Memory inflation is confirmed as the most commonly dysregulation of host immunity with antigen-independent manner in mammals after viral infection. By generating large numbers of effector/memory and terminal differentiated effector memory CD8⁺ T cells with diminished naïve subsets, memory inflation is believed to play critical roles in connecting the viral infection and the onset of multiple diseases. Here, we reviewed the current understanding of memory inflated CD8⁺ T cells in their distinct phenotypic features that different from exhausted subsets; the intrinsic and extrinsic roles in regulating the formation of memory inflation; and the key proteins in maintaining the expansion and proliferation of inflationary populations. More importantly, based on the evidences from both clinic and animal models, we summarized the potential mechanisms of memory inflation to trigger autoimmune neuropathies, such as Guillain-Barré syndrome and multiple sclerosis; the correlations of memory inflation between tumorigenesis and resistance of tumour immunotherapies; as well as the effects of memory inflation to facilitate vascular disease progression. To sum up, better understanding of memory inflation could provide us an opportunity to beyond the acute phase of viral infection, and shed a light on the long-term influences of CD8⁺ T cell heterogeneity in dampen host immune homeostasis.

In the early embryonic stages, Lin-28 homologue A (Lin28a) is highly expressed and declines as the embryo matures. As an RNA-binding protein, Lin28a maintains some adult muscle stem cells (MuSCs) in an embryonic-like state, but its RNA metabolism regulation mechanism remains unclear. BioGPS analysis revealed that Lin28a expression is significantly higher in muscle tissues than in other tissues. Lin28a-positive muscle stem cells (Lin28a+ MuSCs) were sorted from Lin28a-CreERT2; LSL-tdTomato mouse skeletal muscle tissue, which exhibited a higher proliferation rate than the control group. Lin28a-bound transcripts are enriched in various biological processes such as DNA repair, cell cycle, mitochondrial tricarboxylic acid cycle and oxidative stress response. The expression of insulin-like growth factor 2 mRNA-binding protein 3 (Igf2bp3) was markedly elevated in the presence of Lin28a. Co-immunoprecipitation analysis further demonstrated that Lin28a associates with Igf2bp3. Immunofluorescence analyses confirmed that Lin28a, Igf2bp3 and G3bp1 colocalize to form stress granules (SG), and N6-methyladenosine (m⁶A) modification promotes the formation of Lin28a-SG. Sequencing of the transcriptome and RNAs immunoprecipitated by Lin28a, Igf2bp3 and m⁶A antibodies in Lin28a+ MuSCs further revealed that Lin28a and Igf2bp3 collaboratively regulate the expression of DNA repair-related genes, including Fancm and Usp1. Lin28a stabilises Igf2bp3, Usp1, and Fancm mRNAs, enhancing DNA repair against oxidative or proteotoxic stress, thus promoting MuSCs self-renewal. Understanding the intricate mechanisms through which Lin28a and Igf2bp3 regulate MuSCs provides a deeper understanding of stem cell self-renewal, with potential implications for regenerative medicine.

Blood vessels play a crucial role in maintaining the stem cell niche in both tumours and developing organs. Cell competition is critical for tumour progression. We hypothesise that blood vessels may act as a regulator of this process. As a pioneer, the secretions of blood vessels regulate the intensity of cell competition, which is essential for tumour invasion and developmental organ extension. Brd4 expresses highly in endothelial cells within various tumours and is positively correlated with numerous invasive genes, making it an ideal focal point for further research on the relationship between blood vessels and cell competition. Our results indicated that the absence of endothelial Brd4 led to a reduction in neural stem cell mortality and compromised cell competition. Endothelial Brd4 regulated cell competition was dependent on Testican2. Testican2 was capable of depositing Sparc and acted as a suppressor of Sparc. Compromised cell competition resulted in the depletion of neural stem cells and accelerated brain ageing. Testican2 could rescue the run-off of neural stem cells and accelerate the turnover rate of neurons. AD patients show compromised cell competition. Through the cloning of a point mutant of Brd4 identified in a subset of AD patients, it was demonstrated that the mutant lacked the ability to promote cell competition. This study suggests a novel approach for treating age-related diseases by enhancing the intensity of cell competition.

GSDMB-mediated pyroptosis facilitates a pro-inflammatory immune microenvironment and needs to be tightly regulated to avoid excessive inflammation. Here, we provide evidence that itaconate and its cell-permeable derivative 4-octyl itaconate (4-OI) can significantly inhibit GSDMB-rendered pyroptotic activity independent of Nrf2. 4-OI interferes proteolytic process of GSDMB by directly modifying Cys54, Cys148 and Ser212 on granzyme A (GrzA), a serine protease that site-specifically cleaves the inter-domain linker of GSDMB, instead of interaction with GSDMB, thereby blocking pyroptosis and exerts anti-inflammatory effects. Moreover, 4-OI alleviates inflammation by suppressing GSDMB-induced pyroptotic cell death during acute colitis models in intestinal epithelial GSDMB conditional transgenic mice. Our data expand the role of 4-OI as a crucial immunometabolic derivative that regulates innate immunity and inflammation through a newly identified posttranslational modification, and targeting of pyroptosis by 4-OI therefore holds potent therapeutic potential for primarily inflammatory and/or autoimmune diseases.

