BackgroundFat metabolism in pigs is controlled by tissue-specific molecular mechanisms that ultimately affect growth performance and meat quality. Understanding how epigenetic modifications interact with gene expression across key metabolic and fat-depositing tissues is essential for identifying regulatory processes and potential biomarkers to improve pork quality traits. Therefore, this study aimed to elucidate tissue specific epigenetic regulation of fat metabolism by integrating DNA methylation and gene expression profiles from liver, backfat, and loin (longissimus dorsi) tissues at two physiologically developmental stages (10 and 26 weeks), representing the early post-weaning growth phase and near-market weight, respectively. By explicitly comparing these ages and tissues, the study was designed to capture the transition from muscle-dominated growth to increased lipid deposition and to identify tissue- and stage-specific regulatory signatures that may serve as biomarkers for pork quality.ResultsGenome-wide DNA methylation exhibited weak clustering by tissue, whereas gene expression showed clear tissue separation. The liver harbored fewer genes with differential methylation across stage and tissue but a greater number of genes with differential expression than backfat and loin, suggesting distinct regulatory modes. Integrative analysis of the overlap genes between methylation and expression signals highlighted epigenetically mediated regulation of extracellular matrix organization, lipid metabolism, and muscle development pathways. Furthermore, weighted gene co-expression network analysis revealed distinct tissue-specific correlations between co-methylated and co-expressed modules, with enrichment in cholesterol biosynthesis, muscle contractility, and extracellular matrix remodeling. Together, these findings suggest that methylation changes are more subtle than transcriptional shifts, yet they are aligned with key functional pathways, consistent with a role for methylation as a fine-tuning mechanism that shapes tissue-specific transcriptional networks during growth.ConclusionsAcross liver, backfat, and loin, DNA methylation modulates transcriptional programs in a tissue-dependent manner, prioritizing pathways central to lipid handling, extracellular matrix remodeling, and muscle function. This integrated multi-omics framework highlights candidate epigenetic markers and regulatory modules with potential utility for improving pork quality traits through selection or management strategies.

Placenta is a focal point of cellular interactions of maternal and fetal immune systems, fostering immune tolerance essential for a successful pregnancy. However, the mechanisms underlying immune dysregulation in disorders such as preeclampsia remain poorly understood. We constructed a multilayered atlas of the human placenta by performing single-cell RNA sequencing across distinct placental layers from both normal and severe preeclamptic pregnancies. Rather than focusing on specific regions of placenta, such as the villi and decidua, we explore placental architecture by collecting pair-matched tissues from individual pregnancies to suggest appropriate placental-wide immune atlas. To interpret intercellular communication in those unexplored placental layers, we designed two conceptual models: one based on immune interaction frequency (IIF) and another on immune tolerance influencers (IT). We applied machine learning classifiers to identify gene signatures associated with preeclampsia. We observed extensive admixture of semi-allogeneic fetal and maternal cells across all placental layers, regardless of disease status. This contradicts the IIF-based model, which is premised on that such intermixing frequency is a pathologic feature specific to preeclampsia. Instead, analysis under the IT framework revealed key molecular determinants of preeclampsia. Notably, classifier-prioritized genes associated with preeclampsia were enriched for ligands and receptors supporting a role for intercellular immune interactions. Among them, the ligand–receptor pair SPP1–CD44 between fetus and maternal immune cells emerged as peculiarly associated influencers of preeclampsia. Spatial image analysis confirmed co-localization of SPP1–CD44 expression within immune cell populations in preeclamptic placental tissue. Our study provides a comprehensive map of the human placenta and identifies disease-specific immune signaling pathways in preeclampsia using the divide and rule approach. The findings highlight SPP1–CD44 as a putative target of immune dysregulation, offering new insight into the cellular basis of maternal–fetal tolerance and its breakdown in pregnancy-related disorders. Not applicable.

