believed to have activity in reducing collagen synthesis both in vitro/vivo and has shown some effectiveness in treating proliferative scars and keloids 5 . However, these therapies face challenges in the absence of high-level, large-scale clinical trial evidence. Specifically, cytokine therapy or glucocorticoid-based immunomodulation therapy may be based on small clinical cohorts and lack specificity targeting the pathogenesis of keloids. As a significant advancement in the clinical application of immunology, treatment based on immune checkpoints has demonstrated promising therapeutic effects in the field of cancer therapy. This achievement was recognized with the 2018 Nobel Prize in Physiology or Medicine 6 . In fact, compared to previous immunotherapies, immune checkpoint-based treatment approaches offer two advantages. 1. Selective immunomodulatory characteristics: The PD-L1/PD-1 signaling pathway serves as a specific communication mode between proliferating abnormal cells and the immune system. Blocking this pathway can reactivate immune cells without affecting normal anti-tumor immunity, thereby facilitating the elimination of cancer cells 7 . Immunoregulatory measures with better selectivity are expected to inhibit unfavorable immune responses while preserving necessary immune responses to control abnormal proliferation and recurrence. 2. Potential for long-term cure: The efficacy of immunotherapy is limited to a subset of cancer patients; however, those who exhibit a positive response can attain enduring clinical remission, underscoring the potential for sustained success in this therapeutic approach 8 . In this study, in order to understand the potential of immune checkpoint inhibitor therapy in reducing the risk of keloid formation, we utilized a genetic technique called Mendelian randomization, which has been widely used in recent research to infer causal relationships between exposure factors and disease outcomes 9 . Previous studies have confirmed the ability of Mendelian randomization to obtain evidence at the level of randomized clinical trials, referred to as natural randomized clinical trials. This is because Mendelian randomization research is based on genetic traits that are initially randomly distributed in the population, minimally influenced by confounding factors such as smoking, pollution, and other environmental factors, thus naturally eliminating interference from confounding factors 10–12 . Compared to purely observational studies and randomized controlled trials, Mendelian randomization has the ability to infer causal relationships at a lower cost, with previous studies demonstrating its sufficient capability to provide evidence at the level of randomized controlled trials and high consistency with preclinical studies and clinical trial results. An illustrative example is the revelation by Mendelian randomization that PCSK9 inhibitors can reduce the risk of ASCVD in high-risk individuals 13 , subsequently confirmed by clinical trials 14 . Mendelian randomization also supports various ASCVD guidelines by identifying risk factors and excluding false positive results influenced by confounding factors (risk factors supported by observational studies but subsequently excluded in randomized controlled trials, such as HDL cholesterol, vitamin D, vitamin E, etc.) 15–19 . The programmed death ligand 2 (PD-L2), a PD-1 ligand, facilitates immune evasion in cancer cells and serves as a prognostic indicator for patients with specific tumor types. The advancement of novel and efficacious immune combination therapy strategies directed towards PD-L2 holds the potential to enhance the clinical effectiveness of anti-PD-1, thereby showcasing promising prospects for cancer treatment through the utilization of PD-L2-targeting drugs. The proliferative and invasive capabilities of keloids are comparable to those of malignant tumors. The utilization of immune-targeted therapy could potentially serve as a novel approach in the management of keloids. Reaching a sufficient sample size for large-scale clinical trials in keloid patients can pose significant challenges due to the relatively low incidence rate of this condition. In this study, we employed two clinical cohorts comprising East Asian and European populations to simulate the perturbation of PD-L2 (Programmed cell death ligand 2) and explore its potential role in mitigating the risk of keloid formation 20 . Methods 3.1 Study design and participants Clinically, we used a two-sample MR analysis model to assess the causal effects of PD-L2 on Keloids. The flow chart of our study is shown in Figure 1. 