技术资料

Asthma is a widespread chronic lung disease characterized by airway inflammation and hyperresponsiveness. This airway inflammation is classified by either the presence (T2-high) or absence (T2-low) of high levels of eosinophils. Because most therapies for asthma target eosinophils and related pathways,treatment options for T2-low disease are limited. New pathophysiologic targets are needed. Oxidant stress is a common feature of T2-low disease. Airway epithelial expression of the antioxidant enzyme Paraoxonase 2 (PON2) is decreased in a well-recognized population of people with T2-low asthma and people with obesity and asthma. As a potential mechanism of increased oxidant stress,we measured the role of PON2 in lung oxidant responses using an environmentally relevant in vivo murine oxidant exposure (i.e.,ozone) and in vitro studies with an immortalized human airway epithelial cell line BEAS-2B. Pon2-deficient (Pon2−/−) mice developed increased airway hyper-responsiveness compared to wild-type controls. Despite reduced alveolar macrophage influx,Pon2−/− mice exhibited increased nitrite production. In human airway epithelial cells incubated with hydrogen peroxide,PON2 knockdown (PON2KD) decreased mitochondrial function and inner mitochondrial membrane potential. These findings suggest that PON2 functions in defending against airway epithelial oxidant stress. Further studies are needed to elucidate the mechanisms linking PON2,oxidant stress,and asthma pathogenesis. View Publication

VX-770 is a small-molecule CFTR potentiator that is highly efficacious in individuals with cystic fibrosis caused by mutations in CFTR that result in a defect in channel gating. While studies have reported on the mechanism of action of VX-770,there is still more to learn about the impact that it has on CFTR function in various contexts. The aim of the present study was to examine the longevity and stability of the effect of VX-770 on CFTR function in cultured airway epithelia and to measure the consequences of this interaction. The responses to acute and chronic VX-770 exposure were measured in cultures of expanded and re-differentiated primary human nasal epithelial cells. Acute VX-770 exposure resulted in an increase in CFTR-mediated currents in the absence of exogenous compounds that induce the phosphorylation/activation of CFTR,with acute exposure having the same effect as chronic exposure. The functional impact of VX-770 on CFTR was long-lasting in cultured airway epithelia,as they maintained an electrophysiological profile consistent with the saturation of CFTR with VX-770 over time periods of up to 4 days following a short (0.5 min) or low-dose (100 nM) exposure to VX-770 during an analysis in an Ussing chamber. Rinsing the apical surface prior to VX-770 exposure or exposure during the analysis in the Ussing chamber increased the interaction between VX-770 and the CFTR. Importantly,after short,low-dose exposures to VX-770,the CFTR channels in cultured epithelia appeared to remain saturated with VX-770 for extended periods of time,despite the repetitive rinsing of the apical surface. This finding has implications for patients discontinuing the use of VX-770-containing therapies. View Publication

Macrophage polarization critically contributes to a multitude of human pathologies. Hence,modulating macrophage polarization is a promising approach with enormous therapeutic potential. Macrophages are characterized by a remarkable functional and phenotypic plasticity,with pro-inflammatory (M1) and anti-inflammatory (M2) states at the extremes of a multidimensional polarization spectrum. Cell morphology is a major indicator for macrophage activation,describing M1(-like) (rounded) and M2(-like) (elongated) states by different cell shapes. Here,we introduced cell painting of macrophages to better reflect their multifaceted plasticity and associated phenotypes beyond the rigid dichotomous M1/M2 classification. Using high-content imaging,we established deep learning- and feature-based cell painting image analysis tools to elucidate cellular fingerprints that inform about subtle phenotypes of human blood monocyte-derived and iPSC-derived macrophages that are characterized as screening surrogate. Moreover,we show that cell painting feature profiling is suitable for identifying inter-donor variance to describe the relevance of the morphology feature ‘cell roundness’ and dissect distinct macrophage polarization signatures after stimulation with known biological or small-molecule modulators of macrophage (re-)polarization. Our novel established AI-fueled cell painting analysis tools provide a resource for high-content-based drug screening and candidate profiling,which set the stage for identifying novel modulators for macrophage (re-)polarization in health and disease. View Publication

