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The zinc containing matrix metalloproteinase enzyme regulates a diverse array of biological processes in health and disease, including ADAM17 (a disintegrin and metalloproteinase domain 17) enzyme. Due to its large substrate profile, ADAM17 is known to regulate diverse pathways of inflammation and adaptive immunity. However, the role of ADAM17 in modulating the pathogenesis of type 2 allergic asthma is largely unknown. To determine the in vivo contribution of ADAM17 in house dust mite (HDM)-induced airway inflammation and adaptive immune response, we assessed the deletion of ADAM17 in mice conventional dendritic cells (ΔDC) and employed a complementary chemical biology approach using small-molecule novel ADAM17 inhibitor (2155-17). DC-specific ADAM17 ablation (ΔDC) suppressed type 2/ eosinophilic polarized HDM allergic responses and is protected from developing AHR. DC isolated from ΔDC mice showed a reduced state of metabolic activity, immune priming function and suppressed allergen-specific type 2 cell polarizations. Intranasal administration of 2155-17 protected WT mice against type2/ eosinophilic polarized HDM allergic responses. These concurrent results from two independent approaches identify a novel role for ADAM17 as an upstream site in airway inflammation. Furthermore, targeting ADAM17 with a selective small-molecule inhibitor might be harnessed as a potential drug target for type 2-high allergic asthma.

Bipolar disorder (BD) is increasingly recognized as a disease with both mitochondrial dysfunction and heightened inflammatory reactivity, yet contribution to neuronal activity remains unclear. To address these gaps, this study utilizes iPSC-derived cerebral organoids (COs) from BD patients and healthy controls to model disease-specific metabolic and inflammatory dysfunction in a more physiologically relevant system. BD COs exhibited mitochondrial impairment, dysregulated metabolic function, and increased nod-leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activation sensitivity. Treatment with MCC950, a selective NLRP3 inhibitor, effectively rescued mitochondrial function and reduced inflammatory activation in both BD and control COs. The effect of a Bioactive Flavonoid Extract (BFE), a potential therapeutic, was also explored and yielded a partial rescue of inflammasome activation. These findings highlight a mitochondria-inflammasome axis in BD pathophysiology and establish a novel platform for studying BD-associated cellular mechanisms, ultimately bridging the gap between molecular dysfunction and therapeutic development.

AbstractCentral adiposity and poor cardiorespiratory fitness are modifiable risk factors for various diseases. This study investigated their impact on CD4+ differentiated T regulatory (Treg) cell responses. Thirty‐eight young adults were classified into high cardiorespiratory fitness/low visceral adipose tissue (High V̇O2–Low VAT, n = 20) and low cardiorespiratory fitness/high VAT (Low V̇O2–High VAT, n = 18). Body composition was assessed using DXA and ultrasound, while cardiorespiratory fitness and physical activity were measured via treadmill testing and accelerometry. CD4+ cells were cultured in Treg differentiation medium with 2 ng/mL TGF‐β, with or without 100 nM rapamycin or 50 nM Torin‐1, for 96 h. Differentiated Treg from Low V̇O2–High VAT participants exhibited no significant changes in IL‐10 or IL‐6 production with rapamycin or Torin‐1. Conversely, differentiated Treg from High V̇O2–Low VAT participants showed significantly lower IL‐10 production with rapamycin (p < 0.001, adjusted p < 0.001) and Torin‐1 (p < 0.001, adjusted p < 0.001). These findings indicate that low cardiorespiratory fitness and high VAT contribute to an altered inflammatory response, influencing peripheral blood mononuclear cell immunophenotypes and exhaustion markers. Furthermore, mTORC1 and mTORC2 inhibition modulate cytokine production, emphasizing the role of metabolic status in immune regulation. High cardiorespiratory fitness and low visceral adiposity modulate CD4+ T regulatory cells. Fit participants shows be more responsible to mTORC1/2 inhibition via rapamycin or Torin‐1indicating a fitness‐ and adiposity‐dependent immunometabolic regulation.