Age-related dysfunction of salivary glands (SGs) leading to xerostomia or dry mouth is typically associated with increased dental caries and difficulties in mastication, deglutition or speech. Inflammaging-induced hyposalivation plays a significant role in aged SGs; however, the mechanisms by which ageing shapes the inflammatory microenvironment of SGs remain unclear. Here, we show that reduced salivary secretion flow rate in aged human and mice SGs is associated with impaired autophagy and increased M1 polarization of macrophages. Our study reveals the crucial roles of SIRT6 in regulating macrophage autophagy and polarization through the PI3K/AKT/mTOR pathway, as demonstrated by generating two conditional knock out mice. Furthermore, triptolide (TP) effectively rejuvenates macrophage autophagy and polarization via targeting this pathway. We also design a local delivery of TP-loaded apoptotic extracellular vesicles (ApoEVs) to improve age-related SGs dysfunction therapeutically. Collectively, our findings uncover a previously unknown link between SIRT6-regulated autophagy and macrophage polarization in age-mediated hyposalivation, while our locally therapeutic strategy exhibits potential preventive effects for age-related hyposalivation.

The bone marrow (BM) niches are the complex microenvironments that surround cells, providing various external stimuli to regulate a range of haematopoietic stem cell (HSC) behaviours. Recently, it has been proposed that the fate decision of HSCs is often correlated with significantly altered biophysical signals of BM niches. To thoroughly elucidate the effect of mechanical microenvironments on cell fates, we constructed 2D and 3D cell culture hydrogels using polyacrylamide to replicate the mechanical properties of heterogeneous sub-niches, including the inherent rigidity of marrow adipose tissue (2 kPa), perivascular tissue (8 kPa) and endosteum region (35 kPa) in BM. Our observations suggest that HSCs can respond to the mechanical heterogeneity of the BM microenvironment, exhibiting diversity in cell mechanics, haematopoietic pool maintenance and differentiated lineages. Hydrogels with higher stiffness promote the preservation of long-term repopulating HSCs (LT-HSCs), while those with lower stiffness support multi-potent progenitors (MPPs) viability in vitro. Furthermore, we established a comprehensive transcriptional profile of haematopoietic subpopulations to reflect the multipotency of haematopoietic stem and progenitor cells (HSPCs) that are modulated by niche-like stiffness. Our findings demonstrate that HSPCs exhibit completely distinct downstream differentiated preferences within hydrogel systems of varying stiffness. This highlights the crucial role of tissue-specific mechanical properties in HSC lineage decisions, which may provide innovative solutions to clinical challenges.

Aerobic glycolysis is involved in the pathogenesis of pulmonary hypertension (PH). The mechanisms by which glycolysis is increased and how it contributes to pulmonary vascular remodelling are not yet fully understood. In this study, we demonstrated that elevated lipocalin-2 (LCN2) in PH significantly enhances aerobic glycolysis in human pulmonary artery smooth muscle cells (PASMCs) by up-regulating LDHA expression. Knockout of Lcn2 or having heterozygous LDHA deficiency in mice significantly inhibits the progression of hypoxic PH. Our study reveals that LCN2 stimulates LDHA expression by activating Akt-HIF-1α signalling pathway. Inhibition of Akt or HIF-1α reduces LDHA expression and proliferation of PASMCs. Both Akt and HIF-1α play critical roles in the development of PH and are suppressed in the pulmonary vessels of hypoxic PH mice lacking LCN2. These findings shed light on the LCN2-Akt-HIF1α-LDHA axis in aerobic glycolysis in PH.

Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), a subunit of the heterotrimeric G protein EIF2, is involved in the initiation of translation. Our findings demonstrate that the depletion of Eif2s2 in premeiotic germ cells causes oocyte arrest at the pachytene and early diplotene stages at 1 day postpartum (dpp) and 5 dpp, respectively, and eventually leads to oocyte apoptosis and failure of primordial follicle formation. Further studies reveal that Eif2s2 deletion downregulates homologous recombination-related and mitochondrial fission-related protein levels, and upregulates the integrated stress response-related proteins and mRNA levels. Consistently, Eif2s2 deletion significantly decreases the expression of dictyate genes and compromises mitochondrial function, characterized by elongated shapes, decreased ATP levels and mtDNA copy number, along with an excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Furthermore, DNA damage response and proapoptotic protein levels increase, while anti-apoptotic protein levels decrease in Eif2s2-deleted mice. An increase in oocytes with positive cleaved-Caspase-3 and TUNEL signals, alongside reduced Lamin B1 intensity, further indicates oocyte apoptosis. Collectively, Eif2s2 deletion in premeiotic germ cells causes oocyte meiotic arrest at the early diplotene stage by impairing homologous recombination, and eventually leads to oocyte apoptosis mainly through the downregulation of mitochondrial fission-related proteins, ROS accumulation and subsequent DNA damage.

