{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:48:36Z","timestamp":1790182116389,"version":"4.1.0"},"reference-count":56,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,18]],"date-time":"2025-06-18T00:00:00Z","timestamp":1750204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>\n                    Bacteria and associated products are factors in the pathogenesis of bovine endometrial inflammation, contributing to reproductive dysfunction. While peptidoglycan derived from\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    (PGN-Sa) has been demonstrated to induce pro-inflammatory responses and disrupt sperm\u2013immune interactions in bovine endometrial epithelial cells (BEECs) via Toll-like receptor 2\/1 (TLR2\/1), the immunomodulatory potential of peptidoglycan from lactic acid bacteria (LAB) within the female reproductive tract remains unexplored. This study investigated the\n                    <jats:italic>in vitro<\/jats:italic>\n                    immunomodulatory effects of LAB-derived peptidoglycan (PGN-L) on TLR2\/1-mediated inflammation in BEECs, with the specific TLR2\/1 agonist PAM3CSK4 (PAM3) as an inflammatory stimulus. PGN-L was extracted and characterized from\n                    <jats:italic>Lacticaseibacillus rhamnosus<\/jats:italic>\n                    (PGN-Lr) and\n                    <jats:italic>Lactobacillus acidophilus<\/jats:italic>\n                    (PGN-La), and its structural composition was compared to that of commercial PGN-Sa. Subsequently, BEECs were pre-incubated with PGN-L (Lr, La) or PGN-Sa (1 ng\/mL) for 24 h before stimulation with PAM3 (100 ng\/mL) for 3 h. The expression of inflammatory genes (\n                    <jats:italic>TNF<\/jats:italic>\n                    ,\n                    <jats:italic>CXCL8<\/jats:italic>\n                    ,\n                    <jats:italic>IL1B<\/jats:italic>\n                    , and\n                    <jats:italic>PTGES<\/jats:italic>\n                    ) and TLRs (\n                    <jats:italic>TLR1<\/jats:italic>\n                    ,\n                    <jats:italic>TLR2<\/jats:italic>\n                    ,\n                    <jats:italic>TLR4<\/jats:italic>\n                    , and\n                    <jats:italic>TLR6<\/jats:italic>\n                    ) was quantified by RT-qPCR. The protein expression of TNF, PTGES, and TLR2 was detected using immunofluorescence, while PGE\n                    <jats:sub>2<\/jats:sub>\n                    concentrations in the culture media were measured by ELISA. PGN-Lr and PGN-La shared the GlcNAc-MurNAc backbone with PGN-Sa, while PGN-L had a unique modification. PGN-L and PGN-Sa contained lysine at the cross-bridge stem, composed of glycine in PGN-Sa and likely modified D-aspartate in PGN-L. While PGN-Sa and PAM3 significantly upregulated the expression of inflammatory mediators, neither PGN-Lr nor PGN-La alone induced a pro-inflammatory response in BEECs. Importantly, pretreatment with both PGN-Lr and PGN-La significantly reduced PAM3-induced inflammatory gene expression and reduced PGE\n                    <jats:sub>2<\/jats:sub>\n                    secretion.\n                    <jats:italic>In silico<\/jats:italic>\n                    molecular findings suggested a potential mechanism whereby PGN-L may act as a TLR2\/1 antagonist, contrasting with the agonistic effects of PGN-Sa and PAM3, which promoted TLR2\/1 heterodimerization. These findings suggest that PGN-Lr and PGN-La can suppress TLR2\/1-mediated uterine inflammation\n                    <jats:italic>in vitro<\/jats:italic>\n                    , by potentially modulating TLR2\/1 signaling in BEECs. Further investigation of PGN-L holds promise for the development of therapeutic strategies to enhance bovine reproductive efficiency.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2025.1622307","type":"journal-article","created":{"date-parts":[[2025,6,18]],"date-time":"2025-06-18T00:15:48Z","timestamp":1750205748000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Peptidoglycan derived from Lacticaseibacillus rhamnosus and Lactobacillus acidophilus suppress TLR2\/1-mediated inflammation in bovine endometrial epithelial cells"],"prefix":"10.3389","volume":"16","author":[{"given":"Elham","family":"Waehama","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenji","family":"Fukuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alireza","family":"Mansouri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Malinda","family":"Hulugalla","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ihshan","family":"Akthar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed Samy","family":"Yousef","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akio","family":"Miyamoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,6,18]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.17485\/ijst\/2015\/v8i23\/52386","article-title":"Bovine