The link between glutaminolysis and osteoarthritis (OA) has only recently begun to be elucidated. Here, we report the association of obesity- and injury-induced cartilage damage with impaired glutaminolysis in chondrocytes. Defective glutaminolysis triggered the onset and progression of OA, with enhanced catabolism and decreased anabolism. Supplementation of α-ketoglutarate (αKG), a key component in glutaminolysis and an epigenetic factor, effectively protected cartilage against degradation in vivo via a TCA cycle- and HIF-1α-independent manner. Mechanistically, OA pathogenic factors increased H3K27me3 deposition on promoters of key glutaminolysis genes, including Slc1a5 and Gls1, leading to impaired glutaminolysis. Conversely, αKG facilitated Kdm6b-dependent H3K27me3 demethylation of not only glutaminolysis genes to rescue Gln metabolism but also Ube2o to reverse OA. Elevated Ube2o expression led to TRAF6 ubiquitination and subsequent inhibition of NF-κB signaling, thereby reversing the pathological reprogramming of glycolysis and oxidative phosphorylation and protecting against cartilage destruction. Collectively, these results demonstrated that OA pathogenic factors impair glutaminolysis through epigenetic regulation, which further exacerbate OA. Moreover, αKG restores metabolic homeostasis and alleviates OA through H3K27me3 demethylation.

Transplantation of midbrain dopaminergic neurons (mDANs) derived from human pluripotent stem cells (hPSCs) represents a promising therapeutic approach for Parkinson’s disease (PD), with several clinical trials currently in progress. However, the poor survival of grafted mDANs, in part due to inflammation triggered by surgical injury, poses significant challenges to both therapeutic efficacy and safety. In recent years, mesenchymal stem cells (MSCs) and their derivatives have played an important role in the field of stem cell therapy due to their easy accessibility and immunomodulatory properties. However, their potential role as an adjunct in cell therapy remains unclear. Therefore, this study aims to investigate the protective effects of conditioned medium (CM) derived from MSCs and the mechanisms of action by which it attenuates surgery-induced neuroinflammation and enhances the survival of transplanted mDANs. To mimic the neuroinflammatory response caused by the surgical aspect of cell transplantation, transplantation medium alone (i.e. without cells) was injected into the striatum of male NOD SCID mice. The anti-inflammatory effects of MSC-CM in vivo were evaluated by assessing the activation of microglia and expression levels of pro-inflammatory cytokines. Next, an in vitro cell death model was established using IFN-γ and TNF-α stimulation. The protective effects of MSC-CM against IFN-γ and TNF-α-induced inflammatory injury in mDANs were assessed using immunofluorescence staining and TUNEL assay. To identify the active components and underlying mechanisms for the protective effects of MSC-CM, inhibitory treatments, miRNA sequencing, dual-luciferase reporter assay, and subsequent validation by qPCR and Western blotting were performed. Finally, MSC-CM was administered intranasally into male NOD SCID mice following mDAN transplantation to evaluate its protective effects on the grafted mDANs in vivo. MSC-CM suppressed neuroinflammation and significantly protected mDANs from inflammation-induced cell death via inhibition of TLR3/NF-κB/p53 signaling pathway in this model system. This protective effect was largely reproduced by treatment with a specific component of the MSC-CM, miR-143-3p. Overexpression of miR-143-3p further enhanced the protective effects and mechanisms of action of MSC-CM. This study demonstrates that MSC-CM or its component miR-143-3p exert protective effects on mDANs against inflammatory injury, primarily by inhibiting the TLR3/NFκB/p53 signaling pathway.

Dry eye (DE) is a multifactorial ocular surface disease characterized primarily by tear film instability and ocular discomfort. Nearly all forms of DE exhibit elevated inflammatory markers in tear fluid, accompanied by clinical signs of tear dysfunction including reduced secretion and shortened breakup time. Although the pathophysiology of dry eye disease remains incompletely understood, accumulating evidence implicates neutrophil extracellular traps (NETs) as key pathogenic drivers. Robust clinical associations and mechanistic studies have established causal links between NETs and ocular surface pathology. This review summarizes current research on the role of NETs in the development of dry eye, aiming to identify potential therapeutic targets.