Abstract Introduction Virtual memory (TVM) CD8+ T cells exhibit memory-like features without prior antigen exposure and provide protection. We previously identified a CD44s-hiCD49dlo subset that acquires pathogenic properties upon IL-12, IL-15, and IL-18 stimulation and mediates alopecia areata (AA) via NKG2D-dependent cytotoxicity. These findings reveal functional heterogeneity within the TVM compartment, suggesting that subsets can transition to a disease-promoting phenotype under inflammation. Based on these findings, we aimed to define the molecular and epigenetic heterogeneity of TVM cells and identify transcriptional regulators driving pathogenic CD44s-hiCD49dlo CD8+ T cell differentiation. Methods We performed joint single-cell RNA sequencing and ATAC sequencing on conventional TVM and CD44s-hiCD49dlo CD8+ T cells isolated from the lymph nodes and skin of AA mice. UMAP projection and clustering defined transcriptional heterogeneity, and pseudotime trajectory analysis inferred differentiation pathways. Integration of transcriptomic and chromatin accessibility data enabled the identification of transcription factors associated with pathogenic transition. Results Single-cell analyses revealed that conventional TVM and CD44s-hiCD49dlo CD8+ T cells form transcriptionally distinct populations. Clustering identified multiple subpopulations within the TVM pool, including a transitional subset located along a pseudotime trajectory leading toward the pathogenic state. Integrated gene expression and epigenomic profiling highlighted RUNX2, RUNX3, and RORα as candidate regulators orchestrating this differentiation program. Conclusion Taken together, these findings delineate a previously unrecognized trajectory from conventional TVM cells to CD44s-hiCD49dlo CD8+ T cells that drive inflammation in AA. The identified regulatory factors define the transcriptional and epigenetic framework underlying this pathogenic transition and represent potential molecular targets for therapeutic modulation of cytotoxic T-cell responses. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract The global success of mRNA vaccines has underscored the pivotal role of lipid nanoparticles (LNPs), yet how subtle chemical variations in ionizable lipids and their formulation parameters orchestrate complex immune landscapes remains largely elusive. Here, we report a novel ionizable lipid, N4Z, and demonstrate that its distinct chemical signature selectively intensifies early innate immune programs compared to its structural analogue, N4Y. Single-cell transcriptomic profiling at the injection site reveals that N4Z-based LNPs uniquely prime inflammatory and type I interferon-related transcriptional programs, accompanied by a rapid influx of B and CD4 + T cells. Beyond lipid chemistry, we show that formulation-level tuning, that is independent of the ionizable lipid structure, can reshape the systemic biodistribution from hepatic dominance toward lymphoid tissues. This optimization substantially enhances macrophage-associated antigen expression, which in turn amplifies polyfunctional CD4 + T cell responses, T follicular helper cell differentiation, and germinal center reactions. Our findings establish that the coordinated interplay between lipid engineering and formulation design provides a programmable platform for precision mRNA vaccination, achieving superior protective efficacy and neutralizing activity over clinically validated benchmarks.

Abstract Introduction Immune cells play multifaceted roles in the body, ranging from systemic and local pathogen surveillance to pathogenic auto-reactivity in conditions such as autoimmune diseases. While we can detect these immune abnormalities using transcriptomic methods, the complete cellular landscape of these diseases has not yet been comprehensively explored. Methods We profiled blood immune cells with single-cell RNA sequencing method. Results We generated single-cell profiles for 2,098,547 circulating immune cells from 231 individuals spanning 22 distinct immune-related conditions. We delineated five major immune lineages and characterized their transcriptomic alterations. We examined cross-lineage correlations in cellular responses and their associations with disease contexts. Conclusion This study present overall cellular architecture of blood immune cells, and provide broad landscape of systemic multicellular responses in immune-related diseases. Funding Source The Single Cell Atlas of Immune Diseases (SCAID) project Topic Categories Immune Mechanisms of Human Disease (HUM)

ABSTRACT Mainland Southeast Asia (MSEA) remains under-represented in global immunogenomic references despite its extensive genetic heterogeneity. We present the first single-cell immune atlas of an MSEA population, utilizing Thai individuals from the Asian Immune Diversity Atlas (AIDA) as a representative cohort. We demonstrate that the Thai population is highly genetically diverse, reflecting its history as a geographic nexus for Asian admixture. By integrating single-cell transcriptomics with high-resolution genotyping, we show that genetic ancestry significantly shapes innate immune profiles, specifically CD14 + monocytes, highlighting potential evolutionary adaptations to regional pathogens. We identify specific sex-by-ancestry interactions that may drive the baseline activation of pro-inflammatory pathways in females, providing a long-sought cellular rationale for the high prevalence of autoimmune disorders observed in Southeast Asian populations. Ultimately, our study reveals that population-specific genetic architecture dictates immune heterogeneity often missed by self-reported ethnicity or country of origin, providing a critical immunogenomic reference for precision medicine in MSEA regions.

