Molecular Medicine

Abstract Diabetic retinopathy (DR) is a major cause of vision loss in working-age adults. Accumulating evidence suggests that retinal photoreceptors contribute to the initiation and progression of diabetic retinopathy. In this study, we investigated whether hyperglycemia directly alters photoreceptor signaling and whether photoreceptor-derived inflammatory mediators activate downstream Muller glial cells. Primary photoreceptors were isolated from adult mice and cultured with normal glucose, high D-glucose, or high L-glucose as an osmotic control. Photoreceptorconditioned media were analyzed for inflammatory and growth factors and used to stimulate primary Muller glia. High glucose exposure increased photoreceptor production of TNF-a, IL-6, and VEGF. Photoreceptorconditioned media from high glucose-treated photoreceptors induced Muller glial expression of IL-1b, TNF-a, and IL-6. Muller glia exposed to photoreceptor-conditioned media increased VEGF expression and secretion and enhanced MMP-9 expression, secretion, and gelatinase activity. Together, these findings support a direct role for photoreceptors as glucose-responsive neuronal cells that can initiate and amplify neuroglial inflammatory signaling in hyperglycemic conditions.

Human tissue transcriptomic studies frequently rely on biopsies obtained during surgery under general anesthesia, yet the impact of perioperative medications on gene expression has not been systematically evaluated. Using paired liver biopsies from patients with obesity at risk for metabolic dysfunction-associated steatohepatitis (MASH), we compared RNA sequencing profiles from biopsies collected under local anesthesia months before surgery with matched intraoperative biopsies obtained under general anesthesia. Intraoperative samples exhibited striking transcriptional changes enriched for canonical glucocorticoid receptor target genes, implicating perioperative dexamethasone administration as the principal driver. In a matched cohort in which dexamethasone was administered immediately after biopsy collection, we directly demonstrate that dexamethasone profoundly alters hepatic gene expression during surgery. These findings were independently validated and shown to substantially distort associations between liver fibrosis stage and inflammatory gene expression, obscuring biologically relevant immune signatures. Similar glucocorticoid-responsive transcriptional changes were observed in adipose tissue, indicating a systemic effect. Our findings identify perioperative dexamethasone exposure as a previously unrecognized but readily modifiable confounder in human tissue transcriptomics. As biopsy-based bulk and single-cell transcriptomic studies continue to expand, careful documentation and consideration of perioperative medication exposure will be essential to improve the reproducibility, interpretability, and biological validity of human gene expression studies.

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

A population-specific missense variant, rs373863828 (G > A, p.Arg457Gln), has been identified in Pacific Islanders and is associated with increased body mass index and lower odds of type 2 diabetes. However, the mechanism underlying these associations is unknown. The variant is located in exon 5 of CREBRF, a gene which is rapidly upregulated in response to nutrient deprivation. This study aimed to assess the effect of rs373863828 on gene expression in adipose tissue from Samoan adults from the Soifua Manuia (''Good Health'') study. Needle subcutaneous adipose (upper buttock) biopsies were obtained in Apia, Samoa, from fasting Samoans without diabetes. RNA isolated from adipose tissue (n=91, 65.9% female) was subjected to bulk paired-end mRNA-sequencing (2x150 base pair). Differential gene expression among the three rs373863828 genotypes (GG, AG, AA) was assessed using a likelihood-ratio test implemented in DESeq2, and overrepresentation analysis for significant differentially expressed genes (DEGs) (padj < 0.1) was performed with clusterProfiler and oRG.DB. Bulk expression was deconvoluted to determine the cell type composition of the adipose tissue. Twenty-two DEGs were associated with rs373863828 genotype including CBX7 (padj=0.026 ) and SELENOM (padj= 0.004), both of which have roles in adipogenesis and metabolism. A single expression profile comprising 13 of the 22 DEGs corresponding to CREBRF genotype was identified, and overrepresentation analysis supported this finding, with terms such as ''keratinocyte differentiation'' and ''skin development'' enriched among significant DEGs. However, PERMANOVA of adipose cell type proportions showed no difference (p > 0.05) among the three genotypes. rs373863828 genotype was associated with expression of 22 genes in whole subcutaneous adipose tissue of this sample of fasting Samoan adults without diabetes. Future work should assess cell- and tissue-specific rs373863828 genotype effects using a combination of single-cell approaches and human cell models with perturbations associated with nutritional stress.