Mixed connective tissue disease (MCTD) is a rare autoimmune disease, and little is known about its pathogenesis. Furthermore, MCTD, systemic lupus erythematosus (SLE), and primary Sjögren’s syndrome (pSS) share many clinical, laboratory, and immunological manifestations. This overlap complicates early diagnosis and accurate treatment. The transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) from MCTD patients was performed using both bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) for the first time. Additionally, we applied MCTD scRNA-seq data, along with datasets from SLE (GSE135779) and pSS (GSE157278) from the Gene Expression Omnibus database, to characterize and compare the similarities and heterogeneity among MCTD, SLE, and pSS. We first resolved transcriptomic changes in peripheral blood immune cells of MCTD, and then revealed the shared and unique features among MCTD, SLE, and pSS. Analyses showed that the percentage of CD8+ effector T cells was increased, while mucosal-associated invariant T cells were decreased in all three diseases. Genes related to the ‘interferon (IFN) γ response’ and ‘IFN α response’ were significantly upregulated. SCENIC analysis revealed activation of STAT1 and IRF7 in disease states, targeting IFN-related genes. The IFN-II signaling network was notably elevated in all three diseases. Unique features of MCTD, SLE, and pSS were also identified. We dissected the immune landscape of MCTD at single-cell resolution, providing new insights into the development of novel biomarkers and immunotherapies for MCTD. Furthermore, we offer insights into the transcriptomic similarities and heterogeneity across different autoimmune diseases, while highlighting prospective therapeutic targets.

Muscle regeneration hinges on the proliferation and differentiation of satellite cells. FBXL3, a member of the F-box protein family known for its role as a negative regulator of the circadian clock, is implicated in myogenesis. In this study, we demonstrate the expression of FBXL3 in satellite cells of adult mice, where it acts as a negative regulator of myogenic regeneration. This regulation occurs through the promotion of ubiquitination and degradation of TCF12, a transcription factor crucial for differentiation. Loss of FBXL3 activates MyoD and myogenin, thereby augmenting myogenic differentiation and regeneration. The role of FBXL3 in muscle regeneration was also confirmed using the tamoxifen-inducible Pax7-CreER recombination system. To unravel the regulatory mechanism of MyoD and myogenin by FBXL3, we conducted RNA sequencing on Fbxl3+/+and Fbxl3-/- primary myoblasts. Gene set enrichment analysis (GSEA) revealed that FBXL3 deficiency enriches the gene set associated with striated muscle cell development, including MEF2C, a regulator of myogenin expression. Through a search in the ChEA3 database, TCF12 emerged as the downstream candidate gene regulated by FBXL3 to modulate MEF2C. ChIP-PCR assays confirmed the enrichment of TCF12 on MEF2C promoter at three consensus sites. Dual-luciferase reporter assay validated that TCF12 activates the MEF2C promoter. This comprehensive study underscores the crucial role of FBXL3 in satellite cell-mediated myogenic regeneration and provides insights into the intricate regulatory network involving TCF12 and MEF2C.

The presence or absence of adult neural stem cells in the mammalian forebrain ependyma has been debated for two decades. In this study, we performed single-cell RNA sequencing to investigate the cellular composition of the ependymal surface of the adult mouse forebrain using whole mounts of lateral walls of lateral ventricles. We identified 12 different cell subtypes in the ependymal surface. Immunocytochemical analyses revealed that CD133+ multi-ciliated cells comprised 67.6% of ependymal cells, while the remaining 32.4% were CD133-. CD133+ ependymal cells can be further classified into FOXJ1+/SOX2+/ACTA2+ cells, FLT1+/CD31+/CLDN5+ endothelial-like cells, and PDGFRB+/VTN+/NG2+ pericyte-like cells, as well as endothelial-pericyte-like cells and Foxj1+ endothelial-like cells. CD133- ependymal cells can be further divided into endothelial-like cells, Foxj1+ ependymal cells, Foxj1+ endothelial-like cells, pericyte-like cells, endothelial-pericyte-like cells, VIM+ cells, and cells negative for all of these markers. This comprehensive profiling confirms the heterogeneity of the ependymal surface in the adult mouse forebrain. Debate regarding whether adult ependymal cells contain neural stem cells has arisen because different researchers have examined different populations of ependymal cells. Our study provides a new perspective for investigation of clinical endogenous neural stem cells, ultimately paving the way for stem cell therapies in neurological diseases.

