Lactiplantibacillus plantarum lysate isolated from green tea leaves alleviates the effects of Malassezia restricta on primary human scalp cells

Lactiplantibacillus plantarum lysate isolated from green tea leaves alleviates the effects of Malassezia restricta on primary human scalp cells | 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 Lactiplantibacillus plantarum lysate isolated from green tea leaves alleviates the effects of Malassezia restricta on primary human scalp cells Kilsun Myoung, Seunghyun Shin, Suna Kim, Heung Soo Baek, Hyoung-June Kim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6132321/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Scalp seborrheic dermatitis (SSD) is characterized by excessive sebum production, flaking, and itching. This condition is associated with an imbalance in the scalp microbiome, particularly the dominance of Malassezia restricta (MR). Antifungal treatments for SSD often fail to address root causes and can lead to side effects, recurrence, and resistant strains with long-term use. This highlights the need for new, more effective solutions to manage the condition. In this study, we investigated whether the lysate of Lactiplantibacillus plantarum APsulloc 331261 (APsulloc), isolated from green tea leaves, and its lipoteichoic acid (LTA), a bacterial cell wall component, have the potential to serve as natural solutions for SSD. The lysate of APsulloc had no direct effect on MR growth or biofilm formation. However, the lysate improved gene expression of tight junctions and inflammatory cytokines, and upregulated differentiation marker proteins in heat-killed MR-treated scalp keratinocytes. On the other hand, heat-killed MR stimulates differentiation signaling in sebocytes and enhances the expression of lipogenesis-related proteins. APsulloc’s lysate alleviated these effects and inhibit lipid production by sebocytes caused by heat-killed MR. LTA from APsulloc was also found to reduce lipogenesis and the secretion of hair loss-related cytokines in human primary sebocytes that were induced by heat-killed MR. Furthermore, both the lysate and LTA protected outer root sheath cell viability against heat-killed MR-induced damage while promoting dermal papilla cell growth. These finding demonstrate the potential of APsulloc’s cell wall components as natural solutions for improving SSD in vitro . scalp seborrheic dermatitis scalp cells Malassezia restricta Lantiplantibacillus plantarum lipoteichoic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Scalp health is crucial for overall well-being, yet 1–3% of the population suffers from scalp seborrheic dermatitis (SSD), which is characterized by the appearance of red, flaking, and greasy areas on scalp [ 1 ]. While the exact pathogenesis of SSD is not fully understood, microbiome imbalance, particularly involving Malassezia restricta (MR), have been linked to its development [ 2 , 3 ]. Malassezia accounts for approximately 45% of scalp-resident microorganisms, making it the predominant fungal genus of the scalp microbiome [ 3 , 4 ]. MR has been implicated in skin barrier disruption and tissue toxicity by enzyme secretion. MR enzymes break down sebum, producing fatty acids that can trigger skin inflammation and disrupt the epidermal barrier [ 5 ]. Moreover, Malassezia causes oxidative stress, and negatively impact hair quality and growth [ 6 ]. Current SSD treatments typically include topical antifungal and anti-inflammatory agents, such as zinc pyrithione, climbazole, and ketoconazole [ 7 ]. However, these treatments provide temporary relief for some individuals, they fail to address the underlying causes of SSD. Long-term use of antimicrobial chemicals can lead to skin irritation, resistance strains, and potential side effects, especially in sensitive individuals. Therefore, researchers are exploring natural solutions for SSD, aiming to address its root cause and provide safer, more effective alternatives to conventional treatments. Lactic acid bacterial lysates, a mixture of substances from cell lysis, have beneficial effects on skin health. Topical application of Limosilactobacillus reuteri lysate reduces inflammatory responses induced by ultraviolet B radiation in ex vivo skin [ 8 ] and Lacticaseibacillus rhamnosus lysate enhances the skin barrier against the cytotoxic effects of surfactant [ 9 ]. Our previous studies also demonstrated that lysate of Lantiplantibacillus plantarum APsulloc 331261 (APsulloc) promotes keratinocyte differentiation and prevents Staphylococcus aureus -induced inflammatory cytokines from keratinocytes and reconstructed human epidermis [ 10 , 11 ]. Both live APsulloc and its culture supernatant inhibit the growth of five skin pathogenic bacteria, including MR [ 12 ]. However, there is still a lack of research on the efficacy and mechanisms of probiotic substances in relation to human scalp health. This research assessed the potential of APsulloc’s lysate as a natural solution for SSD through in vitro experiments using human scalp cells and MR. We also investigated a key component of bacterial cell wall, lipoteichoic acid (LTA), which accounts for more than 20% of the Lactobacillus cell mass [ 13 ]. Our experiments were designed to determine whether APsulloc lysate could directly the growth of MR and protect scalp keratinocytes and sebocytes against heat-killed MR. We also examined the effects of these components on outer root sheath (ORS) cells and dermal papilla (DP) cells, which are involved in hair loss and growth. Materials and Methods Preparation bacterial samples The APsulloc lysate was prepared as previously described [ 14 ]. L. plantarum APsulloc 331261 (deposit number: KCCM11179P) was grown in BD™ de Man Rogosa and Sharp broth (Difco™, Becton Dickinson, Sparks, MD, USA) at 35°C for 24 h, followed by subculturing. The bacterial cell was harvested, washed, and lysed under high pressure (> 1,500 bars, three times) and filtered through a 0.45-µm membrane filter (Hyundai Micro, Seoul, South Korea) to create the lysate. LTA was extracted by Kyung Hee University Skin Biotechnology Center (Suwon, South Korea). Bacterial cells were disrupted and mixed with an equal volume of n-butanol for 1 h, followed by centrifugation at 13,000 × g for 20 min to obtain the aqueous phase. The aqueous phase containing LTA was purified using hydrophobic interaction chromatography. Column fractions were collected after the phosphate assay and dialyzed. MR (American Type Culture Collection 96810) was cultured in anaerobically for 5 days at 35 ℃ on modified Leeming and Notman agar (MLNA). The cultured MR was scraped and suspended in PBS and then incubated at 80°C for 40 min to prevent further bacterial growth. The heat-killed MR, the lysate, and LTA of APsulloc were lyophilized and stored at -80°C until use. Cell culture Hair follicle cells were isolated from the scalp skin after obtaining approval from the Medical Ethical Committee at Dankook University Hospital (Cheonan, Korea; PRE20240815-001) following previously described methods [ 15 ]. Human scalp keratinocytes were separated by incubating the scalp tissue in 0.25% trypsin/EDTA solution at 37°C for 30 min to separate the epidermis and then incubated in 0.25% trypsin/EDTA solution for 10–15 min. The trypsin activity was neutralized by adding a medium containing fetal bovine serum (FBS). The keratinocytes were cultured in EpiLife medium (Gibco, Thermo Fisher Scientific, Inc.) at 37°C in 5% CO 2 . Sebocytes were purchased from Celprogen (Torrance, CA, USA) and cultured in Human Primary Sebocyte Complete Growth Medium (Celprogen). Each cell type was seeded and incubated for 24 h, after which heat-killed MR and either the lysate or LTA of APsulloc were added to the cell culture medium. ORS cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% FBS after removing the lower bulb region of the follicle and upper portion of the sebaceous gland. On the third day of culture, the medium was replaced with the EpiLife medium. Primary human DP cells were obtained from the Kyungpook National University (Daegu, Korea) and cultured in low-glucose DMEM (Clytia, England, SH30021.01) containing 10% FBS (Welgene, Korea, S001-01) and 1% antibiotic-antimycotic solution (Sigma-Aldrich, A5955). Growth and biofilm formation of MR MR was inoculated in MLNA medium without agar and cultured under anaerobic condition for 5 days at 35 ℃. The cultures were then diluted in fresh medium (1:100), and sub-cultured with or without varying concentrations of APsulloc lysate for an additional 3 days at 35 ℃. The growth of MR was measured by comparing the absorbance at 600 nm using a microplate