Retinal organoids (ROs) model human retinogenesis but remain avascular and lack immune components, limiting their utility for studying vascular retinopathies. We generated vascular organoids (VOrs) from human pluripotent stem cells and evaluated their integration into developing ROs. Dissociated VOr cells (day 12 or day 30) were seeded onto 2D retinal neuroepithelium before 3D transition, followed by long-term culture to day 210. VOr derived cells migrated into ROs and transiently persisted. V30 ROs preserved retinal lamination, major neuronal subtypes and light evoked retinal ganglion cell responses comparable to non vascularised controls, whereas media-only ROs cultured in proangiogenic conditions showed structural disorganisation and reduced neuronal survival. Single-cell RNA sequencing revealed an endogenous, inflammatory endothelial-like population with partial endothelial-to-mesenchymal features and a perivascular fibroblast-like cluster in pro-angiogenic media only RO controls. The endothelial-like cell cluster was significantly enriched for genes upregulated in human retinal endothelial cells exposed to high glucose, indicating a diabetic retinopathy like transcriptional state. Microglia like and border associated macrophage populations were present across all conditions, with developmental shifts in morphology and marker expression. Our findings define conditions under which vascular cells can be incorporated into human ROs without disrupting retinal architecture, while uncovering an intrinsic capacity for inflammatory endothelial activation that partially recapitulates diabetic retinopathy. This work advances the engineering of vascularised, immunocompetent retinal models and highlights endogenous vascular immune programmes that must be considered when modelling retinal disease in vitro.

Background Genome-wide association studies have identified >200 loci associated with IBD, yet the causal gene for most remains unknown. As multiple epithelial functions have been linked with susceptibility to IBD, there is a need to prioritize candidate causal genes for functional studies in this cellular context. Methods Using a standardized definition of regions implicated by index SNPs from three GWAS studies, we categorized regions as containing: (1) a known casual gene, (2) a single gene or (3) multiple genes. We then developed an IBD Priority Score to rank genes based on genetic, genomic and functional data. We next developed and applied an Epithelial Priority Score, based on expression patterns and quantitative traits, to prioritize genes for functional validation in epithelial models. Two candidate genes identified through this approach were tested for their impact on viral response pathways in HT-29 cells. Results The IBD Priority Score prioritized a single gene in 71 of the 104 regions containing multiple genes. The Epithelial Priority Score identified 31 epithelial candidates. Functional studies demonstrated that IRF6 enhanced, whereas IRF8 suppressed, antiviral responses in intestinal epithelial cells stimulated with Poly(I:C). Conclusions Combining multiple genetic, genomic, and functional data is a useful approach for prioritizing the most likely causal gene within IBD GWAS loci, and for prioritizing functional validation studies in epithelial cells and tissues. Moreover, we provide functional evidence for two IBD genes playing a role in the regulation of anti-viral responses in intestinal epithelial cells.

