Although endurance exercise benefits liver health, sex-specific adaptive trajectories remain unclear. This study mapped dynamic liver adaptation in males and females during prolonged training and identified underlying molecular programs. Using publicly available time-resolved liver multi-omics data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we established a computational pipeline for differential analysis of transcriptomic, proteomic, phosphoproteomic, and metabolomic data with FDR correction, followed by FGSEA pathway enrichment. Kinase activities were inferred through ortholog mapping and PhosphoSitePlus. Cross-omics co-expression networks were constructed using WGCNA and topological overlap to link omics features with physiological phenotypes. For experimental validation, liver tissues were collected from endurance-trained Sprague-Dawley rats, and key nodes were confirmed by Western blotting, qRT-PCR, and immunofluorescence/immunohistochemical staining. Public scRNA-seq data were further integrated to map multi-omics signals to single-cell resolution and assess functional changes in specific cell types. The hepatic response to exercise stress was stage-specific, shifting from early transcriptional activation to later proteomic and metabolic remodeling. Multi-omics integration revealed distinct sex-associated adaptive trajectories: males were more strongly associated with energy metabolism, redox-related programs, and amino acid/organic acid catabolism, whereas females showed prominent membrane lipid remodeling, proteostasis -related programs, and mitochondrial/ribosomal translational features. Single-cell analysis showed that tissue remodeling occurred without major lineage turnover, instead involving altered communication among pre-existing cell communities. Validation of PPP1R3G identified a protein-dominant exercise-responsive marker, supporting the contribution of post-transcriptional or protein-level regulation. Hepatic adaptation to endurance stress follows a cross-omics evolutionary pattern with sex-specific reprogramming of energy supply and homeostatic maintenance. This time-resolved framework clarifies how exercise improves liver function and supports sex-oriented metabolic interventions and therapeutic target discovery. Temporal multi-omics maps stage-specific liver adaptation to endurance training. Exercise shifts hepatic responses from transcription to metabolic remodeling. Males favor energy/redox adaptation, females favor lipids and proteostasis. Single-cell analysis links remodeling to altered cell communication, not turnover. PPP1R3G validation supports post-transcriptional control of liver adaptation.
Abstract Unlike adult hearts, neonatal hearts can regenerate and adapt to stress. To identify both cardioprotective and regenerative mechanisms, we compared activated genes and related signaling pathways in the neonatal mouse heart after myocardial infarction (MI) and after exposure to pressure overload using a neonatal model of transverse aortic constriction (nTAC) at postnatal day 1 (P1). We identified three immune-related genes—Ccl4, S100a8, and C1qa—of high interest, as they encode secreted factors, are highly expressed in the neonatal mouse heart in both injury types, and their receptors are expressed on neonatal cardiomyocytes (nCM) and cardiac endothelial cells (EC). We investigated their effects on primary mouse EC and nCM in vitro. Our study found that the combination of these secreted factors (Pool3) enhances EC and nCM cell cycle re-entry and reduces the rate of apoptosis. Combined in vivo and in vitro transcriptome analyses revealed that Toll-like receptor 2 (TLR2) activation in nCM induces a shared pro-survival and cell cycle re-entry-associated signaling, including upregulation of Bcl2 and Birc5, respectively. The direct and pivotal role of TLR2 in enhancing nCM cell cycle re-entry and survival was further confirmed using TLR2 knockout (KO) nCM, a TLR2 inhibitor, and a TLR2 agonist (zymosan). Moreover, the importance of TLR2 in mediating the adaptive response to pressure overload in neonatal mice was confirmed in TLR2 KO mice, which failed to adapt to pressure overload and exhibited high mortality together with maladaptive cardiac remodeling as early as 7 days post-surgery. Collectively, these findings demonstrate that TLR2 is essential for mediating nCM cell cycle re-entry, survival, and adaptive/regenerative response to injury. Graphical Abstract Schematic image of immune-cardiomyocyte crosstalk via TLR2 during cardiac injury in neonatal mice. TLR2 activation in response to secreted immune cytokines such as CCL4, C1QA, and S100A8 during cardiac injury promotes nCM survival and cell cycle re-entry via upregulation of Bcl2 and Birc5, respectively
