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BackgroundDaphnetin has demonstrated various pharmacological activities. The current study evaluated the potential of daphnetin in alleviating unexplained recurrent spontaneous abortion (URSA) and explored underlying mechanisms.MethodsMice with URSA were gavaged with 1 mg/kg, 10 mg/kg, and 20 mg/kg of daphnetin, or infected with adeno-associated viruses harboring knockdown of NR4A1 or overexpression of BACH2 before modeling. Human peripheral blood T lymphocytes were induced into CD4+ T cells, followed by lentivirus infection and daphnetin treatment. The influence of daphnetin on CD4+ T cell viability and Treg and Th17 cell differentiation in cells was analyzed. The concentrations of Treg cells-associated cytokines (TGF-β, IL-10) and Th17 cells-associated cytokines (IL-17, IL-23) in the supernatants of CD4+ T cells were assessed. The regulation of NR4A1 on BACH2 was analyzed by ChIP and dual-luciferase assays.ResultsDaphnetin resulted in fewer immature, resorbed, or dead embryos in mice with URSA, with the most pronounced therapeutic effect of 10 mg/kg. Daphnetin attenuated decidual hemorrhage, with a gain in the percentage/number of Treg cells and a loss of the percentage/number of Th17 cells in the spleen and decidual tissues. Daphnetin enhanced the expression of FoxP3, TGF-β, and IL-10, and suppressed the expression of RORγt, IL-17, IL-23, and the contents of TNF-α, IL-6, and IL-1β in CD4+ T cells. Overexpression of BACH2 further alleviated URSA deterioration caused by NR4A1 knockdown. Daphnetin mediated the transcriptional activation of BACH2 by upregulating NR4A1.ConclusionsUpregulation of NR4A1 by daphnetin mediates BACH2 transcription and Th17/Treg cell homeostasis to improve URSA.

The remodeling of mammary glands during pregnancy is essential for initiating lactation. In dairy animals, the overlap of pregnancy and mammary involution triggers a unique process, regenerative remodeling, which is critical for extending lactation duration and enhancing milk production. Unlike the complete regression of lobuloalveolar structures during involution, the regenerative remodeling preserves alveolar structures and promotes rapid mammary gland renewal. However, the cellular and molecular mechanisms underlying such process remain elusive. Here, taking dairy goats (Capra hircus) as a ruminant model, we identified four luminal cell populations through single-cell RNA-sequencing and found a significant reduction in luminal hormone-responsive (LumHR) cells and an increase in luminal secretory precursors (LumSecP) during regenerative remodeling. A reduction of LumHR cells during regenerative remodeling is essential for promoting the accumulation of LumSecP. Goat mammary organoids and in vivo genetic ablation assays suggested that LumHR cells function as a crucial switch for the differentiation of LumSecP to LumSec cells through the prolactin receptor pathway. Furthermore, high levels of IRF1 inhibited while downregulation of IRF1 stimulated the proliferation of LumHR cells. We showed that IRF1 regulated the dynamics of LumHR cells through hormonal signaling targets, including ESRRB. Our findings identified a key cell type responsible for the dynamics of luminal lineages during regenerative remodeling in large mammals and highlighted the potential for accelerating tissue regeneration through targeted modulation of lineage stage-specific regulators.

AbstractCell-based immunotherapy utilizing regulatory T cells (Tregs) has recently advanced into clinical applications, demonstrating promising results in phase I/II trials to prevent transplant rejection and treat autoimmune diseases. We have completed a clinical trial in renal transplant patients in which the significant biological effect was the increase of B cells with a regulatory phenotype in the blood of kidney transplant patients. The mechanisms by which Tregs regulate B cells and the specific molecules involved in this process remained poorly understood. In this study, we employed an in vitro system of co-culture of peripherally purified B cells and expanded Tregs to show that Tregs can induce a population of memory B cells that express IL-10 and CD25. This subset of B cells has been previously identified as one of humans’ regulatory B cell populations. Notably, these expanded Tregs’ regulation of B cells was found to be independent of IL-10 and reliant on direct cell contact. We established that TIM3 expression by Tregs was crucial for the induction of IL-10-producing CD25+ memory B cells. Our findings suggest that TIM3 is a critical molecule for the induction of regulatory B cells by Tregs, indicating that TIM3 expression by adoptively transferred Tregs is vital in diseases where B cells play a pathogenic role. Key Messages Expanded Tregs induce IL-10+ CD25+ B cells.TIM3 expression on Tregs is crucial for IL-10+ B cell induction.Tregs require direct cell contact to regulate B cells.Blocking TIM3 reduces IL-10+ B cells but increases IFN-γ, TNF-α, IL-17.Tregs enhance regulatory B cell differentiation, promoting tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00109-025-02606-0.

