Investigating the Expression of LL-37 Antimicrobial Peptide in Autoimmune Bullous Disorders: Implications for Pemphigus

Investigating the Expression of LL-37 Antimicrobial Peptide in Autoimmune Bullous Disorders: Implications for Pemphigus | 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 Investigating the Expression of LL-37 Antimicrobial Peptide in Autoimmune Bullous Disorders: Implications for Pemphigus Fatma Dhaffouli, Nesrine Elloumi, Khadija Sellami, Emna Bahloul, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6254185/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 Objective: This study aimed to investigate the expression of the antimicrobial peptide cathelicidin LL-37 and discuss its role as a critical factor in developing pemphigus. Methods: The expression of LL-37 mRNA was assessed in individuals with pemphigus foliaceous (PF) and pemphigus vulgaris (PV) at different stages of the disease, in comparison to healthy controls (HCs), using RT-PCR and ELISA. The LL-37 expression profile was differentially dysregulated in pemphigus. Results: a high LL-37 expression level was shown in newly diagnosed patients compared to healthy controls. The short-term LL-37 gene expression follow-up showed a significant up-regulation after three months of cortical therapy treatment and a strong negative correlation with anti-Dsg1 Abs in PF-monitored patients, suggesting that LL-37 could be implicated in remission induction. In line with this hypothesis, the long-term follow-up results showed a significant increase in LL-37 expression according to disease severity when devising treated patients in the retrospective study, according to their PDAI. Conclusion: Our preliminary finding suggests that the timing and the cellular context change the expression of LL-37 according to the disease severity, which determines the direction of the cellular response. We propose this small peptide as a point of debate requiring deep investigations in pemphigus. antimicrobial peptides Cathelicidin LL-37 immunobullous disorders gene expression Figures Figure 1 Figure 2 Figure 3 1. Introduction Pemphigus is an immunobullous disorder characterised by flaccid blisters and erosions of skin/mucous membranes that are clinically significant with high morbidity and mortality if left untreated [ 1 ]. Because an autoimmune process drives its pathophysiology, the autoantibodies are the basis of diagnostic investigations and treatment strategies. The critical target antigens, desmoglein (Dsg)1 and Dsg3, are essential components of desmosomes; the ‘rivets’ hold keratinocytes in the epidermis together. Once desmosomes fail, the keratinocytes split from one another, leading to blistering [ 2 ]. Infections resulting from fragile skin barrier, dysfunction of immunity, and systemic corticosteroids and other immunosuppressing agent’s use are the most frequent complications of patients with pemphigus and account for 34.3–55.5% of all deaths [ 3 ]. Cells produce anti-microbial peptides (AMPs) to cope with microbial exposure, which inhibits the growth and invasion of pathogens. Keratinocytes, neutrophils, monocytes, and macrophages can produce these AMPs. In normal skin, the production of AMPs occurs constitutively, but a significant increase is noticed when skin is injured because of trauma, inflammation, or infection [ 4 ]. Defensins and cathelicidins are human skin's most studied AMP families [ 5 ]. β defensin (HBD) was the first AMP characterized in human skin; HBD2 with high effectiveness against gram-negative bacteria, and HBD3 with a broader spectrum of antimicrobial action [ 6 ]. While several classes of AMPs exist, LL-37 is the unique cathelicidin peptide [ 7 ]. LL-37 is encoded by the CAMP gene and produced by various cells, including epithelial cells, natural killer cells, neutrophils, T cells, monocytes and dendritic cells [ 8 ]. This peptide has piqued the research community's interest because of its pleiotropic functions as an antimicrobial peptide and its numerous immune system-modulating properties [ 9 ]. The antimicrobial activity of most AMPs results from their unique structural properties, which allow them to disrupt the microbial membrane without attacking human cell membranes. Furthermore, these peptides also act on host cells to regulate immune responses, cytokine production, cell migration, proliferation, maturation, and extracellular matrix synthesis [ 4 ]. This ability to maintain equilibrium plays a vital role in resisting pathogens while maintaining the stability of the immune system. If defects in the expression or processing of LL-37 break this balance, it will result in abnormalities in the body [ 10 ]. LL-37 is also involved in the pathogenesis of multiple autoimmune and inflammatory diseases, such as psoriasis and discoid lupus, where its dysregulation is associated with the onset and progression of the disease [ 11 , 12 ]. The scarcity of studies in autoimmune bullous diseases was the rationale for the design of our study, which aimed to offer a concise general view of the expression of antimicrobial peptides LL-37 and briefly discuss the role of this small peptide as a key factor in the development of pemphigus. 2. Material and Methods 2.1 Subjects All participants provided written informed consent for the study, which was approved by the Human Research Ethics Committee of the Habib Bourguiba University Hospital of Sfax (protocol number 4/12). The diagnosis of pemphigus (PF and PV) is confirmed by clinical presentation, histopathology and Immunological tests for circulatory anti-Dsg1 (cut-off value was set at 20 UI/mL) and/or Dsg3 Abs (cut-off value was set at 14 UI/mL). The first-line pemphigus treatment relies on systemic corticosteroids according to the recent recommendations of Murrell et al. Pemphigus management considered two main phases: remission induction and remission maintenance. During the follow-up assessment, disease activity of both the skin and mucosal surfaces was monitored using the pemphigus disease area index (PDAI), including different skin, scalp, and mucosal lesion scores. On the other hand, a high PDAI score and/or persistent high anti-Dsg1/Dsg3 Abs values define severe chronic pemphigus. The classification strategy followed the disease stage and treatment management (treated/untreated) as described by Shimizu et al [ 13 ]. Therefore, the patient group was divided into three groups: newly diagnosed untreated patients and treated patients with varying disease progression, divided into remitted patients’ group (mild form of the disease) (PDAI ≤ 8) and chronic patients’ group (moderate to severe form of the disease) (PDAI ≥ 9). Based on patient approval, six newly diagnosed patients were subjected to a follow-up at 3 months HCs with no signs of autoimmune disorders were recruited as a control group. 