BackgroundKnee osteoarthritis (KOA) is a prevalent degenerative joint disease in which synovial inflammation and fibrosis are closely linked to pain, stiffness, and functional limitation. Growing evidence suggests that metabolic dysregulation, particularly in lipid metabolism, is involved in KOA pathogenesis, but the underlying mechanisms remain incompletely defined. MethodsSprague Dawley rats underwent bilateral anterior cruciate ligament transection to establish a KOA model; sham-operated rats served as controls. RNA sequencing of synovial tissues was performed to identify differentially expressed genes (DEGs) and enriched pathways, followed by GO/KEGG and GSEA analyses. In vivo, adeno-associated virus vectors were used to overexpress or knock down PPAR-{gamma} and phosphoenolpyruvate carboxykinase 1 (PCK1) via intra-articular injection. Ex vivo, primary rat fibroblast-like synoviocytes (FLSs) were stimulated with IL-1{beta} and transfected with PPAR-{gamma} or PCK1 siRNA/overexpression plasmids. synovitis and fibrosis were evaluated by HE, Masson, and Sirius Red staining, immunofluorescence, ELISA, RT-qPCR, and Western blotting. ResultsRNA-seq revealed 621 up-regulated and 228 down-regulated genes in KOA synovium versus sham, with DEGs significantly enriched in PPAR signaling, adipocytokine, and AMPK pathways. Metabolism-related genes including Fabp5, Plin1, Adipoq, Lep, and Pck1 were up-regulated. GSEA indicated downregulation of PPAR-{gamma} signaling in KOA synovium. In vivo and ex vivo, PPAR-{gamma} expression was reduced in KOA, whereas PCK1, FABP5, and ADIPOQ were increased. PPAR-{gamma} overexpression alleviated synovial inflammation, collagen I deposition, and fibrosis, and suppressed FABP5, ADIPOQ, and PCK1 expression; PPAR-{gamma} knockdown produced the opposite effects. Functional studies showed that PCK1 overexpression aggravated synovial inflammatory cell infiltration and fibrosis, elevated IL-1{beta}, IL-18, and TGF-{beta}, and decreased TIMP1 levels in serum, synovial tissue, and FLSs supernatants, whereas PCK1 silencing reversed these changes. ConclusionsThe PPAR-{gamma}/PCK1 metabolic axis modulates synovitis and fibrosis in KOA. Downregulation of PPAR-{gamma} and consequent upregulation of PCK1 promote synovitis and fibrotic remodeling. These findings identify the PPAR-{gamma}/PCK1 pathway as a potential therapeutic target for KOA.

Inflammatory bowel disease (IBD) often involves disrupted intestinal epithelial barrier, but therapies specifically targeting this barrier are limited. We found that downregulated AUF1 (HNRNPD) contributes to defective barrier integrity in ulcerative colitis (UC). Compared to controls, its expression level was decreased and inversely correlated with clinical severity. Knocking down AUF1 in human and mouse colonic organoids led to impaired barrier function, with reduced Occludin and upregulated Claudin-2, mimicking characteristic IBD-associated mucosal alterations. Distinct RNA-binding activity of AUF1 protein isoforms contributed to these changes: p37 stabilized Occludin mRNA and blocked microRNA-122/Ago2-mediated repression, whereas p40 promoted Claudin-2 mRNA degradation via ubiquitin-proteasome pathway. Restoring AUF1 expression in organoids enhanced epithelial properties and, when transplanted into mice with established colitis, accelerated mucosal healing and epithelial regeneration in recipient mice and decreased fibrosis. Our study unravelled a post-transcriptional mechanism important for intestinal homeostasis and demonstrated a concept of using engineered organoids for treating IBD.

Peripheral neurons exhibit robust regeneration after nerve injury in adulthood; a process linked to the activation of regeneration-associated genes (RAGs). However, the mechanisms underlying nerve regeneration during early postnatal development remain poorly understood. In a previous work, we observed that motoneurons axotomized at early postnatal stages regenerate without overexpressing RAGs, contrasting with the expected RAG activation observed in postnatal day 30 (P30) mice. To determine whether this phenomenon extends to sensory neurones, we investigated two paradigmatic dorsal root ganglia populations: proprioceptive (PV) and nociceptive (TrpV1) neurones, which exhibit distinct regenerative capacities in adult mice. Following sciatic nerve crush injury at P4, P10, and P30, both neuronal subtypes demonstrated robust axonal regeneration across all postnatal stages, with nociceptors exhibiting significantly superior regeneration compared to proprioceptors. Remarkably, no substantial neuronal death was observed at any developmental timepoint. However, RNA sequencing revealed that P4 and P10 sensory neurones failed to upregulate RAGs following injury, whereas P30 neurones exhibited the expected pro-regenerative transcriptional programme. Pathway enrichment analysis confirmed that classical regeneration-associated signalling pathways were activated exclusively in young adults, whilst developmental and inflammatory pathways predominated in early postnatal stages. Using the conditioning lesion paradigm in primary dorsal root ganglia cultures, we demonstrated that only P30 neurones exhibited enhanced neurite outgrowth following conditioning lesion, whilst P4 and P10 neurones showed no conditioning effect or reduced outgrowth, respectively. These findings indicate that early postnatal sensory neurones possess intrinsic growth capacity independent of RAG activation, suggesting fundamentally distinct regenerative mechanisms across developmental stages.

