{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T11:24:51Z","timestamp":1789471491862,"version":"build-2803163510"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,2,7]],"date-time":"2025-02-07T00:00:00Z","timestamp":1738886400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Enhancing an organism\u2019s survival hinges on the development of balanced and adaptable stress response systems. While the initial stress-response set-points in the hypothalamus may be genetically determined, they are further influenced by epigenetic factors during embryonic development. A debate persists regarding the heritability of such behavioral traits. The chick <jats:italic>in ovo<\/jats:italic> heat conditioning model offers a unique insight into this fundamental question, where manipulation during embryonic development can induce heat resilience and even cross-tolerance to promote immunological resilience. In this study, we conducted an analysis of thermal manipulation during embryogenesis to demonstrate paternal heredity and investigate its transmission through sperm DNA methylation in coordination with miR-200a action.<\/jats:p><\/jats:sec><jats:sec><jats:title>Result<\/jats:title><jats:p>First-generation embryos underwent <jats:italic>in ovo<\/jats:italic> heat conditioning (EHC), creating a cohort of embryonic EHC and control chicks. These chicks were then subjected to an intracranial lipopolysaccharide (LPS) challenge. Conditioning rendered the chicks immune resilient, as evidenced by their fibril effect. Male offspring were raised to maturity, and their sperm was analyzed for methylome patterns, revealing significant differences between treatments, particularly in immune and development related genes. Additionally, sperm from EHC males was used for artificial insemination of na\u00efve Cobb hens, resulting in untreated offspring that displayed immune resilience upon LPS challenge, indicating transgenerational effects. Overlap analysis of sperm methylome and differentially methylated sites (DMS) of offspring hypothalamus revealed inheritance of altered methylation associated with specific genes. Several of these genes are potential effectors of miR-200a, whose expression profile in the hypothalamus during LPS challenge was conserved across both generations. To evaluate the role of miR-200a in cross-tolerance acquisition, miR-200a was intracranially injected, and RNA-seq analysis of the hypothalamus revealed genes involved in the regulation of developmental and metabolic processes, stress, and immune response.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study demonstrates paternal trait heredity by revealing that EHC induces cross-tolerance with the immunological system, rendering chicks resilient to LPS that transgenerationally transmit this to untreated offspring. Additionally, analysis of sperm methylation patterns in EHC mature chicks led to identification of genes associated with neuronal development and immune response, indicating potential neural network reorganization. Finally, miR-200a emerges as a regulator potentially involved in mediating the cross-tolerance effect.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1487135","type":"journal-article","created":{"date-parts":[[2025,2,7]],"date-time":"2025-02-07T06:53:04Z","timestamp":1738911184000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Embryonic heat conditioning induces paternal heredity of immunological cross- tolerance: coordinative role of CpG DNA methylation and miR-200a regulation"],"prefix":"10.3389","volume":"16","author":[{"given":"Padma Malini","family":"Ravi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatiana","family":"Kisliouk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shelly","family":"Druyan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amit","family":"Haron","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark A.","family":"Cline","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth R.","family":"Gilbert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noam","family":"Meiri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,2,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1038\/nn.4086","article-title":"Mechanisms of stress in the brain","volume":"18","author":"McEwen","year":"2015","journal-title":"Nat Neurosci"},{"key":"B2","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1210\/en.2017-00896","article-title":"Prenatal stress, glucocorticoids, and developmental programming of the stress response","volume":"159","author":"McGowan","year":"2018","journal-title":"Endocrinology"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/j.yfrne.2018.02.002","article-title":"Behavioral and structural adaptations to stress","volume":"49","author":"Cameron","year":"2018","journal-title":"Front