2.2 Blood sampling and PBMC isolation For the transcriptomic study, a total of 10 ml of peripheral blood were collected from newly diagnosed PV patients (n = 4), treated PV patients (n = 18), newly diagnosed PS patients (n = 12), and treated PS patients (n = 16) recruited at the Department of Dermatology in the Hedi Chaker University Hospital of Sfax, Tunisia, in addition to HC (n = 16) (Fig. 1 ). PBMCs separation was performed by low-density Ficoll-Hypaque gradient centrifugation, the leukocyte-enriched plasma was layered onto low-density Ficoll-Hypaque and centrifuged for 30 min. Then, their viability and purity were checked and evaluated by trypan and truck blue; respectively in light microscopy. 2.3 RNA isolation and Reverse transcription Total RNA was extracted from the Trizol suspension according to the manufacturer’s instructions. The RNA purity and integrity in each sample were assessed using a NanoDrop system (NanoDrop Technologies®, Wilmington, NC, USA) and using standard agarose gel electrophoresis. cDNA was generated using the PrimeScript RT Reagent Kit (TAKARA Bio Inc.®, Japan). 2.4 LL-37 gene expression analysis Relative real-time quantitative RT-PCR of LL-37 was performed using Gene-specific primers (F: 5’-TCGGATGCTAACCTCTACCG-3’/R: 5’-GGGTACAAGATTCCGCAAAA-3’), by SYBR Green Dye detection system analysis. All reactions were performed in duplicate. For verification of the quality of PCR products, melting curves were generated. The relative quantification was performed using the standard curve method, and normalized to the average housekeeping gene GAPDH (F: 5’-GCTCTCTGCTCCTCCTGTTC-3’/R: 5’-CGCCCAATACGACCAAATCC-3’). Data were analyzed by the comparative 2 − ΔCt method. 2.5 Serum concentrations of antimicrobial peptide cathelicidin LL-37 Sandwich ELISA was performed with a Human Antibacterial Protein LL-37 ELISA Kit from CUSABIO (CSB-EL004476HU). In brief, LL-37 standards and patient serum were added to the wells coated with antibodies specific for LL-37, and a biotin-conjugated reagent was added to the wells and incubated. Tetramethylbenzidine 6 substrate was used to quantify the HRP enzymatic reaction. Optical density (OD) was measured spectrophotometrically at 450 nm. 2.6 Statistical analysis The results were analyzed using SPSS software 2.0. The differences in expression between groups were analyzed using the Kruskal-Wallis nonparametric tests and the Mann-Whitney independent sample test. For the results analysis of the same patient at different time points, we used the non-parametric paired-sample tests, the Wilcoxon test. Spearman's test was used for correlation studies. Statistical significance was defined as a value of p < 0.05. 3. Results LL-37 expression profiling was performed and compared in blood samples of pemphigus patients; among clinical disease groups: 20 PV (sex ratio M/F: 8/12,) and 27 PF (sex ratio M/F: 3/24), and 16 HCs. LL-37 gene, as well as serum expressions, were abnormally up-regulated in pemphigus patients compared to HC (Gene expression: Newly diagnosed groups mean =0.106 ± 0.07 and HC mean =0.011 ± 0.003, p = 0.012, Serum level: Newly diagnosed groups mean =10.06 ± 1.006 ng/ml and HC mean =6.22 ± 1.32 ng/ml, p = 0.048) (Fig. 2 .A-B, Fig. 3 ). In detail, LL-37 mRNA gene expression was significantly enhanced in newly diagnosed PV (n = 4) and PF (n = 11) compared to HCs; (PV mean =0.324 ± 0.28, PF mean =0.027 ± 0.007 and HC mean =0.011 ± 0.003, with p = 0.046, p = 0.036; respectively) (Fig. 2 .A). There was no statistically significant difference in LL-37 gene expression levels between samples of PV and PF. To understand the impact of short-term corticosteroid therapy on LL-37 gene expression, six patients were followed after 3 months of treatment. Our data revealed that the LL-37 mRNA expression changed significantly after 3 months of treatment (Newly diagnosed patients mean =0.039 ± 0.014 vs 3M treated mean =0.408 ± 0.24, p = 0.023). A notable negative correlation was revealed when analyzing Dsg1-Abs level and LL-37 mRNA expression in the same group gathering newly diagnosed patients and short-term treated patients (3 months) (r=-0,615; p = 0,025) (Fig. 2 .C). Interestingly, when focusing on patients concerned by follow-up, this relationship was revealed more pronounced with a higher correlation coefficient (r= -0,7; p = 0,020) (Fig. 2 .D). By stratifying PV and PF patients according to their clinical disease stage into chronic and remittent groups, LL-37 gene expression was followed up in a group of six-year average treatment period patients. A notable down-regulation of LL-37 expression was identified in a remittent group compared to the chronic group (remittent group mean =9.57 ± 1.80 vs Chronic group mean =14.24 ± 1.73, p > 0.05). A similar profile was observed when studying LL-37 serum expression, without any statistical significance (remittent group mean =0.025 ± 0.077vs Chronic group mean =0.172 ± 0.05, p > 0.05). This significance was maintained even when studying the two separate classes of pemphigus (remittent PV mean =0.049 ± 0.011 vs Chronic PV mean =0.225 ± 0.073, p = 0.039 and remittent PF mean =0.007 ± 0.003 vs chronic PF mean =0.074 ± 0.034, p = 0.028; respectively) (Fig. 1 .E). There was also a decrease in LL-37 gene expression in remittent PF patients when compared to newly diagnosed ones, p = 0.013. 4. Discussion Cathelicidin LL-37 plays an important role in the early host response against invading pathogens via its antimicrobial activity. In addition, it is an important effector molecule of innate immunity in the skin [ 6 ]. In the present study; we examined the implication of LL-37 in the occurrence and the severity of intra-epidermal autoimmune bullous diseases. Overall, LL-37 gene expression and serum level profiles were differentially dysregulated in pemphigus. Newly diagnosed pemphigus patients showed, whether in peripheral blood cells or serum, a significant upregulation of LL-37 compared to HCs. It seems that the over-expression of LL-37 is important in the disease onset. Yet, there is a lack of studies analyzing the LL-37 expression on a systemic level; particularly on PBMC. An up-regulation was largely reported in various biological specimens. Indeed, in the inflammatory skin disorder context, previous studies showed similar results in psoriatic cutaneous biopsies [ 14 ] and in cutaneous lupus lesions biopsies including discoid lupus erythematosus, subacute cutaneous