Background: The gut–lung axis could be a potential therapeutic target for improving post-acute COVID-19 symptoms,and probiotics have been proposed as possible modulators. Aim: We conducted a pilot study to understand alterations in the gut–lung axis and to explore the effects of a probiotic in post-acute COVID-19 disease. Methods: We included patients after severe COVID-19 disease (sCOV,n = 21) in a randomized,placebo-controlled trial to test the effect of a probiotic (Pro-Vi 5,Institute Allergosan,Graz,Austria) in a six-month intervention and used patients after mild disease (mCOV,n = 10) as controls,to compare the intestinal microbiome,metabolome,and patient-reported outcomes and biomarkers along the gut–lung axis at baseline and throughout probiotic intervention. Results: Compared to mCOV patients,sCOV patients showed lower microbial richness,which was significantly improved by probiotic intervention. A reorganization of Ruminococcaceae and Lachnospiraceae taxa was observed in sCOV patients but remained unaffected by the intervention. Serum metabolome showed a dysregulation of lipoproteins in accordance with higher BMI and comorbidities in sCOV patients. HDL and LDL fractions/components were temporarily decreased in the probiotic group. Stool metabolome was altered at baseline in sCOV patients and an increase in L-DOPA after 3 months and butyrate after 6 months of intervention could be observed. Probiotics partially improved reduced quality of life and modulated altered immune responses in sCOV patients. Increased intestinal permeability at baseline remained unaffected. Conclusion: The study provides evidence of long-term alterations of the gut–lung axis after severe COVID-19 infection and suggests that probiotics can modulate the biomarkers of the gut–lung axis. View Publication

Recent studies have identified SLC2A3 as being abnormally upregulated in multiple tumor types,correlating with poor survival and disrupted microenvironments. However,its prognostic significance in head and neck squamous cell carcinoma (HNSC) remains underexplored. In this study,SLC2A3 was screened as a potential risk gene influencing both immune and tumor components within the tumor microenvironment (TME) of 504 HNSC patients from the TCGA database. Immune infiltration analyses and clinical significance on SLC2A3 were conducted using ESTIMATE,CIBERSORT,ssGSEA,TIMER and clinical prognosis parameters. Additionally,the single-cell dataset is used to analyze the expression of SLC2A3 in various subpopulations. The magnetic activated cell sorting (MACS) is used to isolate CD8+ T cells from PBMCs or tumor tissues. Flow cytometry is used to identify purified and activated CD8+ T cells. GSEA and WB were used to investigate the molecular mechanism of SLC2A3 in CD8+ T cells. The co-culture system of CD8+ T cells and TU686 was used to investigate the effects of SLC2A3 on immune cells and tumor development. In this study,SLC2A3 was identified as a potential risk gene affecting both immune cells and tumor components within the TME of 504 HNSC patients derived from the TCGA database. We conducted immune infiltration analyses and assessed the clinical significance of SLC2A3 using various bioinformatics tools,including ESTIMATE,CIBERSORT,ssGSEA,and TIMER,along with clinical prognosis parameters. The single-cell RNA sequencing dataset was utilized to examine SLC2A3 expression across different cellular subpopulations. Magnetic activated cell sorting (MACS) was employed to isolate CD8+ T cells from peripheral blood mononuclear cells (PBMCs) or tumor tissues. Flow cytometry was implemented to confirm the purity and activation state of the isolated CD8+ T cells. GSEA and Western blot were applied to explore the molecular mechanisms underlying SLC2A3’s role in CD8+ T cells. Lastly,a co-culture system involving CD8+ T cells and TU686 tumor cells was established to study the impact of SLC2A3 on immune cell function and tumor progression. SLC2A3 emerges as an actively variable gene within the immune and stromal components of the TME,linked to aggravated immune infiltration and poor clinical outcomes. The upregulated expression of SLC2A3 is predominantly enriched in immune-related biological processes and linked to the suppression of CD8+ T cells,which are crucial for the survival of HNSC patients. Furthermore,SLC2A3 exhibits specific overexpression in CD8+ T cells and may potentially trigger ferroptosis. Knockdown of SLC2A3 led to a significant increase in the proliferation of CD8+ T cells compared to those without knockdown. In co-culture systems,CD8+ T cells with SLC2A3 knockdown demonstrated an enhanced ability to eliminate tumor cells compared to those without the knockdown. SLC2A3 is associated with changes in the TME and prognostic indicators. Moreover,high SLC2A3 expression in CD8+ T cells may drive cell death through ferroptosis,fostering tumor progression. View Publication