Ancient traditional Chinese medicine (TCM) doctrine says "The superior doctor prevents illnesses," pointing out preventative medicine as the ultimate goal for medical care. TCM recognizes that genetic predisposition and environmental and lifestyle influences contribute to diseases. It divides people into eight constitutions in addition to one normal/healthy kind. People with one of the eight subhealth constitutions are prone to develop different kinds of corresponding illnesses. The goal for this type of categorization is to help people take preemptive measures to prevent or delay disease onset. As the peripheral immune system through surveying the body, it can capture information from essentially all organs and reflect anomalies occurring in each organ. Thus, the detailed profiling of the peripheral immune-system function can generally reflect a person's overall heath state. In this study, we performed the single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from individuals with Tanshi (phlegm dampness) constitution. They were prone to develop metabolic disorders including diabetes. scRNA-seq revealed greatly reduced mucosal-associated invariable T cell content and heightened TNFα-NFκB, JAK-STAT, and interferon signaling. These findings indicated heightened chronic inflammation, as well as increased hypoxia/apoptosis responses, likely resulting from frequent sleep apnea that Tanshi individuals experienced. Altogether, this pilot study demonstrated effectiveness in using scRNA-seq to reveal molecular-immunological bases for constitution categorization, thereby substantiating that preventative medicine originated from TCM.

Disuse-induced bone loss occurs in long-term bed-ridden patients and in astronauts during spaceflight. The underlying mechanisms are poorly understood. In a rodent model of disuse-induced bone loss (called hindlimb unloading (HU)), we observed that decreased numbers of leptin receptor (LepR) positive mesenchymal stem cells (MSCs) in adult bone marrow, contribute to bone loss. MicroRNA-337-3p (miR-337) was upregulated in MSCs upon HU and inhibited MSC proliferation by directly targeting IRS-1 to suppress the PI3kinase-Akt-mTOR pathway. Piezo1 was the upstream receptor for sensing mechanical stress and regulated miR-337 through the Hippo-YAP signaling pathway. Remarkably, the knockout of miR-337 significantly attenuated HU-induced, but not ovariectomy-induced, bone loss by increasing MSC proliferation and osteogenesis. Finally, the transplantation of miR-337-/- MSCs into wild-type HU mice was sufficient to mitigate bone loss. These findings reveal the cellular and molecular mechanisms underlying disuse-induced bone loss and highlight a feasible therapeutic strategy to prevent disuse- or microgravity-induced bone loss on Earth and during spaceflight.

Chimeric antigen receptor (CAR)-modified NK (CAR-NK) cells are candidates for next-generation cancer immunotherapies. Here we generated CD19-specific CAR-NK cells with 4-1BB and CD3ζ signaling endo-domains (CD19-BBz CAR-NK) by transduction of cord blood-derived NK cells using baboon envelope pseudotyped lentiviral vectors and demonstrated their antitumor activity in preclinical B cell lymphoma models in female mice. We next conducted a phase 1 dose-escalation trial involving repetitive administration of CAR-NK cells in 8 patients with relapsed/refractory large B cell lymphoma (NCT05472558). Primary end points were safety, maximum tolerated dose, and overall response rate. Secondary end points included duration of response, overall survival, and progression-free survival. No dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. No cases of cytokine release syndrome, neurotoxicity, or graft-versus-host disease were observed. Results showed an overall response rate of 62.5% at day 30, with 4 patients (50%) achieving complete response. The median progression-free survival was 9.5 months, and the median overall survival was not reached. A post hoc exploratory single-cell RNA sequencing analysis revealed molecular features of CAR-NK cells associated with therapeutic efficacy and efficacy-related immune cell interaction networks. This study met the pre-specified end points. In conclusion, CD19-BBz CAR-NK cells were feasible and therapeutically safe, capable of inducing durable response in patients with B cell lymphoma.

