AAK1 and BMP2K are serine/threonine kinases traditionally known for phosphorylating AP2 during clathrin-mediated endocytosis (CME), but their broader roles remained incompletely defined. Here, using motif-guided in silico, biochemical, and phosphoproteomic screens, we identify PDLIM5 and Talin1 as direct AAK1/BMP2K substrates. Despite high kinase-domain similarity, only AAK1 promotes cell migration and potentiates focal adhesion (FA) turnover. Live-cell imaging shows that AAK1 recruitment to FAs peaks as disassembly begins. The conserved AAK1 C-terminal PDZ-binding motif mediates direct, low-affinity binding to PDLIM5, providing a plausible mechanism for localized substrate access. Dynamic analyses of phospho-mimetic and phospho-null mutants support a model in which AAK1-dependent phosphorylation promotes timely release of PDLIM5 and Talin1 during FA disassembly. These findings reveal a kinase-driven contribution to FA turnover distinct from protease- and phosphatase-based mechanisms and suggest that functional divergence between AAK1 and BMP2K may provide a strategy to modulate cell migration with reduced impact on CME.

Childhood cancer survivors (CCS) are at increased risk of developing heart disease due to the cardiotoxic effects of oncological treatment. This study aimed to investigate the long-term cardiotoxic effects of cancer therapy in CCS using a multimodal approach combining cardiac magnetic resonance (CMR) imaging and circulating blood biomarkers. A total of 117 CCS (mean age 24.7 ± 5.2 years), at least five years post-treatment and in complete remission, were prospectively enrolled. All participants underwent CMR, including T1 mapping, and blood analysis for biomarkers of endothelial damage and oxidative stress. Parameters were compared with sex- and age-matched healthy control groups. Anthracycline treatment was administered in 82.9

Sepsis is a life-threatening condition characterised by an overwhelming immune response and high fatality. While most research has focused on its acute phase, many sepsis survivors remain immunologically weakened leaving them susceptible to serious complications from even mild infections. The mechanisms underlying this prolonged immune dysregulation remain unclear, limiting effective interventions. Here, we analysed whether sepsis induced long-term “training” in hematopoietic stem and progenitor cells (HSPCs), imprinting changes that persist in their myeloid progeny. Peripheral blood analysis of 8 sepsis survivors, 12 patients with septic shock, and 10 healthy donors revealed a significant expansion of CD38 + progenitors in survivors, with increased megakaryocyte-erythroid progenitors and a near significant reduction in mature neutrophil counts. This shift suggests impaired granulopoiesis, favouring immature, immunosuppressive granulocytes. Differentiated macrophages from survivors’ HSPCs exhibited impaired metabolic pathways after lipopolysaccharide stimulation, with downregulation of tricarboxylic acid cycle and glycolysis genes, indicating altered immune metabolism. Pathway analysis revealed enhanced type-I interferon (IFN) and JAK-STAT signalling in survivors’ macrophages, reflective of potentially tolerance-prone reprogramming. Finally, exposing healthy donor HSPCs to IFNβ during macrophage differentiation reduced HSPC proliferation, increased apoptosis, and induced a metabolic shift towards glycolysis over mitochondrial respiration. Together, these findings suggest that sepsis induces lasting reprogramming in HSPCs leading to myeloid progeny with altered immune memory that might drive immune dysregulation in survivors. These data open avenues to explore potential targets to better manage long-term immune alterations in sepsis survivors.

Natural killer (NK) cells are central to cancer immunosurveillance and immunotherapy. Their ability to engage in killing is critical for efficient target elimination and depends on tightly regulated Ca2+ signaling controlling granzyme degranulation. Toll-like receptor (TLR) engagement has been linked to Ca2+ signaling in other immune cell types, while a specific role in NK cells remains unresolved. Given that NK cells express a broad range of TLRs, and that presence of TLR ligands-including damage- and pathogen-associated molecular patterns-can influence clinical scenarios including adoptive NK cell therapy, investigating TLR-driven Ca2+ signaling in NK cells is particularly relevant. Here, we examined how stimulation with selected TLR ligands influences Ca2+ signaling and NK cell activity. Immediate stimulation induced a rapid elevation of cytosolic Ca2+ in expanded NK cells. Functionally, TLR stimulation increased degranulation and enhanced cytotoxicity at high effector-to-target ratios. Under conditions of target excess, however, TLR-treated NK cells displayed impaired killing, likely due to unbalanced Ca2+ levels. These findings demonstrate that TLR signaling directly modulates Ca2+ flux in NK cells and can either potentiate or impair cytotoxic activity depending on context. Although clinical implications remain hypothetical, such TLR-driven dysregulation affects NK cell killing activity in the inflammatory or pathogen-rich environments frequently encountered after chemotherapy. Our findings suggest that TLR-Ca2+ impact on cytotoxicity should be considered in adoptive transfer therapies where previous treatment affects systemic levels of TLR ligands, such as in patients with acute myeloid leukemia, where NK cell immunotherapy is frequently tested in trials.