Therapeutic interventions for diseases such as leukemia and autoimmune disorders are increasingly designed to selectively target and deplete specific immune cell subsets over prolonged periods. This can disrupt the homeostasis of the adaptive immune system, with consequences not only for peripheral blood but also for the “cradle of the immune system”, the bone marrow. This study hypothesized that such immune imbalance due to immune cell depletion therapies impairs the differentiation capacity of mesenchymal stromal cells (MSCs), which are key progenitors of bone cells and are thus essential for maintaining bone health. To validate this hypothesis, a series of cell culture experiments were conducted in which MSCs were stimulated with immune-conditioned media derived from various immune cell subsets. A comprehensive analytical approach was employed to evaluate the differentiating MSCs, including their associated supernatants, deposited collagen, mineralized matrix, and lipid deposition at defined time points during their maturation. Therefore, fluorescence and nonfluorescent histological staining, enzyme-linked immunosorbent assays (ELISAs), and expression analyses with Ribonucleic acid (RNA) were performed. This study demonstrated that, compared with immune-conditioned media from T cells or peripheral blood mononuclear cells (PBMCs), B cell-immune-conditioned media enriched with interleukin 4, bone morphogenetic protein 2 and Dickkopf 1 enhanced the osteogenic differentiation of MSCs in vitro. This positive osteogenic effect is driven primarily by elevated cytokine secretion by stimulated B cells, which in vivo in turn potentially stimulate bone turnover and modulate immune system function. In summary, these data demonstrate an pro-osteogenic and pro-adipogenic influence of B cells on differentiating MSCs in vitro, suggesting that an imbalance in immune cells in the bone marrow perturbs bone homeostasis.
High mammographic breast density is a strong independent risk factor for sporadic breast cancer, yet involved mechanisms remain poorly defined. The extracellular compartment plays a critical role in the intercellular communication during tumor initiation and progression. Although several mechanosensitive pathways have been described, the role of platelets (PLTs) in stiffness-driven signaling in the breast is unknown. Extracellular soluble proteins were sampled in situ from live breast tissue using microdialysis. A total of 108 postmenopausal women were included: women with nondense or dense breasts, women with dense breasts randomized to low-dose acetylsalicylic acid (ASA; 160 mg/day) or no treatment, and patients with estrogen receptor–positive (ER+) breast cancer. Breast density was assessed by magnetic resonance imaging. High-dimensional proteomic profiling of 1,158 proteins was performed using proximity extension assays. To investigate stiffness-dependent PLT responses, cells were cultured in a 3D in vitro system with tunable matrix stiffness generated by cross-linked hyaluronic acid, modeling nondense and dense breast tissue. Dense breast tissue exhibited a distinct extracellular proteomic signature enriched for proteins associated with platelet activation, along with alterations in several immunomodulatory pathways. Several PLT-associated proteins were also elevated in ER+ breast cancers, supporting their clinical relevance. Post hoc exploratory proteomic analysis of samples from women treated with low-dose ASA did not reveal modulation of these proteins, suggesting that stiffness-induced PLT activation may occur via mechanotransduction rather than biochemical pathways in vivo. Consistently, in the 3D in vitro model, increased matrix stiffness representative of dense breasts promoted a procoagulant PLT phenotype without corresponding changes in classical activation markers. Tissue stiffness is a critical regulator of PLT mechanotransduction in dense breast tissue, contributing to a microenvironment permissive for cancer progression. These findings highlight PLT mechanobiology as a potential target for breast cancer prevention and therapy. EudraCT: 2017-000317-22.