reader. The biofilm formation was assessed using a 1% crystal violet solution (Sigma-Aldrich). MR grown in MLNA cultures were distributed into a 96-well plate at 100 µL per well, with the addition of APsulloc’s lysate at various concentrations, and then incubated for 3 more days. To remove planktonic bacteria, the culture medium was discarded, and the wells were washed with PBS. For staining the biofilm, 1% crystal violet solution was added to each well and allowed to react for 20 min. Crystal violet was removed, and the wells were washed with PBS, and then the stained biofilm was dissolved using ethanol for measuring the absorbance at 600 nm. The formation of biofilms was compared as a percentage. Quantitative real-time polymerase chain reaction (qPCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and 2 µg of RNA was reverse‑transcribed into complementary DNA using SuperScript® III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, CA, USA) according to the manufacturer's protocol. qPCR was performed using the ABI 7500HT Fast System (Applied Biosystems; Thermo Fisher Scientific) and the Taqman™ Universal PCR Master Mix (Applied Biosystems) to determine the expression of the following genes: filaggrin, occludin, interleukin (IL)-1a , and IL-1b . Ribosomal protein L13a ( RPL13A ) was selected as a reference gene to normalize cDNA levels. Relative differences in gene expression were calculated based on the threshold cycle values. Western blot Western blot Total intracellular proteins were extracted using 1X RIPA buffer (Cell signaling Technology, Danvers, MA, USA) with protease inhibitors. Protein concentration measured using Pierce BCA protein assay kit (ThermoFisher). Proteins were denatured, resolved on 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 3% BSA, membranes were probed with primary and HRP-linked secondary antibodies (Invitrogen). Evaluation of lipid production of human scalp sebocytes Lipid production by heat-killed MR was analyzed using staining with fluorescent BODIPY® (4,4-difluoro-3a,4adiaza-s-indacene) (Invitrogen). BODIPY diluted in PBS to final concentration of 2 µM and 10% paraformaldehyde fixed co-cultured cells were stained for 30 min, washed with PBS, and then covered with fresh PBS. Fluorescence was measured using a Synergy H2 Multi-Mod Microplate Reader (BioTek) with a 485-nm bandpass excitation filter and compared to the control. Cytokine array Proteins were collected from culture supernatants after treatment with heat-killed MR, lysate, and LTA. Proteins were blotted using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA). The blots were imaged using a chemiluminescent imager (Fusion FX7 imaging system; Peqlab, Erlangen, Germany). Densitometric analysis was performed using ImageJ (version 1.54d; March 30, 2023). Cell viability test of ORS and DP cells Human ORS cells were seeded at a density of 2 × 10 4 cells/well in 96-well plates. After 24 h of incubation, heat-killed MR (10 µg/mL) were added or the same volume of PBS as a control. Simultaneously, lysate or LTA of APsulloc (10 µg/mL) was also added to heat-killed MR. Additionally, in the ORS cells, three hair loss-inducing factors (33 µM Hydrogen Peroxide [H 2 O 2 ], 33 ng/mL Dickkopf-related protein [DKK-1], and 33 ng/mL Transforming Growth Factor Beta 2 [TGFβ2]) were administered in combination to induce cell death. Cell viability was assessed 24 h after treatment using a Cell Counting kit-8 (Dojindo, Kumamoto, Japan). DP cells were seeded in 24-well plates at a density of 3.5 × 10 4 cells/mL. The cells were treated with 5 or 10 µg/mL of APsulloc’s lysate or LTA for 72 h, following which cell viability was also determined using the Cell Counting kit-8 assay. Statistical analysis Data are presented as mean ± standard deviation (SD). Comparisons among three or more groups were performed using one-way ANOVA, followed by Tukey's honest significant difference test. Comparisons between the two groups were performed using a paired two-tailed Student's t-test. The results were analyzed using SPSS for Windows (version 22.0; IBM Corp.). p < 0.05 was considered to indicate a statistically significant. Results Evaluation of APsulloc lysate on growth and biofilm formation of MR We examined whether the APsulloc lysate directly affected the growth or biofilm formation of MR. As shown in Fig. 1 a, the lysate did not affect the growth of MR. We also confirmed whether the APsulloc lysate influences biofilm formation. As Fig. 1 b illustrates, the lysate did not affect biofilm formation. These results suggest that the APsulloc lysate does not have a direct impact on MR. Protective effects of the APsulloc lysate against heat-killed MR on scalp keratinocytes The scalp keratinocytes were treated with heat-killed MR (10 µg/mL) in the presence or absence of the APsulloc lysate for 24 h. When subjected to treat heat-killed MR, substantial reduction of the filaggrin gene expression by a factor of 0.35-fold (± 0.04, p < 0.001) was observed, whereas the addition of APsulloc lysate resulted in significant improvement of 0.55 ± 0.05 (1 µg/mL, p < 0.01) and 1.01 ± 0.27 (5 µg/mL, p < 0.05) (Fig. 2 a). Similarly, occludin gene expression was suppressed by 0.22-fold (± 0.04, p 0.05) at 1 µg/mL and 0.60-fold (± 0.17, p < 0.05) at 5 µg/mL of APsulloc lysate. When treated with heat-killed MR alone, IL-1a and IL-1b mRNA levels were 1.95 ± 0.29 and 4.91 ± 0.56-fold that of the control, respectively (Fig. 2 b). Administering the APsulloc lysate at a concentration of 10 µg/mL effectively attenuated the inflammatory cytokine expression (IL-1α; 1.45 ± 0.02, IL-1β; 3.51 ± 0.19, fold of control) compared with that in the heat-killed MR-only treated group. Heat-killed MR decreased differentiation-related proteins KRT 1 and 10 in scalp keratinocytes, while APsulloc lysate alone had no effect. However, when combined, the lysate mitigated the MR-induced changes in these differentiation markers. (Fig. 2 c). Lysate and lipoteichoic acid of APsulloc modulates lipogenesis in sebocytes induced by heat-killed MR APsulloc lysate protected scalp keratinocytes from a pathogen. We then studied its effects on sebocytes, key cells in SSD symptoms. Treatment with 50 µg/mL of heat-killed MR significantly increased cytoplasmic lipid accumulation, as evidenced by a 278.4 ± 32.5% ( p < 0.001) enhancement in fluorescence intensity of lipid droplets compared to the control group (Fig, 3a, b). Simultaneous treatment with APsulloc’s cell wall components and heat-killed MR resulted in a marked reduction of lipid production in sebocytes. The fluorescence analysis showed clear inhibitory effects of the lysate and LTA (196.4 ± 16.3% with 10 µg/mL of lysate; 147.9 ± 13.3% with 10 µg/mL of LTA compared with the % in the control). Lipid production-related protein expression also showed the same results (Fig. 3 c). Heat-killed MR upregulated lipogenesis protein expression, whereas APsulloc lysate alone showed similar like control. Importantly, combined treatment with APsulloc lysate effectively attenuated heat-killed MR-induced protein expression changes. We used a cytokine antibody array to assess the effects of heat-killed MR and the APsulloc lysate. Heat-killed MR induced the secretion of angiopoietin-2 (ANGPT2) and dickkopf-1 (DDK-1) compared with those in the control group (Fig. 4 ). Although we expected an increase in IL-1α and IL-1β as in scalp keratinocytes, this was not the case. When APsulloc lysate or LTA was administered along with heat-killed MR, the elevated levels of ANGPT2 and DDK-1 induced by heat-killed MR were reduced. The protective effect of APsulloc lysate and LTA on ORS cells against heat-killed MR and promoting DP cell viability When the ORS cells were treated with 50 µg/mL of heat-killed MR and hair loss-inducing factors (H 2 O 2 , DKK-1, and TGFβ2), cell viability was significantly decreased by 48.3 ± 1.6% (Fig. 5 a, p < 0.001). However, combined treatment with 10 µg/mL of APsulloc lysate improved the cell viability by 68.0 ± 3.2% and 10 µg/mL of LTA of APsulloc improved the cell viability by 66.4 ± 9.2% compared with that of control. We also conducted experiments to determine whether the lysate and LTA from APsulloc could improve the viability of DP cells. As shown in Fig. 5 b, both lysate and LTA at concentrations of 5 or 10 µg/mL induced proliferation of DP cells (112.3 ± 7.4 and 119.8 ± 6.3% with 5 and 10 µg/mL of lysate, respectively; 116.1 ± 9.2% and 120.7 ± 2.9% with 5 and 10 µg/mL of LTA, respectively). Our results revealed that APsulloc lysate and LTA can protect ORS cells from apoptosis and increase DP cell viability, indicating that these components likely contribute