postpartum metritis and its therapeutics: A review","volume":"8","author":"Deori","year":"2015","journal-title":"Indian J Sci Technol"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2005.08.021","article-title":"Defining postpartum uterine disease in cattle","volume":"65","author":"Sheldon","year":"2006","journal-title":"Theriogenology"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3168\/jds.2016-12058","article-title":"Genetic and functional analysis of the bovine uterine microbiota. Part I: Metritis versus healthy cows","volume":"100","author":"Bicalho","year":"2017","journal-title":"J Dairy Sci"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/0093-691X(86)90045-2","article-title":"Mechanisms and therapy for retained fetal membranes and uterine infections of cows: A review","volume":"25","author":"Paisley","year":"1986","journal-title":"Theriogenology"},{"key":"B5","doi-asserted-by":"publisher","first-page":"684","DOI":"10.1016\/j.ajog.2016.09.075","article-title":"Evidence that the endometrial microbiota has an effect on implantation success or failure","volume":"215","author":"Moreno","year":"2016","journal-title":"Am J Obstet Gynecol"},{"key":"B6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1155\/2022\/4570320","article-title":"Beneficial effects of lactic acid bacteria on animal reproduction function","volume":"2022","author":"Nur Mahendra","year":"2022","journal-title":"Vet Med Int"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00057","article-title":"Benefits and inputs from lactic acid bacteria and their bacteriocins as alternatives to antibiotic growth promoters during food-animal production","volume":"10","author":"Vieco-Saiz","year":"2019","journal-title":"Front Microbiol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0143294","article-title":"Vaginal microbiome characterization of nellore cattle using metagenomic analysis","volume":"10","author":"Laguardia-Nascimento","year":"2015","journal-title":"PLoS One"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0093631","article-title":"Characterization of fecal microbiota across seven chinese ethnic groups by quantitative polymerase chain reaction","volume":"9","author":"Kwok","year":"2014","journal-title":"PLoS One"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1038\/90609","article-title":"Toll-like receptors: critical proteins linking innate and acquired immunity","volume":"2","author":"Akira","year":"2001","journal-title":"Nat Immunol"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.619408","article-title":"Peptidoglycan switches off the TLR2-mediated sperm recognition and triggers sperm localization in the bovine endometrium","volume":"11","author":"Elesh","year":"2021","journal-title":"Front Immunol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2012.00079","article-title":"The role of TLR2 in infection and immunity","volume":"3","author":"Oliveira-Nascimento","year":"2012","journal-title":"Front Immunol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.111.092718","article-title":"Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium1","volume":"86","author":"Cronin","year":"2012","journal-title":"Biol Reprod"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.109.077370","article-title":"Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle1","volume":"81","author":"Sheldon","year":"2009","journal-title":"Biol Reprod"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1177\/1753425920949917","article-title":"Peptidoglycan derived from Lactobacillus rhamnosus MLGA up-regulates the expression of chicken \u03b2-defensin 9 without triggering an inflammatory response","volume":"26","author":"Huang","year":"2020","journal-title":"Innate Immun"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1016\/j.carbpol.2015.04.026","article-title":"Peptidoglycan diversity and anti-inflammatory capacity in Lactobacillus strains","volume":"128","author":"Wu","year":"2015","journal-title":"Carbohydr Polym"},{"key":"B17","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1038\/mi.2009.124","article-title":"The role of TLRs, NLRs, and RLRs in mucosal innate immunity and homeostasis","author":"Lavelle","year":"2010","journal-title":"Mucosal Immunol"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1530\/REP-19-0414","article-title":"Sperm enter glands of preovulatory bovine endometrial explants and initiate inflammation","volume":"159","author":"Akthar","year":"2020","journal-title":"Reproduction"},{"key":"B19","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1262\/jrd.2022-124","article-title":"Sperm induce proinflammatory responses in the uterus and peripheral blood immune cells of artificially