Despite growing evidence of ochratoxin A (OTA)-induced kidney toxicity, the underlying mechanisms remain elusive. Emerging evidence suggests that small extracellular vesicles (sEVs) act as mediators of intercellular communication to recipient cells during various physiological and pathological conditions. Given the distinctive properties of sEVs, it is hypothesized that OTA-induced sEVs might mediate the OTA-induced kidney pathogenesis. To explore the involvement of sEVs in OTA-induced kidney toxicity, sEVs were isolated and characterized from OTA-exposed rat kidney epithelial cells (NRK52E). Later, these sEVs were used to treat NRK52E cells and Wistar rats to assess the impact of OTA-induced sEVs on kidney toxicity. Label-free proteomics was also performed on OTA-induced sEVs, and key proteins were identified and validated. The biodistribution of sEVs in rats was also assessed using live imaging. The role of validated protein/s in kidney toxicity was further confirmed via a gene silencing and overexpression study. OTA exposure increased sEV secretion into conditioned media of NRK52E cells and into the urine of Wistar rats. Interestingly, we found that OTA-induced sEVs cause similar kidney toxicity in vitro and in vivo systems as OTA exposure, and blocking of sEV secretion markedly alleviated OTA-mediated kidney toxicity. Proteomics analysis identified annexin A2 and fibrinogen-ɣ as common proteins detected in sEVs derived from OTA-exposed NRK52E cells or rat urine. However, immunoblotting validated that annexin A2 was the only sEV-associated protein, expressed significantly in both NRK52E and rat urine following OTA exposure. Notably, silencing of annexin A2 attenuated the ability of OTA-induced sEVs to cause kidney toxicity, whereas overexpression exacerbates it. Our findings identify the annexin A2-enriched sEVs as key mediators of OTA-induced kidney toxicity. Annexin A2, along with other kidney injury markers, offers a promising non-invasive translational biomarker for early detection and monitoring of OTA-induced kidney toxicity.
Trichinellosis is a globally prevalent parasitic disease caused by infection of Trichinella spiralis. B1 cells are critically involved in immune defense against multiple parasitic infections, yet their functional role in trichinellosis remains uncharacterized. Wild-type, Btk conditional knockout and S100a4 knockout mice were used to construct the infection model of T. spiralis. The proportions and BCR signaling of peritoneal B1 cells in mice infected with T. spiralis were detected with flow cytometry and immunofluorescence. RNA sequencing was applied to analyze the possible mechanisms of B1 cell activation. ELISA was used to detect the production of antibodies. Peritoneal B1 cells exhibited obvious accumulation with enhanced B cell receptor (BCR) signaling at the muscle phase of murine T. spiralis infection. B1 cell deficiency impaired host immune responses against T. spiralis at the muscle stage, confirming the essential contribution of B1 cells to anti-trichinella immunity. RNA sequencing analysis revealed significant upregulation of S100A4 in peritoneal B1 cells following infection. Mechanistically, S100A4 regulated the biological functions of peritoneal B1 cells through modifying cell relocation, BCR signaling, and antibody production. Our findings reveal a previously unrecognized role of S100A4-mediated B1 cell responses in host defense against T. spiralis infection. The data provide new mechanistic insights into the immune regulation of trichinellosis.
Abstract Background The coordinated development of the mesenchymal and epithelial primordia of the murine ureter requires intense tissue communication. Previous studies have revealed the crucial role of the FGFR2-SHH-FOXF1-BMP4 signaling axis in the proliferation and differentiation programs of both the epithelium and the mesenchyme. Canonical (β-Catenin/CTNNB1-dependent) WNT signaling in the mesenchymal primordium has been reported to influence mesenchymal development by enhancing proliferation and favoring the smooth muscle cell fate over adventitial fibrocytes. Methods To investigate the role of mesenchymal WNT signaling in early ureter development further, we analyzed the ureters of Ctnnb1-deficient mouse mutants for cellular and molecular changes during embryogenesis. Using pharmacological inhibition and activation approaches in explant cultures of embryonic ureters, we assessed the contribution of altered signaling activities to differentiation changes in Ctnnb1-deficient ureters. Transcriptional profiling of embryonic ureters with short-term WNT signaling inhibition allowed us to identify the primary targets of this signaling pathway in the ureteric mesenchyme. Results We demonstrate that both mesenchymal and epithelial differentiation are impaired in ureters lacking mesenchymal Ctnnb1. This defect is linked to an inability to activate pro-differentiation transcription factors in either tissue primordia. Epithelial changes can be attributed, at least in part, to the loss of BMP4 signaling and the gain of canonical WNT signaling in the epithelial primordium. Mesenchymal WNT signaling directly activates transcription of multiple components of the SHH-FOXF1-BMP4 signaling axis, thereby enhancing this module in the inner region of the ureteric mesenchyme. Conclusions Our work improves the understanding of the signaling network that coordinates cytodifferentiation in the early ureter. Mesenchymal WNT signaling plays a central role in mesenchymal fate decisions but also in promoting epithelial differentiation through enhancement of the SHH-FOXF1-BMP4 axis and inhibition of epithelial WNT-signaling.