Purpose: Retinal ganglion cell (RGC) death following optic nerve injury is driven by transcriptional programs involved in apoptosis, neuroinflammation, and other regulated cell death pathways; however, the early transcriptomic changes initiating these processes remain poorly defined. We used rRNA-depleted total RNA sequencing to characterize early alterations in protein-coding genes, long noncoding RNAs (lncRNAs), and alternative splicing events in mouse retinas following optic nerve crush (ONC) and silicone oil-induced ocular hypertension under-detected (SOHU), prior to substantial RGC loss. Methods: Whole retinas were collected 6 hours after ONC and 5 days after SOHU induction. Differentially expressed protein-coding genes and lncRNA-classified transcripts were identified using total RNA sequencing, followed by functional enrichment, lncRNA-associated pathway analysis, alternative splicing analysis, and fluorescent in situ hybridization (FISH) validation. Results: In SOHU retinas, hundreds of protein-coding genes and lncRNA-classified transcripts were differentially expressed, with enrichment of immune activation, extracellular matrix remodeling, complement, phagocytic signaling, and suppression of mitochondrial energy metabolism pathways. Similarly, in ONC retinas, many protein-coding genes and lncRNA-classified transcripts were differentially expressed, with a prominent chemokine, cytokine-dominant inflammatory response. FISH validation supported injury-associated upregulation of Prss56 and Lif. Both injury models exhibited substantial transcript-level alterations, with widespread alternative splicing changes detected. Conclusions: Early optic nerve injury induces rapid transcriptional changes involving protein-coding genes, lncRNAs, and transcript isoform regulation before substantial RGC loss. These findings highlight early alterations in both coding and noncoding RNA landscapes following retinal injury and provide a valuable resource of candidate genes and pathways for future validation in RGC degeneration and neuroprotection.

The periodontal ligament (PDL) is a mechanically active connective tissue whose fibroblasts and resident progenitor cells drive tooth support, remodeling, and regeneration. Although PDL cell populations are known to be heterogeneous, the composition of expanded primary PDL cultures and the extent to which anatomical origin shapes their transcriptome remain incompletely defined. Here we applied single-cell RNA sequencing to cultured primary PDL cells isolated from matched upper (maxilla) and lower (mandible) jaw sites of four donors, yielding 43,423 cells that were integrated across donors and analyzed by unsupervised clustering. The expanded cultures were uniformly mesenchymal and fibroblastic, essentially devoid of endothelial and immune signatures, and resolved into ten functionally distinct fibroblast substates rather than discrete cell types, including proliferating, collagen- and extracellular-matrix-high, contractile, and progenitor-like populations. Donor identity was the dominant source of transcriptional variation, with several substates restricted to individual donors. A donor-controlled comparison of maxilla versus mandible revealed only a small set of differentially expressed genes, headed by craniofacial positional-identity transcription factors (PITX1 higher in the mandible; BARX1, ALX1, and NKX6-1 higher in the maxilla), indicating that cultured PDL cells retain embryonic positional memory. In contrast, curated wound-healing gene modules did not differ significantly between jaws, and the transcriptional data therefore did not support the clinical impression of faster healing in the maxilla, suggesting that the healing asymmetry more likely reflects vascular, mechanical, and inflammatory signaling factors acting beyond the steady-state transcriptome. These findings characterize the cellular composition of expanded PDL cultures and identify positional identity as the principal jaw-dependent transcriptional feature.