lupus erythematosus, and lupus erythematosus tumidus [ 15 ]. On another hand, the salivary concentration of LL-37 was increased in inflammatory ulcerating diseases in both oral lichen planus and periodontitis [ 16 , 17 ]. Data relating to cathelicidin LL-37 expression in diseases, particularly skin diseases are scarce. Taking together, cathelicidin expression dysregulation appears to occur in inflammatory and auto-immune disorders, as in the case of pemphigus; PF, and PV. However, the physiological roles and precise functions of LL-37 in pemphigus remain to be elucidated. Indeed, both pro- and anti-inflammatory functions have been assigned to LL-37 in a dependent manner on the microenvironment and disease background. Some studies have speculated that these AMPs may provide a protective effect from cutaneous infection in cutaneous lupus erythematosus patients [ 14 ]. However, a pro-inflammatory phenotype of LL-37 on macrophages in SLE was also suggested [ 11 ]. Pemphigus management is divided into two main phases; remission induction and remission maintenance [ 18 ]. In active pemphigus, multiple blisters and erosions trigger the release of inflammatory cytokines IL-6 and IL-17 [ 19 ], thus, the cornerstone of pemphigus treatment remains systemic corticosteroids due to their immunosuppressive and anti-inflammatory properties [ 20 ]. We, therefore, investigated the impact of corticosteroids on LL-37 gene expression change after 3 months of cortico-therapy treatment. The follow-up showed a significant up-regulation in LL-37 gene expression which was negatively correlated with anti-Dsg1 Abs in pemphigus-monitored patients. Interestingly, when subdividing treated patients according to PDAI score, we noted the persistence of LL-37 expression levels in chronic pemphigus groups, compared to the remittent ones. Based on these findings, it seems logical to assume that corticosteroids impact the LL-37 expression according to the disease context. Previous research of Marin-Luevano and her collaborators indicated that the glucocorticoid; dehydroepiandrosterone, promotes the production of LL-37 and β-defensin [ 21 ]. Corticosteroids play a critical role in remission induction 20 ; their interaction with the cytoplasmic corticosteroid receptor, results in the up-regulation of anti-inflammatory proteins and downregulation of those pro-inflammatory [ 22 ]. Therefore, LL-37 up-regulation may contribute to suppressing the inflammatory responses and mediating tissue repair. Indeed, LL-37 possesses a critical role in modulating cytokine production and chemoattracting various immune effector cells leading to stimulation angiogenesis and wound healing [ 23 ]. It was shown that the topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization [ 24 ]. Alongside our results, the up-regulation of LL-37 gene expression was suggested to be implicated in tissue repair in the recovery phase of septic [ 25 ]. It is generally believed that the disease-inducing immune response is initiated at distant sites, followed by a migration of immune cells to the skin and autoantibody binding to Dsg3/Dsg1 of the epidermis [ 26 ]. So, the LL-37 systemic regulation could be tightly linked to the skin level. On the other hand, there was evidence that LL-37 can perform two distinct functions in different tissues and different microenvironments [ 23 ], this peptide has been shown to regulate monocyte/macrophage differentiation [ 27 ]. Macrophages can thus exhibit pro- and anti-inflammatory properties to a degree that is determined by stimuli from their local microenvironment [ 28 ]. It is therefore necessary to examine the immune functions of LL-37 from both sides. Thus, we could suggest that cellular context in persistent chronic phases of the disease may stimulate the pro-inflammatory effect of LL-37. LL-37 exacerbated LPS-induced septic shock in rats when administered 2 hours after LPS treatment [ 29 ]. This is in line with the hypothesis suggesting that at a high concentration or under specific conditions, LL-37 can aggravate host-damaging effects induced by inflammation. This take as to another speculation suggesting that the persistence of high levels of LL-37 gene expression in chronic pemphigus can aggravate damaging effects induced by inflammation. Taking together, we suggest that the timing and the cellular context change the expression of LL-37 mRNA according to the disease severity which determines the direction of the cellular response. On the one side, LL-37 seems to promote immune response and exerts its anti-inflammatory and wound healing effects for remission induction and persistence; on the other side, it seems to have the ability to stimulate inflammation and promote a pro-inflammatory response in the persistent chronic phase of pemphigus. 5. Conclusion In conclusion, the LL-37 gene expression profile was differentially dysregulated in pemphigus according to the disease status which allows us to suggest its contribution to the pathogenesis of the disease. The cellular environment and the timing appear to have an impact on LL-37 expression. Further functional studies on skin cell culture are needed for a more comprehensive understanding. Declarations Ethical approval The study was approved by the Human Research Ethics Committee of the Habib Bourguiba University Hospital of Sfax (protocol number of the ethical committee, 4/12). Consent to participate Written informed consent was obtained from all participants. Acknowledgment The Ministry of High Education and Scientific Research of Tunisia supported this work. We would like to thank the patients and volunteers for their participation Funding Not applicable. Author contribution statement The authors confirm their contribution to the paper as follows: study conception and design: DF, EN, AO; data collection: DF, SK, BE, TS, TH, HH, AO; analysis and interpretation of results and draft manuscript preparation: DF, EN, AO. All authors reviewed the results and approved the final version of the manuscript. Competing Interests The authors declare no conflict of interest. Data availability The data presented in this study are available upon request from the corresponding author References Leshem YA, Gdalevich M, Ziv M, David M, Hodak E, Mimouni D. Opportunistic infections in patients with pemphigus. J Am Acad Dermatol. 2014 Aug;71(2):284-92. doi: 10.1016/j.jaad.2014.03.020. Epub 2014 May 6. PMID: 24815564. Melchionda, V, Harman, K.E. 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Prostate cancer-derived cathelicidin-related antimicrobial peptide facilitates macrophage differentiation and polarization of immature myeloid progenitors to protumorigenic macrophages. Prostate. 