to hair growth. Discussion We investigated the effects of MR, a major cause of SSD, on human scalp cells and evaluated the potential of APsulloc lysate and its cell wall component LTA, as natural solution for SSD management. Heat-killed MR significantly impacted scalp cells, suppressing tight junction and differentiation markers in keratinocytes while upregulating inflammatory cytokines. In sebocytes, MR increased lipid production and accumulation, and elevated expression of hair growth inhibitors. APsulloc lysate and LTA mitigated these MR-induced changes in gene and protein expression, suggesting their potential in managing SSD symptoms through multiple cellular mechanisms. Unlike live probiotics that directly affect microorganisms, bacterial lysates and cell wall components like LTA work indirectly by interacting with host cells. This approach also differs from traditional antimicrobial treatments, potentially enhancing natural defenses against pathogens. LTA has gained research attention for its role in immune regulation, acting as a ligand for TLR2 on human cells and triggering immune responses [ 16 , 17 ]. The structural diversity of LTA is believed to considerably influence its immunomodulatory activity, contributing to its varied biological interactions. In the human epidermis, TLR2 is expressed in key cell types, such as keratinocytes and sebocytes [ 18 ]. TLR2 is also involved in recognizing the components of Malassezia species, inducing a pro-inflammatory response characterized by the release of various cytokines, chemokines, and antimicrobial peptides by keratinocytes [ 19 ]. Previous studies have shown that Malassezia furfur -infected keratinocytes exhibit upregulation of TLR2 , human beta-defensin ( HBD ) 2, and IL-8 gene expression [ 20 ]. Furthermore, hair follicle stem cell TLR2 is essential for maintaining hair homeostasis and regeneration. TLR2 endogenous ligands, such as carboxyethylpyrrole, a metabolite of polyunsaturated fatty acids, promote hair growth [ 21 ]. ORS cells, crucial for hair growth, are predicted to protect against hair loss when their survival is maintained. Sebaceous glands are also known to affect hair growth, with their normal development and function being important for correct hair development and cycling [ 22 ]. DDK-1 can inhibit the growth of ORS cells and trigger apoptotic cell death, and ANGPT2 exhibits estradiol activity to improve hair loss in ovariectomized mice [ 23 , 24 ]. Interestingly, the lysate and LTA from APsulloc reduced the expression of DDK-1 and ANGPT2 that were increased by heat-killed MR in sebocytes and inhibited ORS cell death. We can hypothesize that LTA form APsulloc competitively binds to TLR with the cellular components of MR, thereby modulating the immune response. This hypothesis not only highlights the potential of APsulloc-derived LTA in therapeutic applications but also underscores the complexity of host-pathogen interactions at the molecular level. However, the use of heat-killed MR may not fully represent the complex living scalp microbiome. In an actual scalp environment, MR survived with other microorganisms in a balanced state. And managing live microorganisms or the scalp microbiome with cosmetic or personal care products remains challenging. Further study is needed to confirm whether the immune modulation occurs through competitive binding to TLR2, and the implications of ORS and DP cells viability on hair loss inhibition should be re-examined. Most of all, clinical evidence is required to demonstrate the changes in MR within the complex scalp ecosystem due to treatment with lysate and LTA. These comprehensive studies will significantly contribute to the development of targeted scalp improvement methods in the future and help find natural approaches to potentially reduced hair loss associated with SSD. Conclusions We evaluated the effects of APsulloc lysate and its cell wall component, LTA, on human scalp cells. Although APsulloc lysate could not directly prevent the growth or biofilm formation of MR, the lysate and LTA of APsulloc had a significant indirect protective effect against heat-killed pathogens. These effects are mediated by the modulation of gene expression and the alteration of cytokine release in human scalp cells. Our findings support the potential of natural substances to enhance scalp health and propose new strategies for managing SSD. Declarations Impact statement While recent studies have been published on the effects of lactic acid bacteria lysate on skin health, it is difficult to find content related to scalp health. It is even more challenging to determine which components of the lysate are responsible for these effects. In this study, we examined the impact of lactic acid bacteria lysate and LTA on scalp primary cells. Although more research is needed to managing SSD, we can say that we have found initial clues. Ethics statement All authors declare that they have no conflict of interests. Neither ethical approval nor informed consent was required for this study. Funding statement The authors received no financial support for the research, authorship, and/or publication of this articles. References Clark GW, Pope SM, Jaboori KA (2015) Diagnosis and treatment of seborrheic dermatitis. Am Fam Physician 91(3): 185-190. Tao R, Li R, Wang R (2021) Skin microbiome alterations in seborrheic dermatitis and dandruff: A systematic review. Exp Dermatol (10):1546-1553. https://doi.org/10.1111/exd.14450 Park T, Kim HJ, Myeong NR et al. (2017) Collapse of human scalp microbiome network in dandruff and seborrhoeic dermatitis. Exp Dermatol (9):835-838. https://doi.org/10.1111/exd.1329 Grimshaw SG, Smith AM, Arnold DS et al. (2019) The diversity and abundance of fungi and bacteria on the healthy and dandruff affected human scalp. PLoS One;14(12): e0225796. https://doi.org/10.1371/journal.pone.0225796. Park M, Park S, Jung WH (2021) Skin commensal fungus Malassezia and its lipases. J Microbiol Biotechnol.31(5):637–644. https://doi.org/10.4014/jmb.2012.12048 Trueb RM, Henry JP, Davis MG et al . (2018) Scalp condition impacts hair growth and retention via oxidative stress. Int J Trichology 10(6): 262-270. https://doi.org/10.4103/ijt.ijt_57_18 Borda LJ, Wikramanayake TC (2015) Seborrheic Dermatitis and Dandruff: A Comprehensive Review. J Clin Investig Dermatol. 3(2). https://doi.org/10.13188/2373-1044.1000019 Khmaladze I, Butler E. Fabre S et al. (2019) Lactobacillus reuteri DSM 17938-A comparative study on the effect of probiotics and lysates on human skin. Exp Dermatol. 28(7): 822-828. https://doi.org/ 10.1111/exd.13950 Jung YO, Jeong H, Cho Y et al . (2019) Lysates of a probiotic, Lactobacillus rhamnosus , can improve skin barrier function in a reconstructed human epidermal model. Int J Mol Sci. (17): 4289. https://doi.org/10.3390/ijms20174289. Myoung K, Choi EJ, Kim H et al . (2021) Fermented lysate of Lactiplantibacillus isolated from green tea leaves protects keratinocytes against stress hormone and Staphylococcus aureus . China Detergent & Cosmetics 6(3): 54-60. Myoung K, Choi EJ, Kim S et al . (2024) Nano-sized lysate of Lactiplantibacillus plantarum isolated green tea leaves as a potential skin care ingredient. Biotech Bioprocess Engineering. 29: 1071-1080. https://doi.org/ 10.1007/s12257-024-00132-3 Chae M, Kim BJ, Na J et al . (2021) Antimicrobial activity of Lactiplantibacillus plantarum APsulloc 331261 and APsulloc 331266 against pathogenic skin microbiota. Front Biosci (Elite Ed) 13(2):237–248. https://doi.org/10.52586/E881 Lebeer S, Vanderleyden J, Keersmaecker SCJ (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72(4): 728-764. https://doi.org/10.1128/MMBR.00017-08 Kim W, Lee EJ, Bae IH et al . (2020) Lactobacillus plantarum -derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro. J Extracell Vesicles 9(1):1793514. https://doi.org/10.1080/20013078.2020.1793514 Choi H, Lee Y, Shin SH et al . (2022) Induction of hair growth in hair follicle cells and organ cultures upon treatment with 30 kHz frequency inaudible sound via cell proliferation and antiapoptotic effects. Biomed Rep 16(3):16. https://doi.org/10.3892/br.2022.1499. Triantafilou M, Manukyan M, Mackie A et al . (2004) Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi and lipid raft-dependent. J Biol Chem 279(39): 40882-40889. https://doi.org/10.1074/jbc.M400466200 Saito S, Okuno A, Cao DY et al . (2020) Bacterial lipoteichoic acid attenuates toll-like receptor dependent dendritic cells activation and inflammatory response. Pathogens 9(10). 