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Background: The pathology of Alzheimer’s Disease (AD) is characterized by aggregates of amyloid beta (Aβ) peptides and neurofibrillary tau tangles. Increased blood-brain barrier (BBB) permeability and reduced Aβ clearance,which signal neurovascular dysfunction,have also been proposed as early markers of AD. Despite intense scrutiny,the mechanisms of AD remain elusive and novel treatments that address core symptoms of dementia are limited. New alternative methods (NAMs) aim to develop in-vitro translational models that recapitulate human pathology more accurately than previous models and could contribute to the development of new therapies. Methods: Here,we developed a NAM model of the cortical neurovascular unit (NVU) using brain cells derived from human induced pluripotent stem cells (hiPSCs) from a patient with AD and a healthy individual. Differentiated neurons,astrocytes,pericytes,microglia,and brain-like microvascular endothelial cells were cultured in a microphysiological system to create a brain-chip model to evaluate NVU-related endpoints. Results: Compared to control,AD brain-chips had reduced claudin-5 and ZO-1 expression and increased paracellular permeability. AD brain-chips also had decreased activity of the efflux transporter P-glycoprotein (P-gp),but its expression was unchanged. In AD brain-chips,levels of Aβ42,total tau,and p-tau 181 were decreased in protein lysates from the brain channel,while levels of total tau and p-tau 181 were increased in protein lysates from the vascular channel. Finally,AD brain-chips had increased levels of the proinflammatory markers IL-6 and MCP-1 in effluent from both brain and vascular channels. Conclusion: In this brain-chip model,we showed Aβ-independent NVU dysfunction that was related to neuroinflammation and vascular tau accumulation. This study demonstrates the utility of the brain-chip model to evaluate changes in NVU functions induced by AD-like pathology and highlights donor-specific responses associated with the use of hiPSC-derived models. View Publication

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi sarcoma,primary effusion lymphoma and multicentric Castleman disease. A vaccine that prevents KSHV infection or serves in the treatment of KSHV-related diseases represents a critical unmet need,however,the types of immune responses a vaccine should elicit have not been well defined. The gH/gL glycoprotein complex is an important target of KSHV-neutralizing antibodies,but the epitope specificities targeted by these antibodies remain unknown. Here,we isolated 12 gH/gL-specific monoclonal antibodies (mAbs) from KSHV-infected donors and performed structure/function analyses. These mAbs bind recombinant gH/gL with nanomolar affinities and epitope binning analyses revealed that the mAbs bind to 5 epitope clusters on gH/gL. Seven mAbs were able to neutralize KSHV infection of epithelial cell lines. Two potent neutralizing mAbs mapped to the EphA2 binding site as determined by inhibition of the receptor-ligand interaction and negative stain electron microscopy (nsEM) of the mAb/gH/gL complex. The epitopes of other neutralizing mAbs targeting novel sites of vulnerability were determined by a combination of cryogenic electron microscopy and nsEM. Together,these mAbs help to define the relevant epitope targets for KSHV vaccine design,have utility in understanding the role of antibodies in preventing KSHV infection,enable the development of immunotherapy approaches,and provide valuable tools to understand the molecular details of the KSHV entry process. Author summaryKSHV is an oncogenic virus that can cause cancer in infected individuals. The virus is most prevalent in sub-Saharan Africa and in men who have sex with men. It is possible this virus could be prevented with an effective vaccine,however,the immune response to this virus has not been well defined. gH/gL,a protein essential for viral fusion,plays an important role in infection and could be a possible vaccine target. To better understand the antibody response to this protein,we sought to isolate and characterize monoclonal antibodies that can bind gH/gL and neutralize viral infection. In this study,we isolate and characterize twelve monoclonal antibodies that could bind to five different regions of the gH/gL protein. Seven of these antibodies can neutralize infection,with two being able to block the gH/gL EphA2 receptor-ligand interaction. View Publication

Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation,driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response,leading to hepatocyte damage,senescence,and impaired liver regeneration. While the underlying mechanisms remain incompletely understood,increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here,we identify iRhom2 as a key regulator of immune-mediated liver injury,orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages,yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts,particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress,preserved mitochondrial function,and attenuated apoptosis,indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators,with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably,HMGB1 release occurs independently of TACE catalytic activity,suggesting an alternative unexplored regulatory mechanism. Furthermore,co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition,driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly,HMGB1 neutralization enhances hepatocyte viability and mitigates senescence,underscoring its pathogenic role. Additionally,HMGB1 knockdown in macrophages protects hepatocytes,though p21 expression remains unaffected,hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival. View Publication

Multiple myeloma (MM) is the second most common hematological malignancy,characterized by a clonal expansion of malignant plasma cells in bone marrow. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition of MM. The tumor microenvironment is thought to influence the progression from premalignant conditions. Myeloid‐derived suppressor cells (MDSCs) are a heterogenous group of different cellular subsets with myeloid origin,characterized by their ability to inhibit T‐cell responses. MDSC are thought to play an important immunoregulatory role in different diseases,and in many cancers their levels seem to correlate with a poor prognosis. There are three different subsets,the neutrophil‐like polymorphonuclear (PMN)‐MDSC,the monocyte‐like (M)‐MDSC,and the immature early (e)MDSC. In this study,we investigate the levels and functions of all MDSC subsets in the bone marrow of both MGUS and MM patients and compare it to blood MDSC. We found that MDSC levels are not increased in neither the blood nor bone marrow of MGUS or MM patients,and they lack strong T‐cell suppressive abilities. Blood PMN‐MDSC seems to have a small inhibitory effect,but mature neutrophils were more suppressive. Interestingly,eMDSC levels were decreased in the blood of MM patients. Our data indicate that MDSC are not key players in the pathogenesis of MM,but that mature neutrophils may be more important as they have a stronger immunoregulatory effect. View Publication

Metabolic dysfunction–associated steatotic liver disease (MASLD) is reversible at early stages,making early identification critical. We previously demonstrated that patient-derived induced pluripotent stem cells (iPSCs) carrying MASLD-associated genetic risk variants exhibit greater oleate-induced intracellular lipid accumulation than those without these variants. This study aimed to develop an iPSC-based MASLD risk predictor using functional lipid accumulation assessments. We quantified oleate-induced lipid accumulation in iPSCs from three cohorts: (1) CIRM (22 cases,20 controls),(2) POST (18 cases,16 controls),and (3) UCSF (4 cases,8 controls). Data from the CIRM cohort was used to define an iPSC-based MASLD risk score,which was subsequently validated in the POST and UCSF cohorts. Lipid accumulation was consistently higher in MASLD iPSCs across cohorts. The risk score achieved 44% sensitivity/75% specificity in POST and 75%/100% in UCSF. These findings suggest that oleate-induced lipid accumulation in iPSCs may be a predictor of MASLD risk. Larger studies incorporating additional cellular phenotypes,clinical,and genetic data could enhance predictive accuracy for MASLD surveillance and prevention. View Publication

Background/objectives: Interactions between colorectal tumours and their immune microenvironment critically influence disease progression and response to immunotherapy. However,most organoid systems fail to preserve the complex architecture and immune composition of the original tissue. Here,we applied the air-liquid interface (ALI) organoid model to paired tumour and perilesional colon tissues from colorectal cancer patients to evaluate its ability to retain immune and genetic features and to reproduce responses to chemotherapy and immune checkpoint blockade. Methods: Fresh human tumour and matched healthy colon tissues were processed to generate ALI organoids. Their histological organization,immune cell composition (including CD45+ subsets),and genomic profiles were compared with those of the parental tissues and with conventional Matrigel organoids,either alone or co-cultured with peripheral blood mononuclear cells (PBMCs). Organoids were exposed to 5-FU and nivolumab (anti-PD-1) to assess local immune modulation. Results: ALI organoids faithfully preserved the three-dimensional architecture,native immune infiltrates,and somatic mutational landscape of the source tissues. Importantly,upon PD-1 blockade with nivolumab,ALI organoids consistently exhibited a local expansion of regulatory T cells (Tregs),a phenomenon that could contribute to adaptive immune resistance. This response was not reproduced in PBMC-Matrigel co-culture systems,highlighting the importance of preserving endogenous tumour-immune interactions. Conclusions: Patient-derived ALI organoids represent a physiologically relevant platform that conserves key structural,immunological,and genomic hallmarks of colorectal cancer. By capturing clinically relevant immune remodeling events,such as Treg expansion following PD-1 blockade,this model provides a powerful tool for dissecting tumour-immune interactions. View Publication