2016 May;76(7):624-36. doi: 10.1002/pros.23155. Epub 2016 Feb 9. PMID: 26856684; PMCID: PMC5551898. Rasheed A, Rayner KJ. Macrophage Responses to Environmental Stimuli During Homeostasis and Disease. Endocr Rev. 2021 Jul 16;42(4):407-435. doi: 10.1210/endrev/bnab004. PMID: 33523133; PMCID: PMC8284619. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6254185","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436441186,"identity":"7855f3ba-d55f-4f7f-86bf-052526841124","order_by":0,"name":"Fatma Dhaffouli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBAC9gYQyXaAgb35AAPDAwMbBgYJkIgNbi08B5ghWniOJTAwJBikQbWkEa2F4TARWtjPH93wo+yOPA8b8zGJhILzif2zmw8+YEi4h1sLTzLbzZ5zzwx72NjSJBIMbifOuHMs2YAhoRinFnuGZLYbvG2HGffL95iBtTTcyDGTYPyRgNsW/sdsN/+2HbbvYeMBaTmXOB+khSEBjxaJZLbbQFsSoVoOJG4grOWx2W2Zc4eTgX5JtkgwSDbeeCMt2SABnxb+xGc335Qdtu1hYz5448MfO9l5N5IPPviARwsGcGwAkSRoAAXhKBgFo2AUjAI0AABku1moml1KLAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Sfax","correspondingAuthor":true,"prefix":"","firstName":"Fatma","middleName":"","lastName":"Dhaffouli","suffix":""},{"id":436441187,"identity":"14a1dec3-9c9b-4b9c-a370-63aec17f2067","order_by":1,"name":"Nesrine Elloumi","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Nesrine","middleName":"","lastName":"Elloumi","suffix":""},{"id":436441188,"identity":"0adcc9de-d249-4d6f-9085-df6c0aa8a5c5","order_by":2,"name":"Khadija Sellami","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Khadija","middleName":"","lastName":"Sellami","suffix":""},{"id":436441189,"identity":"9ed92c88-4268-4c58-b6ec-8af38cee1095","order_by":3,"name":"Emna Bahloul","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Emna","middleName":"","lastName":"Bahloul","suffix":""},{"id":436441190,"identity":"8f8a4799-87e2-4bba-9aca-893b5a5ae158","order_by":4,"name":"Safa Tahri","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Safa","middleName":"","lastName":"Tahri","suffix":""},{"id":436441191,"identity":"137995b9-abf9-429d-8987-b4243f15e958","order_by":5,"name":"Hamida Turki","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Hamida","middleName":"","lastName":"Turki","suffix":""},{"id":436441192,"identity":"abe896fe-ab8a-4a95-816e-8e1f1c7fbc43","order_by":6,"name":"Hend Hachicha","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Hend","middleName":"","lastName":"Hachicha","suffix":""},{"id":436441193,"identity":"b8729172-0b44-46c0-9300-db169fa1925f","order_by":7,"name":"Olfa Abida","email":"","orcid":"","institution":"University of Sfax","correspondingAuthor":false,"prefix":"","firstName":"Olfa","middleName":"","lastName":"Abida","suffix":""}],"badges":[],"createdAt":"2025-03-18 14:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6254185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6254185/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79750960,"identity":"f0b09810-5e54-4c41-9878-08d5fb1936bc","added_by":"auto","created_at":"2025-04-02 09:21:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart for the studied groups\u003c/strong\u003e, DIF: direct immunofluorescence; Dsg: desmoglein, IIF: indirect immunofluorescence, PF: pemphigus Foliaceus, PV: pemphigus vulgaris, HC: healthy control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6254185/v1/f307a3d5f1c78c0dbc25e7f5.png"},{"id":79750963,"identity":"f50407e1-9c25-4c17-b7af-aefc1012cebf","added_by":"auto","created_at":"2025-04-02 09:21:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLL-37 expression change in different studied groups. \u003c/strong\u003e(A) Differential expression level of LL-37 in newly diagnosed pemphigus patients (PV; \u003cem\u003en\u003c/em\u003e=4, PF; \u003cem\u003en\u003c/em\u003e=11) compared to HC (n=16); (PV\u003csub\u003emean\u003c/sub\u003e=0.324±0.28, PF\u003csub\u003emean\u003c/sub\u003e=0.027±0.007 and HC\u003csub\u003emean\u003c/sub\u003e=0.011±0.003, with p=0.046, p=0.036; respectively). (B) Differential expression of LL-37 and (C) Dsg1 values after 3M of treatment. LL-37 mRNA expression changed significantly after 3 months of treatment (Newly diagnosed patients\u003csub\u003emean\u003c/sub\u003e=0.039±0.014 vs 3M treated\u003csub\u003emean\u003c/sub\u003e=0.408±0.24, p=0.023) with a notable negative correlation between Dsg1-Abs level and LL-37 mRNA expression. (D) Differential expression level of LL-37 in chronic PV patients (\u003cem\u003en\u003c/em\u003e=8) compared to remittent (n=6) and newly diagnosed; (remittent PV\u003csub\u003emean\u003c/sub\u003e=0.049±0.011 vs Chronic PV\u003csub\u003emean\u003c/sub\u003e=0.225±0.073, p=0.039). (E) LL-37 mRNA expression change in chronic PF patients (\u003cem\u003en\u003c/em\u003e=6), compared to remittent (\u003cem\u003en\u003c/em\u003e=8); (remittent PF\u003csub\u003emean\u003c/sub\u003e=0.007±0.003 vs chronic PF\u003csub\u003emean\u003c/sub\u003e=0.074±0.034, p=0.028; respectively) and newly diagnosed groups (p=0.013). Comparison of two independent samples using the non-parametrical Mann-Witney test, p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6254185/v1/6f79bd0f9da6c8d558c9cffa.png"},{"id":79750961,"identity":"761a034b-272a-49a8-8831-8e265796ebcf","added_by":"auto","created_at":"2025-04-02 09:21:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLL-37 serum expression changes in different studied groups.\u003c/strong\u003e LL-37 serum concentration comparison between Newly diagnosed patients (n=15) and HC (n=11) (Newly diagnosed patientsmean= 10.06±1ng/ml, HCmean=6.22±1.3 ng/ml, p=0.048) and between remittent patients (n=11) and chronic patients (n=14) (Remittent patientsmean= 9.5±1.8 ng/ml, chronic patientsmean= 14.2 ±1.7 ng/ml, p\u0026gt;0.05). Comparison of two independent samples using the non-parametrical Mann-Witney test, p-value \u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6254185/v1/b9ab350876439b5dc0e22d1a.png"},{"id":80984400,"identity":"cc5fff99-d29c-419f-aef2-9be1b60e0266","added_by":"auto","created_at":"2025-04-20 21:46:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1031319,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254185/v1/86fbdda0-54a8-4b5b-b83c-17ceca679862.