825. https://doi.org/10.3390/pathogens9100825 Hari A, Flach T, Shi Y et al. (2010) Toll-like receptors: role in dermatological disease. Mediators Inflamm: 437246 https://doi.org/10.1155/2010/437246 Sparber F, LeibundGut-Landmann S (2017) Host responses to Malassezia spp. In the mammalian skin. Front Immunol 8: 1614. https://doi.org/10.3389/fimmu.2017.01614 Baroni A, Orlando M, Donnarumma G et al . (2006) Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur . Arch Dermatol Res 297(7): 280-288. https://doi.org/10.1007/s00403-005-0594-4 Xiong L, Zhevlakova I, West XZ et al. (2023) TLR2 regulates hair follicle cycle and regeneration via BMP signaling. bioRxiv [Preprint]. https://doi.org/10.1101/2023.08.14.553236 Lee WJ, Cha HW, Lim HJ et al . (2012) The effect of sebocytes cultured from nevus sebaceus on hair growth. Exp Dermatol 21(10): 796-798. https://doi.org/10.1111/j.1600-0625.2012.01572.x Kwack M, Sung Y, Chung E et al . (2008) Dihydrotestosteron-inducible Dickkopf 1 from balding dermal papilla cells causes apoptosis in follicular keratinocytes. J Invest Dermatol 128(2): 262-269. https://doi.org/10.1038/sj.jid.5700999 Endo Y, Obayashi Y, Ono T et al . (2018) Reversal of the hair loss phenotype by modulating the estradiol-ANGPT2 axis in the mouse model of female pattern hair loss. J Dermatol Sci 91(1): 43-51 (2018). https://doi.org/10.1016/j.jdermsci.2018.04.001 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-6132321","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":423817811,"identity":"67762ea2-7d86-4dbc-8a07-b1b429cd5997","order_by":0,"name":"Kilsun Myoung","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Kilsun","middleName":"","lastName":"Myoung","suffix":""},{"id":423817812,"identity":"771e75ff-6482-410e-8629-ece66c6c791d","order_by":1,"name":"Seunghyun Shin","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Seunghyun","middleName":"","lastName":"Shin","suffix":""},{"id":423817813,"identity":"c484dcef-fa36-4691-bac1-f36f92df382e","order_by":2,"name":"Suna Kim","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Suna","middleName":"","lastName":"Kim","suffix":""},{"id":423817814,"identity":"60daf44c-8c72-45b1-a5cc-656b1a8d1266","order_by":3,"name":"Heung Soo Baek","email":"","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":false,"prefix":"","firstName":"Heung","middleName":"Soo","lastName":"Baek","suffix":""},{"id":423817815,"identity":"c03374d7-8c3c-448a-994a-dd3b14fcd28c","order_by":4,"name":"Hyoung-June Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACPiA2AGI5uAgb88EGvFrYoFqMESJsiYS1gACSMrYE/A5jY28+UMy7wya9f9rhYx8+7rCT52NjbvzAUGMTjVMLz7EEY94zabkzbqclz5x5JtmwjY2xWYLhWFouLuexSeQYGPO2Hc5tuJ1jzMzbxpzAJt/YxsDYcBi3Fvk3IC3/0+VBWv621SewsTES0CLBA9JyIMEApIWx7TARWnjSEgzntiUbbgT6hbH3zHGIXxLw+IWf/fAxg7dtdvJyt5MPM/zcUS0v38b+8MOHGhucWkAWGcCZjDBlCbiVgwDzA0wto2AUjIJRMAqQAACjWVECOt4D+QAAAABJRU5ErkJggg==","orcid":"","institution":"Amorepacific (South Korea)","correspondingAuthor":true,"prefix":"","firstName":"Hyoung-June","middleName":"","lastName":"Kim","suffix":""},{"id":423817816,"identity":"01c8ebfc-b0da-4dd5-95b0-4df2a3ee16fe","order_by":5,"name":"Jae Sung Hwang","email":"","orcid":"","institution":"Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Sung","lastName":"Hwang","suffix":""}],"badges":[],"createdAt":"2025-03-01 04:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6132321/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6132321/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77906513,"identity":"c27eb052-e2d5-4837-bb01-34763f9a7874","added_by":"auto","created_at":"2025-03-06 16:39:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217911,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the effect of APsulloc lysate on \u003cem\u003eM. restricta. M. restricta\u003c/em\u003e was incubated with the lysate of APsulloc at concentrations of 1, 5, and 10 μg/mL. After incubation at 35 ℃ for 72 h, (a) bacterial growth were measured an optical density at 600 nm and (b) biofilm formation was assessed by crystal violet staining. All data represent the mean ± standard deviation (SD) of at least three sample experiments.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/2dc4b029f096bfa0751f03cd.png"},{"id":77905708,"identity":"536e8d4f-8319-49b4-b327-c012debae2fe","added_by":"auto","created_at":"2025-03-06 16:31:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2326270,"visible":true,"origin":"","legend":"\u003cp\u003eThe protective effect of APsulloc lysate on human scalp hair follicle keratinocytes against heat-killed \u003cem\u003eM. restricta\u003c/em\u003e (MR)\u003cem\u003e.\u003c/em\u003e The scalp keratinocytes were incubated with heat-killed MR or the APsulloc lysate. After incubation at 37 ℃ for 24 h, (a) the gene expression of barrier function of keratinocytes and (b) inflammatory cytokines were measured. (c) APsulloc lysate alleviated heat-killed MR-induced changes in differentiation protein expression of scalp keratinocytes (KRT 1; keratin 1, KRT 10; keratin 10, GAPDH; Glyceraldehyde-3-phophate-dehydroganase). All data represent the mean ± SD of at least triplicate sample experiments. \u003csup\u003e##\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e###\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus control group; \u003csup\u003e*\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus heat-killed MR-only treated group.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/8030f995dc8d2d8d6b159edc.png"},{"id":77905703,"identity":"aee00527-d743-44ac-9dbf-ebe39dbf7c37","added_by":"auto","created_at":"2025-03-06 16:31:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4025038,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of lysate and LTA on lipogenesis induced by heat-killed MR of human sebocytes. The cells were treated with heat-killed MR and following treatment of lysate or LTA of APsulloc. (a) Microscopic images acquired BODIPY fluorescence analysis. Original magnification, x100. (b) The lipid production of sebocytes were analyzed using fluorescence intensity. (c) Lipogenesis protein expressions were also altered by heat-killed MS and improved by the lysate (SREBP-1; sterol regulatory element-binding protein 1, PPAR-γ; peroxisome proliferator-activated receptor γ, LXR-α; liver X receptor-α, FAS; fatty acid synthase, S100p; S100 calcium-binding protein P, GAPDH; Glyceraldehyde-3-phophate-dehydroganase). All data represent the means ± SD of at least triplicate assays expressed as percentages of the control. \u003csup\u003e###\u003c/sup\u003e \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 compared to the control. \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005, \u003csup\u003e***\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to the heat-killed MR-only treated group. LTA, lipoteichoic acid; MR, \u003cem\u003eM. restricta\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/d84ca52392fdca37fa2ca31a.png"},{"id":77905705,"identity":"90f1a73b-b7a5-467a-9781-f2fa062c2c0e","added_by":"auto","created_at":"2025-03-06 16:31:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":521755,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine secretion pattern of sebocytes assessed in the culture supernatants after treatment with heat-killed MR. Supernatant proteins were then blotted using a Human Cytokine Antibody Array. The blots were captured via chemiluminescent imager. LTA, lipoteichoic acid; MR, \u003cem\u003eM. restricta\u003c/em\u003e; DDK-1, dickkopf-1; IL, interleukin\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/0757e573122464797d0cec3c.png"},{"id":77905700,"identity":"e5634cf8-79e9-49ad-a385-81bfba9cecd0","added_by":"auto","created_at":"2025-03-06 16:31:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303198,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of lysate and LTA of APsulloc on human outer root sheet cell and dermal papilla cell viability. (a) The cells were treated with heat-killed MR with/without lysate or LTA overnight. The cell viability reduced by heat-killed MR and improved by lysate or LTA of APsulloc. (b) The lysate or LTA increased the DP cell viability. Values are presented as mean ± SD of four independent experiments. \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005, \u003csup\u003e***\u003c/sup\u003e \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 versus heat-killed MR-only treated group; \u003csup\u003e###\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 versus control group. LTA, lipoteichoic acid; MR, \u003cem\u003eM. restricta\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/f0ebab2fb770bcf5a501b215.png"},{"id":77906806,"identity":"c63c4a04-f072-4866-9de7-2d31e847115f","added_by":"auto","created_at":"2025-03-06 