The remaining unacceptably high mortality of influenza-induced acute respiratory distress syndrome underscores the urgent need to identify key cellular drivers of host responses. Endothelial cells (ECs) are increasingly recognized for their immunomodulatory roles, but whether they function as antigen-presenting cells (APCs) following respiratory viral infection remains unknown. Here, we show that influenza A virus H1N1 restrictively infects pulmonary microvascular ECs (PMVECs) during late-stage acute lung injury, triggering robust MHC class I (MHC-I) upregulation in vitro, in vivo, and in ex vivo human precision-cut lung slices. Infected PMVECs present H1N1 antigens via MHC-I and co-stimulatory CD40 to lung-resident CD8+ T cells, driving their proliferation and effector function (Granzyme B, IFNγ) to promote viral clearance and resolve inflammation. This process is IFNγ-dependent and STAT1-regulated, forming a positive feedback loop that enhances PMVEC antigen presentation and CD8+ T cells activation. By contrast, the emerging H5N1 (A/Texas/37/2024) infect pulmonary ECs earlier and more broadly but elicits weaker pulmonary EC-driven CD8+ T cell responses, potentially contributing to its higher pathogenicity. These findings reveal PMVECs as active APCs in antiviral defense and highlight new avenues for immunotherapeutic intervention.

Cellular stiffness profoundly impacts cancer metastasis at multiple levels, but mechanisms that regulate cancer cells' stiffness remain poorly understood. Here, we identified potassium efflux and KCNMB1, an auxiliary subunit of the large conductance potassium efflux (BK) channels, as regulators of cellular stiffness downstream of myocardin related transcription factor A (MRTFA). In primary pericytes, KCNMB1 knockdown increased cellular stiffness, which is consistent with the role of potassium efflux in promoting relaxation during excitation-contraction coupling. In a striking contrast, however, KCNMB1 knockdown decreased cellular stiffness in cancer cells. Softer cancer cells were resistant to NK cell mediated cytotoxicity and the low KCNMB1 expression was associated with worse survival in breast cancer patients. Importantly, pharmacological activation of BK channels reduced metastatic burden in mice and improved lysis of cancer cells by cytotoxic T-lymphocytes. These results highlight the unique ionic regulation of stiffness in cancer cells and point to BK channel agonism as a new therapeutic approach in cancer.

BackgroundExtracellular vesicles (EVs) derived from dental pulp stem cells (DPSCs) exhibit immunomodulatory activity in multiple inflammatory models. However, the limited replicative lifespan of primary DPSCs and passage-dependent functional variability restrict scalable and reproducible EV production. Approaches that stabilize EV bioactivity during extended cell expansion are therefore needed.MethodsHuman primary DPSCs were immortalized via expression of human telomerase reverse transcriptase (hTERT) to mitigate senescence-associated limitations. EVs isolated from naïve and hTERT-immortalized DPSCs were characterized for size distribution, morphology, marker expression, and cellular endocytosis. Immunomodulatory activity of EVs isolated from multiple passages of immortalized DPSCs was evaluated in vitro on lipopolysaccharide (LPS)/interferon-γ–activated primary mouse bone marrow–derived macrophages in comparison with primary DPSC EVs. In vivo efficacy was assessed in a murine model of LPS-induced acute lung injury.ResultshTERT expression increased telomerase levels without altering the multi-lineage differentiation potential of the cells. No significant changes in EV size, morphology, or expression of canonical EV markers was observed. EV uptake by macrophages was unchanged between naïve and immortalized DPSC-derived EVs. Both EV sources similarly suppressed pro-inflammatory cytokine expression, including TNF-α, IL-1β, and IL-6, in inflamed primary macrophages. Notably, EVs derived from hTERT-immortalized DPSCs retained immunomodulatory activity through at least passage 15. In vivo, EVs from naïve and immortalized DPSCs comparably reduced pulmonary edema, inflammatory gene expression, and neutrophil accumulation following LPS challenge. Similar preservation of EV activity was observed using EVs derived from immortalized human bone marrow derived mesenchymal stem cells.ConclusionhTERT-mediated immortalization of parent DPSCs retains the immunomodulatory function of derivative EVs during extended culture without altering key physicochemical EV characteristics. Immortalized DPSCs represent a reproducible source of EVs with consistent anti-inflammatory activity, supporting their use as a translationally-relevant platform for EV-based immunomodulatory applications.