The pancreatic islet, the only type of tissue that secretes insulin in response to elevated blood glucose, plays a vital role in diabetes development and treatment. While various islet vascularization strategies have been developed, they have been hindered by major limitations such as relying on pre-patterning and the inability to span long distances. Furthermore, few strategies have demonstrated robust enough vascularization in vivo to support therapeutic subcutaneous islet transplantation. Using adaptive endothelial cells (ECs) reprogrammed by transient expression of the ETS Variant Transcription Factor 2 (ETV-2) gene, we have physiologically vascularized human islets within a generic microchamber and have achieved functional engraftment of human islets in the subcutaneous space of mice. Such adaptive ECs, which we term reprogrammed vascular ECs (R-VECs), have been proven to be a suitable tool for both in vitro disease modeling and in vivo transplantation of not only islets but also other organoids. Key features • This protocol contains two parts: the in vitro and in vivo parts, both utilizing adaptable endothelial cells to functionally vascularize human islets.• The in vitro portion of this protocol describes the method to culture human islets in a vascular bed within a large and commercially available microchamber.• The in vivo portion of this protocol provides a step-by-step procedure to reverse hyperglycemia in streptozotocin-induced diabetic mice.

AbstractIntroductionPain in patients with rheumatoid arthritis (RA) is an unmet clinical need. Targeting joint inflammation with disease-modifying antirheumatic drugs has not resulted in the anticipated reduction in pain for many patients. This can partly be explained by the concept of central sensitisation whereby spinal and supraspinal pathways have a lower threshold of activation, leading to increased perception of pain. Synovial stromal cells, such as fibroblasts, are also thought to play a role through peripheral sensitisation of nerves in the joint. Synovial fibroblasts are known to produce pro-algesic mediators such as interleukin 6 and nerve growth factor at the messenger RNA level. These pro-algesic mediators could activate sensory nerve fibres that send signals from the joint to the spinal cord, thereby driving persistent pain in RA. The purpose of this study is to evaluate which pro-algesic mediators are produced by lining versus sub-lining fibroblasts and whether the level of these mediators correlates with clinical measures of pain in patients with RA.Methods and analysisFiND-Pain RA is a multicentre observational study which will recruit 50 patients with seropositive RA who attend the rheumatology department of Guy’s and St Thomas’ Hospital, London, and the Nuffield Orthopaedic Centre, Oxford. Clinical examination, pain-focused patient-reported outcome measures, ultrasound examination and ultrasound-guided synovial biopsy of the knee will be performed. The levels of known and putative pro-algesic mediators will be measured in fibroblasts from the lining and sub-lining layer of the synovium. The location and spatial morphology of sensory nerve fibres and their proximity to lining and sub-lining fibroblasts will be characterised. The primary outcome will be to determine whether the knee pain scores of participants correlate with the level of leukaemia inhibitory factor, a novel putative pain-mediator expressed in sub-lining fibroblasts. The secondary outcomes will be to determine whether other pro-algesic mediators produced by lining or sub-lining fibroblasts correlate with clinical measures of pain and to assess the location and proximity of sensory nerve fibres to lining versus sub-lining fibroblasts.Ethics and disseminationThe study is a sub-study of the PUMIA (Pain Phenotypes and their Underlying Mechanisms in Inflammatory Arthritis) study, which has been approved by the Bromley Research Ethics Committee (REC: 21/LO/0712). The findings of this study will be disseminated through open-access publications, as well as scientific and clinical conferences.