16:47:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6600073,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6132321/v1/206e53c4-bf95-4bc1-8f94-2c9361fd59bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactiplantibacillus plantarum lysate isolated from green tea leaves alleviates the effects of Malassezia restricta on primary human scalp cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eScalp health is crucial for overall well-being, yet 1\u0026ndash;3% of the population suffers from scalp seborrheic dermatitis (SSD), which is characterized by the appearance of red, flaking, and greasy areas on scalp [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While the exact pathogenesis of SSD is not fully understood, microbiome imbalance, particularly involving \u003cem\u003eMalassezia restricta\u003c/em\u003e (MR), have been linked to its development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eMalassezia\u003c/em\u003e accounts for approximately 45% of scalp-resident microorganisms, making it the predominant fungal genus of the scalp microbiome [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. MR has been implicated in skin barrier disruption and tissue toxicity by enzyme secretion. MR enzymes break down sebum, producing fatty acids that can trigger skin inflammation and disrupt the epidermal barrier [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, \u003cem\u003eMalassezia\u003c/em\u003e causes oxidative stress, and negatively impact hair quality and growth [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Current SSD treatments typically include topical antifungal and anti-inflammatory agents, such as zinc pyrithione, climbazole, and ketoconazole [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, these treatments provide temporary relief for some individuals, they fail to address the underlying causes of SSD. Long-term use of antimicrobial chemicals can lead to skin irritation, resistance strains, and potential side effects, especially in sensitive individuals. Therefore, researchers are exploring natural solutions for SSD, aiming to address its root cause and provide safer, more effective alternatives to conventional treatments.\u003c/p\u003e \u003cp\u003eLactic acid bacterial lysates, a mixture of substances from cell lysis, have beneficial effects on skin health. Topical application of \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e lysate reduces inflammatory responses induced by ultraviolet B radiation in \u003cem\u003eex vivo\u003c/em\u003e skin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e lysate enhances the skin barrier against the cytotoxic effects of surfactant [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our previous studies also demonstrated that lysate of \u003cem\u003eLantiplantibacillus plantarum\u003c/em\u003e APsulloc 331261 (APsulloc) promotes keratinocyte differentiation and prevents \u003cem\u003eStaphylococcus aureus\u003c/em\u003e-induced inflammatory cytokines from keratinocytes and reconstructed human epidermis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Both live APsulloc and its culture supernatant inhibit the growth of five skin pathogenic bacteria, including MR [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, there is still a lack of research on the efficacy and mechanisms of probiotic substances in relation to human scalp health.\u003c/p\u003e \u003cp\u003eThis research assessed the potential of APsulloc\u0026rsquo;s lysate as a natural solution for SSD through \u003cem\u003ein vitro\u003c/em\u003e experiments using human scalp cells and MR. We also investigated a key component of bacterial cell wall, lipoteichoic acid (LTA), which accounts for more than 20% of the \u003cem\u003eLactobacillus\u003c/em\u003e cell mass [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our experiments were designed to determine whether APsulloc lysate could directly the growth of MR and protect scalp keratinocytes and sebocytes against heat-killed MR. We also examined the effects of these components on outer root sheath (ORS) cells and dermal papilla (DP) cells, which are involved in hair loss and growth.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation bacterial samples\u003c/h2\u003e \u003cp\u003eThe APsulloc lysate was prepared as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e APsulloc 331261 (deposit number: KCCM11179P) was grown in BD\u0026trade; de Man Rogosa and Sharp broth (Difco\u0026trade;, Becton Dickinson, Sparks, MD, USA) at 35\u0026deg;C for 24 h, followed by subculturing. The bacterial cell was harvested, washed, and lysed under high pressure (\u0026gt;\u0026thinsp;1,500 bars, three times) and filtered through a 0.45-\u0026micro;m membrane filter (Hyundai Micro, Seoul, South Korea) to create the lysate. LTA was extracted by Kyung Hee University Skin Biotechnology Center (Suwon, South Korea). Bacterial cells were disrupted and mixed with an equal volume of n-butanol for 1 h, followed by centrifugation at 13,000 \u0026times; g for 20 min to obtain the aqueous phase. The aqueous phase containing LTA was purified using hydrophobic interaction chromatography. Column fractions were collected after the phosphate assay and dialyzed. MR (American Type Culture Collection 96810) was cultured in anaerobically for 5 days at 35 ℃ on modified Leeming and Notman agar (MLNA). The cultured MR was scraped and suspended in PBS and then incubated at 80\u0026deg;C for 40 min to prevent further bacterial growth. The heat-killed MR, the lysate, and LTA of APsulloc were lyophilized and stored at -80\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHair follicle cells were isolated from the scalp skin after obtaining approval from the Medical Ethical Committee at Dankook University Hospital (Cheonan, Korea; PRE20240815-001) following previously described methods [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Human scalp keratinocytes were separated by incubating the scalp tissue in 0.25% trypsin/EDTA solution at 37\u0026deg;C for 30 min to separate the epidermis and then incubated in 0.25% trypsin/EDTA solution for 10\u0026ndash;15 min. The trypsin activity was neutralized by adding a medium containing fetal bovine serum (FBS). The keratinocytes were cultured in EpiLife medium (Gibco, Thermo Fisher Scientific, Inc.) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Sebocytes were purchased from Celprogen (Torrance, CA, USA) and cultured in Human Primary Sebocyte Complete Growth Medium (Celprogen). Each cell type was seeded and incubated for 24 h, after which heat-killed MR and either the lysate or LTA of APsulloc were added to the cell culture medium. ORS cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% FBS after removing the lower bulb region of the follicle and upper portion of the sebaceous gland. On the third day of culture, the medium was replaced with the EpiLife medium. Primary human DP cells were obtained from the Kyungpook National University (Daegu, Korea) and cultured in low-glucose DMEM (Clytia, England, SH30021.01) containing 10% FBS (Welgene, Korea, S001-01) and 1% antibiotic-antimycotic solution (Sigma-Aldrich, A5955).\u003c/p\u003e\n\u003ch3\u003eGrowth and biofilm formation of MR\u003c/h3\u003e\n\u003cp\u003eMR was inoculated in MLNA medium without agar and cultured under anaerobic condition for 5 days at 35 ℃. The cultures were then diluted in fresh medium (1:100), and sub-cultured with or without varying concentrations of APsulloc lysate for an additional 3 days at 35 ℃. The growth of MR was measured by comparing the absorbance at 600 nm using a microplate reader. The biofilm formation was assessed using a 1% crystal violet solution (Sigma-Aldrich). MR grown in MLNA cultures were distributed into a 96-well plate at 100 \u0026micro;L per well, with the addition of APsulloc\u0026rsquo;s lysate at various concentrations, and then incubated for 3 more days. To remove planktonic bacteria, the culture medium was discarded, and the wells were washed with PBS. For staining the biofilm, 1% crystal violet solution was added to each well and allowed to react for 20 min. Crystal violet was removed, and the wells were washed with PBS, and then the stained biofilm was dissolved using ethanol for measuring the absorbance at 600 nm. The formation of biofilms was compared as a percentage.