ABSTRACTEmbryonic stem (ES) cell research has uncovered different requirements for WNT/β-catenin signalling in human naïve pluripotent cells compared to the mouse paradigm. It is therefore important to study WNT/β-catenin signalling directly in models that recapitulate early human development. Since TCF/LEF transcription factors mediate regulation of target genes downstream of WNT/β-catenin signalling, we examined the regulation, expression and protein localisation of the four TCF/LEF genes by analysing in vitro ‘snapshots’ of human development, leveraging naïve and primed pluripotent cells, blastoids and preimplantation blastocysts. Strikingly, we comprehensively confirm clear differences between mouse and human pluripotent stem cells, suggesting their differential requirements for WNT signalling reflects a pluripotent state-dependent manner. Human naïve ES cells express considerably lower levels of TCF7L1, unlike their mouse counterparts. TCF7L2 is robustly expressed in the trophectoderm derived from naïve ES cells, in blastoids and human preimplantation blastocysts. In primed pluripotent stem cells, active WNT/β-catenin signalling induces the expression of both TCF7 and LEF1, concomitant with hallmark gastrulation markers. The expression of human TCF/LEF genes indicates a differential requirement for WNT/β-catenin signalling throughout early human embryo development that warrants further investigation. Summary: Analysis of TCF/LEF factors unearths differences between mouse and human pluripotent stem cells, indicating differential requirements between species for WNT/β-catenin signalling in early embryo development.

AbstractGlioblastoma (GBM) tumors remain a challenge for immunotherapy owing to their heterogeneous and immunologically cold properties. GBM cells change the composition of the neural extracellular matrix (ECM), affecting the mobility, survival, and function of immune cells such as tumor-associated microglia and infiltrated macrophages (TAM). The ECM protein fibulin-3/EFEMP1 is a pericellular component uniquely upregulated in GBM compared with the normal brain, which promotes tumor growth and invasion. In this study, we demonstrate that fibulin-3 indirectly modulates TAM behavior and can be targeted to restore innate immune responses. Intracranial tumors initiated by fibulin-3–deficient GBM cells showed increased presence of TAMs with decreased immunosuppression markers (arginase-1, CD206), whereas the opposite effects were observed in tumors overexpressing fibulin-3. In silico analyses revealed a positive correlation between fibulin-3 and an immunosuppressive signature that was validated in GBM stem cells and in vivo. We further demonstrated that fibulin-3 regulates the expression of immunosuppressive signals (CSF-1, TGF-β1, and CD47) by autocrine activation of NF-κB signaling. Immunosuppressive signals were downregulated by knockdown of fibulin-3 in GBM stem cells and by inhibition of this protein using an anti–fibulin-3 antibody. Myeloid cells exhibited higher phagocytic activity and killing of GBM cells in presence of this antibody. Furthermore, locoregional delivery of anti–fibulin-3 in mice with intracranial GBM increased the presence of proinflammatory TAMs, thereby reducing tumor viability. Our findings show that anti–fibulin-3 approaches, which affect the ECM surrounding tumor and immune cells, can diminish immunosuppression in GBM and boost innate immune responses against the tumor.Significance:Inhibition of the ECM protein fibulin-3, which is highly upregulated in glioblastoma tumors, decreases immunosuppressive signals produced by the tumor cells and exposes them to increased attack by TAMs.

BackgroundThe tumor microenvironment (TME) plays a pivotal role in the development and progression of colorectal cancer (CRC), yet the complex crosstalk among its components remains incompletely understood. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) have emerged as key regulators of CRC progression, but their specific contributions, particularly given their heterogeneity, are not fully elucidated. This study identifies podoplanin (PDPN), a transmembrane glycoprotein enriched in CAFs, as highly expressed in the CRC TME, in particular surrounding the tumor, and associated with macrophage infiltration and cancer progression.MethodsWe performed mass spectrometry-based proteomic analysis on matched CRC and adjacent normal tissues from patients to identify altered signaling pathways and protein expression. The clinical relevance of PDPN expression was evaluated in CRC samples from two independent cohorts using immunohistochemistry and immunofluorescence analysis. Publicly available data from the Gene Expression Omnibus (GEO) database were analyzed to assess the association between PDPN expression and patient survival. Functional assays using direct and indirect co-culture systems investigated the influence of macrophage infiltration on stromal PDPN expression and its effect on colon adenocarcinoma cell growth.ResultsPDPN expression was significantly elevated in the stroma of the colorectal tumor tissues compared to normal tissues and correlated with M2-like macrophage infiltration. High PDPN expression was associated with reduced relapse-free survival in CRC patients. Stromal cells pre-conditioned with M2-like macrophages upregulated PDPN and more effectively supported the growth of three colon adenocarcinoma cell lines. PDPN depletion impaired the ability of stromal cells to promote tumor cell proliferation. Mechanistically, M2-like macrophage pre-conditioning induced a TGF-β1–dependent increase in YAP/TAZ nuclear localization, RhoA/ROCK/myosin-driven cytoskeletal contractility, and extracellular matrix (ECM) production in stromal cells. Inhibition of TGF-β1 signaling or ROCK activity reduced stromal support for cancer cell growth.ConclusionThis study reveals a novel mechanism by which the TME facilitates CRC progression and highlights PDPN as a potential prognostic biomarker and therapeutic target in CRC.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03496-3.