The existence of human hippocampal neurogenesis has long been disputed1-12 and its relevance in cognition remains unknown. Recent studies have established the presence of proliferating progenitors and immature neurons and a reduction in the latter in Alzheimer's disease (AD)11,13. However, their origin and the molecular networks that regulate neurogenesis and function are poorly understood. Here we studied human post-mortem hippocampi obtained from different cohorts: young adults with intact memory, aged adults with no cognitive impairments, aged adults with extraordinary memory capacity (SuperAgers)14,15, adults with preclinical intermediate pathology or adults with AD. Using multiomic single-cell sequencing (single-nucleus RNA sequencing and single-nuclei assay for transposase-accessible chromatin with sequencing), we analysed the profiles of 355,997 nuclei isolated from the hippocampus samples and identified neural stem cells, neuroblasts and immature granule neurons. Dysregulated neurogenesis was largely associated with changes in chromatin accessibility. Analyses of transcription factors and target gene signatures that distinguished each of the groups revealed early alterations in chromatin accessibility of neurogenic cells from individuals with preclinical AD, and such changes were even more evident in samples from individuals with AD. We identified a distinct profile of neurogenesis in SuperAgers that may reflect a 'resilience signature'. Finally, alterations in the profile of astrocytes and CA1 neurons govern cognitive function in the ageing hippocampus. Together, our study points to a multiomic molecular signature of the hippocampus that distinguishes cognitive resilience and deterioration with ageing.

Eukaryotic mitochondria are characterized by several features that represent vestiges of their prokaryotic ancestry. One such feature is the N-terminal formylation of proteins encoded by mitochondrial DNA that undergo translation by mitochondrial ribosomes. N-formylated proteins are also released by bacteria and trigger activation of immune cells, such as neutrophils. Growing evidence indicates that circulating levels of mitochondrial formyl proteins are elevated in the serum of patients with excessive inflammatory responses. However, the mechanisms by which they are released into circulation are not known. In this study, we have identified vascular endothelial cells as a source of Pink1-dependent release of mitochondrial formyl proteins in response to inflammatory mediators. Mechanistically, the mitophagy mediator Pink1 is stabilized by inflammatory activation of endothelial cells, promoting mitophagy and mitochondrial formyl peptide release both in mice and primary human endothelial cells. Using nanoparticle delivery of Pink1 -targeting sgRNA in mice expressing endothelial-specific Cas9, we developed a mouse model in which Pink1 is specifically depleted in the endothelium. Deletion of endothelial Pink1 decreased circulating formyl peptide levels, lowered lung neutrophil infiltration, and reduced mortality in mice. We thus propose that endothelial cells upregulate pro-inflammatory mitophagy in response to inflammation, leading to the release of mitochondrial formyl peptides and detrimental neutrophil recruitment into the lung.

Spatial proteomics uncovers the molecular basis of cellular function in intact tissues. Laser capture microdissection coupled with mass spectrometry enables comprehensive proteomic profiling of selected tissue regions but typically does not support cell-type-specific proteomic analysis. We present NicheProt, a 3D optical microscopy-guided and photobleaching-mediated cell barcoding approach for isolating intact, specific cell types from defined microanatomical tissue compartments or niches. Using sequential bottom-up proteomic analysis, we defined two distinct phenotypes of CD11c+ dendritic cells based on their spatial locations in the inflamed mouse spleen. These two compartment-specific dendritic cell populations were characterized by proteomic signatures differing in the levels of 54 proteins. This 3D tissue microscopy-guided method offers cell-type and microregion-resolved proteomic analysis, facilitating the proteomic discovery of previously unrecognized cell subtypes and their functional roles in distinct tissue compartments.

Cellular stiffness impacts multiple steps of cancer metastasis, but mechanisms that regulate the stiffness of cancer cells remain poorly understood. Here, we identified potassium efflux and potassium calcium-activated channel subfamily M regulatory beta subunit 1 (KCNMB1), an auxiliary subunit of the large conductance calcium-activated potassium (BK) channels, as regulators of cellular stiffness downstream of myocardin-related transcription factor A (MRTFA). In primary pericytes, KCNMB1 knockdown increased cellular stiffness, which is consistent with the role of potassium efflux in promoting relaxation during excitation-contraction coupling. In a striking contrast, however, KCNMB1 knockdown decreased cancer cells' stiffness. Softer cancer cells were resistant to natural killer (NK) cell mediated cytotoxicity and the low KCNMB1 expression was associated with reduced survival in breast cancer patients. Importantly, pharmacological activation of BK channels reduced metastatic burden in mice and improved lysis of cancer cells by cytotoxic T lymphocytes. These results highlight the ionic regulation of stiffness in cancer cells and point to BK channel agonism as a therapeutic approach.