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction (qPCR)\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was isolated using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and 2 \u0026micro;g of RNA was reverse‑transcribed into complementary DNA using SuperScript\u0026reg; III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, CA, USA) according to the manufacturer's protocol. qPCR was performed using the ABI 7500HT Fast System (Applied Biosystems; Thermo Fisher Scientific) and the Taqman\u0026trade; Universal PCR Master Mix (Applied Biosystems) to determine the expression of the following genes: filaggrin, occludin, interleukin \u003cem\u003e(IL)-1a\u003c/em\u003e, and \u003cem\u003eIL-1b\u003c/em\u003e. Ribosomal protein L13a (\u003cem\u003eRPL13A\u003c/em\u003e) was selected as a reference gene to normalize cDNA levels. Relative differences in gene expression were calculated based on the threshold cycle values.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eTotal intracellular proteins were extracted using 1X RIPA buffer (Cell signaling Technology, Danvers, MA, USA) with protease inhibitors. Protein concentration measured using Pierce BCA protein assay kit (ThermoFisher). Proteins were denatured, resolved on 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 3% BSA, membranes were probed with primary and HRP-linked secondary antibodies (Invitrogen).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of lipid production of human scalp sebocytes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLipid production by heat-killed MR was analyzed using staining with fluorescent BODIPY\u0026reg; (4,4-difluoro-3a,4adiaza-s-indacene) (Invitrogen). BODIPY diluted in PBS to final concentration of 2 \u0026micro;M and 10% paraformaldehyde fixed co-cultured cells were stained for 30 min, washed with PBS, and then covered with fresh PBS. Fluorescence was measured using a Synergy H2 Multi-Mod Microplate Reader (BioTek) with a 485-nm bandpass excitation filter and compared to the control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytokine array\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProteins were collected from culture supernatants after treatment with heat-killed MR, lysate, and LTA. Proteins were blotted using the Proteome Profiler Human XL Cytokine Array Kit (R\u0026amp;D Systems, Minneapolis, MN, USA). The blots were imaged using a chemiluminescent imager (Fusion FX7 imaging system; Peqlab, Erlangen, Germany). Densitometric analysis was performed using ImageJ (version 1.54d; March 30, 2023).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell viability test of ORS and DP cells\u003c/h3\u003e\n\u003cp\u003eHuman ORS cells were seeded at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plates. After 24 h of incubation, heat-killed MR (10 \u0026micro;g/mL) were added or the same volume of PBS as a control. Simultaneously, lysate or LTA of APsulloc (10 \u0026micro;g/mL) was also added to heat-killed MR. Additionally, in the ORS cells, three hair loss-inducing factors (33 \u0026micro;M Hydrogen Peroxide [H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e], 33 ng/mL Dickkopf-related protein [DKK-1], and 33 ng/mL Transforming Growth Factor Beta 2 [TGFβ2]) were administered in combination to induce cell death. Cell viability was assessed 24 h after treatment using a Cell Counting kit-8 (Dojindo, Kumamoto, Japan). DP cells were seeded in 24-well plates at a density of 3.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL. The cells were treated with 5 or 10 \u0026micro;g/mL of APsulloc\u0026rsquo;s lysate or LTA for 72 h, following which cell viability was also determined using the Cell Counting kit-8 assay.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons among three or more groups were performed using one-way ANOVA, followed by Tukey's honest significant difference test. Comparisons between the two groups were performed using a paired two-tailed Student's t-test. The results were analyzed using SPSS for Windows (version 22.0; IBM Corp.). \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was considered to indicate a statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of APsulloc lysate on growth and biofilm formation of MR\u003c/h2\u003e \u003cp\u003eWe examined whether the APsulloc lysate directly affected the growth or biofilm formation of MR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the lysate did not affect the growth of MR. We also confirmed whether the APsulloc lysate influences biofilm formation. As Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb illustrates, the lysate did not affect biofilm formation. These results suggest that the APsulloc lysate does not have a direct impact on MR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtective effects of the APsulloc lysate against heat-killed MR on scalp keratinocytes\u003c/h2\u003e \u003cp\u003eThe scalp keratinocytes were treated with heat-killed MR (10 \u0026micro;g/mL) in the presence or absence of the APsulloc lysate for 24 h. When subjected to treat heat-killed MR, substantial reduction of the filaggrin gene expression by a factor of 0.35-fold (\u0026plusmn;\u0026thinsp;0.04, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed, whereas the addition of APsulloc lysate resulted in significant improvement of 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (1 \u0026micro;g/mL, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 (5 \u0026micro;g/mL, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similarly, occludin gene expression was suppressed by 0.22-fold (\u0026plusmn;\u0026thinsp;0.04, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) following heat-killed MR exposure, whereas the APsulloc lysate treatment recovered the expression by 0.25-fold (\u0026plusmn;\u0026thinsp;0.09, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) at 1 \u0026micro;g/mL and 0.60-fold (\u0026plusmn;\u0026thinsp;0.17, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 5 \u0026micro;g/mL of APsulloc lysate. When treated with heat-killed MR alone, \u003cem\u003eIL-1a\u003c/em\u003e and \u003cem\u003eIL-1b\u003c/em\u003e mRNA levels were 1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 and 4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56-fold that of the control, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Administering the APsulloc lysate at a concentration of 10 \u0026micro;g/mL effectively attenuated the inflammatory cytokine expression (IL-1α; 1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, IL-1β; 3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, fold of control) compared with that in the heat-killed MR-only treated group. Heat-killed MR decreased differentiation-related proteins KRT 1 and 10 in scalp keratinocytes, while APsulloc lysate alone had no effect. However, when combined, the lysate mitigated the MR-induced changes in these differentiation markers. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLysate and lipoteichoic acid of APsulloc modulates lipogenesis in sebocytes induced by heat-killed MR\u003c/h2\u003e \u003cp\u003eAPsulloc lysate protected scalp keratinocytes from a pathogen. We then studied its effects on sebocytes, key cells in SSD symptoms. Treatment with 50 \u0026micro;g/mL of heat-killed MR significantly increased cytoplasmic lipid accumulation, as evidenced by a 278.4\u0026thinsp;\u0026plusmn;\u0026thinsp;32.5% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) enhancement in fluorescence intensity of lipid droplets compared to the control group (Fig, 3a, b). Simultaneous treatment with APsulloc\u0026rsquo;s cell wall components and heat-killed MR resulted in a marked reduction of lipid production in sebocytes. The fluorescence analysis showed clear inhibitory effects of the lysate and LTA (196.4\u0026thinsp;\u0026plusmn;\u0026thinsp;16.3% with 10 \u0026micro;g/mL of lysate; 147.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3% with 10 \u0026micro;g/mL of LTA compared with the % in the control). Lipid production-related protein expression also showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Heat-killed MR upregulated lipogenesis protein expression, whereas APsulloc lysate alone showed similar like control. Importantly, combined treatment with APsulloc lysate effectively attenuated heat-killed MR-induced protein expression changes.\u003c/p\u003e \u003cp\u003eWe used a cytokine antibody array to assess the effects of heat-killed MR and the APsulloc lysate. Heat-killed MR induced the secretion of angiopoietin-2 (ANGPT2) and dickkopf-1 (DDK-1) compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although we expected an increase in IL-1α and IL-1β as in scalp keratinocytes, this was not the case. When APsulloc lysate or LTA was administered along with heat-killed MR, the elevated levels of ANGPT2 and DDK-1 induced by heat-killed MR were reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eThe protective effect of APsulloc lysate and LTA on ORS cells against heat-killed MR and promoting DP cell viability\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen the ORS cells were treated with 50 \u0026micro;g/mL of heat-killed MR and hair loss-inducing factors (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, DKK-1, and TGFβ2), cell viability was significantly decreased by 48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, combined treatment with 10 \u0026micro;g/mL of APsulloc lysate improved the cell viability by 68.