The physical microenvironment regulates cancer cell behavior through mechanical and geometrical cues. While surface topography is known to influence cell adhesion and proliferation, its effects on population-level behaviors and time-dependent adaptation remain unclear. Here, we developed a simple particle-coating method to generate isotropic topographies (0.7 μm and 2.26 μm) directly in standard culture wells and applied it to melanoma cell lines. Topography did not affect viability but induced a more contractile morphology, reduced migration, and increased proliferation, with stronger effects for 2.26 μm particles. Topography also induced a biphasic proliferative response, with an initial increase followed by a later decline after serial passaging. Transcriptomic analysis revealed upregulation of metabolic and adhesion pathways, supporting early increases in proliferation and adhesion while prolonged activation may cause metabolic stress and reduced proliferation. Overall, these topographies modulate melanoma behavior via adhesion and metabolism changes, offering a simple platform to study time-dependent cellular responses.
 

BACKGROUND AND PURPOSE: Cannabidiol (CBD) and cannabigerol (CBG) are non-psychoactive phytocannabinoids with emerging therapeutic potential in metabolic dysfunction-associated steatotic liver disease (MASLD). However, the molecular mechanisms underlying their beneficial effects remain incompletely understood. In this study, we assessed the metabolomic and lipidomic impact of CBD and CBG in a mouse model of diet-induced obesity and MASLD. EXPERIMENTAL APPROACH: Male C57Bl/6 mice fed on a high-fat diet for 14 weeks were treated for 4 weeks with daily intraperitoneal CBD, CBG or vehicle. Assessments included body composition, indirect calorimetry, glucose tolerance, serum biochemistry and VLDL-triglyceride profiling. Hepatic mechanisms were examined by metabolomics, lipidomics, creatine kinase activity, cathepsin activity-based probes and gene/protein expression, with a choline-deficient diet cohort to test phospholipid-dependence of CBG. KEY RESULTS: CBD or CBG treatment improved glycaemic control, reduced hepatic triglycerides and normalised serum lipids, without affecting energy expenditure. Metabolomics revealed increased hepatic phosphocreatine and creatine with enhanced creatine kinase activity, indicating phosphocreatine-based energy buffering independent of fatty acid oxidation changes. Lipidomics showed reduced triglycerides and ceramides, with increased phospholipids and lysobisphosphatidic acids, correlating with restored hepatic cathepsin activity and improved lysosomal lipid degradation. CBG was ineffective in choline-deficient MASLD, indicating phospholipid pathway dependence. CONCLUSIONS AND IMPLICATIONS: These findings identify a novel, endocannabinoid system-independent mechanism by which CBD and CBG enhance hepatic energy buffering and lysosomal function, contributing to improved liver lipid handling and supporting phytocannabinoids as promising MASLD therapeutics.

Accurate and rapid identification of aggressive cancer cells remains a major clinical challenge. Here, we present a simple, label-free mechanophenotyping platform that integrates controlled colloidal topographies with particle-uptake measurements to reveal biophysical traits associated with metastatic progression. Non-close-packed polystyrene bead arrays were formed on cell culture plates by controlled deposition and stabilized with a thin silicon oxide coating. These arrays display micro- and nano-features with a size range of 0.23-2.3 μm at diverse densities and were used to assess adhesion across cancer cells exhibiting different levels of malignancy. Particle uptake differences were most pronounced for particle diameters above 0.5 μm, whereas adhesion differences emerged predominantly on particles $\geq$0.7 μm and increased progressively with larger particle sizes. Colloidal topographies were fabricated at particle deposition concentrations of 500 μg/mL and 1000 μg/mL, and adhesion differences were observed under both conditions, with more potent effects at the higher concentration. At the metastatic site, cells exhibited increased particle uptake, stronger adhesion, and a larger morphological engagement on colloid-coated substrates, characterized by extensive actin-rich protrusions wrapping individual particles. AFM force mapping confirmed higher adhesion forces to a colloidal probe, while transcriptomic profiling revealed enrichment of adhesion and ECM-remodeling pathways in the adhesive metastatic state. We also find that lymphatically selected cells exhibit reduced adhesion on colloid-coated surfaces but higher particle uptake compared to the primary tumor cells. These results indicate that after leaving the primary tumor, metastatic cells have reduced adhesive potential, which is only regained upon reaching secondary sites. By exposing adhesion differences that are undetectable on flat substrates and linking them to particle uptake assays, this platform produces functional signatures of metastatic potential. This method is technically accessible, compatible with imaging and molecular workflows, and adaptable for high-throughput or clinical analysis, offering a potential route for label-free detection and classification of cancer cells by their aggressiveness.