Neuroendocrinology"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1111\/jne.12175","article-title":"The consequences of early-life adversity: Neurobiological, behavioral and epigenetic adaptations","volume":"26","author":"Maccari","year":"2014","journal-title":"J Neuroendocrinology"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.psj.2021.101164","article-title":"Chicken embryo as a model in epigenetic research","volume":"100","author":"Bednarczyk","year":"2021","journal-title":"Poultry Science"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1038\/s41380-018-0280-5","article-title":"Early-life epigenetic changes along the corticotropin-releasing hormone (CRH) gene influence resilience or vulnerability to heat stress later in life","volume":"24","author":"Cramer","year":"2019","journal-title":"Mol Psychiatry"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1002\/dneu.22252","article-title":"The balance between stress resilience and vulnerability is regulated by corticotropin-releasing hormone during the critical postnatal period for sensory development","volume":"75","author":"Cramer","year":"2015","journal-title":"Dev Neurobiol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1186\/1297-9686-45-16","article-title":"Epigenetics and phenotypic variability: Some interesting insights from birds","volume":"45","author":"Fr\u00e9sard","year":"2013","journal-title":"Genet Selection Evolution"},{"key":"B9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1096\/fj.202101948R","article-title":"Embryonic heat conditioning in chicks induces transgenerational heat\/immunological resilience via methylation on regulatory elements","volume":"36","author":"Rosenberg","year":"2022","journal-title":"FASEB J"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.14202\/vetworld.2016.260-268","article-title":"Impact of heat stress on health and performance of dairy animals: A review","volume":"9","author":"Das","year":"2016","journal-title":"Veterinary World"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.3382\/ps.2008-00030","article-title":"Thermal manipulations during broiler embryogenesis: Effect on the acquisition of thermotolerance","volume":"87","author":"Piestun","year":"2008","journal-title":"Poult Sci"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0227700","article-title":"Embryonic thermal manipulation has short and long-term effects on the development and the physiology of the Japanese quail","volume":"15","author":"Carvalho","year":"2020","journal-title":"PloS One"},{"key":"B13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12864-016-2661-y","article-title":"Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge","volume":"17","author":"Loyau","year":"2016","journal-title":"BMC Genomics"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.3382\/ps.2013-03177","article-title":"Transgenerational epigenetic effects on innate immunity in broilers: An underestimated field to be explored","volume":"92","author":"Berghof","year":"2013","journal-title":"Poultry Science"},{"key":"B15","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/j.coi.2020.02.002","article-title":"The epigenetically-encoded memory of the innate immune system","volume":"65","author":"Sun","year":"2020","journal-title":"Curr Opin Immunol"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.3382\/ps\/pey460","article-title":"Effect of embryonic thermal manipulation on heat shock protein 70 (HSP70) expression and subsequent immune response to post-hatch lipopolysaccharide challenge in Pekin ducklings","volume":"98","author":"Shanmugasundaram","year":"2019","journal-title":"Poult Sci"},{"key":"B17","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1165\/ajrcmb.17.1.f132","article-title":"The role of nuclear factor-B in cytokine gene regulation","volume":"17","author":"Blackwell","year":"1997","journal-title":"Am J Respir Cell Mol Biol"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1006\/cyto.2001.0959","article-title":"Over-expression of HSP-70 inhibits bacterial lipopolysaccharide-induced production of cytokines in human monocyte-derived macrophages","volume":"16","author":"Ding","year":"2001","journal-title":"Cytokine"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1016\/j.tig.2019.12.001","article-title":"Small RNAs in the transgenerational inheritance of epigenetic information","volume":"36","author":"Duempelmann","year":"2020","journal-title":"Trends Genet"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3695","article-title":"Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice","volume":"17","author":"Gapp","year":"2014","journal-title":"Nat Neurosci"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.3389\/fendo.2018.00402","article-title":"Overview of microRNA biogenesis, mechanisms of actions, and circulation","volume":"9","author":"O\u2019Brien","year":"2018","journal-title":"Front