Traumatic optic neuropathy refers to optic nerve (ON) injury by trauma, including explosion and traffic accident. Retinal ganglion cell (RGC) death is the critical pathological cause of irreversible visual impairment and blindness in ON injury. We previously investigated the patterns of 11 modes of cell death in mouse retina post-ON injury. Here we aimed to identify additional signalling pathways regulating RGC survival in rodents post-ON injury. RNA sequencing analysis identified the upregulation of inflammation and cellular senescence-related genes in retina post-ON injury, which were confirmed by immunoblotting and immunofluorescence analyses. Increased expression of senescence-associated β-galactosidase (SA-βgal) in RGCs and activation of microglia were also found. Transforming growth factor-β receptor type II inhibitor (LY2109761) treatment suppressed p15^Ink4b and p21^Cip1 protein and SA-βgal expression and promoted RGC survival post-ON injury with decreasing the expression of cell death markers in retina. Consistently, senolytics (dasatinib and quercetin) treatments can promote RGC survival and alleviate the reduction of ganglion cell complex thickness and pattern electroretinography activity post-ON injury with reducing SA-βgal, p15^Ink4b, p21^Cip1, microglial activation and cell death marker expression. In summary, this study revealed the activation of cellular senescence in rodent retina post-ON injury and contribute to RGC survival regulation. Targeting cellular senescence can promote RGC survival after ON injury, suggesting a potential treatment strategy for traumatic optic neuropathy.

Intervertebral discs (IVDs) are rhythmic tissues that experience daily low-load recovery. Notably, aging and abnormal mechanical stress predispose IVDs to degeneration due to dysrhythmia-induced disordered metabolism. Meanwhile, Rev-erbα acts as a transcriptional repressor in maintaining biorhythms and homeostasis; however, its function in IVD homeostasis and degeneration remains unclear. This study assessed the relationship between low Rev-erbα expression levels and IVD degeneration. Rev-erbα deficiency accelerated needle puncture or aging-induced IVD degeneration, characterized by increased extracellular matrix (ECM) catabolism and nucleus pulposus (NP) cell apoptosis. Mechanistically, Rev-erbα knockdown in NP cells aggravated rhIL1β-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, exacerbating the imbalanced ECM and NP cell apoptosis. Meanwhile, blocking NLRP3 inflammasome activation mitigated Rev-erbα deficiency and needle puncture-induced IVD degeneration. Particularly, Rev-erbα mediated the transcriptional repression of the NLRP3 inflammasome via the ligand heme-binding of nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3) complex. Thus, the increased expression of Rev-erbα in NP cells following short-term rhIL1β treatment failed to inhibit NLRP3 transcription in vitro owing to heme depletion. Pharmacological activation of Rev-erbα in vivo and in vitro alleviated IVD degeneration by altering the NLRP3 inflammasome. Taken together, targeting Rev-erbα may be a potential therapeutic strategy for alleviating IVD degeneration and its related diseases.

The ability to visualise microRNA in situ is crucial for studying microRNAs, their microRNA-associated biological functions and disease diagnosis. Traditional fluorescence in situ hybridisation methods based on paraformaldehyde fixation of microRNAs suffer from release of microRNAs from cells, which limits the sensitivity of in situ hybridisation, making them unsuitable for the detection of small, low-abundance microRNAs. To reduce the loss, microRNAs were covalently cross-linked to proteins within cells by combining EDC and paraformaldehyde, and the target microRNA was used as the initiator chain for a branched hybridisation chain reaction to detect microRNA expression levels in situ. A simplified branched hybridisation chain reaction can be realised by coupling two hybridisation chain reaction circuits with a hairpin linker. Upon forming the primary hybridisation chain reaction product with extended sequence, this sequence reacts with the linker hairpin H3 to release the initiator sequence, resulting in the formation of numerous dendritic branched hybridisation chain reaction products. Imaging results show that this technique can detect microRNAs with high sensitivity and selectivity at both the single-cell and single-molecule levels. Compared with the traditional fluorescence in situ hybridisation technique, this method greatly improves the sensitivity and image resolution of in situ imaging detection. Therefore, we believe that the target-initiated branched hybridisation chain reaction based in situ detection method provides a reliable assay platform for analysing disease-related microRNA expression.