Muscle stem cells (MuSCs) are parenchymal cells in skeletal muscle regeneration and maintenance. With aging, MuSCs experience a decline in their regenerative function and reduction in their number. However, recent evidence points to substantial heterogeneity within the aged MuSC population, raising questions about the underlying mechanisms of age-associated dysfunction. Here, we used Pax7CreERT2;RosaYFP mice (MuSCYFP) to label Pax7-expressing MuSCs and chronologically traced MusCs until geriatric age. Genetic labeling and chronological tracing revealed that the number of YFP+ MuSC remained comparable between young, middle and geriatric ages. At geriatric age, YFP+ MuSCs exhibited reduced expression of traditional MuSC markers such as VCAM1 and PAX7. A previously unrecognized subpopulation emerged, characterized by loss of VCAM1 and low or absent PAX7. Despite their altered marker profile, these cells retained transcriptional signatures of quiescence and myogenic potential, but displayed significantly reduced proliferative and regenerative capacities. They displayed gene expression patterns indicative of senescence-like state and were selectively ablated by senolytic treatment. DHT restored regenerative function in aged mice and re-induced VCAM1 expression in YFP+/Pax7-/low/VCAM1- cells, indicating responsiveness to rejuvenation. Based on their emergence with aging, functional impairment and responsiveness to rejuvenation, we termed this population GERI-MuSCs (Geriatric Emerging Rejuvenation-responsive and Impaired MuSCs). CD63 and CD200 were identified as novel surface markers that together with VCAM1, reliably detect GERI-MuSCs as well as classical Pax7+/VCAM1High MuSCs, providing a tool for comprehensive isolation of MuSCs from aged wild-type mice. Together, our findings provide a refined framework for studying MuSC aging and offer new tools for isolating functionally distinct MuSC subsets from aged skeletal muscle.

This study aimed to investigate the effects of different aging methods on the free amino acid (FAA) composition and flavor characteristics of beef striploin (longissimus lumborum) using an electronic tongue (e-tongue). Beef samples from Hanwoo steers (n = 6, grade 2) were subjected to wet, dry, and dry-bag aging for 15, 30, 45, and 60 days. FAA profiles were quantified and classified into sweet, savory, bitter, and functional groups, while e-tongue analysis was conducted to evaluate multiple taste modalities, including umami, sourness, bitterness, astringency, saltiness, and richness. Wet aging retained higher sourness throughout the storage period, whereas dry and dry-bag aging resulted in progressive increases in umami and richness, with dry aged beef showing the highest umami intensity after 45 days. FAA analysis revealed that glutamine decreased markedly in dry and dry-bag aging, accompanied by increased glutamic acid and ammonia. Correlation analysis indicated that functional amino acids (taurine, β-alanine, ethanolamine, and ammonia) were strongly associated with umami, richness, and saltiness, whereas classical umami amino acids (glutamic and aspartic acids) showed no significant correlation with e-tongue values. These results suggest that functional amino acids play a more critical role than total FAA content in shaping beef flavor perception. Taken together, this study demonstrates the potential of combining amino acid profiling with e-tongue evaluation to provide an objective and comprehensive understanding of flavor development in aged beef.

Climate change creates major challenges in livestock industry, making chickens vulnerable to heat stress because they can tolerate a narrow range of temperatures. Heat stress disrupts metabolic and physiological homeostasis, leading to reduced growth, productivity, reproduction, and immune function, thereby threatening the economic viability of poultry farming. This review explores the multifaceted impacts of heat stress on poultry, including physiological responses, production performance, and immune function. Recent advances in transcriptomic and genomic research have shed light on the molecular mechanisms underlying heat stress resilience in poultry. Key genes such as HSP70, HSP90, HSP27, and HSP47 are significantly upregulated under heat stress, playing vital roles in protein folding, preventing aggregation, and protecting cellular integrity. Additionally, genes like SOD and CAT enhance antioxidant defenses, mitigating oxidative damage. Genes such as RB1CC1, BAG3, and TRMT1L regulate apoptosis and oxidative stress, promoting cell survival. In the liver, CCK, DIO3, and ANGPTL4 improve energy homeostasis and reduce metabolism-related heat production, while BMP10 and MYH7 in the heart contribute to cardiac adaptation during thermal stress. Genetic adaptations such as the Naked neck, Frizzle, and Dwarf gene provide intrinsic thermotolerance by reducing feather mass, altering feather structure, and minimizing body size, thereby improving heat dissipation. These genetic traits, combined with transcriptomic insights into heat resilience genes, offer opportunities for developing heat-tolerant chicken breeds. By integrating molecular genetics, transcriptomics, and management strategies, this review highlights the importance of selective breeding programs to enhance poultry thermotolerance. Future research should focus on leveraging indigenous breeds, advanced molecular tools, and nutritional interventions to mitigate the effects of rising global temperatures. Enhancing heat stress resilience in poultry is imperative to ensure sustainable production and global food security in this climate change.