Horizontal transfer of small non-conjugative plasmids is primarily attributed to transformation, transduction or comobilization with conjugative elements; however, transfer through intercellular membranous nanotube conduits can also occur. Here we show that nanotube-dependent plasmid exchange (NPex) operates bidirectionally between bacteria, enabling plasmid donation and, to a lesser extent, plasmid acquisition. We identified a Bacillus subtilis isolate, BSB1, deficient in NPex and show that a prophage-encoded factor, YokF, blocks plasmid transmission. YokF is an endonuclease that localizes to the membrane of donor bacteria, where it interacts with the nanotube component, FlhA, to impede plasmid transfer through DNA degradation. We further show that YokF provides an advantage to donor bacteria by restricting the sharing of beneficial plasmids with competing neighbouring cells. Bioinformatics and functional analyses revealed that YokF homologues are widespread across Gram-positive bacteria, representing a conserved family of gatekeepers that restrict plasmid flow via NPex.

Gestational diabetes mellitus (GDM) is defined as hyperglycemia first identified during pregnancy and can lead to adverse maternal and neonatal outcomes. The molecular mechanisms leading to these outcomes are currently poorly understood. While transcriptomics of GDM placentas has been previously studied, the effect on precursor mRNA splicing remains largely unknown. This study explores the impact of GDM on placental splicing and identifies its regulatory mechanisms. Using RNA sequencing data from Norwegian and Chinese cohorts, we uncovered thousands of differential splicing events. Pathway enrichment analysis revealed significant associations with metabolic and diabetes-related pathways. Splicing factor motif and cross-linking and immunoprecipitation sequencing analyses highlighted serine/arginine-rich splicing factor 10 (SRSF10) as a key regulator in this process, with its binding enriched at misspliced exons. Silencing SRSF10 in placental cells mirrored GDM-associated missplicing in key genes. These findings underscore splicing dysregulation as a critical process in GDM pathogenesis, suggesting that targeting SRSF10 could be a potential therapeutic approach to mitigate the deleterious effects of GDM. ARTICLE HIGHLIGHTS: Gestational diabetes mellitus (GDM) causes hyperglycemia during pregnancy and adverse maternal and neonatal outcomes. Bulk placental gene expression has been reported largely unchanged. RNA sequencing of Norwegian and Chinese GDM placentas reveals hundreds of differential splicing events enriched for metabolic- and diabetes-related pathways. Motif enrichment and cross-linking and immunoprecipitation sequencing integration identify serine/arginine splicing factor 10 as a key regulator of GDM-associated missplicing. Silencing serine/arginine splicing factor 10 in placental models recapitulates the GDM-associated missplicing program.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst formation, inflammation, and metabolic dysregulation. The endocannabinoid system (ECS), particularly the cannabinoid-1 receptor (CB(1)R), regulates renal metabolism and inflammatory signaling, yet its role in ADPKD remains largely unexplored. METHODS: We analyzed publicly available human kidney transcriptomic datasets (bulk microarray GSE7869; single-nucleus RNA-sequencing from ADPKD GSE185948, and diabetic kidney disease cohorts GSE195460) and validated findings in ADPKD patient kidney tissue versus non-cystic controls using quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blotting. Longitudinal disease progression was evaluated in Pkd1(RC/RC) mice at 3, 6, 9, and 12 months, with comprehensive assessment of ECS components, endocannabinoid (eCB) levels, and kidney function parameters. Correlation examined associations between ECS markers and disease severity. RESULTS: Human ADPKD kidneys demonstrated consistent upregulation of CNR1 transcripts across platforms, with single-nucleus analysis revealing enrichment in proximal tubule-derived populations including failed-repair proximal tubule cells. ADPKD tissue exhibited significant reductions in key ECS-metabolizing enzymes (FAAH, NAPEPLD, MGLL) and marked depletion of eCB ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG). In contrast, diabetic kidney disease showed minimal ECS alterations, indicating ADPKD-specific dysregulation. Pkd1(RC/RC) mice recapitulated human findings, with Cnr1 upregulation beginning at 6 months and significant AEA/N-oleoylethanolamine (OEA) depletion at 9–12 months. CB(1)R protein elevation preceded ligand depletion, suggesting progressive receptor sensitization. Correlation analyses revealed robust associations between CB(1)R/enzyme expression, eCB depletion, and declining kidney function (kidney weight-to-body weight ratio, blood urea nitrogen, and creatinine clearance). CONCLUSIONS: ADPKD kidneys exhibit disease-specific dysregulation of the ECS, characterized by increased CB(1)R expression accompanied by paradoxical depletion of eCB ligands. These alterations correlate with cyst burden and functional decline across human and murine disease stages, identifying the ECS as a prominently affected pathway during ADPKD progression. While our findings establish a strong association between ECS dysregulation and disease severity, whether altered CB(1)R signaling represents a causal driver of cystogenesis or a secondary, yet therapeutically targetable component of the cystic and injury response will require direct genetic or pharmacologic modulation of CB(1)R/ECS signaling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10020-026-01457-w.