The amyloid precursor protein (APP) plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). While the production of Amyloid beta (Aβ) has traditionally been considered the primary cause of AD, the role of the APP intracellular domain (AICD) remains largely elusive. In this study, we established a novel model in the adult fly wing by expressing human APP, recapitulating AD-associated axon degeneration. Using this model, we discovered that ectopic APP expression in Drosophila wing margin neurons led to age-dependent axon degeneration. APP's effect depended on AICD production, and AICD overexpression alone was sufficient to induce axon degeneration in adult wings. Further investigations indicated that APP- or AICD-induced axon degeneration could be alleviated by blocking autophagy, but not apoptosis. Additionally, we identified a FoxO/Snail-Atg1 axis as an essential mediator of APP/AICD-induced autophagy-dependent axon degeneration. Finally, we demonstrated that administration of chloroquine, an autophagy inhibitor, effectively ameliorates APP- or AICD-induced axon degeneration. Our findings provide crucial insights into how APP induces autophagy-dependent axon degeneration through AICD production, laying a foundation for future investigations into AD pathogenesis.

Acute ischemic stroke (AIS) is associated with a high incidence and significant rates of disability, making it a critical focus of clinical research. The current review investigates the role of serum inflammatory markers in the pathogenesis and prognosis of AIS. By quantitatively analyzing specific inflammatory markers, this study aims to enhance the understanding of the pathophysiological mechanisms underlying AIS, support early diagnosis, improve disease assessment, and establish a scientific foundation for targeted treatment strategies to optimize clinical outcomes. From a pathophysiological perspective, multiple inflammatory markers are involved in the inflammatory response that occurs within brain tissue following cerebral ischemia. The serum levels of various inflammatory markers were measured in individuals with AIS, revealing strong correlations between these markers and disease severity. The findings indicate that these markers can serve as reliable indicators of disease progression. Further analysis demonstrated their prognostic value in predicting functional recovery and the risk of recurrence. Notably, during a 3-month follow-up, each 0.32 ng/mL increase in matrix metalloproteinases-9 levels was associated with a 16 % increase in the risk of disability and mortality after AIS. The findings of this review contribute to a more comprehensive understanding of the pathological and physiological mechanisms of AIS and offer a foundation for advancing early diagnostic methods, disease assessment tools, and personalized treatment strategies. Monitoring inflammatory marker levels may enable clinicians to more accurately evaluate disease severity and develop tailored therapeutic interventions, potentially reducing disability and recurrence rates while improving quality of life for individuals with AIS. The findings highlight the potential of precision medicine approaches based on inflammatory markers to shape future AIS treatment paradigms.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a critical condition resulting from impaired oxygen and blood flow to the brain during birth, leading to neuroinflammation, neuronal apoptosis, and long-term neurological deficits. Despite the use of therapeutic hypothermia, current treatments remain inadequate in fully preventing brain damage. Recent advances in mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) offer a novel, cell-free therapeutic approach, as these EVs can cross the blood-brain barrier (BBB) and deliver functional microRNAs (miRNAs) to modulate key pathways involved in inflammation and neuroprotection. This review examines how specific miRNAs encapsulated in MSC-EVs—including miR-21, miR-124, miR-146, and the miR-17-92 cluster—target the complex inflammatory responses that drive HIE pathology. By modulating pathways such as NF-κB, STAT3, and PI3K/Akt, these miRNAs influence neuroinflammatory processes, reduce neuronal apoptosis, and promote tissue repair. The aim is to assess the therapeutic potential of miRNA-loaded MSC-EVs in mitigating inflammation and neuronal damage, thus addressing the limitations of current therapies like therapeutic hypothermia.