Acute myeloid leukemia is an aggressive malignancy with limited treatment options and high relapse rates. Cellular immunotherapy using natural killer (NK) cells offers a promising approach to improve treatment outcomes by employing their innate cytotoxic potential. However, the results of clinical studies show inconsistent efficacy, which could be explained by various aspects, including variable donor-host combinations or differences in cryopreservation steps affecting the viability and cytotoxicity of the final immunotherapeutic product. Here we introduce a novel strategy involving an intermediate cryopreserved product prepared on day 7 of NK-cells expansion, followed by an additional 10-12 days of culture. This approach allowed substantial recovery of NK-cell function after recultivation. Furthermore, since NK cells from different donors display considerable functional and proliferation variability, we performed detailed functional analyses of multiple donors to identify functional and molecular signatures linked to cytotoxicity and expansion potential, combining cytotoxicity assays, immunophenotyping and global transcriptomic profiling. This revealed a distinct gene expression signature distinguishing NK cell donors with superior cytotoxicity and expansion potential, linked to activation and cellular stress responses. Our findings demonstrate that our current Good Manufacturing Practice-compliant expansion method with intermediate cryopreservation supports improved therapeutic timing and preserves NK-cell quality. In parallel, the proposed donor selection approach may enhance manufacturing efficiency by identifying donors whose NK cells have greater cytotoxic and expansion potential.

Acute myeloid leukemia (AML) is an aggressive malignancy with limited treatment options and high relapse rates. Cellular immunotherapy using natural killer (NK) cells offers a promising approach to improve treatment outcomes by employing their innate cytotoxic potential. However, the results of clinical studies show inconsistent efficacy, which could be explained by various aspects, including variable donor-host combinations or differences in cryopreservation steps affecting the viability and cytotoxicity of the final immunotherapeutic product. Here we introduce a novel strategy involving an intermediate cryopreserved product prepared on day 7 of NK-cells expansion, followed by an additional 10–12 days of culture. This approach allowed substantial recovery of NK-cell function after recultivation. Furthermore, since NK cells from different donors display considerable functional and proliferation variability, we performed detailed functional analyses of multiple donors to identify functional and molecular signatures linked to cytotoxicity and expansion potential, combining cytotoxicity assays, immunophenotyping, and global transcriptomic profiling. This revealed a distinct gene expression signature distinguishing NK cell donors with superior cytotoxicity and expansion potential, linked to activation and cellular stress responses. Our findings demonstrate that our current Good Manufacturing Practice-compliant expansion method with intermediate cryopreservation supports improved therapeutic timing and preserves NK-cell quality. In parallel, the proposed donor selection approach may enhance manufacturing efficiency by identifying donors whose NK cells have greater cytotoxic and expansion potential.