Long COVID (LC) or Post-Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome represents a widespread health challenge that necessitates the development of novel diagnostic approaches and targeted therapies that can be readily deployed. Immune dysregulation has been reported as one of the hallmarks of LC, but the extent of LC immune dysregulation in patients over time remains unclear. We therefore assessed SARS-CoV-2-specific antibody responses, peripheral immune cell profiles, autoantibody profiles and circulating cytokines for up to 6 months in participants with a SARS-CoV-2 infection who either convalesced or developed LC. Compared to convalescent, LC participants with a broad range of LC phenotypes exhibited persistently elevated IgG titers for SARS-CoV-2 Envelope and Nucleocapsid proteins over the 6 months of study duration. In contrast, the IgG responses to Spike protein were significantly lower in the LC cohort with predominantly IgG1 and IgG3 class-switched bias. Using CyTOF analysis we show elevated numbers of circulating T follicular helper cells (cTFH) and mucosa-associated invariant T cells (MAIT), which also correlated with high anti-Envelope IgG titers. Persistent immune activation was accompanied by augmented serum cytokine profiles with LIF, IL-11, Eotaxin-3, and HMGB-1 in LC participants, who also demonstrated significantly higher rates of autoantibodies. These findings highlight the persistence of immune dysregulation in LC, underscoring the need to explore targeted therapies addressing viral persistence, dysregulated antibody production, and autoimmunity.

Rare and common genetic variants contribute to the risk of atrial fibrillation (AF). Although ion channels were among the first AF candidate genes identified, rare loss-of-function variants in structural genes, such as TTN, have also been implicated in AF pathogenesis, partly through the development of atrial myopathy; however, the underlying mechanisms are poorly understood. While TTN truncating variants (TTNtvs) have been causally linked to arrhythmia and cardiomyopathy syndromes, the role of missense variants (mvs) remains unclear. We show that rare TTNmvs are associated with worse clinical outcomes in a single-center ethnic minority clinical cohort and uncover a pathogenic mechanism by which the T32756I variant drives AF. Modeling the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) revealed that the mutant cells display aberrant contractility, increased activity of a cardiac potassium channel (KCNQ1, Kv7.1), and dysregulated calcium homeostasis without compromising the sarcomeric integrity of the atrial cardiomyocytes. We also show that a titin-binding protein, the Four-and-a-Half Lim domains 2 (FHL2), has increased binding with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I-iPSC-aCMs, enhancing the slow delayed rectifier potassium current (Iks). Suppression of FHL2 in mutant iPSC-aCMs normalized the Iks, supporting FHL2 as an Iks modulator. Our findings demonstrate that a single amino acid substitution in titin not only impairs its function but also remodels ion channels, contributing to AF. These findings underscore the importance of high-throughput screening to assess the pathogenicity of TTNmvs and establish a mechanistic connection between titin, potassium ion channels, and sarcomeric proteins, which may represent a novel therapeutic target.

Macrophages across different tissues exhibit remarkable functional diversity while maintaining core innate immune-cell programming. These complex programs are governed by gene regulatory networks, in which precise transcription factor activity tunes the expression of functionally relevant gene modules. Although the contributions of individual transcription factors have been well characterized, the higher-order regulatory interactions that coordinate tissue-resident macrophage identity and inflammatory response regulation remain poorly understood. Here, we integrate single-cell RNA-seq data with ATAC-seq profiling and deep-learning-based chromatin accessibility modeling to infer gene regulatory network architectures in tissue-resident versus recruited monocyte-derived alveolar macrophages under inflammatory stress. Our results suggest that inflammatory responses are more restrained in tissue-resident alveolar macrophages compared with recruited macrophages due to a stabilizing regulatory network architecture involving PU.1 and CEBP/β. This work advances our understanding of functional plasticity in tissue-resident macrophages and their role in host defense.

Human lung organoids (hLOs) derived from induced pluripotent stem cells (iPSCs) are of great interest, as they inform lung development, such as differentiation of lung epithelial subtypes in the distal alveolar unit. An unaddressed question is whether introducing endothelial cells (ECs) and vascularization provides a better representation of hLOs. Here we describe a method in which vessels become integrated with hLOs. hLOs were generated by combining human iPSC-derived lung progenitor cells (LPs) with ECs at varying LP:EC ratios. At the optimal combination of both cells, we observed vessel infiltration of hLOs compared to without ECs. Red blood cells were seen in hLOs implanted into kidney capsules of NOD/SCID mice. Both human and mouse ECs conjoined to form chimeric vessels in hLOs. The vascularized hLOs showed alveolar type II epithelial (ATII) cells and ATI cells, although there was no difference in 1:1 ATII/ATI ratio. We observed primitive airway sacs with alveolar epithelial cells lining the lumen of vascularized hLOs. Electron microscopy revealed surfactant production in ATII cells of vascularized hLOs in contrast to absence of vessels. The vascularized hLOs also mounted a robust inflammatory response characterized by influx of mouse neutrophils after challenging mice with LPS. Thus, interactions of ECs with LPs generated vascularized hLOs that induced ATII and ATI differentiation, although not reaching to the ratio of 1:9 seen in mature human lungs. hLOs also showed the LPS induced inflammatory response upon transplantation into recipient mice. Our results show the potential of vascularized hLOs for studying human lung development and inflammatory lung injury.