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the Expression of LL-37 Antimicrobial Peptide in Autoimmune Bullous Disorders: Implications for Pemphigus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePemphigus is an immunobullous disorder characterised by flaccid blisters and erosions of skin/mucous membranes that are clinically significant with high morbidity and mortality if left untreated [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Because an autoimmune process drives its pathophysiology, the autoantibodies are the basis of diagnostic investigations and treatment strategies. The critical target antigens, desmoglein (Dsg)1 and Dsg3, are essential components of desmosomes; the \u0026lsquo;rivets\u0026rsquo; hold keratinocytes in the epidermis together. Once desmosomes fail, the keratinocytes split from one another, leading to blistering [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Infections resulting from fragile skin barrier, dysfunction of immunity, and systemic corticosteroids and other immunosuppressing agent\u0026rsquo;s use are the most frequent complications of patients with pemphigus and account for 34.3\u0026ndash;55.5% of all deaths [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Cells produce anti-microbial peptides (AMPs) to cope with microbial exposure, which inhibits the growth and invasion of pathogens.\u003c/p\u003e \u003cp\u003eKeratinocytes, neutrophils, monocytes, and macrophages can produce these AMPs. In normal skin, the production of AMPs occurs constitutively, but a significant increase is noticed when skin is injured because of trauma, inflammation, or infection [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Defensins and cathelicidins are human skin's most studied AMP families [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. β defensin (HBD) was the first AMP characterized in human skin; HBD2 with high effectiveness against gram-negative bacteria, and HBD3 with a broader spectrum of antimicrobial action [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While several classes of AMPs exist, LL-37 is the unique cathelicidin peptide [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. LL-37 is encoded by the CAMP gene and produced by various cells, including epithelial cells, natural killer cells, neutrophils, T cells, monocytes and dendritic cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This peptide has piqued the research community's interest because of its pleiotropic functions as an antimicrobial peptide and its numerous immune system-modulating properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The antimicrobial activity of most AMPs results from their unique structural properties, which allow them to disrupt the microbial membrane without attacking human cell membranes. Furthermore, these peptides also act on host cells to regulate immune responses, cytokine production, cell migration, proliferation, maturation, and extracellular matrix synthesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis ability to maintain equilibrium plays a vital role in resisting pathogens while maintaining the stability of the immune system. If defects in the expression or processing of LL-37 break this balance, it will result in abnormalities in the body [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. LL-37 is also involved in the pathogenesis of multiple autoimmune and inflammatory diseases, such as psoriasis and discoid lupus, where its dysregulation is associated with the onset and progression of the disease [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe scarcity of studies in autoimmune bullous diseases was the rationale for the design of our study, which aimed to offer a concise general view of the expression of antimicrobial peptides LL-37 and briefly discuss the role of this small peptide as a key factor in the development of pemphigus.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Subjects\u003c/h2\u003e \u003cp\u003e All participants provided written informed consent for the study, which was approved by the Human Research Ethics Committee of the Habib Bourguiba University Hospital of Sfax (protocol number 4/12).\u003c/p\u003e \u003cp\u003eThe diagnosis of pemphigus (PF and PV) is confirmed by clinical presentation, histopathology and Immunological tests for circulatory anti-Dsg1 (cut-off value was set at 20 UI/mL) and/or Dsg3 Abs (cut-off value was set at 14 UI/mL). The first-line pemphigus treatment relies on systemic corticosteroids according to the recent recommendations of Murrell et al. Pemphigus management considered two main phases: remission induction and remission maintenance. During the follow-up assessment, disease activity of both the skin and mucosal surfaces was monitored using the pemphigus disease area index (PDAI), including different skin, scalp, and mucosal lesion scores. On the other hand, a high PDAI score and/or persistent high anti-Dsg1/Dsg3 Abs values define severe chronic pemphigus.\u003c/p\u003e \u003cp\u003eThe classification strategy followed the disease stage and treatment management (treated/untreated) as described by Shimizu et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, the patient group was divided into three groups: newly diagnosed untreated patients and treated patients with varying disease progression, divided into remitted patients\u0026rsquo; group (mild form of the disease) (PDAI\u0026thinsp;\u0026le;\u0026thinsp;8) and chronic patients\u0026rsquo; group (moderate to severe form of the disease) (PDAI\u0026thinsp;\u0026ge;\u0026thinsp;9).\u003c/p\u003e \u003cp\u003eBased on patient approval, six newly diagnosed patients were subjected to a follow-up at 3 months\u003c/p\u003e \u003cp\u003eHCs with no signs of autoimmune disorders were recruited as a control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Blood sampling and PBMC isolation\u003c/h2\u003e \u003cp\u003eFor the transcriptomic study, a total of 10 ml of peripheral blood were collected from newly diagnosed PV patients (n\u0026thinsp;=\u0026thinsp;4), treated PV patients (n\u0026thinsp;=\u0026thinsp;18), newly diagnosed PS patients (n\u0026thinsp;=\u0026thinsp;12), and treated PS patients (n\u0026thinsp;=\u0026thinsp;16) recruited at the Department of Dermatology in the Hedi Chaker University Hospital of Sfax, Tunisia, in addition to HC (n\u0026thinsp;=\u0026thinsp;16) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePBMCs separation was performed by low-density Ficoll-Hypaque gradient centrifugation, the leukocyte-enriched plasma was layered onto low-density Ficoll-Hypaque and centrifuged for 30 min. Then, their viability and purity were checked and evaluated by trypan and truck blue; respectively in light microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA isolation and Reverse transcription\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the Trizol suspension according to the manufacturer\u0026rsquo;s instructions. The RNA purity and integrity in each sample were assessed using a NanoDrop system (NanoDrop Technologies\u0026reg;, Wilmington, NC, USA) and using standard agarose gel electrophoresis. cDNA was generated using the PrimeScript RT Reagent Kit (TAKARA Bio Inc.