inseminated cows","volume":"69","author":"Marey","year":"2023","journal-title":"J Reprod Dev"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1210\/en.2005-1113","article-title":"Expression and function of toll-like receptor 4 in the endometrial cells of the uterus","volume":"147","author":"Herath","year":"2006","journal-title":"Endocrinology"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1016\/j.tiv.2014.04.005","article-title":"Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells","volume":"28","author":"Magata","year":"2014","journal-title":"Toxicol In Vitro"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0214516","article-title":"TLR2\/4 signaling pathway mediates sperm-induced inflammation in bovine endometrial epithelial cells in vitro","volume":"14","author":"Ezz","year":"2019","journal-title":"PLoS One"},{"key":"B23","article-title":"A new morphologically characterized cell wall preparation (Whole peptidoglycan) from Bifidobacterium infantis with a higher efficacy on the regression of an established tumor in mice","author":"Sekine","year":"1985","journal-title":"Cancer Res"},{"key":"B24","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1262\/jrd.2024-093","article-title":"Semen extender triggers a mild physiological inflammatory response in the uterus without disrupting sperm-uterine immune crosstalk in vitro in cattle","volume":"71","author":"Hulugalla","year":"2025","journal-title":"J Reprod Dev"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod62.5.1116","article-title":"Production of prostaglandin F2\u03b1 by cultured bovine endometrial cells in response to tumor necrosis factor \u03b1: cell type specificity and intracellular mechanisms","volume":"62","author":"Skarzynski","year":"2000","journal-title":"Biol Reprod"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1002\/mrd.22955","article-title":"A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro","volume":"85","author":"Elweza","year":"2018","journal-title":"Mol Reprod Dev"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1158090","article-title":"Sperm activate TLR2\/TLR1 heterodimerization to induce a weak proinflammatory response in the bovine uterus","volume":"14","author":"Mansouri","year":"2023","journal-title":"Front Immunol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1002\/0471142735.ima03bs111","article-title":"Trypan blue exclusion test of cell viability","volume":"111","author":"Strober","year":"2015","journal-title":"Curr Protoc Immunol"},{"key":"B29","doi-asserted-by":"crossref","first-page":"1 p","DOI":"10.1002\/9780470649367","volume-title":"Culture of animal cells: a manual of basic technique and specialized applications","author":"Freshney","year":"2010"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2008.73","article-title":"Analyzing real-time PCR data by the comparative CT method","volume":"3","author":"Schmittgen","year":"2008","journal-title":"Nat Protoc"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/29.9.e45","article-title":"A new mathematical model for relative quantification in real-time RT-PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acids Res"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3389\/fendo.2023.1134868","article-title":"Hyaluronan regulates sperm-induced inflammatory response by enhancing sperm attachment to bovine endometrial epithelial cells via CD44: in-silico and in-vitro approaches","volume":"14","author":"Ezz","year":"2023","journal-title":"Front Endocrinol"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.3390\/ijms25031496","article-title":"Homology modeling, molecular dynamics simulation, and prediction of bovine TLR2 heterodimerization","volume":"25","author":"Mansouri","year":"2024","journal-title":"Int J Mol Sci"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1021\/ja808971c","article-title":"Staphylococcus aureus peptidoglycan tertiary structure from carbon-13 spin diffusion","volume":"131","author":"Sharif","year":"2009","journal-title":"J Am Chem Soc"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1186\/1475-2859-13-S1-S9","article-title":"Cell wall structure and function in lactic acid bacteria","volume":"13","author":"Chapot-Chartier","year":"2014","journal-title":"Microb Cell Factories"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J Comput Chem"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21256","article-title":"AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility","volume":"16","author":"Morris","year":"2009","journal-title":"J Comput Chem"},{"key":"B38","doi-asserted-by":"publisher","first-page":"16082","DOI":"10.1038\/s41598-020-73225-7","article-title":"A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups","volume":"10","author":"Kowsar","year":"2020","journal-title":"Sci