The intestine hosts the largest immune system and peripheral nervous system in the human body. The gut‒brain axis orchestrates communication between the central and enteric nervous systems, playing a pivotal role in regulating overall body function and intestinal homeostasis. Here, using a human three-dimensional in vitro culture model, we investigated the effects of serotonin, a neuromodulator produced in the gut, on immune cell and intestinal tissue interactions. Serotonin attenuated the tumor necrosis factor-induced proinflammatory response, mostly by affecting the expression of chemokines. Serotonin affected the phenotype and distribution of tissue-migrating monocytes, without direct contact with the cells, by remodeling the intestinal tissue. Collectively, our results show that serotonin plays a crucial role in communication among gut–brain axis components and regulates monocyte migration and plasticity, thereby contributing to gut homeostasis and the progression of inflammation. In vivo studies focused on the role of neuromodulators in gut inflammation have shown controversial results, highlighting the importance of human experimental models. Moreover, our results emphasize the importance of human health research in human cell-based models and suggest that the serotonin signaling pathway is a new therapeutic target for inflammatory bowel disease. The gut–brain axis involves communication between the brain and the gut, which is important for maintaining gut health. Here the authors explored this by studying serotonin’s role in gut inflammation using a three-dimensional human cell model. They used intestinal organoids to mimic human gut conditions. These organoids were treated with serotonin and TNF to study their effects on gut cells and immune responses. The researchers found that serotonin reduced TNF-induced inflammation by altering gene expression related to immune cell movement. The study showed that serotonin can decrease the production of certain inflammatory signals in the gut, potentially reducing inflammation. This suggests that targeting serotonin could help treat inflammatory bowel disease (IBD). In conclusion, serotonin plays a role in controlling gut inflammation, offering insights into new treatments for IBD. Future research could explore serotonin’s broader impact on other inflammatory diseases. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, in which epithelial-to-mesenchymal transition (EMT) plasticity is required for successful metastasis. ERK3 has been implicated in promoting breast cancer migration and invasion, but the mechanisms remain elusive. Here, we investigated ERK3 expression across patient-derived datasets and explored its role in promoting EMT plasticity using different 2D and 3D in vitro models to investigate cell-extracellular matrix adhesion, migration and invasion, anchorage-independent growth, extravasation and colonization. We have established an association between ERK3 overexpression and aggressive breast cancer phenotypes, higher tumour plasticity, as informed by its grade, and poor clinical outcomes. Based on the hypothesis that ERK3 contributes to TNBC progression by supporting a partial-EMT state, we showed that ERK3 contributes to different steps of the metastatic process, especially by enabling collective migration but also by modulating other functional aspects related to an active EMT program. In conclusion, our results demonstrate that ERK3 contributes to TNBC progression and potentially metastasis by promoting EMT plasticity and collective migration.

Objectives:Several years after the COVID-19 pandemic, the impact of SARS-CoV-2 on immunity and the potential protective role of Bacillus Calmette-Guérin (BCG) vaccination through trained immunity remain a subject of investigation. This study aimed to determine the long-term impact of SARS-CoV-2 on immune cells and the association between BCG vaccination, latent infections and COVID-19 severity and sepsis progression. Methods:We conducted a prospective analysis of patients who recovered from mild/severe/critical COVID-19 (n = 97, 3-17 months after COVID-19) and sepsis patients (n = 64). First, we assessed the impact of COVID-19 and its severity on immune cell frequencies and expression of functional markers. Further, we analysed plasma titres of anti-Toxoplasma gondii/cytomegalovirus/BCG antibodies and their association with COVID-19 severity and sepsis outcome. To examine monocyte responses to secondary challenge, monocytes isolated from COVID-19 convalescent patients, BCG vaccinated and unvaccinated volunteers were stimulated with SARS-CoV-2 and LPS. Results:Post-COVID-19 patients showed immune dysregulation regardless of disease severity characterised by altered expression of activation and functional markers in myeloid (CD39, CD64, CD85d, CD11b) and lymphoid cells (CD39, CD57, TIGIT). Strikingly, post-critical COVID-19 patients showed elevated expression of CD57 in CD8+ T cells compared to other severity groups. A trend toward improved outcomes in BCG-seropositive COVID-19/sepsis patients was observed, although this may be confounded by age differences between groups. In contrast, the monocyte response to stimulation appeared unaffected by COVID-19 severity. Conclusion:These findings highlight the long-term alterations of immune cells in post-COVID-19 patients, emphasising the substantial impact of COVID-19 on immune function.