Immune checkpoint inhibitors (ICIs) have transformed the treatment landscape of advanced renal cell carcinoma (RCC), yet only a subset of patients derives durable clinical benefit and no robust predictive biomarkers have been implemented in routine clinical practice. As T-cell receptor (TCR)-mediated antigen recognition underlies effective antitumor immunity, characterization of the intratumoral TCR repertoire may provide clinically relevant information for patient stratification. Here, we evaluated the predictive value of the baseline intratumoral TCR{beta} and TCR{gamma} repertoires in a real-world cohort of patients with advanced RCC treated predominantly with first-line immune checkpoint inhibitor-based regimens. Responders exhibited a significantly more even TCR{beta} repertoire together with increased TCR{gamma} diversity and clonal richness, whereas non-responders showed greater TCR{gamma} clonal dominance. In addition, the preferential usage of specific TRBV genes, including TRBV5.7, TRBV7.1, and TRBV18, was associated with clinical response and longer progression-free survival. In this exploratory cohort, integration of the most informative TCR{beta} and TCR{gamma} variables into a combined random forest model yielded an area under the receiver operating characteristic curve of 0.90 in leave-one-out cross-validation, with 100% sensitivity and 81.8% specificity at the selected threshold. To our knowledge, this is the first study to simultaneously characterize baseline intratumoral TCR{beta} and TCR{gamma} repertoires in patients with advanced RCC predominantly receiving first-line immune checkpoint blockade. These findings support baseline intratumoral TCR repertoire features as candidate tissue biomarkers for pretreatment patient stratification and provide a rationale for the prospective validation of TCR repertoire profiling as a predictor of immunotherapy benefit in RCC.

Both severe (Hurler syndrome; MPS IH) and attenuated (Hurler-Scheie or Scheie syndrome; MPS IA) forms of mucopolysaccharidosis I (MPS I) arise from the same underlying enzyme deficiency; however, they differ in the onset, persistence, and severity of key clinical features. These include skeletal abnormalities, joint contractures, cardiac disease, and neurocognitive and neurobehavioral impairment, which is not fully alleviated with either enzyme replacement (ERT) or hematopoietic cell transplantation (HCT). The objective of this study was to identify metabolic differences between MPS IH and MPS IA that could result in meaningful biomarkers and targeted adjunctive therapies to address unmet clinical needs in MPS IH treated with HCT. We performed plasma metabolomics in patients with MPS IH treated with HCT (N=17) or MPS IA treated with ERT (N=8). Welchs two-sample t-test was used to identify metabolites that differed significantly between groups. Statistical significance was evaluated based on p<0.05. After controlling for multiple testing, we used a false discovery rate of q<0.05. We identified 125 compounds that were significantly different between MPS IH and MPS IA. Of those metabolites, 14 had a q<0.05. Individuals with MPS IH had increased metabolites in the sphingolipid, beta-oxidation, amino acid catabolism, and glycosaminoglycan pathways compared to MPS IA. Persistent metabolic differences were observed in MPS IH treated with HCT compared to MPS IA treated with ERT, pointing towards additional biological processes that may contribute to disease progression in MPS IH after HCT. Although these results require further confirmation, they provide a foundation to guide future investigations towards potential biomarkers or targeted adjunctive therapies.