IGF2BP1 is an oncofoetal RNA binding protein (RBP) expressed in many tumors. Interest has focused of late on the role of RBPs in cancer, although their mechanism of action is not always well understood. Using a newly described small molecule inhibitor of IGF2BP1, termed AVJ16, we have analyzed the effects of this inhibition on RNA binding, RNA expression, and protein expression. AVJ16 treatment downregulates RNAs encoding members of several pro-oncogenic signaling pathways, including Hedgehog, Wnt, and PI3K-Akt, and there is a strong correlation between IGF2BP1 RNA binding, RNA expression, and protein expression. At the cellular level, colony formation, invasion, and spheroid growth are all strongly reduced by exposure to AVJ16, while apoptosis and cell death are enhanced. All of these effects are limited to cells expressing IGF2BP1. In syngeneic LUAD xenografts in mice, IP injection of AVJ16 prevents tumor growth, and incubation with AVJ16 induces cell death in human organoids derived from IGF2BP1-expressing LUADs but not from healthy lung tissue. These results demonstrate that AVJ16 is a promising candidate for targeted therapy directed against tumors expressing IGF2BP1.

BACKGROUND AND AIMS: Interleukin-6 (IL-6) performs multiple roles in regulating metabolic pathways in both mice and man. Here, we examined the age-dependent metabolic phenotype of SGP mice-mice overexpressing sgp130, a factor that specifically blocks IL-6 trans-signaling-that were housed in distant vivaria. METHODS: Transgenic SGP mice engineered to block IL-6 trans-signaling and wild-type littermates were raised in a Jerusalem animal facility to up to 14 months of age and assessed for weight gain, body composition, and metabolic determinants of energy expenditure in young versus aged mice. Proteomic and RNA-seq analyses were performed on liver samples as a function of age and genotype. RESULTS: At ∼6 months of age, weight gain, body fat accumulation, hepatosteatosis, hyperglycemia, and macrophage recruitment to adipose tissue emerged and progressed with age in SGP mice maintained in the Jerusalem animal facility, but not in 3 other vivaria. IL-6/sIL-6R blockade strongly reduced signal transducer and activator of transcription 3 phosphorylation in the liver, and hepatocyte-targeted ablation of signal transducer and activator of transcription 3 recapitulated the IL-6 trans-signaling blockade phenotype. Multiomics analyses of mouse livers revealed age- and genotype-related changes in gene expression profiles attributable to bacterial byproducts. Depletion of the gut microbiota by antibiotic treatment from the age of 6 months reversed the obese phenotype in transgenic mice, confirming the crucial role of the microbiome in the phenotype. Accordingly, the microbiome of mice from the Jerusalem animal facility differed significantly from that of mice from animal facilities in Kiel and Hamburg, Germany, where the same mice did not develop a metabolic phenotype. CONCLUSION: These findings reveal the crucial functions of IL-6 trans-signaling in preventing mature-onset body fat accumulation induced by certain intestinal microbiota.

