Motaharehsadat Heydarian

Neutrophils are essential frontline immune cells in the defence against bacterial pathogens such as Staphylococcus aureus , but their killing machinery also causes host tissue damage. How neutrophils regulate these processes remains incompletely understood. Here, we profiled the proteome and phosphoproteome of human neutrophils across a two-hour exposure to S. aureus , alongside five simultaneous functional readouts, before investigating how an initial encounter modulates the response to a second. The phosphoproteomic response resolved into qualitatively distinct early and late windows: the early landscape drives particle uptake, while by the late window this machinery is deactivated, with a broadly supressed kinome, and cells geared towards inflammatory output. Correlating protein phosphorylation and functional kinetics — an approach we term KINETIC — implicates putative mediators of key neutrophil antimicrobial functions. A genome-wide CRISPR knockout screen in neutrophil-like PLB-985 cells, sorted on phagocytic load, recovered canonical engulfment machinery among the strongest facilitators of phagocytosis, additionally identifying the lysosomal Ragulator-mTORC1 nutrient sensing pathway and HOPS complex corresponding to the KINETIC-identified pathways. Conversely, the NRF2 repressor KEAP1 was the strongest phagocytic brake, positioning oxidative burden as a regulator of phagocytic appetite. Neutrophils in the late window exhibited impaired phagocytosis and phagosomal acidification on S.aureus re-challenge. Underlying these functional deficits, cells in the late window were refractory across multiomic layers: a second S. aureus meal altered no phosphoprotein, protein, or transcript, whereas the chemoattractants PAF and fMLP evoked a largely consistent phosphoproteomic programme, delineating the phenotype as receptor-specific rather than a global loss of signalling capacity. Consistent with de-repressing the antioxidant response via genetic loss of KEAP1 increasing phagocytosis, reducing ROS production via inhibition of the NADPH oxidase resulted in a partial rescue of phagocytosis of the re-exposed S.aureus . Together, these findings describe a ROS-gated phagocytic rheostat, enabling neutrophils to meter their phagocytic appetite according to their exposure history, likely limiting uptake of pathogens they lack the capacity to effectively digest. Inappropriate activation of this rheostat under conditions of physiological stress or severe illness may underpin the defects in phagocytosis identified in such patients.

Clinical monitoring in the most vulnerable patients such as newborns relies on invasive and costly procedures and/or wired sensor surveillance, increasing discomfort and risk for undetected events. Addressing this critical need, we present a noninvasive, biocompatible silk-based sensor capturing multiple critical body functions via colorimetric analysis of body fluids, optimized for its use in critically ill preterm neonates where transepidermal fluid mirrors the interstitial compartment, thereby opening new avenues for future clinical monitoring. The sensor introduces twelve different colorimetric inks that stabilize labile bioresponsive molecules to a simple, wax-printed paper microfluidic wearable patch to facilitate real-time tracking of biomarkers (temperature (T), pH, sodium (S), and glucose (G)) on a miniaturized surface. Colorimetric responses showed high precision and reproducibility in sensing ranges relevant for neonatal care (T: 32-41 [°C]; pH: 3-9; S: 2.92-29.20 [mg/mL], G: 0.039-0.625 [mg/mL]). Deep learning for color response quantification achieved automated measurement (T:0.455 [°C]; pH:0.416; S:0.857 [mg/mL], G:0.019 [mg/mL]; mean absolute error). The sensor’s optimized interface for sample collection on skin and its performance under clinically relevant conditions, e.g., increased humidity (80%), tracking on moving object (0.986 AP @ IoU=0.5), sensor shear/rotation, uncontrolled light, as well as successful capture of physiological effects in biological fluids together with its miniaturized design caters to the critical needs of intensified monitoring in high-end patient populations such as neonates. Significance Statement We designed a noninvasive, wearable and miniaturized paper sensor patch capturing critical body functions via colorimetric analysis of body fluids (perspiration and saliva) adapted to the clinical needs of neonatal monitoring. Through twelve different bioresponsive inks stabilized in silk-fibroin into a simple, wax-printed paper microfluidic patch, the sensor facilitates simultaneous real-time tracking of multiple biomarkers under clinically relevant conditions using deep learning models for automated sensor detection and parameter estimation.

Introduction: Environmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised.Methods: We delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air–liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation.Results: COPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC⁺) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub⁺), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5⁺(P=0.018) and LAMB3⁺(P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells.Conclusion: We identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.

Background: Pulmonary vascular disease (PVD) affects the majority of preterm neonates with bronchopulmonary dysplasia (BPD) and significantly determines long-term mortality through undetected progression into pulmonary hypertension. Our objectives were to associate characteristics of pulmonary artery (PA) flow and cardiac function with BPD-associated PVD near term using advanced magnetic resonance imaging (MRI) for improved risk stratification.Methods: Preterms i.e.increased net forward volume right/left ratio, decreased mean relative area change and pathological right end-diastolic volume, to sensitively detect BPD-associated PVD while correcting for PMA (leave-one-out area under the curve 0.88, sensitivity 0.80 and specificity 0.81). We linked these changes to increased right ventricular (RV) afterload (RV–arterial coupling (p=0.02), PA mid-systolic notching (t2; p=0.015) and cardiac index (p=1.67×10⁻⁸)) and correlated echocardiographic findings. Identified in moderate/severe BPD, we successfully applied the PA flow model in heterogeneous mild BPD cases, demonstrating strong correlation of PVD probability with indicators of BPD severity,i.e.duration of mechanical ventilation (r_s=0.63, p=2.20×10⁻⁴) and oxygen supplementation (r_s=0.60, p=6.00×10⁻⁴).Conclusions: Abnormalities in MRI PA flow and cardiac function exhibit significant, synergistic potential to detect BPD-associated PVD, advancing the possibilities of risk-adapted monitoring.

Vitamin D-enriched diet during gestation resulted in reduced lung inflammation and matrix remodeling in neonatal mice exposed to clinically relevant, postnatal injury. The results underscore the need to monitor the subclinical effects of vitamin D insufficiency that impact health in the offspring when other risk factors come into play.

Introduction Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes. Methods We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice. Results We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo. Conclusion We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.

Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.

Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell^® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.