Dengue virus (DENV), the etiological agent of dengue fever, remains a global health concern, leading to severe illness and death in the absence of any definitive cure. Research has shown that vitamin D may reduce DENV replication in vitro and that dengue patients with low or deficient vitamin D levels are at higher risk of severe dengue. Studies indicate that viral replication is inhibited in human monocyte-derived macrophages (MDM) differentiated in the presence of vitamin D (D3MDM), suggesting that vitamin D may prevent DENV entry into host cells. However, despite these findings, the role of vitamin D in regulating the temporal expression patterns of genes as early, mid, and late transcriptional profile of DENV-infected macrophages remains unclear. Therefore, utilizing a kinetic transcriptomic profile is crucial. This approach provides detailed insights into the dynamic changes in gene expression over time, helping to clarify how vitamin D can modulate the immune response at critical stages of DENV infection. To address the transcriptional dynamics, we conducted a comprehensive analysis of gene expression patterns in MDM and D3MDM infected with Dengue virus serotype 2 (DENV-2). Utilizing bulk RNA sequencing alongside a standard viral growth curve, we systematically analyzed transcriptional kinetics by selecting key time points: 1.5, 3, 5.5, and 10 hours post-infection (h.p.i.) to monitor early viral entry and replication events and 24 h.p.i. to assess gene expression during peak viral particle production. Our temporal analysis revealed a progressive increase in cellular transcripts within the first hour of infection, with a more pronounced gene expression pattern in DENV-2-infected MDM compared to DENV-2-infected D3MDM at this early stage. Enrichment analysis indicated a reduced inflammatory response in DENV-2-infected D3MDM. Additionally, transcription factor analysis suggested diminished NF-κB signaling, but enhanced IRF5 activity was elevated in the DENV-2-infected D3MDM. High-dimensional clustering analysis identified nine unique gene clusters across both macrophage types, with notable upregulation of genes associated with antiviral activity, including IDO1, ISG20, OASL, IFI44L, RSAD2, IFIT1, MX1, EPSTI1, CXCL10, and CXCL11 in DENV-2-infected D3MDM at 1.5 h.p.i., suggesting an enhanced early antiviral response. These findings indicate that vitamin D modulates the magnitude and diversity of the early transcriptional responses, highlighting its potential as a therapeutic option to mitigate DENV severity.

Epilepsy is a central nervous system disorder causing uncontrollable seizures. One-third of patients do not respond to current medications, necessitating new treatments. This study targeted epileptogenesis, the process leading to chronic epilepsy, using human mesenchymal stem cells (MSCs) in a rodent model. MSC transplantation can positively affect neurodegenerative diseases by modifying inflammation. Additionally, glial cell line-derived neurotrophic factor (GDNF) may counteract seizures and tissue damage. We transplanted naïve immortalized human adipose-derived MSCs (Ctrl-MSCs) or GDNF-releasing MSCs (GDNF-MSCs, releasing 588.67 ± 20.14 pg/ml/24 h GDNF) into rat hippocampi after kainic acid-induced status epilepticus. Seizure progression was monitored for 5 weeks using video-EEG, behavioral assessments, and histological analysis. Both cell types influenced epileptogenesis. GDNF-MSCs delayed early-stage seizures, while Ctrl-MSCs reduced seizure frequency in later stages. Differences emerged in seizure development and cumulative seizure count, with Ctrl-MSCs showing significant seizure-attenuating effects. Behavioral differences were also noted: Ctrl-MSCs improved short-term memory and reduced anxiety, whereas GDNF-MSCs primarily reduced anxiety without significantly improving memory. This study highlights the therapeutic potential of MSCs, with or without GDNF, in modulating epileptogenesis, offering promising avenues for future clinical treatments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05853-z.