Although immune checkpoint blockade (ICB) therapies have shifted the treatment paradigm for non-small-cell lung cancer (NSCLC), many patients remain resistant. Here we characterize the tumor cell states and spatial cellular compositions of the NSCLC tumor microenvironment (TME) by analyzing single-cell transcriptomes of 232,080 cells and spatially resolved transcriptomes of tumors from 19 patients before and after ICB–chemotherapy. We find that tumor cells and secreted phosphoprotein 1-positive macrophages interact with collagen type XI alpha 1 chain-positive cancer-associated fibroblasts to stimulate the deposition and entanglement of collagen fibers at tumor boundaries, obstructing T cell infiltration and leading to poor prognosis. We also reveal distinct states of tertiary lymphoid structures (TLSs) in the TME. Activated TLSs are associated with improved prognosis, whereas a hypoxic microenvironment appears to suppress TLS development and is associated with poor prognosis. Our study provides novel insights into different cellular and molecular components corresponding to NSCLC ICB–chemotherapeutic responsiveness, which will benefit future individualized immuno-chemotherapy. Samples from 19 patients with non-small-cell lung cancer treated with a combination of chemotherapy and immune checkpoint blockade are profiled with single-cell RNA sequencing and spatial transcriptomics to identify factors associated with treatment resistance.

[This corrects the article DOI: 10.3389/fimmu.2023.1212330.].

Internal tandem duplication mutations of the FMS-like tyrosine kinase-3 (FLT3-ITDs) occur in 25%-30% of patients with acute myeloid leukemia (AML) and are associated with dismal prognosis. Although FLT3 inhibitors have demonstrated initial clinical efficacy, the overall outcome of patients with FLT3-ITD AML remains poor, highlighting the urgency to develop more effective treatment strategies. In this study, we reveal that FLT3 inhibitors reduced protein stability of the anti-cancer protein p53, resulting in drug resistance. Blocking p53 degradation with proteasome inhibitors restores intracellular p53 protein levels and, in combination with FLT3-ITD inhibitors, shows superior therapeutic effects against FLT3-ITD AML in cells, mouse models, and patients. These data suggest that this combinatorial therapeutic approach may represent a promising strategy to target FLT3-ITD AML.

The brain-derived neurotrophic factor (BDNF)/TrkB pathwayplaysa crucial role in neural plasticity and neuronal survival but is oftendeficient in neurodegenerative diseases like Alzheimer's disease(AD). CF3CN acts as a specific TrkB agonist that displays therapeuticeffects in the AD mouse model, but its brain/plasma ratio (B/P ratio)distribution is not satisfactory. To increase its brain exposure,we synthesized several derivatives and employed nanoparticle (NP)formulation to optimize the most potent #2 derivative's invivo PK profiles. We generated stable #2-loaded zein/lactoferrin compositeNPs (#2/zein/LF) using the antisolvent co-precipitation method. Invivo PK studies revealed that nanoencapsulation improved #2'soral bioavailability by approximately 2-fold and significantly enhancedits plasma C (max) and t (1/2), but the brain profiles were comparable. Pharmacodynamicsshowed that #2/zein/LF activates TrkB signaling that phosphorylatesasparagine endopeptidase (AEP) T322 and decreases its enzymatic activity,resulting in reduced AEP-cleaved amyloid precursor protein and Taufragments in the brains of AD mice, correlating with its PK profiles.After 3 months of treatment in 3xTg mice, #2/zein/LF decreased ADpathologies and alleviated cognitive dysfunction. Hence, zein/LF compositenanoencapsulation is a promising drug delivery method for improvingthe PK profiles of a potential preclinical candidate for treatingneurodegenerative diseases.

Abnormal mechanical loading often leads to the progressive degradation of cartilage and causes osteoarthritis (OA). Although multiple mechanoresponsive strategies based on biomaterials have been designed to restore healthy cartilage microenvironments, methods to remotely control the on-demand mechanical forces for cartilage repair pose significant challenges. Here, a magneto-mechanically controlled mesenchymal stem cell (MSC) platform, based on the integration of intercellular mechanical communication and intracellular mechanosignaling processes, is developed for OA treatment. MSCs loaded with antioxidative melanin@Fe3O4 magnetic nanoparticles (Magcells) rapidly assemble into highly ordered cell clusters with enhanced cell-cell communication under a time-varying magnetic field, which enables long-term retention and differentiation of Magcells in the articular cavity. Subsequently, via mimicking the gait cycle, chondrogenesis can be further enhanced by the dynamic activation of mechanical signaling processes in Magcells. This sophisticated magneto-mechanical actuation strategy provides a paradigm for developing mechano-therapeutics to repair cartilage in OA treatment.