IntroductionCystic Fibrosis (CF) is a genetic disease due to loss-of-function mutations of the CFTR channel. F508del is the most frequent mutation (70% of alleles in Italy),while other mutations have much lower frequency. Among them,G85E (0.4% frequency globally,1.13% in Italy) emerges as a mutation characterized by a severe CFTR folding and trafficking defect.MethodsTo investigate the pharmacological responsiveness of the G85E-CFTR variant,we performed a functional and biochemical characterization in heterologous expression systems and ex vivo models based on patient-derived human nasal epithelial cells (HNEC).ResultsOur study demonstrated that treatment of primary airway cells with elexacaftor and tezacaftor causes a significant (although modest) rescue of CFTR function,that reaches 15%–25% of the activity measured in non-CF epithelia. A detrimental effect of chronic treatment with ivacaftor,further limiting G85E rescue,was also observed. A higher rescue of CFTR function,up to 25%–35% of the normal CFTR activity,with no evidence of negative effects upon chronic potentiator treatment,can be achieved by combining elexacaftor with ARN23765,a novel type 1 corrector endowed with very high potency. Importantly,dose-response relationships suggest that G85E might alter the binding of type 1 correctors,possibly affecting their affinity for the target.DiscussionIn conclusion,our studies suggest that novel combinations of modulators,endowed with higher efficacy leading to increased rescue of G85E-CFTR,are needed to improve the clinical benefit in patients for this variant. View Publication

SummaryVγ9Vδ2 T cells constitute a homogeneous effector T cell population that lyses tumors of different origin,including the prostate. We generated a bispecific T cell engager (bsTCE) to direct Vγ9Vδ2 T cells to PSMA+ prostate cancer (PCa) cells. The PSMA-Vδ2 bsTCE triggered healthy donor and PCa patient-derived Vγ9Vδ2 T cells to lyse PSMA+ PCa cell lines and patient-derived tumor cells while sparing normal prostate cells and enhanced Vγ9Vδ2 T cell antigen cross-presentation to CD8+ T cells. Vγ9Vδ2 T cell expressed NKG2D and DNAM-1 contributed to Vγ9Vδ2 T cell activation and tumor lysis at low PSMA-Vδ2 bsTCE concentrations. In vivo models confirmed the antitumor efficacy of the bsTCE and demonstrated a half-life of 6–7 days. Tissue-cross reactivity analysis was in line with known tissue distribution of PSMA and Vγ9Vδ2 T cells. Together these data show the PSMA-Vδ2 bsTCE to represent a promising anti-tumor strategy and supports its ongoing evaluation in a phase 1/2a clinical trial in therapy refractory metastatic castration-resistant PCa. Graphical abstract Highlights•PSMA-Vδ2 bsTCE-activated Vγ9Vδ2 T cells lyse prostate tumor cells while sparing normal cells•PSMA-Vδ2 bsTCE-activated Vγ9Vδ2 T cells enhance antigen cross-presentation to CD8+ T cells•DNAM-1 and NKG2D contribute to Vγ9Vδ2 T cell activation at low PSMA-Vδ2 bsTCE concentrations•Anti-tumor efficacy of PSMA-Vδ2 bsTCE was confirmed in in vivo immunodeficient NCG mice Therapy; Immune response; Cancer View Publication

Cardiac regenerative therapy has recently progressed by reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and advanced by large-scale differentiation-derived cardiomyocytes (hiPSC-CMs). However,repairing damaged cardiac tissues with hiPSC-CMs remains limited due to immune rejection,cardiac arrhythmias,and concerns over tumor formation after hiPSC-CM transplantation. Despite efforts in profiling epigenomic changes during cardiac differentiation,regulatory mechanisms underlying 5-methylcytosine (m5C) deposition in RNA m5C epitranscriptomic landscape during hiPSC-to-cardiomyocyte differentiation remain unclear. Herein,bisulfite RNA-sequencing analysis was conducted in human pluripotent stem cells (hPSCs) from three independent cellular origins,and their derived cardiomyocytes (hPSC-CM),metabolic-maturation of derived cardiomyocytes (hPSC-CM-lac) and biochemical-enhanced derived cardiomyocytes (hPSC-CM-TDI). Integrated analysis of differentially methylated RNA m5C profiles and transcriptome-wide expression facilitated the identification of m5C sites coupled to the cardiomyocyte differentiation and RNA-dependent regulatory mechanisms of stem cell pluripotency. The RNA m5C profiles in this dataset allow the evaluations of the m5C level and distribution of specific m5C loci and facilitate understanding of the m5C epitranscriptomic landscape in biological functions of hPSC-CM beyond in vivo transplantation barriers. View Publication