Endocrinology"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1111\/jnc.14014","article-title":"Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex","volume":"141","author":"Kisliouk","year":"2017","journal-title":"J Neurochem"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1007\/s12192-018-0911-4","article-title":"Identification of differentially expressed microRNAs in Sahiwal (Bos indicus) breed of cattle during thermal stress","volume":"23","author":"Sengar","year":"2018","journal-title":"Cell Stress Chaperones"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1080\/15592294.2020.1795596","article-title":"Cross-tolerance: embryonic heat conditioning induces inflammatory resilience by affecting different layers of epigenetic mechanisms regulating IL6 expression later in life","volume":"16","author":"Rosenberg","year":"2021","journal-title":"Epigenetics"},{"key":"B25","volume-title":"Broiler Management Guide","year":"2018"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2020.00767","article-title":"Embryonic heat conditioning induces TET-dependent cross-tolerance to hypothalamic inflammation later in life","volume":"11","author":"Rosenberg","year":"2020","journal-title":"Front Genet"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-019-0164-4","article-title":"MARS-seq2.0: an experimental and analytical pipeline for indexed sorting combined with single-cell RNA sequencing","volume":"14","author":"Keren-Shaul","year":"2019","journal-title":"Nat Protoc"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.14806\/ej.17.1.200","article-title":"Cutadapt removes adapter sequences from high-throughput sequencing reads","volume":"17","author":"Martin","year":"2011","journal-title":"EMBnet J"},{"key":"B29","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1093\/bioinformatics\/bts635","article-title":"STAR: Ultrafast universal RNA-seq aligner","volume":"29","author":"Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu638","article-title":"HTSeq-A Python framework to work with high-throughput sequencing data","volume":"31","author":"Anders","year":"2015","journal-title":"Bioinformatics"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: A Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2009","journal-title":"Bioinformatics"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1101\/gr.196394.115","article-title":"Metilene: Fast and sensitive calling of differentially methylated regions from bisulfite sequencing data","volume":"26","author":"J\u00fchling","year":"2016","journal-title":"Genome Res"},{"key":"B34","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1007\/s12017-019-08535-9","article-title":"MiR-34 and miR-200: regulator of cell fate plasticity and neural development","volume":"21","author":"Jauhari","year":"2019","journal-title":"Neuromolecular Med"},{"key":"B35","doi-asserted-by":"publisher","first-page":"15","DOI":"10.3892\/mco.2023.2611","article-title":"Role of miR-200 family in brain metastases: A systematic review","volume":"18","author":"Fotakopoulos","year":"2023","journal-title":"Mol Clin Oncol"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1051\/animres:2005004","article-title":"The effect of duration of thermal manipulation during broiler chick embryogenesis on body weight and body temperature of post-hatched chicks","volume":"54","author":"Collin","year":"2005","journal-title":"Anim Res"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2020.610319","article-title":"Effects of thermal conditioning on changes in hepatic and muscular tissue associated with reduced heat production and body temperature in young chickens","volume":"7","author":"Ouchi","year":"2021","journal-title":"Front Vet Sci"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1113\/EP090143","article-title":"Epigenetic responses to heat: From adaptation to maladaptation","volume":"107","author":"Murray","year":"2022","journal-title":"Exp Physiol"},{"key":"B39","doi-asserted-by":"publisher","first-page":"6978936","DOI":"10.1155\/2016\/6978936","article-title":"The role of toll-like receptor 4 in infectious and noninfectious inflammation","volume":"2016","author":"Molteni","year":"2016","journal-title":"Mediators Inflamm"},{"key":"B40","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1186\/s13148-020-00997-0","article-title":"Male obesity impacts DNA methylation reprogramming in sperm","volume":"13","author":"Keyhan","year":"2021","journal-title":"Clin Epigenetics"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3172-20.2022","article-title":"Cocaine-induced changes in sperm cdkn1a methylation are associated with cocaine resistance in male offspring","volume":"42","author":"Swinford-Jackson","year":"2022","journal-title":"J Neurosci"},{"key":"B42","doi-asserted-by":"publisher","first-page":"995","DOI":"10.1038\/mp.2014.84","article-title":"Age-related