Hanwoo (Korean cattle) is an indigenous breed of high economic value in Korea, where improving body weight (BW) remains a priority for both productivity and farm profitability. Because growth traits are shaped by multiple biological layers, including host genetics, rumen microbial ecology, and transcriptomic regulation, a single-omics perspective is often insufficient. To address this complexity, we applied an integrative multi-omics framework, DIABLO (Data Integration Analysis for Biomarker discovery using Latent cOmponents), combining genomic (42,091 SNPs), rumen transcriptomic (RNA-seq), and microbiome (16S rRNA) data from 58 steers. After adjusting BW for age, animals were classified into High- and Low-BW groups. Feature pre-selection yielded 100 discriminant SNPs, 161 large-effect candidate expression features, and 20 candidate microbial features showing nominal abundance differences between groups. Integration of these datasets highlighted exploratory SNP–gene–microbiome associations, including three SNPs, ARS-BFGL-NGS-104362, ARS-BFGL-NGS-43924 and ARS-BFGL-NGS-24293, that remained significantly associated with BW. These variants were linked to epithelial and metabolic genes such as FUT1, KRTDAP, and TCN1, which in turn showed strong correlations with Anaerobutyricum, a butyrate-producing genus identified as an exploratory microbial candidate. Taken together, these results suggest candidate host epithelial and rumen microbial axes associated with BW variation, rather than establishing causal mechanisms. By applying a supervised multi-omics integration strategy, this study identifies exploratory candidate multi-omics features associated with BW variation in Hanwoo steers. Because of the modest sample size and the supervised nature of the integration framework, the resulting signature should be interpreted as hypothesis-generating and requiring validation in larger independent cohorts.

Heterologous vaccination strategies have shown superior efficacy over homologous regimens in clinical studies, but the underlying immunological mechanisms remain incompletely understood. Using a mouse model, we investigated the immune responses induced by heterologous prime-boost vaccination with adenoviral and mRNA vaccines. Heterologous vaccination (adenoviral prime, mRNA boost) elicited higher neutralizing antibody titers and stronger CD8+ T cell responses against Delta and Omicron-BA.5 variants compared to homologous regimens. Single-cell transcriptomic analysis of injection-site tissues revealed that adenoviral priming induced minimal changes in cellular composition but established a pre-conditioned innate immune environment. This effect was further amplified upon mRNA boosting, particularly through fibroblast-driven chemokine responses that promoted immune cell recruitment. These findings suggest that adenoviral priming enhances local immune activation upon boosting, contributing to the heightened adaptive immune response observed in heterologous vaccination. This study provides mechanistic insights into the immunological effects of heterologous prime-boost strategies against SARS-CoV-2 variants.