3.2 Genome-wide association study (GWAS) data In this study, the GWAS European and East Asian data used for genetic analysis were derived from the Saori Sakaue et al. 21 This research project involved approximately 179,000 Japanese volunteers, with an average age of about 63 years and a gender distribution of 46.3% female. The study samples were obtained from the biobank Japan, which collected DNA and serum samples from 12 medical institutions throughout Japan. The biobank aimed to recruit around 200,000 individuals primarily of Japanese descent. Participant data were mainly derived from disease records extracted from unstructured electronic medical records (EMR). Prior to their involvement in the research, all individuals provided written informed consent. The study received approval from the ethics committees of the Institute of Medical Sciences at the University of Tokyo and the RIKEN Center for Integrative Medical Sciences. All research procedures strictly followed the principles outlined in the Declaration of Helsinki by the World Medical Association. The GWAS data of European individuals primarily come from the UK Biobank (UKB) and the FinnGen project. The UKB project is a population-based prospective cohort that includes approximately 500,000 individuals from the United Kingdom, with an average participant recruitment age of 56.8 years, of which 53.8% are female. The study focuses on 158 disease endpoints derived from clinical information in the UKB. The FinnGen project integrates genotype data from Finnish biobanks and digital health record data from Finnish health registries. The mean age of participants at DNA sample collection is 51.8 years, with 56.3% being female 21 . 3.3 Mendelian Randomization Analysis Mendelian Randomization (MR) was utilized to explore the causal links between PD-L2 and the risk of keloids, relying on several key assumptions 22 . The MR analysis hinges on three primary assumptions: (1) strong correlation between instrumental variables (IVs) and the exposure, (2) independence of IVs from confounding factors, and (3) IVs exerting no direct influence on the outcome, with their impact solely mediated through the exposure. SNPs with p-values < 5 × 10^8, indicating significant associations with the phenotype, were selected from each cohort for further investigation. Linkage disequilibrium (LD) exclusion criteria were applied, with an R^2 threshold of 0.001 and a maximum distance threshold of 10,000 kilobases, to address LD issues. SNPs in LD were resolved by selecting the SNP with the lowest p-value for subsequent analysis. To mitigate bias from weak IVs, SNPs with F-statistic < 10, considered weak IVs introducing potential bias, were excluded. In cases where only one SNP could be evaluated for an exposure factor after LD exclusion, the Wald ratio was employed for statistical analysis. The impact of the exposure factor on the outcome was assessed using the inverse variance weighting (IVW) method. Additionally, MR analyses were conducted using MR-Egger 23 and Median Mendelian Randomization methods 24 , known for their robustness against pleiotropy. MR-Egger method, employing weighted regression with an unconstrained intercept, reduces susceptibility to pleiotropic effects by not assuming all genetic variants as valid IVs. Median Mendelian Randomization method, robust against pleiotropy, uses the median instrument variable from all included variants, ensuring resilience even with a proportion of invalid genetic variation. Consistent results from these methods enhance causal inference. The primary outcomes were derived from IVW-MR analysis, considering only positive results meeting the statistical threshold and showing consistent OR estimates across all three methods. Cochrane's Q statistic was calculated to assess result heterogeneity. Fixed-effects model estimation was used in the absence of heterogeneity, assuming no pleiotropy. In cases of detected heterogeneity, a random-effects model was employed, assuming the genetic instrument's association strength with the risk factor is independent of the pleiotropic effect size or has an average effect size of zero. MR-Egger regression and sensitivity analysis were conducted to detect horizontal pleiotropy. The R package "TwoSampleMR" facilitated the MR analysis, with results reported as ORs with 95% confidence intervals (CI) 24–26 . All statistical analyses were performed in R (version 4.3.1). The instrumental variable for PD-L2 is derived from the GWAS study by Arthur Gilly and colleagues, the associations between these genetic markers and circulating proteins were replicated and validated in two independent cohorts with sample sizes of 1605 and 1328, respectively 27 . Results 4.1 Causal factors of keloids associated with PD-L2 in East Asian cohort. We first analyzed the association between PD-L2 levels and the risk of keloids in the Asian population. The results, as shown in Figure 2, indicate that the expression level of PD-L2 is associated with an increased risk of keloids and demonstrates a significant causal relationship. The P-value estimated using the inverse variance weighted method is 0.02, with an odds ratio (OR) between 1.25 (1.04-1.51). The directions of the OR estimated by the other two statistical methods (MR Egger and Weighted median) are consistent with the IVW method, and no significant pleiotropy was detected, suggesting that PD-L2 is a risk factor for keloids. 