The immunoregulatory effects of probiotics have been widely studied,particularly in maintaining immune balance. Conventional in vitro functional screening of probiotics relies on fresh donor-derived primary immune cells,which often exhibit significant inter-individual and temporal variability,limiting reproducibility and interpretation. As an alternative,human-induced pluripotent stem cell (iPSC)-derived dendritic cells were co-cultured with five probiotic strains in the current study to evaluate their immunomodulatory interactions. To assess whether cytokines produced by probiotic-stimulated dendritic cells can influence T cell differentiation,human CD4+ T cells were exposed to the conditioned medium derived from co-cultures. Enzyme-linked immunosorbent assay results demonstrated that iPSC-derived dendritic cells secreted cytokines at distinct concentrations in response to different probiotic strains,suggesting that these cells can distinguish between different microbial stimuli,and supporting their use in functional probiotic screening. Among the five strains tested,Lactiplantibacillus plantarum LPA-56,Limosilactobacillus reuteri RU-23,and Lactobacillus fermentum Fem-99 induced cytokine production levels that promoted the differentiation of the human CD4+ T cells into regulatory T cells. These findings demonstrate that iPSC-derived dendritic cells have immunomodulatory potential,are reliable for in vitro screening of probiotics,and offer a promising strategy for selecting potent immunoregulatory probiotic candidates. View Publication

The Mas-related G protein-coupled receptor (MRGPR) X2 is expressed on skin mast cells and can be stimulated by an unusually broad spectrum of ligands,including specific drugs and even endogenous peptides. MRGPRX2 activation can induce mast cell degranulation and consequently mediator release,leading to inflammatory and hypersensitivity reactions. In addition,MRGPRX2 mediates pain and itching sensations,leading to increased efforts to identify MRGPRX2 inhibitors,including plant-derived compounds. Components within oat extracts have been shown to mediate anti-inflammatory and itch-relieving properties,but a possible inhibitory effect on MRGPRX2 activation has not yet been investigated. We aimed to fill this gap and explored whether an oat kernel extract can modulate MRGPRX2 activation. For this purpose,we established a mast cell model with the human LAD2 cell line and used it to investigate the consequences of exposure to oat extract. While we did not observe any influence on cell viability,we analyzed the impact of oat extract on MRGPRX2-mediated mast cell activation and degranulation initiated by the three confirmed MRGPRX2 ligands c48/80,substance P,and cortistatin 14. Exposure to oat extract resulted in a significant reduction in mast cell degranulation for all three ligands,as assessed by the release of β-hexosaminidase,tryptase,cell surface expression of CD63 and CD107a,and phosphorylation of ERK. All results were confirmed with primary human mast cells. Thus,we demonstrated for the first time that oat extract leads to a significant reduction in MRGPRX2 activation,pointing to a previously unrecognized capacity of natural compounds to modulate this pathway. View Publication

HIV-associated distal sensory polyneuropathy (HIV-DSP) remains prevalent even in the antiretroviral therapy (ART) era. Previously,we identified the upregulation of nociceptive ion channels transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in the dorsal root ganglia (DRG) of simian immunodeficiency virus (SIV)-infected ART-treated macaques. To investigate upstream mechanisms,we performed bulk RNA-seq and pathway analysis on DRGs from uninfected,SIV-infected,and SIV-infected/ART macaques. SIV infection drove strong activation of upstream regulators of interferon γ (IFNγ) and lipopolysaccharide (LPS). Although ART reduced overall IFNγ and LPS pathway activity,the IFNγ-inducible chemokines C-X-C motif chemokine ligand (CXCL)9 and CXCL10 remained significantly upregulated. To determine whether these chemokines influence TRPV1/TRPA1 expression,we treated induced pluripotent stem cell-derived peripheral sensory neurons (iPSC-PSNs) with CXCL9 and CXCL10,which induced a significant increase in TRPV1 but not TRPA1 expression. In parallel experiments,IFNγ but not LPS stimulated monocyte-derived macrophages (MDMs) to release CXCL9 and CXCL10. Conditioned media from IFNγ-treated MDMs modestly increased TRPV1 expression in iPSC-PSNs,and pharmacological inhibition of CXCR3,the receptor of CXCL9/10,did not reduce this effect. Together,these data indicate that persistent IFNγ-driven CXCL9/10 signaling may be one contributor to nociceptor sensitization underlying HIV-DSP,even in the presence of ART. View Publication