BACKGROUND: Core pathology in Alzheimer's disease (AD) includes amyloid-β (Aβ) deposition, gliosis, and eventual neuronal loss. Depression during midlife increases the risk of developing AD at late life. Late-life depression is highly prevalent among AD patients, but its role in AD pathogenesis is unclear, and specifically whether it pushes the brain with established AD pathology towards degeneration. CNS myeloid cells (Microglia and CNS-associated macrophages) clear Aβ early on; however, in advanced disease stages, they adopt a neurotoxic phenotype that exacerbates neurodegeneration. It is unclear whether and how stress and depression influence CNS myeloid cells' dysfunction in AD and the neurodegenerative process. METHODS: To investigate the impact of chronic stress on microglial function and on neurodegeneration, we utilized the 5xFAD mouse model, which exhibits extensive Aβ pathology but no neuronal loss at age 7 months, representing a late preclinical AD stage. We used a six-week chronic mild stress (CMS) paradigm to induce depressive behavior, after which CNS myeloid cell activation state was evaluated by transcriptomic analysis, activation marker expression and oxidation function. Neuronal and microglial densities were assessed histologically. RESULTS: Transcriptomic analysis of freshly isolated CNS myeloid cells showed a basal hyper-activated state in non-stressed 5xFAD mice, whereas CMS suppressed multiple immunologic and metabolic pathways. CMS reduced CD68 expression and reduced oxidative function in CNS myeloid cells. CMS did not induce neurodegeneration in the (behaviorally-relevant) pre-frontal, primary motor, hippocampal and Amygdalar cortices in 5xFAD mice. Rather, CMS protected these regions from microglia-mediated neurodegeneration, caused by a microbial TLR2 agonist. CONCLUSION: Chronic stress and depression attenuate CNS myeloid cells. While this has been shown to promote amyloid pathology at early stages, similar attenuation of CNS myeloid cells at the stage of established AD pathology may interfere with their transition into fully neurotoxic microglia, which cause neurodegeneration. These findings highlight the importance of tailoring microglial-targeted therapies to the stage-dependent roles of these cells during AD progression.

Bone fracture healing requires coordinated interactions between immune cells and skeletal tissues, with flat bones exhibiting unique biomechanical and physiologic characteristics as compared with long bones. The mandible, the lower jawbone that supports the oral cavity, has distinct features due to its proximity to the oral mucosa, which is enriched in immune cells and microbiota, and its exposure to masticatory forces, making it a clinically relevant model for studying immune-skeletal interactions during fracture repair. Moreover, mandibular fractures are common maxillofacial injuries, posing challenges owing to functional and aesthetic considerations, as well as the risk of infection and impaired healing. Recent studies have highlighted conflicting roles for interleukin 17 (IL-17) and γδT cells in bone fracture or defect healing. As the primary source of IL-17 in the oral mucosa, γδT cells play a critical role in alveolar bone remodeling and respond to environmental factors such as the microbiota and age. We thus sought to investigate their role in the repair process of a mandibular defect. Here, we developed a murine model where a 1.5-mm drill hole defect spontaneously healed within 3 wk. Analysis revealed rapid leukocyte infiltration at the defect site by day 3, predominantly neutrophils and inflammatory monocytes, with γδT cells and adaptive leukocytes accumulating later at days 7 and 14. Depletion of γδT cells via Tcrd-GDL mice or genetic ablation of IL-17 in Il17af(-/-) mice accelerated the kinetics of mandibular healing. Bulk RNA sequencing and immunologic analysis revealed that while early recruitment of neutrophils and monocytes was unaffected in Il17af(-/-) mice, later inflammatory responses were diminished, resulting in accelerated repair. These findings suggest that IL-17-producing γδT cells (γδ17T cells) delay mandibular fracture repair by inhibiting inflammation resolution, thus prolonging the reparative phase. Targeting γδ17T cells or IL-17 may thus represent therapeutic strategies to enhance bone regeneration, particularly in challenging clinical settings involving mandibular fractures.

Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease afflict millions of individuals globally and are significant sources of disease mortality. While the molecular mechanisms underlying such diseases are unclear, environmental and social factors, such as cigarette smoke and obesity, increase the risk of disease development. Yet, not all smokers or obese individuals will develop chronic respiratory diseases. The mitogen-activated protein kinase p38α is abnormally active in such maladies, but its contribution, if any, to disease etiology is unknown. To assess whether p38α activation per se in the lung could impose disease symptoms, we generated a transgenic mouse model allowing controllable expression of an intrinsically active variant, p38α(D176A+F327S), specifically in lung alveolar type 2 pneumocytes. Sustained expression of p38α(D176A+F327S) did not appear to induce obvious pathological outcomes or to exacerbate inflammatory outcomes in mice challenged with common respiratory disease triggers. However, mice expressing p38α(D176A+F327S) in alveolar type 2 cells and fed with a high-fat diet exhibited increased numbers of airway eosinophils and lymphocytes, upregulated levels of proinflammatory cytokines and chemokines including interleukin-1β and eotaxin, as well as a reduction in levels of leptin and adiponectin within the lung. Neither high-fat diet nor p38α(D176A+F327S) alone induced such outcomes. Perhaps in obese individuals with associated respiratory diseases, elevated p38α activity which happens to occur is the factor that promotes their development.

BACKGROUND: Bacillus Calmette-Guerin (BCG) therapy is an established immunotherapy for non-muscle invasive bladder cancer (NMIBC); however, the response variability of patients remains a challenge, necessitating insight into immune cell function. Previous studies established that a preexisting Th2 immune microenvironment correlates with a positive BCG therapy outcome. Therefore, in this study, we explored the role of mast cells (MCs) and eosinophils in bladder cancer as a potential predicting tool for BCG immunotherapy response. METHODS: We investigated the effect of MCs and eosinophils on bladder cancer cell viability together with their chemotactic migration towards cancer cells in vitro. The effect of BCG on these immune cells was also evaluated. Moreover, we performed an orthotopic model of bladder cancer in MC- and eosinophil-deficient mice. Finally, to evaluate whether these immune cells predict the therapy response, 26 patient biopsies pre-BCG treatment were analyzed for MC and eosinophil numbers in the tissue and sequenced for gene expression. RESULTS: Eosinophils, but not MCs, reduced bladder cancer cell viability in vitro and inhibited tumor growth in vivo. However, addition of BCG did not increase these effects in vitro. Patient biopsy analysis and mRNA sequencing showed that neither cell type predicted long-term therapy responsiveness. Gene expression analysis suggested that extracellular matrix and epithelial-to-mesenchymal transition factors could influence BCG therapy outcomes. CONCLUSION: Even though eosinophils exhibit anti-tumorigenic effects in bladder cancer, neither MCs nor eosinophils were predictive of the long-term BCG therapy response. However, our findings implicate that matrix-related factors may modulate BCG therapy responses.

BACKGROUND: ATAD3A, a nuclear gene encoding the ATAD3A protein, has diverse roles in mitochondrial processes, encompassing mitochondrial dynamics, mitochondrial DNA maintenance, metabolic pathways and inter-organellar interactions. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. Yet, gaps in knowledge remain regarding the underlying pathogenesis. METHODS: To further investigate the gene function and its implication in health and disease, we utilized CRISPR/Cas9 genome editing to generate a knockout model of the zebrafish ortholog gene, atad3. We characterized the phenotype of the null model, performed mitochondrial and functional tests, and compared the transcriptome of null embryos to their healthy siblings. RESULTS: Analysis of atad3-null zebrafish embryos revealed microcephaly, small eyes, pericardial edema and musculature thinning, closely mirroring the human rare disease phenotype. Larvae exhibited delayed hatching and embryonic lethality by 13 days post-fertilization (dpf). Locomotor activity, ATP content, mitochondrial content, and mitochondrial activity were all reduced in the mutant embryos. Transcriptome analysis at 3 dpf via RNA-sequencing indicated decline in most mitochondrial pathways, accompanied by a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was corroborated in fibroblasts from an affected individual. CONCLUSIONS: The atad3-null zebrafish model emerges as a reliable representation of human ATAD3A-associated disorders, with similarities in differentially expressed pathways and processes. Furthermore, our study underscores mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATAD3A-associated disorders and identifies potential readouts for therapeutic studies.