Highlights What are the main findings? Aggregated Aβ reduces phagocytosis and decreases surface TREM2 expression in monocytes carrying CD33 rs3865444 or SPI1 rs1057233 AD-genetic risk variants.Genetic variants interact with Aβ to reduce myeloid cell fitness in the periphery. What are the implications of the main findings? These findings highlight that peripheral monocytes, like brain-resident microglia, are genetically and functionally linked to AD risk.These results emphasize the importance of studying peripheral myeloid cells to understand genetically influenced immune dysfunction in Alzheimer’s disease.AbstractWhile genetics implicate a central role for dysregulated innate immunity in Alzheimer’s disease (AD), the contributions of peripheral myeloid cells, such as monocytes, have been largely overlooked in favor of microglia. Here, we investigate whether AD-associated loci, specifically rs3865444 in the CD33 locus and rs1057233 in the SPI1 locus, converge on shared functional pathways in monocytes in the context of amyloid-beta peptide 1-42 (Aβ1-42) as an immune stimulus. To do so, we isolated monocytes from peripheral blood mononuclear cells (PBMCs) from healthy individuals and exposed them to aggregated Aβ1-42. In this study, we identify functional convergence of the CD33 and SPI1 AD risk variants in the context of aggregated Aβ, both resulting in reduced phagocytosis and loss of surface TREM2 expression, demonstrating an interaction between genetics and environment to reduce myeloid cell fitness. These findings highlight that peripheral monocytes, like brain-resident microglia, are genetically and functionally linked to AD risk, underscoring their importance as accessible immune cells that contribute to disease susceptibility and progression.

BackgroundAcute myeloid leukemia (AML) remains clinically challenging due to its molecular heterogeneity and poor outcomes, highlighting the urgent need for novel biomarkers and therapeutic targets.PurposeThis study aims to identify and characterize the role of leucine-rich α-2-glycoprotein 1 (LRG1) in AML, evaluating its potential as both a prognostic biomarker and a therapeutic target.MethodsWe conducted an integrated basic and clinical investigation of LRG1 in AML. Methods included analysis of LRG1 expression in patient samples versus controls and pre- versus post-treatment, assessment of its clinical correlations with mutations and subtypes, and evaluation of its prognostic impact. Functional validation was performed using LRG1 knockdown models to assess effects on colony formation, apoptosis, and differentiation. Single-cell RNA profiling was utilized to identify LRG1-enriched cell populations and explore its role in microenvironmental crosstalk.ResultsIntegrated analysis revealed significantly elevated LRG1 expression in AML patients compared to controls (P<0.001), with levels decreasing post-treatment (P<0.001). High LRG1 expression correlated with FLT3 mutations (P<0.01), M3-M5 AML subtypes (M0&M1&M2 VS M3, P<0.001; M0&M1&M2 VS M4, P<0.01; M0&M1&M2 VS M5, P<0.001; M3 VS M5, P<0.05; M4 VS M5, P<0.05), and worse survival (P<0.01). Functionally, LRG1 knockdown impaired colony formation (P<0.001), increased apoptosis (P<0.001), and disrupted differentiation (P<0.01). Single-cell profiling identified LRG1 enrichment in hematopoietic stem and progenitor cells (HSPCs) and myeloid progenitors, where it facilitated microenvironmental crosstalk via Macrophage Migration Inhibitory Factor (MIF), Galactoside-binding lectin (GALECTIN), and Cyclophilin A (CypA) signals.ConclusionOur findings establish LRG1 as a robust prognostic biomarker and a key functional regulator of AML maintenance through myeloid progenitor dysregulation, presenting it as a promising target for new therapeutic strategies.

BackgroundInterleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase involved in T cell activation, differentiation, and receptor signaling. Soquelitinib (SQL) is a selective, covalent inhibitor of ITK. The abnormal activation of T cells plays a key role in the early inflammatory stages of systemic sclerosis (SSc). In the study reported here, we investigated the efficacy of SQL in two mouse models of lung fibrosis, and related pulmonary arterial hypertension (PAH), mimicking lung involvement in SSc.MethodsSQL was evaluated in the bleomycin-induced lung fibrosis mouse model and the Fra-2 mouse model, characterized by interstitial lung disease (ILD) and pulmonary vascular remodeling leading to PAH.ResultsIn the bleomycin-induced lung fibrosis model, SQL significantly reduced leucocyte infiltration into the lungs and attenuated interstitial lung damage, primarily by modulating Th2 and Th17 pathways, although it did not affect collagen deposits. In the Fra-2 model, SQL improved clinical scores, reduced histological scores of interstitial lung damage and inflammatory infiltrates, and significantly downregulated Th17-dependent RORγt and STAT3 expression in lesional lungs. While SQL showed a trend toward improved lung function on imaging and modest effects on collagen deposits, it significantly reduced PAH in the Fra-2 mice, improving key cardiac hemodynamic parameters and showing trends of beneficial effects on vascular remodeling.ConclusionsOur findings highlight the potential of selective ITK inhibition with SQL as a promising therapeutic strategy for lung damage and pulmonary vascular remodeling in SSc.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-025-03687-5.

BackgroundChronic hepatitis B virus (HBV) infection progression is closely associated with repeated inflammatory liver injury. Myeloid-derived suppressor cells (MDSCs), known to suppress inflammatory responses during hepatitis, may play a protective role in mitigating liver damage; nevertheless, the precise functional specialization of MDSC subpopulations and the critical regulator in controlling their suppressive capacity remain incompletely understood. This study investigates the immunoregulatory role of IL-21, a pleiotropic immunomodulatory cytokine, in modulating MDSC function and its implications for HBV-associated liver inflammation prognosis.MethodsThe frequencies of peripheral and intrahepatic MDSCs, along with serum IL-21 levels, in patients with chronic HBV infection were measured with freshly isolated samples. C57BL/6 mice carrying pAAV-HBV1.2 plasmids was established to explore the effect of IL-21 on aminotransferase levels and MDSC function.ResultsPatients with chronic HBV infection demonstrated significantly elevated frequencies of circulating MDSCs compared to healthy controls. Of note, the granulocytic MDSCs (gMDSCs) were markedly more abundant in hepatic tissues than in peripheral blood. Strikingly, a significant inverse correlation was observed between circulating gMDSC frequencies and serum ALT levels specifically in patients with elevated serum IL-21 concentrations, whereas this correlation was absent in the low IL-21 subgroup. In vitro functional assays demonstrated that exogenous IL-21 potentiated the immunosuppressive capacity of MDSCs, significantly attenuating IFN-γ production in co-cultured T cells. Consistent with these findings, hydrodynamic injection of IL-21 plasmid in an HBV mouse model resulted in significantly reduced serum ALT levels concomitant with enhanced arginase I expression in hepatic MDSCs.ConclusionsThese results reveal that IL-21 enhances MDSC-mediated immunosuppression, implicating this regulatory axis in the attenuation of hepatic inflammation during chronic HBV infection. These findings provide a novel perspective on the involvement of IL-21 in the negative regulation of intrahepatic inflammation and provide a novel perspective on the role of IL-21 in developing immunotherapeutic strategies to optimize available anti-inflammatory treatments in chronic HBV infection.Supplementary informationThe online version contains supplementary material available at 10.1186/s12967-025-07406-1.