Interleukin-17 (IL-17) plays a central role in the pathogenesis of various autoimmune diseases. Soluble CD14 (sCD14),a marker of innate immune activation,is elevated in several inflammatory conditions. However,its influence on IL-17 production and the differentiation of Th17 cells remains poorly understood. We found that sCD14 enhances Th17-associated cytokine production and up-regulates critical transcription factors such as STAT3 and RORC. Notably,sCD14's effect on Th17 polarization was mediated indirectly through autologous sCD14-treated peripheral blood mononuclear cell (PBMC) supernatant (sCD14-PBMC-Sup). Additionally,we identified a distinct cytokine profile enriched for pro-inflammatory cytokines and chemokines in sCD14-treated T cells,further reinforcing the Th17-promoting role of sCD14. Interestingly,gamma-aminobutyric acid (GABA),a metabolite elevated in sCD14-treated monocytes,was identified as a potential contributor to Th17 polarization. GABA supplementation in T-cell cultures enhanced IL-17A secretion,indicating its role as a signaling molecule in T-cell differentiation. Our findings also revealed the expansion of innate lymphoid cell (ILC)2/3-like cells in T-cell cultures exposed to sCD14-PBMC-Sup and GABA,highlighting the potential role of monocytes in Th17-mediated immunity. Furthermore,while sCD14 promoted Th17 polarization,it simultaneously impaired T-cell activation and proliferation,suggesting an immunosuppressive effect mediated by soluble factors released from monocytes. These results underscore the dual role of sCD14 in modulating T-cell responses,promoting Th17 differentiation while suppressing T-cell effector functions. This study identifies a previously unrecognized role for sCD14 in promoting Th17 induction,highlighting its contribution to immune regulation and its potential as a therapeutic target in Th17-driven autoimmune conditions. View Publication

Cis-regulatory elements (CREs) are noncoding DNA regions regulating cell-type-specific gene expression programs by interacting with distal gene promoters. Here,we aim to decode the function and spatial organization of CRE-promoter interactions in human cardiomyocytes. We analyzed the epigenome and chromatin interactions of human male atrial,ventricular,and failing cardiomyocytes. Atrial and ventricular cardiomyocytes harbored chamber-specific CRE-promoter interactions modulating gene expression as confirmed by functional epigenetic silencing. These CRE-promoter interactions explain the distinct contribution of non-coding genetic variants to atrial and ventricular diseases,such as dilated cardiomyopathy and arrhythmias. We dissected the prototypic KCNJ2 locus,encoding a potassium channel associated with ventricular arrhythmia susceptibility. Functional epigenetic silencing confirmed that CREs,harboring QT-duration-associated genetic risk factors,modulate KCNJ2 gene expression levels,alter KCNJ2-dependent channel currents,and affect cardiomyocyte repolarization. The presented human CM-specific chromatin interaction analysis provides key insights into regulatory mechanisms and aids in interpreting genetic risk factors. Here the authors functionally test and resolve the spatial genome organization of cis-regulatory elements and genetic variants in atrial,ventricular,and failing human cardiomyocytes and linked them to heart disease traits,including QT syndrome. View Publication

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However,the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes,particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here,we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations,we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems,which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity,we use an arrangement relying on an FEP film,Immersol W,water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution,supports large tile-scan acquisitions,and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost,plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes. View Publication