Multiple myeloma (MM) remains an incurable hematologic malignancy with bortezomib resistance representing a major therapeutic challenge. The mevalonate (MVA) pathway, a crucial metabolic cascade involved in cholesterol synthesis, has emerged as a potential target for cancer therapy. In this study, we first investigated the correlations between the MVA pathway and MM progression. Leveraging public databases, transcriptome sequencing, mass cytometry, as well as in vitro and in vivo experimental models, we further systematically evaluated the therapeutic efficacy and underlying mechanisms of statins, a class of clinically available MVA pathway inhibitors, for the treatment of MM. Using microarray and sequencing datasets, we found that the MVA pathway is dysregulated in MM and correlated with poor prognosis, as core genes in this pathway were significantly dysregulated in MM patients. Statins directly inhibit MM cell viability, induce apoptosis, and up-regulate apoptosis-associated proteins. These effects of statins are associated with the activation of the ERK pathway. They also induce dysregulation of metabolism, oxidative stress, and autophagy in MM cells. Moreover, the combination of statins and bortezomib exerts a synergistic anti-MM effect both in vitro and in vivo. Our preclinical data suggest that the MVA pathway can serve as a therapeutic target for MM and the combination of statins, widely used and safe drugs in the clinic, with bortezomib offers a promising strategy to overcome bortezomib resistance and improve therapeutic outcomes for MM patients.
Megakaryocytes, beyond their classical role in platelet formation, are increasingly recognized as immune modulators. This study aimed to investigate the function and activation mechanisms of megakaryocytes during Pseudomonas aeruginosa (PA) infection, focusing on their interaction with bacterial toxins and innate immune pathways. This study evaluated the response of pulmonary megakaryocytes to PA-induced acute pneumonia in mice. Specifically, the activation status of pulmonary megakaryocytes was determined by flow cytometry and sorting, followed by RNA sequencing, combining Gene Ontology and Gene Set Enrichment Analysis. The expression of pro-inflammatory cytokines and chemokines within megakaryocytes after PA infection were detected by qPCR and fluorescent confocal imaging. The effect of megakaryocytes on the chemotaxis and recruitment of neutrophils and monocytes was demonstrated using chemotaxis assays. Additionally, purified PA toxins were injected into the lungs of mice in vivo or added to flow-sorted cells in vitro in order to induce inflammation and evaluate the response of megakaryocytes to a single PA toxin. The activation status of intracellular signaling pathways was verified through inhibitor-mediated blocking experiments. PA infection significantly activates megakaryocytes, which promote the recruitment of phagocytes and enhance the host’s defense against PA infection. The innate immune response in the lungs was diminished during the early stages of PA infection among the megakaryocytes depleted mice, resulting in increased bacterial load and higher mortality. Flow cytometry sorting followed by RNA sequencing indicated that megakaryocytes exhibit a typical pro-inflammatory phenotype, along with NF-κB pathways activation. Furthermore, the PA toxin PcrV was identified as a key NF-κB activator on megakaryocytes, as demonstrated by increased expression of inflammatory markers and chemokines in response to PcrV. Inhibition of NF-κB signaling attenuated this pro-inflammatory phenotype of megakaryocytes. This study provides new insights into how megakaryocytes shape the cytokine and chemokine landscape in the lung during PA infection. It highlights the intrinsic interactions between megakaryocytes and phagocytes during pathogen invasion, suggests that managing megakaryocyte activity could be a new strategy to enhance immune defense. Megakaryocytes, traditionally known for their role in platelet production, have recently emerged as key players in immune responses during infections. In this study, we explore the involvement of pulmonary megakaryocytes in the response to PA infection, with a specific focus on the mechanistic activation of megakaryocytes via bacterial toxins. We demonstrate that PA infection triggers significant alterations in chemokine signaling and NF-κB pathways in pulmonary megakaryocytes, leading to the upregulation of pro-inflammatory cytokines and chemotactic factors. Activated pulmonary megakaryocytes effectively promote the recruitment of phagocytes, enhancing the host’s resistance to PA infection. we identify PcrV as a key NF-κB activator in megakaryocytes, evidenced by elevated expression of inflammatory markers and chemokines in response to PcrV. Inhibition of NF-κB signaling dampens this inflammatory response, confirming its critical role in megakaryocyte. Together, these findings provide novel insights into the immune functions of megakaryocytes as active modulators during bacterial infections. This work underscores the intricate crosstalk between megakaryocytes and phagocytes and suggests that targeting megakaryocyte activity may represent a promising strategy for controlling bacterial pneumonia.