Triple-negative breast cancer (TNBC) is an aggressive subtype that accounts for 10-15 % of breast cancer. Current treatment of high-risk early-stage TNBC includes neoadjuvant chemo-immune therapy. However, the substantial variation in immune response prompts an urgent need for new immune-targeting agents. This requires a comprehensive understanding of TNBC's tumor microenvironment. We recently demonstrated that Galectin-3 (Gal-3) binding protein/Gal-3 complex secreted by TNBC cells induces immunosuppression, through inhibiting CD45 signaling in T cells. Here, we further investigated the interaction between secreted Gal-3 and T cells in TNBC. Using CRISPR/Cas9 gene editing of the TNBC MDA-MB-231 cell-line, we obtained Gal-3 negative((neg)) clones. We studied these in an in-vitro model, co-cultured with peripheral blood mononuclear cells (PBMC) to imitate immune-tumor interaction, and in an in-vivo model, when implanted in mice. Gal-3(neg) tumors in mice had decelerated tumor growth after PBMC inoculation. In contrast, the Gal-3 positive((pos)) tumors continued growing despite PBMC inoculation, and tumor T regulatory cell (CD4/FoxP3+) infiltration increased. RNA sequencing of T cells from women with TNBC with elevated plasma levels of Gal-3 revealed significantly lower expression of oxidative phosphorylation genes than in T cells from healthy women. Similarly, in our in-vitro model, the decreased expression of oxidative phosphorylation genes and mitochondrial dysfunction resulted in a significant increase in CD8 intracellular reactive oxygen species. Consequently, T exhausted cells (CD8/PD1/Tim3/Lag3+) significantly increased in PBMC co-cultured with Gal-3(pos) TNBCs. To conclude, we revealed a novel TNBC-related Gal-3 suppressor mechanism that involved upregulation of CD4 T regulatory and of CD8 T exhausted cells.

BACKGROUND: Chicken embryos emerge from their shell by the piercing movement of the hatching muscle. Although considered a key player during hatching, with activity that imposes a substantial metabolic demand, data are still limited. The study provides a bioenergetic and transcriptomic analyses during the pre-post-hatching period. METHODS: Weight and morphology alongside content determination of creatine and glycogen were analysed. Transcriptome identified differentially expressed genes and enriched biological processes associated with hatching muscle development, catabolism, and energy provision. Using gene set enrichment, we followed the dynamics of gene-sets involved in energy pathways of oxidative phosphorylation, protein catabolism, glycolysis/gluconeogenesis, and glycogen metabolism. RESULTS: Results show several significant findings: (A) Creatine plays a crucial role in the energy metabolism of the hatching muscle, with its concentration remaining stable while glycogen concentration is depleted at hatch and placement. (B) The hatching muscle has the capacity for de-novo creatine synthesis, as indicated by the expression of related genes (AGAT, GAMT). (C) Transcriptome provided insights into genes related to energy pathways under conditions of pre-hatch oxygen and post-hatch glucose limitations (oxidative phosphorylation: NDUF, MT-ND, SDH, UQCR, COX, MT-CO, ATP5, MT-ATP; glycolysis/gluconeogenesis: FBP,G6PC, PFKM; glycogen metabolism: PPP1, PYGL, GYG1). (D) The post-hatch upregulation of protein catabolic processes genes (PSMA, RNF, UBE, FBX), which align with the muscle's weight dynamics, indicates a functional shift from movement during hatching to lifting the head during feeding. CONCLUSIONS: There is a dynamic metabolic switch in the hatching muscle during embryo-to-hatchling transition. When glycogen concentration depletes, energy supply is maintained by creatine and its de-novo synthesis. Understanding the hatching muscle's energy dynamics is crucial, for reducing hatching failures in endangered avian species, and in domesticated chickens.

In mucosal barriers, tissue cells and leukocytes collaborate to form specialized niches that support host-microbiome symbiosis. Understanding the spatial organization of these barriers is crucial for elucidating the mechanisms underlying health and disease. The gingiva, a unique mucosal barrier with significant health implications, exhibits intricate tissue architecture and likely contains specialized immunological regions. Through spatial transcriptomic analysis, this study reveals distinct immunological characteristics between the buccal and palate regions of the murine gingiva, impacting natural alveolar bone loss. The microbiota primarily affects gingival immunity in the buccal region. Additionally, a significant influence of the microbiota on the junctional epithelium facing the oral biofilm offers new insights into neutrophil recruitment. The microbiota also regulates the proliferation and barrier-sealing function of the gingival epithelium. This underscores the presence of immunological niches in the gingiva, with the microbiota differentially influencing them, highlighting the high complexity of this oral mucosal barrier.