Anaplastic thyroid cancer (ATC) is an aggressive malignancy with a poor prognosis and limited treatment options. Previous studies have shown that selective downregulation of the MADD (MAP-kinase-activating death domain-containing protein) gene isoform increases ATC cell susceptibility to TRAIL-induced apoptosis. However, the existence of multiple MADD gene isoforms raised the possibility of functional compensation. This study aimed to definitively evaluate the role of MADD in ATC by employing CRISPR-Cas9 to Cas9 to target exon 3, a conserved exon expressed in all known MADD isoforms, resulting in functional knockout of MADD expression. CRISPR-Cas9-mediated MADD knockout, performed in three ATC cell lines (8505 C, C643, and HTH7) with distinct mutational backgrounds, significantly impaired ATC cell function in vitro, as indicated by reduced viability, increased apoptosis, decreased migration, and G0/G1 cell cycle arrest. RNA-seq analysis revealed alterations in genes related to cell survival, proliferation, and metastasis. In the orthotopic ATC mouse model, MADD deletion dramatically suppressed tumor growth, reduced lung metastases, and prolonged survival. Our findings demonstrate that MADD plays a crucial role in ATC cell survival, proliferation, and metastasis. The consistent effects observed across multiple cell lines and in vivo models suggest that MADD may represent a promising therapeutic target for this aggressive malignancy.

Translational control shapes the proteome and is particularly important in regulating gene expression under stress. A key source of endothelial stress is treatment with tyrosine kinase inhibitors (TKIs), which lowers cancer mortality but increases cardiovascular mortality. Using a human induced pluripotent stem cell-derived endothelial cell (hiPSC-EC) model of sunitinibinduced vascular dysfunction combined with ribosome profiling, we assessed the role of translational control in hiPSC-ECs in response to stress. We identified staphylococcal nuclease and tudor domain-containing protein 1 (SND1) as a sunitinibdependent translationally repressed gene. SND1 translational repression was mediated by the mTORC1/4E-BP1 pathway. SND1 inhibition led to endothelial dysfunction, whereas SND1 OE protected against sunitinib-induced endothelial dysfunction. Mechanistically, SND1 transcriptionally regulated UBE2N, an E2-conjugating enzyme that mediates K63-linked ubiquitination. UBE2N along with the E3 ligases RNF8 and RNF168 regulated the DNA damage repair response pathway to mitigate the deleterious effects of sunitinib. In silico analysis of FDA-approved drugs led to the identification of an ACE inhibitor, ramipril, that protected against sunitinib-induced vascular dysfunction in vitro and in vivo, all while preserving the efficacy of cancer therapy. Our study established a central role fortranslational control of SND1 in sunitinib-induced endothelial dysfunction that could potentially be therapeutically targeted to reduce sunitinib-induced vascular toxicity.

Macrophages are driven to form distinct functional phenotypes in response to different immunological stimuli, in a process widely referred to as macrophage polarization. Transcriptional regulators that guide macrophage polarization in response to a given trigger remain largely unknown. In this study, we interrogate the programmable landscape in macrophages to find regulatory panels that determine the precise polarization state that a macrophage is driven to. Towards this, we configure an integrative network analysis pipeline that utilizes macrophage transcriptomes in response to 28 distinct stimuli and reconstructs contextualized human gene regulatory networks, and identifies epicentres of perturbations in each case. We find that these contextualized regulatory networks form a spectrum of thirteen distinct clusters with M1 and M2 at the two ends. Using our computational pipeline, we identify combinatorial panels of epicentric regulatory factors (RFs) for each polarization state. We demonstrate that a set of three RFs i.e., CEBPB, NFE2L2 and BCL3, is sufficient to change the polarization destination from M1 to M2. siRNA knockdown of the 3-RF set in THP1 derived M0 cells, despite exposure to an M1 stimulant, significantly attenuated the shift to M1 phenotype, and instead increased the expression of M2 markers. Single knockdown of each RF also showed a similar trend. The siRNA-mediated knockdown of the 3-RF set rendered the macrophages hyper-susceptible to Staphylococcus aureus infection, demonstrating the importance of these factors in modulating immune responses. Overall, our results provide insights into the transcriptional mechanisms underlying macrophage polarization and identify key regulatory factors that may be targeted to modulate immune responses.