\u0026reg;, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 LL-37 gene expression analysis\u003c/h2\u003e \u003cp\u003eRelative real-time quantitative RT-PCR of LL-37 was performed using Gene-specific primers (F: 5\u0026rsquo;-TCGGATGCTAACCTCTACCG-3\u0026rsquo;/R: 5\u0026rsquo;-GGGTACAAGATTCCGCAAAA-3\u0026rsquo;), by SYBR Green Dye detection system analysis. All reactions were performed in duplicate. For verification of the quality of PCR products, melting curves were generated. The relative quantification was performed using the standard curve method, and normalized to the average housekeeping gene \u003cem\u003eGAPDH\u003c/em\u003e (F: 5\u0026rsquo;-GCTCTCTGCTCCTCCTGTTC-3\u0026rsquo;/R: 5\u0026rsquo;-CGCCCAATACGACCAAATCC-3\u0026rsquo;). Data were analyzed by the comparative 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Serum concentrations of antimicrobial peptide cathelicidin LL-37\u003c/h2\u003e \u003cp\u003eSandwich ELISA was performed with a Human Antibacterial Protein LL-37 ELISA Kit from CUSABIO (CSB-EL004476HU). In brief, LL-37 standards and patient serum were added to the wells coated with antibodies specific for LL-37, and a biotin-conjugated reagent was added to the wells and incubated. Tetramethylbenzidine 6 substrate was used to quantify the HRP enzymatic reaction. Optical density (OD) was measured spectrophotometrically at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results were analyzed using SPSS software 2.0. The differences in expression between groups were analyzed using the Kruskal-Wallis nonparametric tests and the Mann-Whitney independent sample test. For the results analysis of the same patient at different time points, we used the non-parametric paired-sample tests, the Wilcoxon test. Spearman's test was used for correlation studies. Statistical significance was defined as a value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eLL-37 expression profiling was performed and compared in blood samples of pemphigus patients; among clinical disease groups: 20 PV (sex ratio M/F: 8/12,) and 27 PF (sex ratio M/F: 3/24), and 16 HCs. LL-37 gene, as well as serum expressions, were abnormally up-regulated in pemphigus patients compared to HC (Gene expression: Newly diagnosed groups \u003csub\u003emean\u003c/sub\u003e=0.106\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 and HC\u003csub\u003emean\u003c/sub\u003e=0.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, p\u0026thinsp;=\u0026thinsp;0.012, Serum level: Newly diagnosed groups \u003csub\u003emean\u003c/sub\u003e=10.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.006 ng/ml and HC\u003csub\u003emean\u003c/sub\u003e=6.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 ng/ml, p\u0026thinsp;=\u0026thinsp;0.048) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A-B, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn detail, LL-37 mRNA gene expression was significantly enhanced in newly diagnosed PV (n\u0026thinsp;=\u0026thinsp;4) and PF (n\u0026thinsp;=\u0026thinsp;11) compared to HCs; (PV\u003csub\u003emean\u003c/sub\u003e=0.324\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, PF\u003csub\u003emean\u003c/sub\u003e=0.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 and HC\u003csub\u003emean\u003c/sub\u003e=0.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, with p\u0026thinsp;=\u0026thinsp;0.046, p\u0026thinsp;=\u0026thinsp;0.036; respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A). There was no statistically significant difference in LL-37 gene expression levels between samples of PV and PF.\u003c/p\u003e \u003cp\u003eTo understand the impact of short-term corticosteroid therapy on LL-37 gene expression, six patients were followed after 3 months of treatment. Our data revealed that the LL-37 mRNA expression changed significantly after 3 months of treatment (Newly diagnosed patients \u003csub\u003emean\u003c/sub\u003e=0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 vs 3M treated \u003csub\u003emean\u003c/sub\u003e=0.408\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, p\u0026thinsp;=\u0026thinsp;0.023). A notable negative correlation was revealed when analyzing Dsg1-Abs level and LL-37 mRNA expression in the same group gathering newly diagnosed patients and short-term treated patients (3 months) (r=-0,615; p\u0026thinsp;=\u0026thinsp;0,025) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C). Interestingly, when focusing on patients concerned by follow-up, this relationship was revealed more pronounced with a higher correlation coefficient (r= -0,7; p\u0026thinsp;=\u0026thinsp;0,020) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.D).\u003c/p\u003e \u003cp\u003eBy stratifying PV and PF patients according to their clinical disease stage into chronic and remittent groups, LL-37 gene expression was followed up in a group of six-year average treatment period patients. A notable down-regulation of LL-37 expression was identified in a remittent group compared to the chronic group (remittent group\u003csub\u003emean\u003c/sub\u003e=9.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80 vs Chronic group\u003csub\u003emean\u003c/sub\u003e=14.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). A similar profile was observed when studying LL-37 serum expression, without any statistical significance (remittent group\u003csub\u003emean\u003c/sub\u003e=0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.077vs Chronic group\u003csub\u003emean\u003c/sub\u003e=0.172\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This significance was maintained even when studying the two separate classes of pemphigus (remittent PV\u003csub\u003emean\u003c/sub\u003e=0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011 vs Chronic PV\u003csub\u003emean\u003c/sub\u003e=0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.073, p\u0026thinsp;=\u0026thinsp;0.039 and remittent PF\u003csub\u003emean\u003c/sub\u003e=0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 vs chronic PF\u003csub\u003emean\u003c/sub\u003e=0.074\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034, p\u0026thinsp;=\u0026thinsp;0.028; respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E). There was also a decrease in LL-37 gene expression in remittent PF patients when compared to newly diagnosed ones, p\u0026thinsp;=\u0026thinsp;0.013.