Objective: Kindlin-2, encoded by FERMT2, is a focal adhesion protein essential for cartilage homeostasis and mechanotransduction. While loss of Kindlin-2 in mice induces osteoarthritis (OA)-like pathology, the upstream mechanisms regulating its expression in human cartilage remain unknown. Methods: A bioinformatics pipeline was applied to the FERMT2 promoter to predict transcriptional regulators, followed by network and enrichment analyses. Predicted candidates were validated by siRNA knockdown in the human C28/I2 chondrocyte cell line and in primary OA chondrocytes. Pathway enrichment highlighted hypoxia-related regulators, which were further tested by hypoxia mimetic treatment (IOX2) and culture under low oxygen (1% O2). Gene expression changes were quantified by qPCR, and effects on extracellular matrix (ECM) markers were assessed. Results: In silico analysis identified 21 candidate transcription factors, of which GATA1, MEF2A, and RBPJ were validated as regulators of FERMT2. Silencing of GATA1 and MEF2A reduced FERMT2 expression, whereas RBPJ knockdown increased FERMT2 in both C28/I2 cells and primary OA chondrocytes. However, RBPJ silencing also reduced ACAN and increased COL1A1, suggesting detrimental effects on ECM homeostasis. Enrichment analysis revealed a strong association between FERMT2 regulation and hypoxia pathways, supported by conserved hypoxia response elements in the promoter. Experimentally, both IOX2 and 1% O2 significantly upregulated FERMT2 expression in primary chondrocytes. Hypoxia also increased anabolic ECM genes and reduced catabolic enzymes, but these effects occurred independently of Kindlin-2. Conclusions: This study identifies hypoxia as a novel regulator of kindlin-2 expression in human cartilage, providing new insight into the upstream control of this molecule in osteoarthritis. While additional transcription factors such as GATA1, MEF2A, and RBPJ contribute to FERMT2 regulation, only hypoxia consistently enhanced FERMT2 expression without adverse ECM effects.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Visceral white adipose tissue (vAT) is more susceptible than subcutaneous white adipose tissue (scAT) to obesity-associated metaflammation, but the metabolic pathways accompanying this depot specific transition remain incompletely defined. We profiled vAT and scAT from mice fed a control diet or a 60% kcal high-fat diet (HFD) for 1 or 8 weeks. Untargeted metabolomics was integrated with targeted endocannabinoid quantification, gene expression and promoter associated chromatin analyses, adipocyte and stromal vascular fraction (SVF) measurements, cross-omics correlation analysis, and cell-culture perturbations. After 1 week, HFD induced relatively few metabolic changes, although vAT showed early alterations associated with sphingomyelin metabolism. By week 8, both depots exhibited extensive but distinct metabolic remodeling. The KEGG retrograde endocannabinoid-signaling pathway was the highest-ranked enriched pathway in vAT, with arachidonoylglycerol (2AG) among the most strongly altered metabolites. Targeted analysis confirmed selective 2AG accumulation in vAT, accompanied by coordinated changes in genes and promoter-associated chromatin marks related to endocannabinoid synthesis, degradation and receptor expression. Cellular fractionation localized excess 2AG predominantly to vAT adipocytes, whereas Cnr2 was enriched in the SVF and induced by HFD specifically in vAT. Cross omics integration further revealed a denser and predominantly inverse transcript metabolite association network in vAT than in scAT. Using macrophage adipocyte co-culture and secretome profiling, we demonstrated that cannabinoid stimulation shifts activated macrophage outputs away from pro-inflammatory cytokines (Il1b, Ccl2) toward extracellular matrix (ECM) proteoglycan remodeling and intercellular signaling.