Diabetic cardiomyopathy (DCM) is a significant complication of diabetes mellitus, characterized by impaired cardiac function and mitochondrial injury. The natural alkaloid 1-deoxynojirimycin (DNJ) has exhibited cardioprotective potential; however, its specific molecular targets in cardiomyocytes are yet to be identified. This work sought to discover the direct targets of DNJ in cardiomyocytes and elucidate the molecular mechanism behind its cardioprotective activity. We first established a cardiomyocyte injury model induced by high glucose. Next, activity-based protein profiling (ABPP) was employed to screen for proteins that directly bind to DNJ. The interactions between DNJ and candidate targets were verified using surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and molecular docking. Finally, the functional outcomes were validated through small interfering RNA (siRNA) knockdown and plasmid overexpression. ABPP screening identified mitochondrial F0 complex subunit B1 (ATP5F1) as the principal binding target of DNJ. SPR analysis confirmed a high-affinity interaction between DNJ and ATP5F1 (Kd = 52.6 nM). Furthermore, CETSA demonstrated that this binding occurred in living cells. Treatment with DNJ (120 µg/mL) significantly ameliorated high glucose-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Knockdown of ATP5F1 via siRNA attenuated the protective effects of DNJ. Conversely, ATP5F1 overexpression potentiated these protective effects. ATP5F1 was identified as a direct functional target of DNJ for the first time. DNJ provides cardioprotection by mitigating mitochondrial malfunction, oxidative stress, and apoptosis via its direct interaction with ATP5F1. These findings collectively clarify a new mode of action for DNJ and offer a scientific basis for its possible therapeutic use in DCM.
The global emergence of monkeypox virus (MPXV) highlights the urgent need for a deeper understanding of host–pathogen interactions. Although transcriptional responses to MPXV infection have been characterized, the role of epitranscriptomic regulation particularly N6‑methyladenosine (m6A) modification remains largely unexplored. We performed an integrated analysis of time‑series transcriptomic and m6A methylome profiles using whole blood samples collected from MPXV‑infected rhesus macaques at 7, 14, and 21 days post‑infection, with distinct animals used at each time point. Host gene expression and m6A modification dynamics were examined over the course of infection. Differential expression and differential m6A modification analyses were conducted, followed by integrative pathway and immune cell signature profiling. MPXV infection induced sustained host reprogramming, characterized by suppression of immune pathways and activation of metabolic processes. A global increase in m6A modifications was observed, accompanied by upregulation of the methyltransferase METTL3 and downregulation of demethylases (FTO, ALKBH5) and readers (YTHDF1-3). Knockdown of METTL3 or YTHDF2 reduced viral replication, suggesting a proviral role for this regulatory circuit. Integrative analysis identified 38 genes with coordinated changes in both transcription and m6A modification across all three time points. Focusing on literature-curated pathogenic pathways, we further identified 11 dual-regulated host factors. Notably, DNAJB1 was the only gene shared between these two independent selection strategies. m6A peaks near transcription start sites and within 5′UTR positively correlated with gene expression, whereas coding region modifications showed weak negative correlations. Immune lineage signatures showed gradual declines in T cell, NK, and monocyte/macrophage signatures with a progressive increase in B cell signatures. Cross‑dataset comparison confirmed core m6A regulatory trends despite heterogeneity across tissues and viral strains. This study reveals m6A epitranscriptomic remodeling as a key correlate of the host response to MPXV infection and nominates DNAJB1 alongside the other 10 dual‑regulated genes as candidate host factors for further mechanistic investigation.