BackgroundHuman mammary epithelial cell (HMEC) cultures encounter a stress-associated barrier termed stasis,during which most cells adopt a senescence-like phenotype. From these cultures,rare variants emerge from the basal epithelial population,re-initiating growth. Variants exhibit pre-malignant properties,including an aberrant epigenetic program that enables continued proliferation and acquisition of genetic changes. Following oncogenic transformation,variants produce tumors that recapitulate the histopathological characteristics of metaplastic breast cancer (MBC),a rare and aggressive subtype marked by the differentiation of neoplastic epithelium into squamous and mesenchymal elements.MethodsUsing a serum-free HMEC culture system,we probed the capacity for phenotypic plasticity inherent to basal epithelial cell populations from human breast tissue as they navigated stasis and emerged as variant populations.ResultsWe observed robust activation of a TGF-β-dependent epithelial-mesenchymal transition (EMT) program in basal epithelial cells during stasis,followed by subsequent attenuation of this program in emerging variants. Inhibition of the TGF-β pathway or depleting the EMT regulators Snail or Slug allowed basal epithelial cells to collectively bypass stasis,demonstrating that cellular dysfunction and arrest resulting from TGF-β and EMT activation are central to this in vitro barrier. The spontaneous emergence of variants from stasis cultures was associated with a restricted EMT trajectory,characterized by the stabilization of hybrid EMT states associated with greater proliferative capacity,rather than progressing to a complete mesenchymal state characterized by irreversible growth arrest. Epigenetic mechanisms,which contributed to the dysregulated growth control characteristic of the variant phenotype,also contributed to the stability of the hybrid EMT program in variants. By overcoming the cellular dysfunction and growth arrest resulting from TGF-β and complete EMT,variants exhibited a higher oncogenic transformation efficiency compared to pre-stasis basal epithelial cells. Inhibiting the TGF-β pathway prior to stasis significantly reduced EMT in the basal epithelial population,alleviated selective pressure driving variant emergence,and also enhanced oncogenic transformation efficiency,resulting in tumors with markedly diminished metaplastic differentiation.ConclusionsThis study reveals how an epigenetic program governs basal epithelial cell fate decisions and contributes to the development of MBC progenitors by restricting access to terminal mesenchymal states that induce growth arrest and,instead,favoring hybrid EMT states with enhanced tumorigenic potential.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-024-01920-8. View Publication

BackgroundCystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel. For people with CF (pwCF) affected by the most common pathogenic variant F508del,a tritherapy,named Trikafta/Kaftrio (ETI: elexacaftor (VX-445) /tezacaftor (VX-661) / ivacaftor (VX-770)) was successfully developed. However,in CF airway epithelial cells the calcium homeostasis is also disturbed; it is observed an increased calcium mobilization in CF cells compared to non-CF cells. Here,we studied the effects of ETI on intracellular calcium levels in F508del-CFTR airway epithelial cells to determine whether these compounds,individually or collectively,could normalize intracellular calcium levels.MethodsWe measured intracellular calcium variations using human airway epithelial cells (hAEC) from pwCF,human bronchial epithelial CFBE41o- F508del-CFTR cells and Chinese Hamster Ovary (CHO) cells using the fluorescent probe Fluo4-AM,in the presence or absence of extracellular calcium. The rescue to the plasma membrane of F508del-CFTR protein by ETI was determined by western blot. The SarcoEndoplasmic Reticulum Calcium ATPase (SERCA),was also analysed by western blotting and by interference assay.ResultsWe show that ETI normalizes calcium homeostasis in our cellular models. However,we also found that (1) each ETI-corrector compound is capable of mobilizing calcium acutely in the absence of CFTR,and (2) tezacaftor mobilizes calcium from the endoplasmic reticulum (ER) probably via inhibition of the SERCA pump.ConclusionsWe show that ETI not only corrects the abnormal trafficking and function of F508del-CFTR but also normalizes calcium homeostasis in our cellular models. Finally,we identified SERCA as a potential intracellular target for tezacaftor.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12931-024-03059-8. View Publication