Objective: Toll like receptor (TLR) activation by cartilage derived damage-associated molecular patterns contributes to osteoarthritis (OA) pathogenesis, but the role of synovial fibroblasts in this process remains incompletely understood. We investigated the TLR responsiveness of human OA synovial fibroblasts and determined how TLR activation influences fibroblast-mediated regulation of chondrocyte function. Design: Primary synovial fibroblasts isolated from OA patients were characterized for TLR expression and stimulated with agonists targeting TLR1/2 to TLR9. Inflammatory mediator production, matrix metalloproteinase expression, mitochondrial respiration, and transcriptomic responses were assessed. Functional effects on cartilage homeostasis were evaluated using autologous chondrocyte spheroid co-cultures. Results: Approximately 50% of cells within OA synovial membranes were fibroblasts (CD45-CD31-PDPN+). Synovial fibroblasts expressed multiple TLRs at both the transcript and protein levels. Activation of TLR1/2, TLR4, TLR5, and TLR2/6 induced robust expression of IL-6, IL-8, G-CSF, MMP3, and MMP10, with minimal effects on mitochondrial respiratory function. Transcriptomic analysis revealed activation of inflammatory pathways together with enrichment of antigen presentation and protein translation programs. In autologous co-culture, TLR1/2-activated synovial fibroblasts promoted inflammatory and catabolic gene expression, suppressed anabolic gene expression, and impaired chondrocyte spheroid growth. Conclusions: Human OA synovial fibroblasts are highly responsive to TLR activation and acquire a pro-inflammatory, cartilage-degrading phenotype that directly impairs chondrocyte homeostasis. These findings identify synovial fibroblasts as key effectors of innate immune signalling within the OA joint and support targeting shared TLR-mediated pathways in joint-resident cells as a potential disease-modifying therapeutic strategy.

Abstract Rationale: Vagal sensory neurons are molecularly and functionally diverse, and they play critical roles in visceral sensation and homeostasis. The vagal system is anatomically divided into jugular and nodose ganglia, which exhibit distinct peripheral innervation patterns and physiological functions. Although recent transcriptomic studies have characterized nodose neuronal subtypes in detail, the molecular profiles of jugular sensory neurons remain poorly defined. Objectives: To define the molecular profiles of genetically identified jugular sensory neurons and determine the transcriptional responses of jugular and nodose airway afferents to allergic airway inflammation. Methods: We performed deep transcriptomic profiling of two genetically defined jugular nociceptor populations, MrgprC11 and MrgprD neurons, and compared them with airway-innervating P2ry1 nodose neurons and Phox2b nodose neurons. We further examined transcriptional changes of MrgprC11 and P2ry1 neurons following OVA-induced allergic airway inflammation. Measurements and Main Results: Transcriptomic analyses revealed distinct molecular signatures of jugular nociceptive neurons and identified subtype-specific genes across diverse functional categories. Following OVA challenge, MrgprC11 and P2ry1 neurons exhibited strikingly divergent transcriptional responses. More than 400 genes that were differentially expressed in both neuronal populations were regulated in opposite directions. GO enrichment analysis suggested that MrgprC11 jugular neurons adopted a metabolically restrained state with reduced translation and bioenergetic activities, whereas P2ry1 nodose neurons exhibited metabolically active state with enhanced translation and bioenergetic activities. Conclusions: These findings provide new insight into vagal sensory neuron diversity and reveal distinct transcriptional adaptations of jugular and nodose airway afferents during allergic inflammation.