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCathelicidin LL-37 plays an important role in the early host response against invading pathogens via its antimicrobial activity. In addition, it is an important effector molecule of innate immunity in the skin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the present study; we examined the implication of LL-37 in the occurrence and the severity of intra-epidermal autoimmune bullous diseases. Overall, LL-37 gene expression and serum level profiles were differentially dysregulated in pemphigus.\u003c/p\u003e \u003cp\u003eNewly diagnosed pemphigus patients showed, whether in peripheral blood cells or serum, a significant upregulation of LL-37 compared to HCs. It seems that the over-expression of LL-37 is important in the disease onset. Yet, there is a lack of studies analyzing the LL-37 expression on a systemic level; particularly on PBMC. An up-regulation was largely reported in various biological specimens. Indeed, in the inflammatory skin disorder context, previous studies showed similar results in psoriatic cutaneous biopsies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and in cutaneous lupus lesions biopsies including discoid lupus erythematosus, subacute cutaneous lupus erythematosus, and lupus erythematosus tumidus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. On another hand, the salivary concentration of LL-37 was increased in inflammatory ulcerating diseases in both oral lichen planus and periodontitis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Data relating to cathelicidin LL-37 expression in diseases, particularly skin diseases are scarce. Taking together, cathelicidin expression dysregulation appears to occur in inflammatory and auto-immune disorders, as in the case of pemphigus; PF, and PV. However, the physiological roles and precise functions of LL-37 in pemphigus remain to be elucidated. Indeed, both pro- and anti-inflammatory functions have been assigned to LL-37 in a dependent manner on the microenvironment and disease background. Some studies have speculated that these AMPs may provide a protective effect from cutaneous infection in cutaneous lupus erythematosus patients [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, a pro-inflammatory phenotype of LL-37 on macrophages in SLE was also suggested [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePemphigus management is divided into two main phases; remission induction and remission maintenance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In active pemphigus, multiple blisters and erosions trigger the release of inflammatory cytokines IL-6 and IL-17 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], thus, the cornerstone of pemphigus treatment remains systemic corticosteroids due to their immunosuppressive and anti-inflammatory properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We, therefore, investigated the impact of corticosteroids on LL-37 gene expression change after 3 months of cortico-therapy treatment. The follow-up showed a significant up-regulation in LL-37 gene expression which was negatively correlated with anti-Dsg1 Abs in pemphigus-monitored patients.\u003c/p\u003e \u003cp\u003e Interestingly, when subdividing treated patients according to PDAI score, we noted the persistence of LL-37 expression levels in chronic pemphigus groups, compared to the remittent ones. Based on these findings, it seems logical to assume that corticosteroids impact the LL-37 expression according to the disease context. Previous research of Marin-Luevano and her collaborators indicated that the glucocorticoid; dehydroepiandrosterone, promotes the production of LL-37 and β-defensin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Corticosteroids play a critical role in remission induction\u003csup\u003e20\u003c/sup\u003e; their interaction with the cytoplasmic corticosteroid receptor, results in the up-regulation of anti-inflammatory proteins and downregulation of those pro-inflammatory [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, LL-37 up-regulation may contribute to suppressing the inflammatory responses and mediating tissue repair. Indeed, LL-37 possesses a critical role in modulating cytokine production and chemoattracting various immune effector cells leading to stimulation angiogenesis and wound healing [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It was shown that the topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Alongside our results, the up-regulation of LL-37 gene expression was suggested to be implicated in tissue repair in the recovery phase of septic [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is generally believed that the disease-inducing immune response is initiated at distant sites, followed by a migration of immune cells to the skin and autoantibody binding to Dsg3/Dsg1 of the epidermis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. So, the LL-37 systemic regulation could be tightly linked to the skin level.\u003c/p\u003e \u003cp\u003eOn the other hand, there was evidence that LL-37 can perform two distinct functions in different tissues and different microenvironments [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], this peptide has been shown to regulate monocyte/macrophage differentiation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Macrophages can thus exhibit pro- and anti-inflammatory properties to a degree that is determined by stimuli from their local microenvironment [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It is therefore necessary to examine the immune functions of LL-37 from both sides. Thus, we could suggest that cellular context in persistent chronic phases of the disease may stimulate the pro-inflammatory effect of LL-37. LL-37 exacerbated LPS-induced septic shock in rats when administered 2 hours after LPS treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This is in line with the hypothesis suggesting that at a high concentration or under specific conditions, LL-37 can aggravate host-damaging effects induced by inflammation. This take as to another speculation suggesting that the persistence of high levels of LL-37 gene expression in chronic pemphigus can aggravate damaging effects induced by inflammation. Taking together, we suggest that the timing and the cellular context change the expression of LL-37 mRNA according to the disease severity which determines the direction of the cellular response. On the one side, LL-37 seems to promote immune response and exerts its anti-inflammatory and wound healing effects for remission induction and persistence; on the other side, it seems to have the ability to stimulate inflammation and promote a pro-inflammatory response in the persistent chronic phase of pemphigus.