Timely resolution of innate immune responses activated by surgical intervention is crucial for patient recovery. While cytokines and innate immune cells are critical in inflammation resolution, the specific role of IL-18 in these processes remains controversial and underexplored. We investigate determinants of successful recovery using peripheral blood samples from orthopedic surgery (ORT) patients (n = 33) at T0 (before surgery), T1 (24 h after surgery) and T2 (3 days after surgery). Monocytes from ORT patients underwent immunophenotyping together with bulk transcriptomic analysis. We found that IL-18 strongly defines the recovery immune signature. These results were further validated in vitro by comparing IL-18 and TNF-α effects on monocytes, and in 3D human intestine organoids together with single cell (sc)-RNAseq analysis. Transcriptomics of ORT monocytes revealed upregulation of ITG family integrins, namely ITGB3 and ITGB5, CXCL family chemokines, notably CXCL1-3, CXCL5, and SCL/TAL1 factor controlling differentiation and migration, but not pro-inflammatory genes. Similar changes were observed in IL-18 stimulated healthy donor monocytes in vitro, including an increase in CD11b, CD64, and CD86 levels, accompanied by increased phosphorylation of Akt but not NFκB. These changes were attenuated in the presence of TNF-α, thus showing a unique role of IL-18 when acting alone without its most frequent paired cytokine TNF-α. We further confirmed that IL-18 induces monocyte-macrophage transition and migration using human intestinal organoids. Finally, TNF-α/IL-18 ratio showed a high predictive value of clinical severity in septic patients. We propose a novel role of IL-18 on monocyte migration and macrophage transition characterizing successful orthopedic surgery recovery, as well as the ratio of IL-18/TNF-α as a novel marker of inflammation resolution, with potential implications for patient monitoring and therapeutic strategies.

Autologous hematopoietic stem and progenitor cell (HSPC) transplantation is performed after myeloablation in cancer treatment to restore blood cell production and support immune recovery. Despite its success in achieving survivorship, many recipients later suffer from recurrent infections and pulmonary complications. The mechanisms driving the complications after HSPC transplantation later in life are unknown. However, the induction and/or maintenance therapeutics might be driving the negative outcomes in treated patients. We investigated the effects of the cancer therapeutics topotecan and 13-cis-retinoic acid (13cisRA) on cell phenotype and functions of HSPCs isolated from cord blood and primary monocytes from adult donors. In HSPCs, 13cisRA reduced autophagy and lysosomal activity, triggered a DNA damage response through increased γH2A.X and CDKN2 expression (including the spliced p14ARF isoform), and upregulated the epigenetic regulator SIRT3. 13cisRA also activated primary monocytes, inducing CXCL8 and CCL2 production. By contrast, topotecan had no effects on mature monocytes but induced DNA damage, metabolic remodeling, and epigenetic alterations in HSPCs. These changes included increased CDKN1A expression, higher γH2A.X-positive cell frequency, autophagy activation, and SIRT1 upregulation. The differential effects of 13cisRA and topotecan on HSPCs and monocytes might underlie the long-term complications of autologous HSPC transplantation. By modulating DNA damage, autophagy, and epigenetic pathways, these therapies could influence hematopoietic recovery and immune reconstitution, offering insights for improving transplantation outcomes.

BackgroundTrauma is a leading global cause of mortality, and systemic inflammatory response syndrome (SIRS) remains a significant complication, contributing to adverse outcomes. Neutrophils, as first responders to tissue injury, undergo substantial phenotypic and functional changes following trauma. This study investigates neutrophil subpopulations defined by CD16 and CD62L expression in trauma patients, focusing on their correlation with clinical biomarkers, trauma severity, and functional properties.MethodsWe included 50 non-infectious trauma patients, categorized into SIRS and Non-SIRS groups, and 43 elective surgery patients as controls. Neutrophil subsets were analyzed at two time points (TP1 and TP2) using flow cytometry. Functional assays evaluated phagocytosis, oxidative burst, mitochondrial function, and degranulation. Correlations between neutrophil subpopulations and clinical markers, including lactate, creatine kinase, Injury Severity Score, and Trauma and Injury Severity Score, were examined.ResultsPatients with SIRS exhibited higher proportions of banded neutrophils and CD16lowCD62Llow neutrophils at TP1, alongside reduced levels of mature neutrophils. Elevated lactate and creatine kinase levels positively correlated with banded neutrophils and CD16lowCD62Llow neutrophils, while negatively correlating with mature neutrophils CD16highCD62Lhigh and hypersegmented neutrophils CD16highCD62Llow. Hypersegmented neutrophils were more prevalent in Non-SIRS patients at TP1 and in SIRS patients at TP2. Banded neutrophils showed a positive correlation with Injury Severity Score and an inverse correlation with Trauma and Injury Severity Score (TRISS), whereas hypersegmented neutrophils were negatively associated with ISS and positively correlated with TRISS. These correlations likely reflect the pro-inflammatory role of banded neutrophils and the inflammation-resolving function of hypersegmented neutrophils. CD16lowCD62Llow neutrophils displayed impaired phagocytosis, oxidative burst, and degranulation capacity, indicating functional deficiencies.ConclusionThis study highlights the dynamic changes in neutrophil subpopulations in trauma and their association with systemic inflammation and clinical severity. Increased banded neutrophils correlate with SIRS and metabolic stress, whereas hypersegmented neutrophils may contribute to resolving inflammation. CD16lowCD62Llow neutrophils exhibit functional impairments, warranting further investigation. Monitoring neutrophil subpopulations could aid in identifying trauma patients at risk for non-infectious SIRS and guide therapeutic interventions.