Trastuzumab-based HER2-targeted therapy remains the cornerstone treatment for HER2-positive breast cancer. However, its clinical efficacy is significantly modulated by the tumor microenvironment (TME). Our single-cell sequencing analysis of clinical samples revealed that patients with poor radiologic response after trastuzumab-based neoadjuvant therapy presented significant enrichment of TIGIT+ NK cells with high immune checkpoint expression, exhausted CD8+ T cells, and immunosuppressive regulatory T cells (Tregs). Further analyses leveraging cell-cell communication, spatial transcriptomics, and multiplex immunofluorescence showed that SPP1+ tumor-associated macrophages (SPP1+ TAMs) enrichment was associated with dysfunctional NK- and T-cell states in tumors from patients with poor radiologic response. Functional validation studies revealed that SPP1+ TAMs actively induced exhaustion phenotypes in both NK cells and CD8+ T cells, thereby impairing trastuzumab-dependent antibody-dependent cellular cytotoxicity (ADCC) and adaptive immune responses. In vivo experiments using humanized NCG murine models further confirmed the SPP1+ TAMs-mediated suppression of NK cell function. Significantly, HER2-positive breast cancer patients with elevated SPP1⁺ TAMs levels experienced both reduced efficacy of trastuzumab neoadjuvant therapy and diminished long-term survival prospects. In summary, our findings provide the first systematic characterization of TME remodeling following trastuzumab therapy, identifying SPP1+ TAMs as a potential driver of trastuzumab resistance. This work advances our understanding of microenvironmental mechanisms underlying trastuzumab resistance and suggests new therapeutic strategies targeting TAM-mediated immunosuppression.
Myopia is increasingly recognized as a complex pathology involving retinal remodeling, yet the cellular mechanisms linking early visual stress to this process remain unclear. This study investigated whether Müller glial (MG) cells undergo a glial-mesenchymal transition (GMT)-like response in early myopia and identified the upstream signaling axis driving this transformation. We integrated single-cell and spatial transcriptomic analyses to construct a high-resolution atlas of retinal cell heterogeneity in a guinea pig model of form-deprivation myopia (FDM). Single-cell analysis revealed an expanded MG population (18.9
The tumor microenvironment (TME) is a dynamic and constantly evolving milieu surrounding cancer cells, comprising numerous components that influence tumor growth. The Carcinoembryonic Antigen-Related Cell Adhesion Molecule (CEACAM) family has been found to be abnormally expressed in various cancers, and recent studies have highlighted its critical role in shaping and regulating the TME. Several antibody drugs targeting the CEACAM family have been developed over the last years. However, due to the physiological expression of CEACAMs in normal tissues, most of these targeted agents have yielded unsatisfactory clinical trial outcomes, characterized by significant toxicity and limited efficacy. In view of this, enhancing therapeutic efficacy while reducing adverse effects has become a focal point of current research. This review systematically summarizes the structural features of the CEACAM family and the intrinsic mechanistic differences underlying their tumor-promoting effects, and discusses the limitations of existing clinical drugs. Based on antibody optimization strategies, we propose a conceptual framework of “dual-recognition” antibodies and elaborate on precision targeting approaches at three levels—epitope, molecular, and environmental—to provide directional guidance for future investigations.
Breast cancer (BC) development is influenced by multifactorial mechanisms. Despite significant advancements in early diagnostic techniques and comprehensive therapeutic approaches, BC continues to pose a substantial threat to women’s health, exhibiting persistently high incidence and mortality rates. The Notch signaling pathway, a highly conserved evolutionary pathway initially identified in Drosophila, has garnered extensive research interest and is implicated in the pathogenesis of diverse malignancies, including BC. However, accumulating evidence reveals a highly context-dependent role for Notch signaling in cancer, with documented functions ranging from oncogenic to tumor-suppressive depending on the specific cellular and microenvironmental context. Current evidence indicates that Notch1–4 display nonredundant functional divergence in BC. In this review, we discuss how receptor-specific regulatory mechanisms shape distinct Notch1–4 signaling outputs and summarize the context-dependent functions and molecular mechanisms of Notch receptors across tumor-cell states, BC molecular subtypes, and tumor microenvironmental components. We also review current advances and limitations in pan-Notch inhibition and receptor-specific targeting strategies, aiming to provide clearer directions for future Notch receptor–targeted therapy in BC.