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% and 10 \u0026micro;g/mL of LTA of APsulloc improved the cell viability by 66.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2% compared with that of control. We also conducted experiments to determine whether the lysate and LTA from APsulloc could improve the viability of DP cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, both lysate and LTA at concentrations of 5 or 10 \u0026micro;g/mL induced proliferation of DP cells (112.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 and 119.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3% with 5 and 10 \u0026micro;g/mL of lysate, respectively; 116.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2% and 120.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% with 5 and 10 \u0026micro;g/mL of LTA, respectively). Our results revealed that APsulloc lysate and LTA can protect ORS cells from apoptosis and increase DP cell viability, indicating that these components likely contribute to hair growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe investigated the effects of MR, a major cause of SSD, on human scalp cells and evaluated the potential of APsulloc lysate and its cell wall component LTA, as natural solution for SSD management. Heat-killed MR significantly impacted scalp cells, suppressing tight junction and differentiation markers in keratinocytes while upregulating inflammatory cytokines. In sebocytes, MR increased lipid production and accumulation, and elevated expression of hair growth inhibitors. APsulloc lysate and LTA mitigated these MR-induced changes in gene and protein expression, suggesting their potential in managing SSD symptoms through multiple cellular mechanisms.\u003c/p\u003e \u003cp\u003eUnlike live probiotics that directly affect microorganisms, bacterial lysates and cell wall components like LTA work indirectly by interacting with host cells. This approach also differs from traditional antimicrobial treatments, potentially enhancing natural defenses against pathogens. LTA has gained research attention for its role in immune regulation, acting as a ligand for TLR2 on human cells and triggering immune responses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The structural diversity of LTA is believed to considerably influence its immunomodulatory activity, contributing to its varied biological interactions. In the human epidermis, TLR2 is expressed in key cell types, such as keratinocytes and sebocytes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. TLR2 is also involved in recognizing the components of \u003cem\u003eMalassezia\u003c/em\u003e species, inducing a pro-inflammatory response characterized by the release of various cytokines, chemokines, and antimicrobial peptides by keratinocytes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous studies have shown that \u003cem\u003eMalassezia furfur\u003c/em\u003e-infected keratinocytes exhibit upregulation of \u003cem\u003eTLR2\u003c/em\u003e, human beta-defensin (\u003cem\u003eHBD\u003c/em\u003e) 2, and \u003cem\u003eIL-8\u003c/em\u003e gene expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, hair follicle stem cell TLR2 is essential for maintaining hair homeostasis and regeneration. TLR2 endogenous ligands, such as carboxyethylpyrrole, a metabolite of polyunsaturated fatty acids, promote hair growth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. ORS cells, crucial for hair growth, are predicted to protect against hair loss when their survival is maintained. Sebaceous glands are also known to affect hair growth, with their normal development and function being important for correct hair development and cycling [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. DDK-1 can inhibit the growth of ORS cells and trigger apoptotic cell death, and ANGPT2 exhibits estradiol activity to improve hair loss in ovariectomized mice [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Interestingly, the lysate and LTA from APsulloc reduced the expression of DDK-1 and ANGPT2 that were increased by heat-killed MR in sebocytes and inhibited ORS cell death.\u003c/p\u003e \u003cp\u003eWe can hypothesize that LTA form APsulloc competitively binds to TLR with the cellular components of MR, thereby modulating the immune response. This hypothesis not only highlights the potential of APsulloc-derived LTA in therapeutic applications but also underscores the complexity of host-pathogen interactions at the molecular level. However, the use of heat-killed MR may not fully represent the complex living scalp microbiome. In an actual scalp environment, MR survived with other microorganisms in a balanced state. And managing live microorganisms or the scalp microbiome with cosmetic or personal care products remains challenging. Further study is needed to confirm whether the immune modulation occurs through competitive binding to TLR2, and the implications of ORS and DP cells viability on hair loss inhibition should be re-examined. Most of all, clinical evidence is required to demonstrate the changes in MR within the complex scalp ecosystem due to treatment with lysate and LTA. These comprehensive studies will significantly contribute to the development of targeted scalp improvement methods in the future and help find natural approaches to potentially reduced hair loss associated with SSD.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe evaluated the effects of APsulloc lysate and its cell wall component, LTA, on human scalp cells. Although APsulloc lysate could not directly prevent the growth or biofilm formation of MR, the lysate and LTA of APsulloc had a significant indirect protective effect against heat-killed pathogens. These effects are mediated by the modulation of gene expression and the alteration of cytokine release in human scalp cells. Our findings support the potential of natural substances to enhance scalp health and propose new strategies for managing SSD.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eImpact statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile recent studies have been published on the effects of lactic acid bacteria lysate on skin health, it is difficult to find content related to scalp health. It is even more challenging to determine which components of the lysate are responsible for these effects. In this study, we examined the impact of lactic acid bacteria lysate and LTA on scalp primary cells. Although more research is needed to managing SSD, we can say that we have found initial clues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeither ethical approval nor informed consent was required for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support for the research, authorship, and/or publication of this articles.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eClark GW, Pope SM, Jaboori KA (2015) Diagnosis and treatment of seborrheic dermatitis. Am Fam Physician 91(3): 185-190. \u003c/li\u003e\n\u003cli\u003eTao R, Li R, Wang R (2021) Skin microbiome alterations in seborrheic dermatitis and dandruff: A systematic review. Exp Dermatol (10):1546-1553. https://doi.org/10.1111/exd.14450\u003c/li\u003e\n\u003cli\u003ePark T, Kim HJ, Myeong NR \u003cem\u003eet al.\u003c/em\u003e (2017) Collapse of human scalp microbiome network in dandruff and seborrhoeic dermatitis. Exp Dermatol (9):835-838. https://doi.org/10.1111/exd.1329\u003c/li\u003e\n\u003cli\u003eGrimshaw SG, Smith AM, Arnold DS \u003cem\u003eet al.\u003c/em\u003e (2019)\u003cem\u003e \u003c/em\u003eThe diversity and abundance of fungi and bacteria on the healthy and dandruff affected human scalp. PLoS One;14(12): e0225796. https://doi.org/10.1371/journal.pone.0225796. \u003c/li\u003e\n\u003cli\u003ePark M, Park S, Jung WH (2021) Skin commensal fungus \u003cem\u003eMalassezia\u003c/em\u003e and its lipases. J Microbiol Biotechnol.31(5):637\u0026ndash;644. https://doi.org/10.4014/jmb.2012.12048\u003c/li\u003e\n\u003cli\u003eTrueb RM, Henry JP, Davis MG \u003cem\u003eet al\u003c/em\u003e. (2018) Scalp condition impacts hair growth and retention via oxidative stress. Int J Trichology 10(6): 262-270. https://doi.org/10.4103/ijt.ijt_57_18\u003c/li\u003e\n\u003cli\u003eBorda LJ, Wikramanayake TC (2015) Seborrheic Dermatitis and Dandruff: A Comprehensive Review. J Clin Investig Dermatol. 