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, the LL-37 gene expression profile was differentially dysregulated in pemphigus according to the disease status which allows us to suggest its contribution to the pathogenesis of the disease. The cellular environment and the timing appear to have an impact on LL-37 expression. Further functional studies on skin cell culture are needed for a more comprehensive understanding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Human Research Ethics Committee of the Habib Bourguiba University Hospital of Sfax (protocol number of the ethical committee, 4/12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ministry of High Education and Scientific Research of Tunisia supported this work. We would like to thank the patients and volunteers for their participation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contribution to the paper as follows: study conception and design: DF, EN, AO; data collection: DF, SK, BE, TS, TH, HH, AO; analysis and interpretation of results and draft manuscript preparation: DF, EN, AO. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available upon request from the corresponding author\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeshem YA, Gdalevich M, Ziv M, David M, Hodak E, Mimouni D. Opportunistic infections in patients with pemphigus. J Am Acad Dermatol. 2014 Aug;71(2):284-92. doi: 10.1016/j.jaad.2014.03.020. Epub 2014 May 6. PMID: 24815564. \u003c/li\u003e\n\u003cli\u003eMelchionda, V, Harman, K.E. Pemphigus vulgaris and pemphigus foliaceus: an overview of the clinical presentation, investigations and management. 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PMID: 23778112. \u003c/li\u003e\n\u003cli\u003ePopescu IA, Statescu L, Vata D, Porumb-Andrese E, Patrascu AI, Grajdeanu IA, Solovastru LG. Pemphigus vulgaris - approach and management. Exp Ther Med. 2019 Dec;18(6):5056-5060. doi: 10.3892/etm.2019.7964. Epub 2019 Aug 30. PMID: 31819769; PMCID: PMC6895778.\u003c/li\u003e\n\u003cli\u003eLee SH, Hong WJ, Kim SC. Analysis of Serum Cytokine Profile in Pemphigus. Ann Dermatol. 2017 Aug;29(4):438-445. doi: 10.5021/ad.2017.29.4.438. Epub 2017 Jun 21. PMID: 28761292; PMCID: PMC5500709.\u003c/li\u003e\n\u003cli\u003eKridin K. Emerging treatment options for the management of pemphigus vulgaris. Ther Clin Risk Manag. 2018 Apr 27;14:757-778. doi: 10.2147/TCRM.S142471. PMID: 29740210; PMCID: PMC5931200. \u003c/li\u003e\n\u003cli\u003eMarin-Luevano SP, Rodriguez-Carlos A, Jacobo-Delgado Y, Valdez-Miramontes C, Enciso-Moreno JA, Rivas-Santiago B. 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Wound healing activity of the human antimicrobial peptide LL37. Peptides. 2011 Jul;32(7):1469-76. doi: 10.1016/j.peptides.2011.06.005. Epub 2011 Jun 13. PMID: 21693141.\u003c/li\u003e\n\u003cli\u003eBarbeiro DF, Barbeiro HV, Zampieri FG, C\u0026eacute;sar Machado MC, Torggler Filho F, Gomes Cunha DM, Goulart AC, Velasco IT, Monteiro da Cruz Neto L, Possolo de Souza H, Pinheiro da Silva F. Cathelicidin LL-37 bloodstream surveillance is down regulated during septic shock. Microbes Infect. 2013 May;15(5):342-6. doi: 10.1016/j.micinf.2013.01.001. Epub 2013 Jan 14. PMID: 23328115.\u003c/li\u003e\n\u003cli\u003eYuan H, Zhou S, Liu Z, Cong W, Fei X, Zeng W, Zhu H, Xu R, Wang Y, Zheng J, Pan M. Pivotal Role of Lesional and Perilesional T/B Lymphocytes in Pemphigus Pathogenesis. J Invest Dermatol. 2017 Nov;137(11):2362-2370. doi: 10.1016/j.jid.2017.05.032. Epub 2017 Jun 22. PMID: 28647348.\u003c/li\u003e\n\u003cli\u003eCha HR, Lee JH, Hensel JA, Sawant AB, Davis BH, Lee CM, Deshane JS, Ponnazhagan S. Prostate cancer-derived cathelicidin-related antimicrobial peptide facilitates macrophage differentiation and polarization of immature myeloid progenitors to protumorigenic macrophages. Prostate. 2016 May;76(7):624-36. doi: 10.1002/pros.23155. Epub 2016 Feb 9. PMID: 26856684; PMCID: PMC5551898.\u003c/li\u003e\n\u003cli\u003eRasheed A, Rayner KJ. Macrophage Responses to Environmental Stimuli During Homeostasis and Disease. Endocr Rev. 2021 Jul 16;42(4):407-435. doi: 10.1210/endrev/bnab004. PMID: 33523133; PMCID: PMC8284619.\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":"antimicrobial peptides, Cathelicidin LL-37, immunobullous disorders, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-6254185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6254185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study aimed to investigate the expression of the antimicrobial peptide cathelicidin LL-37 and discuss its role as a critical factor in developing pemphigus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The expression of LL-37 mRNA was assessed in individuals with pemphigus foliaceous (PF) and pemphigus vulgaris (PV) at different stages of the disease, in comparison to healthy controls (HCs), using RT-PCR and ELISA. The LL-37 expression profile was differentially dysregulated in pemphigus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e a high LL-37 expression level was shown in newly diagnosed patients compared to healthy controls. The short-term LL-37 gene expression follow-up showed a significant up-regulation after three months of cortical therapy treatment and a strong negative correlation with anti-Dsg1 Abs in PF-monitored patients, suggesting that LL-37 could be implicated in remission induction. In line with this hypothesis, the long-term follow-up results showed a significant increase in LL-37 expression according to disease severity when devising treated patients in the retrospective study, according to their PDAI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our preliminary finding suggests that the timing and the cellular context change the expression of LL-37 according to the disease severity, which determines the direction of the cellular response. We propose this small peptide as a point of debate requiring deep investigations in pemphigus.\u003c/p\u003e","manuscriptTitle":"Investigating the Expression of LL-37 Antimicrobial Peptide in Autoimmune Bullous Disorders: Implications for Pemphigus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 09:21:48","doi":"10.21203/rs.3.rs-6254185/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":"8dceb086-a6f5-48af-adde-de6fd8fd452e","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-20T21:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-02 09:21:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6254185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6254185","identity":"rs-6254185","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}