Polycystic ovary syndrome (PCOS) is among the most common disorders affecting up to 15% of the menstruating population globally. It is the leading cause of anovulatory infertility and a major risk factor for type 2 diabetes. Elevated testosterone levels are a core endophenotype. Despite that prevalence, the underlying causes remain unknown. PCOS genome-wide association studies (GWAS) have reproducibly mapped a number of susceptibility loci, including one encompassing a gene regulating androgen biosynthesis, DENND1A. Identifying the causal variants within these loci will provide fundamental insight into the precise biological pathways that are disrupted in PCOS. Here, we report the discovery of gene regulatory mechanisms that help explain genetic association with PCOS in the GATA4, FSHB and DENND1A loci using a combination of high throughput reporter assays, CRISPR-based epigenome editing, and genetic association analysis from PCOS case and control populations. In addition, we find that increasing endogenous DENND1A expression causes elevated testosterone levels in an adrenal cell model, specifically by perturbing candidate regulatory elements. These results further highlight the potential for combining genetic variant analyses with experimental approaches to fine map genetic associations with disease risk. Polycystic ovary syndrome (PCOS) is a common disorder, the causes of which remain incompletely understood. Here the authors report the discovery of gene regulatory mechanisms that help to explain genetic associations with PCOS in the GATA4, FSHB and DENND1A loci.

AbstractWe present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR–Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering. Graphical Abstract Graphical Abstract

ABSTRACTRegulatory T cells (Tregs) are associated with enhanced tumor progression and reduced therapy response rates. Therefore, overcoming the Treg‐mediated immunosuppressive barrier within the tumor to enhance antitumor immune responses is of central interest to advance cancer immunotherapy. To date, no tools exist that can be exploited to dampen Treg function and differentiation in vivo. Here, we show for the first time that the antibiotic thiostrepton exerts a potent inhibitory effect on Tregs. Mechanistically, thiostrepton disrupts Treg differentiation, reduces the expression of Treg activation markers, and inhibits Treg suppressive functions. Accordingly, using an MC38 tumor model, we demonstrate that thiostrepton treatment reduces the number of intratumoral Foxp3+ Treg cells and prevents tumor growth. These effects are conserved in human T cells, as thiostrepton also inhibits the differentiation of human Tregs. Our findings highlight thiostrepton as a promising Treg‐targeting immunomodulatory compound with the potential to enhance antitumor immune responses. Regulatory T cells (Tregs) promote tumor progression and therapy resistance by suppressing antitumor immunity. Here, we identify the antibiotic thiostrepton as a novel Treg‐targeting immunomodulator for cancer immunotherapy. Thiostrepton impairs Treg differentiation and suppressive capacity. In an MC38 model, thiostrepton treatment reduced intratumoral Foxp3⁺ Tregs and significantly inhibited tumor growth.

Cholecystectomy represents the most prevalent biliary surgical procedure for gallbladder abnormalities. Growing evidence suggests that cholecystectomy is associated with an elevated risk of colorectal cancer. However, the underlying mechanism remains elusive. Here we show that cholecystectomy exacerbates colorectal tumorigenesis in both AOM/DSS and APCmin/+ mice models. Metagenomic sequencing and targeted metabolomics show that cholecystectomy leads to a decrease of Bifidobacterium breve (B. breve) and an increase of Ruminococcus gnavus (R. gnavus), along with increased levels of glycoursodeoxycholic acid (GUDCA) in human and tauroursodeoxycholic acid (TUDCA) in mice. Fecal microbiota transplantation, single bacterial colonization and bile acid supplementation demonstrate that cholecystectomy-related gut microbiota perturbations promote the production of TUDCA and facilitate colorectal tumorigenesis. RNA-sequencing and co-immunoprecipitation reveal that the compromised bile acid metabolism inhibits farnesoid X receptor (FXR) signaling, disrupts the FXR/β-catenin interaction, and ultimately exacerbates colorectal tumorigenesis. Significantly, FXR agonist obeticholic acid (OCA) averts cholecystectomy-related colorectal tumorigenesis. The gut microbiota holds a crucial position in cholecystectomy-induced colorectal tumorigenesis, and modulation of the gut microbiota-bile acid-FXR axis represents a promising preventive strategy. Cholecystectomy is associated with an increased risk of colorectal cancer, and the underlying mechanism remains elusive. Here, the authors show that cholecystectomy-related microbiome and bile acid alterations disrupts the FXR/β-catenin interaction, and ultimately exacerbates colorectal tumorigenesis.