Metastatic cancer cells, originating from cancer stem cells with metastatic capacity, utilize nutrient flexibility to navigate the challenges of the metastatic cascade. However, the nutrient required to maintain the stemness potentials of metastatic cancer cells remains unclear. Here, we reveal that metastatic breast cancer cells sustain stemness and initiate metastasis upon detachment by taking up and oxidizing lactate. In detached metastasizing breast cancer cells, lactate is incorporated into the tricarboxylic acid cycle, boosting oxidative phosphorylation, and promoting the stemness potentials via α-KG-DNMT3B-mediated SOX2 hypomethylation. Moreover, lactate is taken up and oxidized in mitochondria by the CD147/MCT1/LDHB complex, which correlates with stemness potentials and tumor metastasis in patients with breast cancer. An intracellularly expressed single-chain variable fragment targeting mitochondrial CD147 (mito-CD147 scFv) effectively disrupts the mitochondrial CD147/MCT1/LDHB complex, inhibits lactate-induced stemness potential, depletes circulating breast cancer cells, and reduces metastatic burden, suggesting promising clinical applications in reducing lactate-fueled metastasis. Metastatic cancer cells rely on metabolic flexibility to survive. Here, the authors show that metastatic breast cancer cells use lactate for mitochondrial oxidation via the CD147/MCT1/LDHB complex to sustain stemness and promote metastasis.

Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis and pneumonia in children under 5 years of age. RSV infection is characterized by lung inflammation, viral expansion, and airway barrier destruction. The crosstalk between neurons and immune and non-immune cells develops from early life onwards. The lungs and airways are densely innervated by sympathetic neurons where they secrete the neurotransmitter noradrenaline (NA) at homeostasis and following injury in adult mice. NA, once secreted into the lungs, can act on immune and non-immune cells through β-adrenergic receptors (β-ARs). However, it is unknown whether NA-β-AR signaling axis regulates antiviral immunity during early-life infections. In a mouse model of neonatal RSV infection, we find that stimulation of adrenergic signaling via β2-AR agonist increases viral clearance, anti-inflammatory responses, and disease recovery. By contrast, loss of sympathetic neurons and β2-AR increases the RSV viral load and immunopathology in neonates. Stimulating β2-AR signaling has several downstream effects, including reduced airway mucus secretion and protection from endothelial barrier disruption, which together prevent airway blockade, respiratory failure, and facilitate control of RSV infection. Collectively, our studies demonstrate that sympathetic neurons and β2-AR signaling protect neonates from viral expansion and lung barrier breakdown, promoting antiviral defense. RSV is the most common cause of respiratory infections in infants and young children. Here, the authors used a neonatal mouse RSV infection model and uncovered a role of sympathetic neurons and β2-adrenergic signaling in protecting neonates from viral expansion and immunopathology to promote antiviral defense.

BackgroundThe landscape of cancer treatment has evolved rapidly within the last 50 years, and whilst radiotherapy, chemotherapy, and surgery remain the mainstay treatment options, there has been a shift towards using immunotherapy alone or in combination with other treatment modalities. There is an emerging paradigm that radiotherapy is immunogenic, driving stimulation of antigen-specific T cells capable of recognising tumour cells at distal sites to the treatment location.MethodsWhole blood samples were collected from patients with primary and oligometastatic cancer before, during, and after treatment with stereotactic ablative radiotherapy (SABR). Using clinical full blood counts, multiparameter flow cytometry, Luminex, and ELISpot assays, this study explored the impact of SABR on systemic immune cell composition, inflammatory markers, and antigen-specific T cell responses.ResultsWe identified striking systemic changes collectively indicating profound SABR-driven immunosuppression. Such changes were characterised by pronounced and sustained lymphopenia which included loss of CD4+ and CD8+ T cells, B cells, and natural killer (NK) cells accompanied by an overall decline in effector T cell responses to common recall and cancer antigens. This loss of lymphocytes drove a rise in the neutrophil-to-lymphocyte ratio (NLR), which was associated with poorer progression-free survival (PFS) if increased from baseline. A higher dosage of radiation and treatment to a larger area were both associated with more pronounced lymphocyte loss, a concomitant NLR increase, and poorer PFS, particularly in individuals with liver lesions.ConclusionsThese findings support a role for lymphocytes in preventing disease progression after SABR and suggest that a change to clinical practice to spare lymphocytes from the toxic effects of irradiation may have beneficial effects for patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04218-6.