Soft microrobots, compared with their rigid counterparts, offer superior adaptability in dynamic and confined biological environments. Here, magnetically-guided liquid metal microrobots composed of gallium-indium alloys embedded with Fe nanoparticles are introduced. The unique combination of magnetic maneuverability, high surface tension, intrinsic radiopacity, and deformability allows liquid metal-based microbots to overcome limitations of both hard microrobots and fragile droplet-based systems. Under magnetic actuation, liquid metal-based magnetic microrobots exhibit controllable rolling and upstream locomotion resembling neutrophil-like navigation, enabling precise maneuvering even against physiological flow. Bridging in vitro with in vivo experiments, quail egg chorioallantoic membrane models are used to demonstrate guided transport of these microrobots through blood vessels, accumulation at tumor xenografts, and migration within subcutaneous tissues. Moreover, their strong X-ray visibility enables real-time fluoroscopic tracking, validated in porcine heart vasculature. Importantly, liquid metal-based magnetic microbots can cross endothelial barriers in a vascular flow-on-a-chip platform, while maintaining endothelial biocompatibility. By integrating deformability, magnetic steerability, and imaging visibility, liquid metal-based microrobots establish a powerful platform for minimally invasive transvascular navigation. This work highlights the potential of liquid metal-based magnetic microrobots for targeted drug delivery, image-guided therapy, and intelligent biomedical interventions.

Background:Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse, yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. Methods:TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. Results:Both leukemic cells and MSCs produced TGF-β, with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity, adhesion, and migration, supporting its immunosuppressive role. In functional assays, TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids, particularly in models incorporating MSCs. Additionally, MSCs themselves provided a protective environment for leukemic cells, further reducing NK cell effectiveness in 2D co-cultures. Conclusion:TGF-β suppresses both NK cell cytotoxicity and migration, limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β-mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials.

Fibroblast growth factors (FGFs) control organ morphogenesis during development as well as tissue homeostasis and repair in the adult organism. Despite their importance, many mechanisms that regulate FGF function are still poorly understood. Interestingly, the thermodynamic stability of 22 mammalian FGFs varies widely, with some FGFs remaining stable at body temperature for more than 24 h, while others lose their activity within minutes. How thermodynamic stability contributes to the function of FGFs during development remains unknown. Here we show that FGF10, an important limb and lung morphogen, exists as an intrinsically unstable protein that is prone to unfolding and is rapidly inactivated at 37 °C. Using rationally driven directed mutagenesis, we have developed several highly stable (STAB) FGF10 variants with a melting temperature of over 19 °C more than that of wildtype FGF10. In cellular assays in vitro, the FGF10-STABs did not differ from wildtype FGF10 in terms of binding to FGF receptors, activation of downstream FGF receptor signaling in cells, and induction of gene expression. In mouse embryonal lung explants, FGF10-STABs, but not wildtype FGF10, suppressed branching, resulting in increased alveolarization and expansion of epithelial tissue. Similarly, FGF10-STAB1, but not FGF10 wildtype, inhibited the growth of mouse embryonic tibias and markedly altered limb morphogenesis when implanted into chicken limb buds, collectively demonstrating that thermal instability should be considered an important regulator of FGF function that prevents ectopic signaling. Furthermore, we show enhanced differentiation of human iPSC-derived lung organoids and improved regeneration in ex vivo lung injury models mediated by FGF10-STABs, suggesting an application in cell therapy.

Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.