BackgroundRenal CD8+ tissue-resident memory T (TRM) cells display prolonged survival and activity in lupus nephritis (LN),exacerbating renal pathology. NLRP3 regulates the T cell response. This study explored the impact of NLRP3 inflammasome activity on the regulatory functions of TRM cells in LN.MethodsNLRP3 inflammasome activity in renal CD8+ TRM cells from lupus-prone MRL/lpr mice and in vitro induced human CD8+CD103+ T cells was assessed by quantifying NLRP3,caspase-1,gasdermin D (GSDMD),and IL-1β levels using flow cytometry,ELISA,and western blotting analysis. The specific NLRP3 inhibitor MCC950,caspase-1 inhibitor Ac-YVAD-cmk,and NF-κB inhibitor JSH23 were utilized to delineate the role of NLRP3 in modulating the pathogenicity of CD8+ TRM cells in LN.ResultsActivation of the NLRP3 inflammasome was confirmed in renal CD8+CD69+CD103+ TRM cells derived from mice with LN and in vitro-induced human CD8+CD103+ TRM-like cells. MCC950 curtailed the infiltration and activity of CD8+CD69+CD103+ TRM cells and enhanced renal outcomes. MCC950 also suppressed the maturation and functional capabilities of CD8+CD103+ T cells in a manner reliant on inflammasome activity in vitro. IL-1β promoted the expression of TGF-βRII in CD8+ T cells via the NF-κB pathway.ConclusionsNLRP3 inflammasome activity in renal CD8+CD69+CD103+ TRM cells contributes to LN pathogenesis by regulating cell differentiation and effector functions. Therapeutically targeting the NLRP3 inflammasome could significantly mitigate CD8+CD69+CD103+ TRM cell-mediated renal damage in LN.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05951-9. View Publication

BackgroundMelanoma cells frequently dedifferentiate in response to inflammation which can increase responses to certain cytokines. Interferon-γ (IFNγ) is an integral part of the anti-tumor immune response and can directly induce both differentiational changes and expression of immunosuppressive proteins in melanoma cells. How the differentiation status of melanoma cells affects IFNγ responses remains unclear.MethodsDedifferentiation of melanoma cells was induced via either siRNA or shRNA mediated MITF knockdown and the cells were subsequently treated with IFNγ. Effects of MITF knockdown and IFNγ treatment on gene expression were evaluated via qPCR and RNA sequencing. A Luminex assay was used to analyze the effects of dedifferentiation and IFNγ treatment on cytokine secretion. Effects on PD-L1 protein expression were analyzed via flow cytometry and western blotting. Inhibition of the JAK kinases,NF-κB and STAT3 with small molecule inhibitors,and siRNA mediated knockdown of STAT1 and IRF1 was applied to investigate the molecular mechanism behind IFNγ induced PD-L1 expression in dedifferentiated melanoma cells. The effects of inhibitor treatments and siRNA mediated knockdowns were evaluated via qPCR and western blotting. Bioinformatic analysis of publicly available RNA sequencing data,consisting of 45 patient derived melanoma cell lines,with or without IFNγ treatment,was conducted to assess the generalizability of the in vitro results.ResultsDedifferentiation renders 624Mel melanoma cells hypersensitive to IFNγ stimulation in a context-dependent manner,resulting in non-additive upregulation of IFNγ-induced genes,increased PD-L1 protein expression and amplified secretion of CCL2,CXCL10 and IL-10. Furthermore,the intensified PD-L1 protein expression occurs through the JAK-STAT1-IRF1 axis. Lastly,dedifferentiated patient derived melanoma cell lines showed enhanced inflammatory signaling in response to IFNγ compared to differentiated cells,and tended to have higher PD-L1 expression,associated with increased IRF1 expression and activity.ConclusionsTogether,these findings indicate the existence of a molecular context linking dedifferentiation and IFNγ signaling in melanoma which may lead to immune evasion. Additionally,the variability in PD-L1 expression among MITFlow and MITFhigh cells suggests that high IFNγ-induced PD-L1 expression associates with enhanced inflammatory gene expression. These results imply that modulating melanoma differentiation may help shape IFNγ responsiveness.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01963-6. View Publication