4.2 Causal factors of keloids associated with PD-L2 in European cohort. Furthermore, in order to determine whether this causal relationship still persists within the European population, we conducted further investigations into the association between PD-L2 and the susceptibility to keloid formation among Europeans. This cohort included 668 patients diagnosed with keloids and 481,244 controls. The results indicate a causal relationship between PD-L2 and keloids, with P of inverse variance weighted = 0.004, OR=1.24 (1.07-1.43). The directions of the OR estimated by the other two statistical methods align with the inverse variance weighted method, and no significant pleiotropy was detected, suggesting that elevated levels of PD-L2 are associated with an increased risk of keloid formation. Discussion The expression of PD-L2, an additional ligand for PD-1, was initially observed in macrophages and dendritic cells. Recent research has demonstrated that PD-L2 exhibits high expression levels in human cancers and can serve as a reliable indicator of cancer stage and response to drug treatment. The characteristics of keloids, such as their propensity for proliferation and invasion, closely resemble those observed in malignant tumors; however, the etiology remains unknown, posing continuous challenges to the treatment of keloids. In this study, we used a two-sample Mendelian randomization approach to determine the causal relationship between PD-L2 and the risk of keloids. Our findings are derived from two cohorts of different ethnicities and were validated using various hypothesis testing methods, ensuring the strength and credibility of our results. A key strength of our study is the inclusion of a large-scale genome-wide association study (GWAS) sample. These two cohorts had sample sizes of 178,726 (1055 cases) and 481,912 (668 cases) respectively, making this study the largest in terms of sample size in keloid-related research, to our knowledge. We validated in these two large-scale clinical cohorts that the simulated perturbation of PD-L1 treatment has an effect in reducing the risk of keloid formation, suggesting that immune therapy strategies targeting the PD-L1/PD-1 axis may be a potential treatment approach for keloids. Among all studies related to keloids, our research has a relatively large sample size 28 . This not only enhances the statistical power of our study but also bolsters the persuasiveness of our conclusions. To our knowledge, previous reports from basic or clinical research have not yet established the potential role of the PD-1/PD-L1 axis in the pathogenesis of keloids. Our study's innovative discovery that the expression of PD1-related genes may serve as a predisposing factor for keloid formation suggests that therapeutic interventions targeting PD-L2 could potentially offer an alternative treatment approach in contrast to conventional corticosteroid injections(Figure 4). While previous studies have shown the performance of Mendelian randomization in providing evidence comparable to randomized clinical trials, further basic experiments, preclinical investigations, and randomized clinical trials are still needed in the future to clarify the roles of immune checkpoints and their related molecules in the pathogenesis of keloids. This study has several limitations. Firstly, the keloids GWAS summary data used in this study did not adjust for age and gender because this information was not available for all individuals in each dataset. Secondly, the MR results reflect lifetime changes in keloids risk due to PD-L2 levels, and further research is needed to clarify its short-term impact on keloids risk. Thirdly, further large-scale clinical trials are needed in the future to clarify the effectiveness or safety of this treatment and whether it could lead to sustained relief in keloid patients and reduce their recurrence rates. Conclusion In conclusion, this study provides strong evidence that PD-L2 is a causal determinant of keloids risk. The implementation of specific targeted immunotherapy may prove to be a viable and efficacious strategy for the treatment of keloids. These findings, based on a large-scale sample, may provide evidence for further treatment and prevention of keloids. Declarations Author Contributions All authors had full access to all the data in this study and take responsibility for the integrity of the data and the accuracy of the data analysis. Ben Wang and Yiwen Deng contributed equally and share the first authorship. Ben Wang: Writing–original draft, Supervision, Methodology, Conceptualization. Yiwen Deng: Writing–original draft, Methodology, Conceptualization,Validation. Zhujie Ran: Supervision, Methodology. Bo Pan: Writing – review & editing, Visualization, Supervision. Haiyue Jiang: Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was funded by the National Natural Science Foundation of China (No. 82371796) Funding sources National Natural Science Foundation of China (No. 82371796) Data availability The clinical data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics statement The data for these studies were approved by the ethics committees of the Institute of Medical Sciences at the University of Tokyo, the RIKEN Center for Integrative Medical Sciences, the UK Biobank (UKB), and the FinnGen project. Supporting Information Additional Supporting Information may be found in the online version of this article on the publisher’s website: References Zhang, M., Chen, H., Qian, H. & Wang, C. Characterization of the skin keloid microenvironment. Cell Commun. Signal. CCS 21 , 207 (2023). Che, K., Lyu, Q. & Ma, G. 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Epidemiol. 