Cells in maternal and fetal immune systems may communicate, leading to immune tolerance during pregnancy; however, this hypothesis remains controversial. Here, we profiled single cell transcriptional signatures in placental layers comprising the maternal–fetal interface and deep placenta, then searched for genes associated with preeclampsia. To investigate the underlying principle of the failure of immune tolerance, we started by clarifying the systemic framework, comprising models of immune interaction frequency (IIF) and specific triggers (i.e., influencers) of tolerance (IT). We generated single cell transcriptional profiles of normal term (Norms) and preeclampsia preterm (PePT) parturitions. Fetal and maternal cells are admixed across the placenta, for both Norms and PePTs, rejecting the IIF model of immune failure during pregnancy posed by excessive interactions between fetomaternal cells. Whereas placental layers are well mixed with maternal cells, we identified a conserved gradual immune transition of fetal T cells in both PePT and Norm, disproving the IIF model. To search for influencers of PePT in the IT model, we established and validated a classification model for PePT and Norm immune cells, including T cells, and then prioritized major contributors to the classifier model, which are highly enriched in ligands and receptors (p = 5.98E–05). Among the prioritized ligand receptors, SPP1 and CD44 are suggested as influencers of inflammation signatures and were experimentally validated by the exclusive colocalization of SPP1– and CD44–expressing cells in the PePT placentas. Different interleukin-4 and interferon-γ levels in the serum and urine of PePTs further support the contribution of SPP1 to associated pathways, including allograft rejection. Our findings provide insight into the influence of specific immune interactions between cells in the human placenta and their influencer-derived impact on PePT. ### Competing Interest Statement The authors have declared no competing interest.

Clustering analysis is a fundamental step in scRNA-seq data analysis. However, its reliability is compromised by clustering inconsistency among trials due to stochastic processes in clustering algorithms. Despite efforts to obtain reliable and consensus clustering, existing methods cannot be applied to large scRNA-seq datasets due to high computational costs. Here, we develop the single-cell Inconsistency Clustering Estimator (scICE) to evaluate clustering consistency and provide consistent clustering results, achieving up to a 30-fold improvement in speed compared to conventional consensus clustering-based methods, such as multiK and chooseR. Application of scICE to 48 real and simulated scRNA-seq datasets, some with over 10,000 cells, successfully identifies all consistent clustering results, substantially narrowing the number of clusters to explore. By enabling the focus on a narrower set of more reliable candidate clusters, users can greatly reduce computational burden while generating more robust results.

Human diseases affect various cell types within the local niche, but the multicellular responses of human diseases have not been collectively assessed across organs. Here, we categorize multicellular responses of human diseases based on a remapped human cell atlas, incorporating 12 million single-cell transcriptome profiles acquired from an automated data collection pipeline. Additional curation expands the collection up to 46 million cells, covering 160 disease conditions across 49 distinct organ identities. From the re-aligned atlas of single-cell transcriptome data, we constructed a hierarchical cell classification framework comprising 32 major cell types and 165 cell subtype annotations. The universal cell annotation framework enabled abundance-based clustering of the blood samples from various disease contexts, which rendered 10 different blood phenotypes including a Covid-19-specific interferon response enriched phenotype. Further, systematic profiling of disease and organ association patterns of cellular subtype reveals archetypes of human diseases repeatedly found across organs, including a multicellular phenotype highly specific to the tumor microenvironment. Collectively, our study provides foundational resources for cross-tissue cell type annotation and presents a comprehensive multicellular overview of disease-associated responses across human tissues. Computational and Systems Immunology (COMP)

Human diseases affect various cell types within the local niche, but the multicellular responses of human diseases have not been collectively assessed across organs. Here, we categorize multicellular responses of human diseases based on a remapped human cell atlas, incorporating 12 million single-cell transcriptome profiles acquired from an automated data collection pipeline. Additional curation expands the collection up to 46 million cells, covering 160 disease conditions across 49 distinct organ identities. From the re-aligned atlas of single-cell transcriptome data, we constructed a hierarchical cell classification framework comprising 32 major cell types and 165 cell subtype annotations. Systematic profiling of disease and organ association patterns of cellular subtype reveals archetypes of human diseases repeatedly found across organs, including a multicellular phenotype highly specific to the tumor microenvironment. The universal cell atlas could serve as a reference for unbiased deconvolution of spatial transcriptome datasets, revealing spatial distribution patterns associated with immunotherapy responses. Collectively, our study provides foundational resources for cross-tissue cell type annotation, presents a comprehensive multicellular overview of disease-associated responses across human tissues, and highlights multicellular responses in tumor microenvironments. Seongryong Kim, Sungmin Cheong, Suho Lee, Yeju Kim, Jong-Eun Park. Cross-tissue atlas of human disease identifies tumor-specific components in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6265.