Rep"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jpcc.7b00010","article-title":"Transport behavior of the enantiomers of lactic acid through the cyclic peptide nanotube: enantiomer discrimination","volume":"121","author":"Farrokhpour","year":"2017","journal-title":"J Phys Chem C"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1021\/ct700200b","article-title":"P-LINCS: A parallel linear constraint solver for molecular simulation","volume":"4","author":"Hess","year":"2008","journal-title":"J Chem Theory Comput"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21287","article-title":"CHARMM: The biomolecular simulation program","volume":"30","author":"Brooks","year":"2009","journal-title":"J Comput Chem"},{"key":"B42","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"2","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21816","article-title":"SwissParam: A fast force field generation tool for small organic molecules","volume":"11","author":"Zoete","year":"2011","journal-title":"J Comput Chem"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jctc.1c00645","article-title":"gmx_MMPBSA: A new tool to perform end-state free energy calculations with GROMACS","volume":"17","author":"Vald\u00e9s-Tresanco","year":"2021","journal-title":"J Chem Theory Comput"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1002\/fft2.44","article-title":"Guidelines for cell viability assays","volume":"1","author":"Kamiloglu","year":"2020","journal-title":"Food Front"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.02691","article-title":"Uterine microbiota of dairy cows with clinical and subclinical endometritis","volume":"9","author":"Wang","year":"2018","journal-title":"Front Microbiol"},{"key":"B47","doi-asserted-by":"publisher","first-page":"295","DOI":"10.1016\/j.anireprosci.2004.04.006","article-title":"Postpartum uterine health in cattle","author":"Sheldon","year":"2004","journal-title":"Anim Reprod Sci"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.3168\/jds.2019-18050","article-title":"Graduate Student Literature Review: Potential mechanisms of interaction between bacteria and the reproductive tract of dairy cattle","volume":"103","author":"Owens","year":"2020","journal-title":"J Dairy Sci"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.3168\/jds.2016-11671","article-title":"A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium","volume":"100","author":"Gen\u00eds","year":"2017","journal-title":"J Dairy Sci"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1262\/jrd.2021-023","article-title":"Association of intrauterine presence of Lactobacillus spp. with inflammation and pathogenic bacteria in the uterus in postpartum dairy cows","volume":"67","author":"Wu","year":"2021","journal-title":"J Reprod Dev"},{"key":"B51","doi-asserted-by":"publisher","first-page":"295","DOI":"10.1016\/j.intimp.2015.04.004","article-title":"Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice","volume":"26","author":"Kawahara","year":"2015","journal-title":"Int Immunopharmacol"},{"key":"B52","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1016\/j.mib.2004.12.008","article-title":"The role of peptidoglycan in pathogenesis","volume":"8","author":"Boneca","year":"2005","journal-title":"Curr Opin Microbiol"},{"key":"B53","doi-asserted-by":"publisher","first-page":"174","DOI":"10.1128\/MMBR.63.1","article-title":"Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope","volume":"63","author":"Navarre","year":"1999","journal-title":"Microbiol Mol Biol Rev"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.165.10.5392","article-title":"Cutting edge: TLR2-deficient and myD88-deficient mice are highly susceptible to Staphylococcus aureus infection","volume":"165","author":"Takeuchi","year":"2000","journal-title":"J Immunol"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2007.09.008","article-title":"Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide","volume":"130","author":"Jin","year":"2007","journal-title":"Cell"},{"key":"B56","doi-asserted-by":"publisher","first-page":"1310","DOI":"10.1038\/s41564-025-01989-7","article-title":"Gut commensal Phascolarctobacterium faecium retunes innate immunity to mitigate obesity and metabolic disease in mice","volume":"10","author":"Li\u00e9bana-Garc\u00eda","year":"2025","journal-title":"Nat Microbiol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1622307\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,18]],"date-time":"2025-06-18T00:15:49Z","timestamp":1750205749000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1622307\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,18]]},"references-count":56,"alternative-id":["10.3389\/fimmu.2025.1622307"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1622307","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,18]]},"article-number":"1622307"}}