40 , 304–314 (2016). Bowden, J., Davey Smith, G. & Burgess, S. Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int. J. Epidemiol. 44 , 512–525 (2015). Hartwig, F. P., Davey Smith, G. & Bowden, J. Robust inference in summary data Mendelian randomization via the zero modal pleiotropy assumption. Int. J. Epidemiol. 46 , 1985–1998 (2017). Gilly, A. et al. Whole-genome sequencing analysis of the cardiometabolic proteome. Nat. Commun. 11 , 6336 (2020). Allen, R. J. et al. Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis. Am. J. Respir. Crit. Care Med. 201 , 564–574 (2020). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5235659","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":364793516,"identity":"335b935d-ee00-4985-af78-2360a011ada3","order_by":0,"name":"Ben Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Wang","suffix":""},{"id":364793517,"identity":"72676330-fbd0-4cd6-97c6-40faa3242a4b","order_by":1,"name":"Yiwen Deng","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yiwen","middleName":"","lastName":"Deng","suffix":""},{"id":364793518,"identity":"dee2c15b-0188-4641-a3bd-e8505224dce0","order_by":2,"name":"Zhujie Ran","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhujie","middleName":"","lastName":"Ran","suffix":""},{"id":364793519,"identity":"ed341f75-fac7-4660-868b-5c858829af29","order_by":3,"name":"Bo Pan","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Pan","suffix":""},{"id":364793520,"identity":"b8032ce8-762a-4f61-8b73-5ab50e6d5c90","order_by":4,"name":"Haiyue Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACxmYQ0cDAA6IeSFRIyPGTooXZwOKMhbFkA1FWQVSxSVS2VSRuIKSFuZ352cOvO+xkDG4kH5C4OU+CcQMD88NHN/A6jM3cWPZMMo/BjbQEw5nbJJjNGdiMjXPw+8VMWrKNGaglxyBZcpsEm2UDD5s0fi3s34Ba6oFa8j8c/jtHgsfgAEEtPGaSH9sOg2xhbJBskJAgRkuZNGPbcR7JM8+MGSSOSRhINhPwi2H/8W2SP9uq7fmOJz//IVFTV9/P3vzwMV4tDcCABsWjwgGYEDMe5SAgD3LcDxCjgYDKUTAKRsEoGLkAAJHgSVNzpSLnAAAAAElFTkSuQmCC","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Haiyue","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-10-10 02:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5235659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5235659/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67202563,"identity":"ae048cb6-450c-4cc3-85c7-a0c566497b79","added_by":"auto","created_at":"2024-10-22 10:19:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":903002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart of the study of Mendelian randomization.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5235659/v1/2ddb3ad43b3c02e233bda9cc.jpg"},{"id":67202562,"identity":"5dbac5de-b811-4284-a04f-28bf6f6a05d8","added_by":"auto","created_at":"2024-10-22 10:19:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe causal effects of PD-L2 on the risk of keloids were estimated. The impact of each risk factor was expressed using odds ratios (OR) and their corresponding 95% confidence intervals (95% CI), along with the IVW P-value to assess statistical significance. The assessment of horizontal pleiotropy is also included in the figure. The \"Consistent\" column represents the consistency between the causal effect estimates from the weighted median model and the MR-Egger model with the IVW method. OR denotes the odds ratio, 95%CI refers to the 95% confidence intervals.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5235659/v1/76e088bde9d38095f3315112.jpg"},{"id":67202565,"identity":"66b55a1e-57ed-4a48-8a9c-719d3c818cac","added_by":"auto","created_at":"2024-10-22 10:19:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe causal effects of PD-L2 on the risk of keloids were estimated. The impact of each risk factor was expressed using odds ratios (OR) and their corresponding 95% confidence intervals (95% CI), along with the IVW P-value to assess statistical significance. The assessment of horizontal pleiotropy is also included in the figure. The \"Consistent\" column represents the consistency between the causal effect estimates from the weighted median model and the MR-Egger model with the IVW method. OR denotes the odds ratio, 95%CI refers to the 95% confidence intervals.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5235659/v1/f43c068f1df2053658467413.jpg"},{"id":67202564,"identity":"72331f74-1332-4725-b079-8d09e87e9e71","added_by":"auto","created_at":"2024-10-22 10:19:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":569033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the mechanism of the effect of PD-L2 on keloid\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5235659/v1/e025d0acc95ccea597572216.jpg"},{"id":67204000,"identity":"61462098-bae2-4c80-a9ba-2661ae740fe5","added_by":"auto","created_at":"2024-10-22 10:35:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2016765,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5235659/v1/71293052-9855-466e-9334-606194c5293e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Evidence Supports the Therapeutic Potential of Immune Checkpoint PD-L2 in the Development of Keloid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pathological characteristics of keloid are excessive collagen deposition and sustained abnormal proliferation of fibroblasts during wound healing\u003csup\u003e1\u003c/sup\u003e. Keloid involves numerous characteristics resembling those of tumors, including unregulated cellular proliferation. Current treatments for keloid mainly involve intralesional corticosteroid injection combined with surgical excision followed by local adjuvant chemotherapy\u003csup\u003e2,3\u003c/sup\u003e. Unfortunately, clinical observations indicate that approximately 50% of keloids do not exhibit a positive response to corticosteroid injections\u003csup\u003e4\u003c/sup\u003e, while 9-50% of initially responsive keloids eventually experience recurrence.