Due to a pivotal role in the post-transcriptional regulation of genes implicated in numerous diseases, miRNAs serve as promising disease biomarkers and therapeutic targets. We introduce a new oligonucleotide probe termed miRNA-trigger, which selectively downregulates newly assigned target mRNAs by hijacking specific miRNAs. By engineering the miRNA-trigger to suppress the anti-apoptotic BCL-xL gene, we induce apoptosis selectively in breast cancer cells overexpressing specific miRNAs and further validate its therapeutic efficacy in vivo, by significantly reducing the tumor volume of the xenograft mouse upon its tail-vein injection. This approach establishes a new platform for self-modulating oligonucleotide therapy by redirecting disease-associated miRNAs.

Genetic variations in CLCN4 , encoding the H+/Cl- exchanger CLC-4, are associated with human neurodevelopmental disorders with highly variable phenotypes. A lack of physiologically relevant models has hampered molecular understanding of pathogenic mechanisms. We now establish engineered brain organoid and neuronal cell systems to examine impacts of patient-relevant CLCN4 genetic variations. We find that CLCN4 variants reduced excitatory neuron numbers due to early-stage cell death, accompanied by altered endo-lysosomal dynamics and disrupted autophagic flux. Transcriptomic profiling showed significant downregulation of long non-coding RNA MEG3 in CLCN4 -variant neurons. Restoring MEG3 expression is sufficient to rescue cellular defects and improve survival of CLCN4 -variant neurons. These findings link CLCN4 dysfunction with impaired autophagy and neuronal cell death, highlighting MEG3 as a potential therapeutic target for neurodevelopmental disorders involving autophagic dysfunction. ### Competing Interest Statement The authors have declared no competing interest. Ministry of Health and Welfare, https://ror.org/00vxgjw72, HI18C1077 Ministry of Science and ICT, https://ror.org/01wpjm123, 2021R1A2C1004884, RS-2024-00398786, RS-2024-00335144, RS-2024-00407383, 25-BR-01-02, 25-BR-05-07 Ministry of Environment, RS-2025-02223058 Institute for Basic Science, IBS-R002-A1 Ministry of Education, NRF-2022R1A6A3A13073152 Korea Advanced Institute of Science and Technology

Eosinophils are major effector cells in type 2 immune responses, contributing to host defense and allergic diseases. They also contribute to maintaining tissue homeostasis by regulating various immune cell types, including neutrophils. Here we show that eosinophils directly associate with neutrophils in the lungs of asthma-induced mice. Eosinophil-specific deficiency of the short-chain fatty acid receptor, GPR43, results in hyperactivation of eosinophils and increases the expression of neutrophil chemoattractants and PECAM-1, thereby enhancing the interaction between eosinophils and neutrophils. This interaction exposes neutrophils to eosinophil-derived IL-4 and GM-CSF, which induce the conversion of conventional neutrophils into more pathogenic, Siglec-Fhi neutrophils capable of enhancing Th17 cell differentiation and aggravating asthma symptoms in mouse models. Our results thus implicate GPR43 as a critical regulator of eosinophils, and describe eosinophil-mediated modulation of neutrophil differentiation and function.

Chickens are one of the most economically and nutritionally important livestock species globally, yet they are highly vulnerable to environmental stressors which compromise their growth and physiological stability. In this study we investigated the effect of wind speed on growth performance, physiological responses, blood profiles, and transcriptomic adaptations in Ross-308 broilers under high-temperature conditions. A total of 240 broilers were divided into three groups: low wind, high temperature (LWH; 33°C, 60% RH, 0 m/s), medium wind, high temperature (MWH; 33°C, 60% RH, 1 m/s), and high wind, high temperature (HWH; 33°C, 60% RH, 2 m/s). The results showed that broilers in the HWH and MWH groups had significantly higher feed intake, body weight gain, and improved thermoregulation, as indicated by lower rectal temperatures and respiration rates compared to the LWH group. Additionally, serum potassium and phosphorus levels were higher in the HWH and MWH groups, indicating enhanced metabolic stability under heat stress. Transcriptomic analysis identified differentially expressed genes associated with the heat stress response, such as HSPB9, THBS1, TLR2, EHHADH, and PDK4. Functional enrichment analysis identified pathways related to vital processes, innate immune responses, and biosynthetic pathways, which contribute to thermal resilience. Upregulated genes like PDK4 and heat shock proteins were associated with improved energy efficiency and stress response, whereas downregulated genes in LWH indicated compromised growth. This study underscores the vital role of wind speed in ameliorating heat stress effects in broilers, laying the groundwork for enhanced environmental management and potential genetic and nutritional strategies in poultry production.