3(2). https://doi.org/10.13188/2373-1044.1000019\u003c/li\u003e\n\u003cli\u003eKhmaladze I, Butler E. Fabre S \u003cem\u003eet al.\u003c/em\u003e (2019) \u003cem\u003eLactobacillus reuteri\u003c/em\u003e DSM 17938-A comparative study on the effect of probiotics and lysates on human skin. Exp Dermatol. 28(7): 822-828. https://doi.org/ 10.1111/exd.13950\u003c/li\u003e\n\u003cli\u003eJung YO, Jeong H, Cho Y \u003cem\u003eet al\u003c/em\u003e. (2019) Lysates of a probiotic, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, can improve skin barrier function in a reconstructed human epidermal model. Int J Mol Sci. (17): 4289. https://doi.org/10.3390/ijms20174289.\u003c/li\u003e\n\u003cli\u003eMyoung K, Choi EJ, Kim H \u003cem\u003eet al\u003c/em\u003e. (2021) Fermented lysate of \u003cem\u003eLactiplantibacillus\u003c/em\u003e isolated from green tea leaves protects keratinocytes against stress hormone and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. China Detergent \u0026amp; Cosmetics 6(3): 54-60. \u003c/li\u003e\n\u003cli\u003eMyoung K, Choi EJ, Kim S \u003cem\u003eet al\u003c/em\u003e. (2024) Nano-sized lysate of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e isolated green tea leaves as a potential skin care ingredient. Biotech Bioprocess Engineering. 29: 1071-1080. https://doi.org/ 10.1007/s12257-024-00132-3\u003c/li\u003e\n\u003cli\u003eChae M, Kim BJ, Na J \u003cem\u003eet al\u003c/em\u003e. (2021) Antimicrobial activity of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e APsulloc 331261 and APsulloc 331266 against pathogenic skin microbiota. Front Biosci (Elite Ed) 13(2):237\u0026ndash;248. https://doi.org/10.52586/E881\u003c/li\u003e\n\u003cli\u003eLebeer S, Vanderleyden J, Keersmaecker SCJ (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72(4): 728-764. https://doi.org/10.1128/MMBR.00017-08\u003c/li\u003e\n\u003cli\u003eKim W, Lee EJ, Bae IH \u003cem\u003eet al\u003c/em\u003e. (2020) \u003cem\u003eLactobacillus plantarum\u003c/em\u003e-derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro. J Extracell Vesicles 9(1):1793514. https://doi.org/10.1080/20013078.2020.1793514\u003c/li\u003e\n\u003cli\u003eChoi H, Lee Y, Shin SH \u003cem\u003eet al\u003c/em\u003e. (2022) Induction of hair growth in hair follicle cells and organ cultures upon treatment with 30 kHz frequency inaudible sound via cell proliferation and antiapoptotic effects. Biomed Rep 16(3):16. https://doi.org/10.3892/br.2022.1499. \u003c/li\u003e\n\u003cli\u003eTriantafilou M, Manukyan M, Mackie A \u003cem\u003eet al\u003c/em\u003e. (2004) Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi and lipid raft-dependent. J Biol Chem 279(39): 40882-40889. https://doi.org/10.1074/jbc.M400466200\u003c/li\u003e\n\u003cli\u003eSaito S, Okuno A, Cao DY \u003cem\u003eet al\u003c/em\u003e. (2020) Bacterial lipoteichoic acid attenuates toll-like receptor dependent dendritic cells activation and inflammatory response. Pathogens 9(10). 825. https://doi.org/10.3390/pathogens9100825\u003c/li\u003e\n\u003cli\u003eHari A, Flach T, Shi Y et al. (2010) Toll-like receptors: role in dermatological disease. Mediators Inflamm: 437246 https://doi.org/10.1155/2010/437246\u003c/li\u003e\n\u003cli\u003eSparber F, LeibundGut-Landmann S (2017) Host responses to \u003cem\u003eMalassezia\u003c/em\u003e spp. In the mammalian skin. Front Immunol 8: 1614. https://doi.org/10.3389/fimmu.2017.01614\u003c/li\u003e\n\u003cli\u003eBaroni A, Orlando M, Donnarumma G \u003cem\u003eet al\u003c/em\u003e. (2006) Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to \u003cem\u003eMalassezia furfur\u003c/em\u003e. Arch Dermatol Res 297(7): 280-288. https://doi.org/10.1007/s00403-005-0594-4\u003c/li\u003e\n\u003cli\u003eXiong L, Zhevlakova I, West XZ \u003cem\u003eet al.\u003c/em\u003e (2023)\u003cem\u003e \u003c/em\u003eTLR2 regulates hair follicle cycle and regeneration via BMP signaling. bioRxiv [Preprint]. https://doi.org/10.1101/2023.08.14.553236\u003c/li\u003e\n\u003cli\u003eLee WJ, Cha HW, Lim HJ \u003cem\u003eet al\u003c/em\u003e. (2012) The effect of sebocytes cultured from nevus sebaceus on hair growth. Exp Dermatol 21(10): 796-798. https://doi.org/10.1111/j.1600-0625.2012.01572.x\u003c/li\u003e\n\u003cli\u003eKwack M, Sung Y, Chung E \u003cem\u003eet al\u003c/em\u003e. (2008) Dihydrotestosteron-inducible Dickkopf 1 from balding dermal papilla cells causes apoptosis in follicular keratinocytes. J Invest Dermatol 128(2): 262-269. https://doi.org/10.1038/sj.jid.5700999\u003c/li\u003e\n\u003cli\u003eEndo Y, Obayashi Y, Ono T \u003cem\u003eet al\u003c/em\u003e. (2018) Reversal of the hair loss phenotype by modulating the estradiol-ANGPT2 axis in the mouse model of female pattern hair loss. J Dermatol Sci 91(1): 43-51 (2018). https://doi.org/10.1016/j.jdermsci.2018.04.001\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"scalp seborrheic dermatitis, scalp cells, Malassezia restricta, Lantiplantibacillus plantarum, lipoteichoic acid","lastPublishedDoi":"10.21203/rs.3.rs-6132321/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6132321/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eScalp seborrheic dermatitis (SSD) is characterized by excessive sebum production, flaking, and itching. This condition is associated with an imbalance in the scalp microbiome, particularly the dominance of \u003cem\u003eMalassezia restricta\u003c/em\u003e (MR). Antifungal treatments for SSD often fail to address root causes and can lead to side effects, recurrence, and resistant strains with long-term use. This highlights the need for new, more effective solutions to manage the condition. In this study, we investigated whether the lysate of \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e APsulloc 331261 (APsulloc), isolated from green tea leaves, and its lipoteichoic acid (LTA), a bacterial cell wall component, have the potential to serve as natural solutions for SSD. The lysate of APsulloc had no direct effect on MR growth or biofilm formation. However, the lysate improved gene expression of tight junctions and inflammatory cytokines, and upregulated differentiation marker proteins in heat-killed MR-treated scalp keratinocytes. On the other hand, heat-killed MR stimulates differentiation signaling in sebocytes and enhances the expression of lipogenesis-related proteins. APsulloc\u0026rsquo;s lysate alleviated these effects and inhibit lipid production by sebocytes caused by heat-killed MR. LTA from APsulloc was also found to reduce lipogenesis and the secretion of hair loss-related cytokines in human primary sebocytes that were induced by heat-killed MR. Furthermore, both the lysate and LTA protected outer root sheath cell viability against heat-killed MR-induced damage while promoting dermal papilla cell growth. These finding demonstrate the potential of APsulloc\u0026rsquo;s cell wall components as natural solutions for improving SSD \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Lactiplantibacillus plantarum lysate isolated from green tea leaves alleviates the effects of Malassezia restricta on primary human scalp cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-06 16:31:40","doi":"10.21203/rs.3.rs-6132321/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-02T20:01:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-01T20:31:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32428104744251746557464750896272323873","date":"2025-04-19T15:42:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226706659684986226494487002118548816442","date":"2025-04-13T08:56:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287516321881658010362026975625032901346","date":"2025-03-18T08:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157871723428166551726006659613461565661","date":"2025-03-12T13:00:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-12T12:53:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-04T07:58:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-04T07:58:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2025-03-01T03:55:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b980bcb7-0abc-42a5-b768-7595452e2fdc","owner":[],"postedDate":"March 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-11T20:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-06 16:31:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6132321","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6132321","identity":"rs-6132321","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}