\u003csup\u003e5\u003c/sup\u003e. Therefore, the treatment of keloids presents a significant challenge, primarily due to the limited understanding of their specific pathogenesis, small sample sizes in previous studies, and a lack of effective research methods. These factors are crucial limitations that hinder the development of more targeted approaches for keloid treatment. In this study, we attempt to identify treatment targets associated with keloids from two large-scale clinical cohorts to advance keloid treatment-related progress.\u003c/p\u003e\n\u003cp\u003eThe effectiveness of glucocorticoid therapy suggests that the abnormal immune microenvironment within keloids may be a driving factor in their persistent growth. Therefore, inhibiting abnormal immune responses has become a potential therapeutic approach for keloids. Some cytokines have been explored for their therapeutic value in keloids, such as interferon-gamma, which is believed to have activity in reducing collagen synthesis both in vitro/vivo and has shown some effectiveness in treating proliferative scars and keloids\u003csup\u003e5\u003c/sup\u003e. However, these therapies face challenges in the absence of high-level, large-scale clinical trial evidence. Specifically, cytokine therapy or glucocorticoid-based immunomodulation therapy may be based on small clinical cohorts and lack specificity targeting the pathogenesis of keloids.\u003c/p\u003e\n\u003cp\u003eAs a significant advancement in the clinical application of immunology, treatment based on immune checkpoints has demonstrated promising therapeutic effects in the field of cancer therapy. This achievement was recognized with the 2018 Nobel Prize in Physiology or Medicine\u003csup\u003e6\u003c/sup\u003e. In fact, compared to previous immunotherapies, immune checkpoint-based treatment approaches offer two advantages. 1. Selective immunomodulatory characteristics: The PD-L1/PD-1 signaling pathway serves as a specific communication mode between proliferating abnormal cells and the immune system. Blocking this pathway can reactivate immune cells without affecting normal anti-tumor immunity, thereby facilitating the elimination of cancer cells\u003csup\u003e7\u003c/sup\u003e. Immunoregulatory measures with better selectivity are expected to inhibit unfavorable immune responses while preserving necessary immune responses to control abnormal proliferation and recurrence. 2. Potential for long-term cure: The efficacy of immunotherapy is limited to a subset of cancer patients; however, those who exhibit a positive response can attain enduring clinical remission, underscoring the potential for sustained success in this therapeutic approach\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, in order to understand the potential of immune checkpoint inhibitor therapy in reducing the risk of keloid formation, we utilized a genetic technique called Mendelian randomization, which has been widely used in recent research to infer causal relationships between exposure factors and disease outcomes\u003csup\u003e9\u003c/sup\u003e. Previous studies have confirmed the ability of Mendelian randomization to obtain evidence at the level of randomized clinical trials, referred to as natural randomized clinical trials. This is because Mendelian randomization research is based on genetic traits that are initially randomly distributed in the population, minimally influenced by confounding factors such as smoking, pollution, and other environmental factors, thus naturally eliminating interference from confounding factors\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. Compared to purely observational studies and randomized controlled trials, Mendelian randomization has the ability to infer causal relationships at a lower cost, with previous studies demonstrating its sufficient capability to provide evidence at the level of randomized controlled trials and high consistency with preclinical studies and clinical trial results. An illustrative example is the revelation by Mendelian randomization that PCSK9 inhibitors can reduce the risk of ASCVD in high-risk individuals\u003csup\u003e13\u003c/sup\u003e, subsequently confirmed by clinical trials\u003csup\u003e14\u003c/sup\u003e. Mendelian randomization also supports various ASCVD guidelines by identifying risk factors and excluding false positive results influenced by confounding factors (risk factors supported by observational studies but subsequently excluded in randomized controlled trials, such as HDL cholesterol, vitamin D, vitamin E, etc.)