The vocal fold is an architecturally complex organ comprising a heterogeneous mixture of various layers of individual epithelial and mesenchymal cell lineages. Here we performed single-cell RNA sequencing profiling of 5836 cells from the vocal folds of adult Sprague–Dawley rats. Combined with immunostaining, we generated a spatial and transcriptional map of the vocal fold cells and characterized the subpopulations of epithelial cells, mesenchymal cells, endothelial cells, and immune cells. We also identified a novel epithelial-to-mesenchymal transition-associated epithelial cell subset that was mainly found in the basal epithelial layers. We further confirmed that this subset acts as intermediate cells with similar genetic features to epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma. Finally, we present the complex intracellular communication network involved homeostasis using CellChat analysis. These studies define the cellular and molecular framework of the biology and pathology of the VF mucosa and reveal the functional importance of developmental pathways in pathological states in cancer.

Ischemic heart disease (IHD) is a prevalent cardiovascular condition that remains the primary cause of death due to its adverse ventricular remodelling and pathological changes in end-stage heart failure. As a complex pathologic condition, it involves intricate regulatory processes at the cellular and molecular levels. The immune system and cardiovascular system are closely interconnected, with immune cells playing a crucial role in maintaining cardiac health and influencing disease progression. Consequently, alterations in the cardiac microenvironment are influenced and controlled by various immune cells, such as macrophages, neutrophils, dendritic cells, eosinophils, and T-lymphocytes, along with the cytokines they produce. Furthermore, studies have revealed that Gata6⁺ pericardial cavity macrophages play a key role in regulating immune cell migration and subsequent myocardial tissue repair post IHD onset. This review outlines the role of immune cells in orchestrating inflammatory responses and facilitating myocardial repair following IHD, considering both macro and micro views. It also discusses innovative immune cell-based therapeutic strategies, offering new insights for further research on the pathophysiology of ischemic heart disease and immune cell-targeted therapy for IHD.

CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48–72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product.

The cytoskeleton is essential for mechanical signal transduction and autophagy. However, few studies have directly demonstrated the contribution of the cytoskeleton to mechanical stress-induced autophagy. We explored the role of the cytoskeleton in response to compressive force-induced autophagy in human cell lines. Inhibition and activation of cytoskeletal polymerization using small chemical molecules revealed that cytoskeletal microfilaments are required for changes in the number of autophagosomes, whereas microtubules play an auxiliary role in mechanical stress-induced autophagy. The intrinsic mechanical properties and special intracellular distribution of microfilaments may account for a large proportion of compression-induced autophagy. Our experimental data support that microfilaments are core components of mechanotransduction signals.

The trophoblast lineage differentiation represents a rate-limiting step in successful embryo implantation. Adhesion, invasion and migration processes within the trophoblast are governed by several transcription factors. Among them, CDX2 is a critical regulator shaping the destiny of the trophoblast. While its altered expression is a linchpin initiating embryo implantation in mice, the precise influence of CDX2 on the functionality and lineage differentiation of early human trophoblast remains unclear. In this study, we employed well-established human trophoblast stem cell (hTSC) lines with CDX2 overexpression coupled with a 3D in vitro culture system for early human embryos. We revealed that the downregulation of CDX2 is a prerequisite for syncytialization during human embryo implantation based on immunofluorescence, transcriptome analysis, CUT-tag sequencing and the construction of 3D human trophoblast organoids. While CDX2 overexpression inhibited syncytialization, it propelled hTSC proliferation and invasive migration. CDX2 exerted its influence by interacting with CGA, PTGS2, GCM1, LEF1 and CDH2, thereby hindering premature differentiation of the syncytiotrophoblast. CDX2 overexpression enhanced the epithelial–mesenchymal transition of human trophoblast organoids. In summary, our study provides insights into the molecular characteristics of trophoblast differentiation and development in humans, laying a theoretical foundation for advancing research in embryo implantation.

Repressing BET proteins’ function using bromodomain inhibitors (BETi) has been shown to elicit antitumor effects by regulating the transcription of genes downstream of BRD4. We previously showed that BETi promoted cell death of triple-negative breast cancer (TNBC) cells. Here, we proved that BETi induce altered mitochondrial dynamics fitness in TNBC cells falling in cell death. We demonstrated that BETi treatment downregulated the expression of BCL-2, and proteins involved in mitochondrial fission and increased fused mitochondria. Impaired mitochondrial fission affected oxidative phosphorylation (OXPHOS) inducing the expression of OXPHOS-related genes, SDHa and ATP5a, and increased cell death. Consistently, the amount of mitochondrial DNA and mitochondrial membrane potential (∆Ψm) increased in BETi-treated cells compared to control cells. Lastly, BETi in combination with Metformin reduced cell growth. Our results indicate that mitochondrial dynamics and OXPHOS metabolism support breast cancer proliferation and represent novel BETi downstream targets in TNBC cells.