A growing body of evidence suggests that enhanced glycolysis profoundly reprograms inflammatory response in acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS), but the underlying mechanisms largely remain unclear. Lactate is the end-product of glycolysis, which has been recently found to function as a bioactive metabolite via G-protein coupled receptor 81 (GPR81). In the present study, the potential roles of lactate-GPR81 axis were investigated in mice with lipopolysaccharide (LPS)-induced ALI. The results indicated that LPS challenge increased the level of lactate in bronchoalveolar lavage fluid (BALF). Pharmacological suppression of glycolysis or inhibition of lactate dehydrogenase decreased lactate level and alleviated lung injury, but supplementation with lactate or a GPR81 agonist exacerbated lung injury. Global deletion of GPR81 or endothelial-specific deletion of GPR81, but not myeloid-specific deletion of GPR81, resulted in alleviated lung injury. In endothelial cells, the differentially expressed genes (DEGs) in RNA-seq after lactate supplementation were enriched in cAMP signaling pathway. Consistently, supplementation with lactate resulted in decline of cAMP, reduction of VE-cadherin and enhanced phosphorylation of myosin light chain 2 (MLC2) in endothelial cells, whereas knockdown of GPR81 reversed these effects. In addition, the modulation of lactate/GPR81 on cAMP, VE-cadherin and MLC2 was validated in GPR81 knockout mice. Importantly, the elevation of lactate is positively correlated with the degree of protein leakage, the level of proinflammatory cytokines in BALF and APACHE II score from patients with ARDS. Taken together, the present study suggests that endothelial GPR81 molecularly bridges glycolysis and inflammation, which drives microvascular hyperpermeability and the development of ALI.
Sepsis is a life-threatening syndrome resulting from a dysregulated host immune response to infection and is frequently accompanied by coagulation abnormalities and multiple organ dysfunction. As central regulators at the intersection of hemostasis, inflammation, and immunity, platelets play a critical role in the initiation and progression of sepsis. Beyond their traditional function in hemostasis, platelets actively participate in immune modulation, inflammatory amplification, and immunothrombosis. This review systematically summarizes the molecular and cellular mechanisms through which platelets contribute to organ injury in sepsis, with particular emphasis on platelet–immune cell interactions. We discuss how these processes drive the development of acute lung injury, myocardial depression, hepatic and renal dysfunction, and disseminated intravascular coagulation. In addition, we examine the dynamic alterations in platelet phenotype and function during sepsis and their clinical implications. Ultimately, we evaluate the therapeutic potential of antiplatelet strategies, addressing current challenges and future directions for translating platelet-targeted interventions into effective treatments for sepsis.
Phagocytosis of surface-bound microbes is essential for host defence and environmental feeding, yet the mechanism by which macrophages remove surface-bound particles has only recently been described. This process involves the formation of an F-actin-rich, force-bearing ring around the surface-attached particle. Here, we identify the IQGAP-related protein IqgD from the professional phagocyte Dictyostelium discoideum as a key regulator of mechanically demanding phagocytosis. IQGAPs are large multidomain scaffold proteins that interact with Rho family GTPases and F-actin. IqgD contains a calponin homology domain (CHD), a GAP-related domain (GRD), a RasGAP C-terminal (RGCT), and an extreme C-terminal (CT) domain. In this study, we used biochemical and imaging approaches with full-length and truncated protein variants to investigate whether IqgD interacts with D. discoideum Rho GTPases and F-actin. We also performed comprehensive phenotypic characterisation of IqgD-deficient cells to determine the cellular function of IqgD. We show that the CHD is essential for F-actin binding and cortical localisation, while the GRD and CT domains mediate interactions with Rac1 GTPases and the actin-bundling proteins cortexillins. Moreover, similar to mammalian IQGAPs, IqgD maintains Rac1 in its active conformation. Although IqgD is enriched in macropinocytic and phagocytic cups, it is not required for fluid uptake or internalisation of bacteria from suspension. However, loss of IqgD markedly reduces growth on bacterial lawns and strongly impairs uptake of surface-attached microbeads and yeast particles. Furthermore, IqgD localises to F-actin-rich ring-like structures that form around surface-bound particles at the basal cell surface. IqgD is not required for all forms of macroendocytosis; rather, it is specifically required for mechanically demanding phagocytosis. This includes the formation of enlarged phagocytic cups during phagocytosis of yeast particles or the generation of greater force, as in phagocytosis of surface-bound particles and bacteria within bacterial lawns. Similar to phagocytosis of surface-attached particles in mammalian macrophages, D. discoideum also forms an F-actin-rich ring around the particle at the cell base, suggesting that force-driven particle detachment and internalisation may be an evolutionarily conserved mode of substrate-dependent phagocytosis. Our findings provide mechanistic insight linking IqgD with Rac1, cortexillins, and F-actin in the regulation of demanding forms of phagocytosis.