\u0026nbsp;\u003csup\u003e15\u0026ndash;19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe programmed death ligand 2 (PD-L2), a PD-1 ligand, facilitates immune evasion in cancer cells and serves as a prognostic indicator for patients with specific tumor types. The advancement of novel and efficacious immune combination therapy strategies directed towards PD-L2 holds the potential to enhance the clinical effectiveness of anti-PD-1, thereby showcasing promising prospects for cancer treatment through the utilization of PD-L2-targeting drugs. The proliferative and invasive capabilities of keloids are comparable to those of malignant tumors. The utilization of immune-targeted therapy could potentially serve as a novel approach in the management of keloids. Reaching a sufficient sample size for large-scale clinical trials in keloid patients can pose significant challenges due to the relatively low incidence rate of this condition. In this study, we employed two clinical cohorts comprising East Asian and European populations to simulate the perturbation of PD-L2 (Programmed cell death ligand 2) and explore its potential role in mitigating the risk of keloid formation\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e3.1 Study design and participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eClinically, we used a two-sample MR analysis model to assess the causal effects of PD-L2 on Keloids. The flow chart of our study is shown in Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;Genome-wide association study (GWAS) data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the GWAS European and East Asian data used for genetic analysis were derived from the Saori Sakaue et al.\u003csup\u003e21\u003c/sup\u003e This research project involved approximately 179,000 Japanese volunteers, with an average age of about 63 years and a gender distribution of 46.3% female. The study samples were obtained from the biobank Japan, which collected DNA and serum samples from 12 medical institutions throughout Japan. The biobank aimed to recruit around 200,000 individuals primarily of Japanese descent. Participant data were mainly derived from disease records extracted from unstructured electronic medical records (EMR). Prior to their involvement in the research, all individuals provided written informed consent. The study received approval from the ethics committees of the Institute of Medical Sciences at the University of Tokyo and the RIKEN Center for Integrative Medical Sciences. All research procedures strictly followed the principles outlined in the Declaration of Helsinki by the World Medical Association.\u003c/p\u003e\n\u003cp\u003eThe GWAS data of European individuals primarily come from the UK Biobank (UKB) and the FinnGen project. The UKB project is a population-based prospective cohort that includes approximately 500,000 individuals from the United Kingdom, with an average participant recruitment age of 56.8 years, of which 53.8% are female. The study focuses on 158 disease endpoints derived from clinical information in the UKB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe FinnGen project integrates genotype data from Finnish biobanks and digital health record data from Finnish health registries. The mean age of participants at DNA sample collection is 51.8 years, with 56.3% being female\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3.3\u0026nbsp;Mendelian Randomization Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMendelian Randomization (MR) was utilized to explore the causal links between PD-L2 and the risk of keloids, relying on several key assumptions\u003csup\u003e22\u003c/sup\u003e. The MR analysis hinges on three primary assumptions: (1) strong correlation between instrumental variables (IVs) and the exposure, (2) independence of IVs from confounding factors, and (3) IVs exerting no direct influence on the outcome, with their impact solely mediated through the exposure. SNPs with p-values \u0026lt; 5 \u0026times; 10^8, indicating significant associations with the phenotype, were selected from each cohort for further investigation. Linkage disequilibrium (LD) exclusion criteria were applied, with an R^2 threshold of 0.001 and a maximum distance threshold of 10,000 kilobases, to address LD issues. SNPs in LD were resolved by selecting the SNP with the lowest p-value for subsequent analysis. To mitigate bias from weak IVs, SNPs with F-statistic \u0026lt; 10, considered weak IVs introducing potential bias, were excluded. In cases where only one SNP could be evaluated for an exposure factor after LD exclusion, the Wald ratio was employed for statistical analysis. The impact of the exposure factor on the outcome was assessed using the inverse variance weighting (IVW) method. Additionally, MR analyses were conducted using MR-Egger\u003csup\u003e23\u003c/sup\u003e and Median Mendelian Randomization methods\u003csup\u003e24\u003c/sup\u003e, known for their robustness against pleiotropy. MR-Egger method, employing weighted regression with an unconstrained intercept, reduces susceptibility to pleiotropic effects by not assuming all genetic variants as valid IVs. Median Mendelian Randomization method, robust against pleiotropy, uses the median instrument variable from all included variants, ensuring resilience even with a proportion of invalid genetic variation. Consistent results from these methods enhance causal inference. The primary outcomes were derived