Osteosarcoma (OS) is one of the top ranking and deadliest primary malignant bone tumor of youngsters and adolescents. OS has poor prognostic features due to its immunosuppressive "cold" tumor microenvironment, obstructing the anti-tumor effector functions of immune cells (including cytotoxic CD8 T lymphocytes). Recent developments have focused on the role of Gut Microbiota in modulating effector immune responses in various cancers. However, the immunomodulatory role of gut microbiota and its derived gut-associated metabolites (GAMs) in OS still remains unclear. Here we report that peripheral CD8 T lymphocytes in osteosarcoma patients are less frequent in circulation, hypo-producers of effector cytokines (IFN-{gamma} and TNF-), and have compromised metabolic fitness. We characterized and investigated the effect of a panel of microbiota-derived GAMs on CD8 T lymphocytes, confirming their immunomodulatory role with respect to CD8 T lymphocytes activation, cellular metabolism and effector functions. Indole-3-lactic acid (ILA; product of tryptophan metabolism), was observed to be the strongest immunostimulant among all the GAMs studied. ILA enhanced the anti-tumor effector functions of CD8 T lymphocytes through increased glucose uptake, a higher mitochondrial bio-mass and increased production of IFN-{gamma} and TNF-. Moreover, ILA-primed cytotoxic T lymphocytes exhibited considerably higher cytotoxicity and increased apoptosis of osteosarcoma cells (U2OS). Altogether, our data demonstrates that ILA acts as a microbial-derived immune modulator to metabolically reprogram and restore dysfunctional CD8 T lymphocytes against osteosarcoma. This study for the first time demonstrates the therapeutic potential of exploiting the nexus between "Gut-Immune-Bone Tumor" ternary as a safe and cost-effective combinatorial immunotherapy against osteosarcoma. Graphical AbstractIndole-3-Lactic Acid (ILA) Reinvigorates CD8+ T-Cell Immunity in Osteosarcoma Circulating CD8+ T cells from osteosarcoma patients exhibit impaired metabolic fitness, reduced effector cytokine production, and diminished tumoricidal activity. Screening of gut-associated metabolites identified the microbial tryptophan metabolite indole-3-lactic acid (ILA) as the most potent immunomodulator. ILA restores glucose uptake and mitochondrial biomass, enhances IFN-{gamma} and TNF- production, promotes polyfunctional CD8+ T-cell responses, and significantly improves CTL-mediated killing of osteosarcoma cells, highlighting its potential as a microbiota-derived immunometabolic therapeutic for osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/741694v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10416a4org.highwire.dtl.DTLVardef@16a99b8org.highwire.dtl.DTLVardef@19201cforg.highwire.dtl.DTLVardef@b4ca26_HPS_FORMAT_FIGEXP M_FIG C_FIG

Abstract Background Liposarcoma is a rare cancer, comprising four major histological subtypes: well-differentiated, dedifferentiated, myxoid and pleomorphic liposarcoma. Therapeutic options remain limited for advanced disease, and additional biomarkers that may support biological characterization, diagnosis, or risk stratification are needed. Methods Bulk RNA sequencing (RNA-seq) was performed on paired tumour and adjacent non-tumour tissues from an analysable subset of 16 patients (22 tumour specimens) within a clinicopathological cohort of 21 patients. Differential gene expression analysis, principal component analysis, gene set enrichment (GSEA) analysis and single-sample GSEA (ssGSEA) were used to define subtype-specific transcriptional programs and imprinted-gene dysregulation. PEG10 expression was validated by RT-qPCR and RNA fluorescence in situ hybridization (FISH), and its cellular localization was explored by combined FISH and immunofluorescence for P53, vimentin, Ki67, and CD34. PEG10 expression in liposarcoma was further investigated in an external single-cell RNA-sequencing dataset of dedifferentiated liposarcoma. An exploratory survival analysis was performed in the pan-sarcoma TCGA-SARC cohort. Results Transcriptomic profiling identified distinct subtype-specific gene expression patterns and broad dysregulation of imprinted genes in liposarcoma compared with adjacent non-tumour tissue. Among all the imprinted genes, PEG10 showed a marked tumour-to-normal expression difference and good discriminatory performance between tumour and normal tissues in the discovery cohort (AUC=0.867). In the TCGA sarcoma cohort high PEG10 expression was associated with shorter overall survival. Immuno-FISH and single-cell analyses revealed that PEG10 was predominantly detected in tumour and proliferating cell populations. Importantly, PEG10 expression was higher in high-grade tumours. In bulk RNA-seq data, PEG10 expression correlated with hypoxia, extracellular matrix remodelling and invasion ssGSEA signatures. In the external single-cell RNA-seq dataset PEG10high tumours showed a reduced immune cell infiltration. Conclusions This study provides an integrated transcriptomic characterisation of liposarcoma and identifies PEG10 as a candidate tumour-associated biomarker linked to aggressive disease features. The diagnostic and prognostic value of PEG10 requires validation in larger, independent, subtype-balanced liposarcoma cohorts, and its potential therapeutic relevance requires direct functional investigation.