Chronic tissue inflammation causes progressive tissue damage, organ dysfunction, and increased susceptibility to inflammatory diseases. Viral infections are major drivers of this process, but the molecular mechanisms linking antiviral immune responses to persistent inflammation and tissue pathology remain poorly understood. The function of TRIM47 was examined using primary macrophages and mouse models of RNA virus infection. Alveolar macrophages, peritoneal macrophages, and bone marrow–derived macrophages (BMDM) from wild-type and Trim47-deficient (Trim47-KO) mice were treated with RNA viruses or RNA mimics, and antiviral cytokine production was quantified. Sublethal reovirus and influenza A virus infection mouse models were used to evaluate survival, cytokine responses, and viral titers in vivo. Liquid chromatography-tandem mass spectrometry (LC-MS) was used to identify TRIM47-interacting proteins. Molecular and biochemical assays were used to examine post-translational modification, aggregation and activation of mitochondrial antiviral-signaling protein (MAVS). TRIM47 was highly expressed in macrophages, and TRIM47 deficiency in BMDM markedly reduced type I interferon (IFN-I) production following RNA virus infection or RNA mimics stimulation. In sublethal RNA virus infection mouse models, Trim47-KO mice exhibited reduced survival, impaired IFN-I responses, higher viral titers, increased inflammatory cell infiltration and tissue inflammation including myocarditis compared to wild-type mice. Mechanistically, TRIM47 interacted with MAVS and promoted SUMO1-mediated MAVS SUMOylation at K297 and K348, thereby enhancing MAVS aggregation and activation and driving robust IFN-I, IL-1β and IL-18 production in macrophages. These findings establish TRIM47 as a molecular switch of MAVS activation and antiviral cytokine responses in macrophages, highlighting its role in restricting RNA virus infection and mitigating inflammation. This study provides mechanistic insight into host pathways that may be therapeutically leveraged in chronic viral inflammation.
Uterine spiral artery remodelling (SAR) is a fundamental developmental process that facilitates optimal placental perfusion and supports fetal growth. Central to SAR is the phenotypic transformation of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic state, directed by invasive trophoblast cells. To advance these findings, we sought to elucidate the epigenetic mechanisms governing trophoblast-induced reprogramming of VSMC identity, enabling plasticity required for uterine vascular adaptation. VSMC dedifferentiation was assessed by qRT-PCR, Western blotting, and immunofluorescence. Epigenetic alterations were evaluated by Western blotting. A chromatin remodelling PCR array was performed and validated by qRT-PCR and Western blotting. Chromatin remodelling factors were downregulated using antisense oligonucleotides (ASOs), and VSMC dedifferentiation was confirmed by Western blotting. E13.5 and E16.5 rat metrial glands were used for in vivo validation. An IUGR rat model was generated by administering dexamethasone from E13.5 to E20.5. In IUGR tissues, trophoblast invasion, VSMC dedifferentiation, and chromatin remodelling factor expression were validated by Western blotting. Co-culture of primary E16.5 rat trophoblast cells with VSMCs revealed a trophoblast-induced shift in the expression landscape of epigenetic regulators, resulting in upregulation of chromatin-modifying “writers” (CBP, DNMT1, DNMT3A), downregulation of “erasers” (HDAC 1,2,3), and an increase in both transcription activation marks (H3K27ac, H3K9ac) and repression marks (H3K27me3, H3K9me3). Targeted real-time PCR array profiling identified coordinated downregulation of 13 chromatin remodelling genes (ARID1B, SMARCAD1, SMARCD1, SMARCD3, BMI1, EZH2, CBX2, CBX5, CBX6, BAZ1B, ZMYND8, CHD1, MBD3,) during VSMC de-differentiation. This change in the epigenetic landscape was recapitulated in vivo within the metrial gland, the entry point of uterine spiral arteries, on E16.5. Knockdown of these factors impaired the phenotypic transition of VSMCs, establishing their mechanistic role in enabling vascular adaptation. Notably, in a model of intrauterine growth restriction (IUGR), the normal expression dynamics of chromatin remodelling factors and VSMC phenotypic markers were reversed, indicating the need for the dynamic epigenetic regulatory axis, which is essential for vascular adaptation in healthy pregnancies. These findings uncover a trophoblast-driven epigenetic axis that governs VSMC plasticity during SAR and highlight its dysregulation as a potential contributor to the pathogenesis of IUGR.