from IVW-MR analysis, considering only positive results meeting the statistical threshold and showing consistent OR estimates across all three methods. Cochrane\u0026apos;s Q statistic was calculated to assess result heterogeneity. Fixed-effects model estimation was used in the absence of heterogeneity, assuming no pleiotropy. In cases of detected heterogeneity, a random-effects model was employed, assuming the genetic instrument\u0026apos;s association strength with the risk factor is independent of the pleiotropic effect size or has an average effect size of zero. MR-Egger regression and sensitivity analysis were conducted to detect horizontal pleiotropy. The R package \u0026quot;TwoSampleMR\u0026quot; facilitated the MR analysis, with results reported as ORs with 95% confidence intervals (CI) \u003csup\u003e24\u0026ndash;26\u003c/sup\u003e.\u0026nbsp;All statistical analyses were performed in R (version 4.3.1).\u003c/p\u003e\n\u003cp\u003eThe instrumental variable for PD-L2 is derived from the GWAS study by Arthur Gilly and colleagues, the associations between these genetic markers and circulating proteins were replicated and validated in two independent cohorts with sample sizes of 1605 and 1328, respectively\u003csup\u003e27\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e4.1 Causal factors of keloids associated with PD-L2 in East Asian cohort.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We first analyzed the association between PD-L2 levels and the risk of keloids in the Asian population. The results, as shown in Figure 2, indicate that the expression level of PD-L2 is associated with an increased risk of keloids and demonstrates a significant causal relationship. The P-value estimated using the inverse variance weighted method is 0.02, with an odds ratio (OR) between 1.25 (1.04-1.51). The directions of the OR estimated by the other two statistical methods (MR Egger and Weighted median) are consistent with the IVW method, and no significant pleiotropy was detected, suggesting that PD-L2 is a risk factor for keloids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Causal factors of keloids associated with PD-L2 in European cohort.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Furthermore, in order to determine whether this causal relationship still persists within the European population, we conducted further investigations into the association between PD-L2 and the susceptibility to keloid formation among Europeans. This cohort included 668 patients diagnosed with keloids and 481,244 controls. The results indicate a causal relationship between PD-L2 and keloids, with P of inverse variance weighted = 0.004, OR=1.24 (1.07-1.43). The directions of the OR estimated by the other two statistical methods align with the inverse variance weighted method, and no significant pleiotropy was detected, suggesting that elevated levels of PD-L2 are associated with an increased risk of keloid formation.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eThe expression of PD-L2, an additional ligand for PD-1, was initially observed in macrophages and dendritic cells. Recent research has demonstrated that PD-L2 exhibits high expression levels in human cancers and can serve as a reliable indicator of cancer stage and response to drug treatment. The characteristics of keloids, such as their propensity for proliferation and invasion, closely resemble those observed in malignant tumors; however, the etiology remains unknown, posing continuous challenges to the treatment of keloids. In this study, we used a two-sample Mendelian randomization approach to determine the causal relationship between PD-L2 and the risk of keloids. Our findings are derived from two cohorts of different ethnicities and were validated using various hypothesis testing methods, ensuring the strength and credibility of our results. A key strength of our study is the inclusion of a large-scale genome-wide association study (GWAS) sample. These two cohorts had sample sizes of 178,726 (1055 cases) and 481,912 (668 cases) respectively, making this study the largest in terms of sample size in keloid-related research, to our knowledge. We validated in these two large-scale clinical cohorts that the simulated perturbation of PD-L1 treatment has an effect in reducing the risk of keloid formation, suggesting that immune therapy strategies targeting the PD-L1/PD-1 axis may be a potential treatment approach for keloids. Among all studies related to keloids, our research has a relatively large sample size\u003csup\u003e28\u003c/sup\u003e. This not only enhances the statistical power of our study but also bolsters the persuasiveness of our conclusions.\u003c/p\u003e\n\u003cp\u003eTo our knowledge, previous reports from basic or clinical research have not yet established the potential role of the PD-1/PD-L1 axis in the pathogenesis of keloids. Our study\u0026apos;s innovative discovery that the expression of PD1-related genes may serve as a predisposing factor for keloid formation suggests that therapeutic interventions targeting PD-L2 could potentially offer an alternative treatment approach in contrast to conventional corticosteroid injections(Figure 4). While previous studies have shown the performance of Mendelian randomization in providing evidence comparable to randomized clinical trials, further basic experiments, preclinical investigations, and randomized clinical trials are still needed in the future to clarify the roles of immune checkpoints and their related molecules in the pathogenesis of keloids.