sperm DNA methylation changes are transmitted to offspring and associated with abnormal behavior and dysregulated gene expression","volume":"20","author":"Milekic","year":"2015","journal-title":"Mol Psychiatry"},{"key":"B43","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41598-023-43484-1","article-title":"Exploring the role of miR-200 family in regulating CX3CR1 and CXCR1 in lung adenocarcinoma tumor microenvironment: implications for therapeutic intervention","volume":"13","author":"Sharma","year":"2023","journal-title":"Sci Rep"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.3390\/ani13030508","article-title":"Expression Profile of miRNA from High, Middle, and Low Stress-Responding Sheep during Bacterial Endotoxin Challenge","volume":"13","author":"Shandilya","year":"2023","journal-title":"Animals"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1196065","article-title":"miR-200a attenuated oxidative stress, inflammation, and apoptosis in dextran sulfate sodium-induced colitis through activation of Nrf2","volume":"14","author":"Peng","year":"2023","journal-title":"Front Immunol"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1016\/j.scitotenv.2019.134399","article-title":"Herbs as thermoregulatory agents in poultry: An overview","volume":"703","author":"Abd-El-Hack","year":"2020","journal-title":"Sci Total Environ"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1016\/j.jtherbio.2018.12.013","article-title":"Stress biomarkers and proteomics alteration to thermal stress in ruminants: A review","volume":"79","author":"Abdelnour","year":"2019","journal-title":"J Therm Biol"},{"key":"B48","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s13567-020-0736-x","article-title":"MicroRNA gga-miR-200a-3p modulates immune response via MAPK signaling pathway in chicken afflicted with necrotic enteritis","volume":"51","author":"Pham","year":"2020","journal-title":"Vet Res"},{"key":"B49","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1093\/noajnl\/vdad160","article-title":"Exploring the multifaceted role of NRF2 in brain physiology and cancer: A comprehensive review","volume":"6","author":"Moubarak","year":"2024","journal-title":"Neurooncol Adv"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1016\/j.jaci.2017.08.034","article-title":"MicroRNA","volume":"141","author":"Lu","year":"2018","journal-title":"J Allergy Clin Immunol"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1111\/j.1460-9568.2010.07493.x","article-title":"MiR-138 inhibits EZH2 methyltransferase expression and methylation of histone H3 at lysine 27, and affects thermotolerance acquisition","volume":"33","author":"Kisliouk","year":"2011","journal-title":"Eur J Neurosci"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1172\/JCI40000","article-title":"Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation","volume":"120","author":"Iskandar","year":"2010","journal-title":"J Clin Invest"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.1002\/mco2.161","article-title":"Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities","volume":"3","author":"Hu","year":"2022","journal-title":"MedComm"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbi.2013.10.016","article-title":"Essential role of IL-10\/STAT3 in chronic stress-induced immune suppression","volume":"36","author":"Hu","year":"2014","journal-title":"Brain Behav Immun"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.1007\/s11055-008-0028-9","article-title":"Stress-induced changes in cellular responses in hypothalamic structures to administration of an antigen (Lipopolysaccharide) (in terms of C-fos protein expression)","volume":"38","author":"Gavrilov","year":"2008","journal-title":"Neurosci Behav Physiol"},{"key":"B56","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.00907","article-title":"MicroRNA-200a inhibits inflammation and atherosclerotic lesion formation by disrupting EZH2-mediated methylation of STAT3","volume":"11","author":"Wang","year":"2020","journal-title":"Front Immunol"},{"key":"B57","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2010.08.004","article-title":"Interleukin-2 receptor signaling: at the interface between tolerance and immunity","volume":"33","author":"Malek","year":"2010","journal-title":"Immunity"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1487135\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,2,7]],"date-time":"2025-02-07T06:53:10Z","timestamp":1738911190000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1487135\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,7]]},"references-count":57,"alternative-id":["10.3389\/fimmu.2025.1487135"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1487135","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,7]]},"article-number":"1487135"}}