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. Firstly, the keloids GWAS summary data used in this study did not adjust for age and gender because this information was not available for all individuals in each dataset. Secondly, the MR results reflect lifetime changes in keloids risk due to PD-L2 levels, and further research is needed to clarify its short-term impact on keloids risk. Thirdly, further large-scale clinical trials are needed in the future to clarify the effectiveness or safety of this treatment and whether it could lead to sustained relief in keloid patients and reduce their recurrence rates.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides strong evidence that PD-L2 is a causal determinant of keloids risk. The implementation of specific targeted immunotherapy may prove to be a viable and efficacious strategy for the treatment of keloids. These findings, based on a large-scale sample, may provide evidence for further treatment and prevention of keloids.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors had full access to all the data in this study and take responsibility for the integrity of the data and the accuracy of the data analysis. Ben Wang and Yiwen Deng contributed equally and share the first authorship.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBen Wang: Writing\u0026ndash;original draft, Supervision, Methodology, Conceptualization.\u003c/p\u003e\n\u003cp\u003eYiwen Deng: Writing\u0026ndash;original draft, Methodology, Conceptualization,Validation.\u003c/p\u003e\n\u003cp\u003eZhujie Ran: Supervision, Methodology.\u003c/p\u003e\n\u003cp\u003eBo Pan: Writing \u0026ndash; review \u0026amp; editing, Visualization, Supervision.\u003c/p\u003e\n\u003cp\u003eHaiyue Jiang: Writing \u0026ndash; review \u0026amp; editing, Visualization, Supervision, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No. 82371796)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China (No. 82371796)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data for these studies were approved by the ethics committees of the Institute of Medical Sciences at the University of Tokyo, the RIKEN Center for Integrative Medical Sciences, the UK Biobank (UKB), and the FinnGen project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional Supporting Information may be found in the online version of this article on the publisher\u0026rsquo;s website:\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, M., Chen, H., Qian, H. \u0026amp; Wang, C. 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Care Med.\u003c/em\u003e \u003cstrong\u003e201\u003c/strong\u003e, 564\u0026ndash;574 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Keloid, PD-L2, Immune checkpoint","lastPublishedDoi":"10.21203/rs.3.rs-5235659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5235659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKeloids are pathological scars that extend beyond the original skin injury, resembling tumor-like growth, with unclear etiology and challenging treatment. This study aims to determine whether there is a causal link between the immune checkpoint PD-L2 and keloid risk, assessing immune checkpoints as potential therapeutic targets. A Mendelian randomization analysis was conducted using genome-wide association study summary statistics. PD-L2 expression levels were analyzed in Asian and European cohorts to explore their association with keloid risk. Two clinical cohorts with different ancestries were used to investigate association between PD-L2 expression and the risk of keloids. The Asian cohort included 178,726 samples (1055 cases), while the European cohort included 481,912 samples (668 cases). Our findings indicate that higher levels of PD-L2 expression in plasma proteome are associated with an increased risk of keloids. This conclusion holds true in both European and Asian large-scale cohorts, with OR (Asia) values of 1.25 (1.04-1.51), P_IVW=0.02, and OR (Europe) of 1.24 (1.07-1.43), P_IVW=0.004. This study reveals for the first time a causal relationship between the expression levels of the immune checkpoint PD-L2 and the risk of keloids. This provides a new perspective for understanding the pathogenesis of keloid and for developing new treatment and prevention strategies.\u003c/p\u003e","manuscriptTitle":"Genetic Evidence Supports the Therapeutic Potential of Immune Checkpoint PD-L2 in the Development of Keloid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-22 10:19:23","doi":"10.21203/rs.3.rs-5235659/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14316aab-2795-4d00-b5dc-5ffe9d406bd8","owner":[],"postedDate":"October 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T10:19:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-22 10:19:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5235659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5235659","identity":"rs-5235659","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}