Metabolic changes in Toxoplasma gondii-infected host cells measured by autofluorescence imaging.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion.IMPORTANCEThis study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide.

Microfluidic device with reconfigurable spatial temporal gradients reveals plastic astrocyte response to stroke and reperfusion.

As a leading cause of mortality and morbidity, stroke constitutes a significant global health burden. Ischemic stroke accounts for 80% of cases and occurs due to an arterial thrombus, which impedes cerebral blood flow and rapidly leads to cell death. As the most abundant cell type within the central nervous system, astrocytes play a critical role within the injured brain. We developed a novel microphysiological platform that permits the induction of spatiotemporally controlled nutrient gradients, allowing us to study astrocytic response during and after transient nutrient deprivation. Within 24 h of inducing starvation in the platform, nutrient deprivation led to multiple changes in astrocyte response, from metabolic perturbations to gene expression changes, and cell viability. Furthermore, we observed that nutrient restoration did not reverse the functional changes in astrocyte metabolism, which mirrors reperfusion injury observed in vivo. We also identified alterations in numerous glucose metabolism-associated genes, many of which remained upregulated or downregulated even after restoration of the nutrient supply. Together, these findings suggest that astrocyte activation during and after nutrient starvation induces plastic changes that may underpin persistent stroke-induced functional impairment. Overall, our innovative device presents interesting potential to be used in the development of new therapies to improve tissue repair and even cognitive recovery after stroke.

Optical coherence tomography of human fetal membrane sub-layers during loading.

Fetal membranes have important mechanical and antimicrobial roles in maintaining pregnancy. However, the small thickness (<800 µm) of fetal membranes places them outside the resolution limits of most ultrasound and magnetic resonance systems. Optical imaging methods like optical coherence tomography (OCT) have the potential to fill this resolution gap. Here, OCT and machine learning methods were developed to characterize the ex vivo properties of human fetal membranes under dynamic loading. A saline inflation test was incorporated into an OCT system, and tests were performed on n = 33 and n = 32 human samples obtained from labored and C-section donors, respectively. Fetal membranes were collected in near-cervical and near-placental locations. Histology, endogenous two photon fluorescence microscopy, and second harmonic generation microscopy were used to identify sources of contrast in OCT images of fetal membranes. A convolutional neural network was trained to automatically segment fetal membrane sub-layers with high accuracy (Dice coefficients >0.8). Intact amniochorion bilayer and separated amnion and chorion were individually loaded, and the amnion layer was identified as the load-bearing layer within intact fetal membranes for both labored and C-section samples, consistent with prior work. Additionally, the rupture pressure and thickness of the amniochorion bilayer from the near-placental region were greater than those of the near-cervical region for labored samples. This location-dependent change in fetal membrane thickness was not attributable to the load-bearing amnion layer. Finally, the initial phase of the loading curve indicates that amniochorion bilayer from the near-cervical region is strain-hardened compared to the near-placental region in labored samples. Overall, these studies fill a gap in our understanding of the structural and mechanical properties of human fetal membranes at high resolution under dynamic loading events.

Inflammatory CD4/CD8 double-positive human T cells arise from reactive CD8 T cells and are sufficient to mediate GVHD pathology.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

Time-domain single photon-excited autofluorescence lifetime for label-free detection of T cell activation.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique, capable of label-free assessment of the metabolic state and function within single cells. The FLIM measurements of autofluorescence were recently shown to be sensitive to the functional state and subtype of T cells. Therefore, autofluorescence FLIM could improve cell manufacturing technologies for adoptive immunotherapy, which currently require a time-intensive process of cell labeling with fluorescent antibodies. However, current autofluorescence FLIM implementations are typically too slow, bulky, and prohibitively expensive for use in cell manufacturing pipelines. Here we report a single photon-excited confocal whole-cell autofluorescence system that uses fast field-programmable gate array-based time tagging electronics to achieve time-correlated single photon counting (TCSPC) of single-cell autofluorescence. The system includes simultaneous near-infrared bright-field imaging and is sensitive to variations in the fluorescence decay profile of the metabolic coenzyme NAD(P)H in human T cells due to the activation state. The classification of activated and quiescent T cells achieved high accuracy and precision (area under the receiver operating characteristic curve, AUC = 0.92). The lower-cost, higher acquisition speed, and resistance to pile-up effects at high photon flux compared to traditional multiphoton-excited FLIM and TCSPC implementations with similar SNR make this system attractive for integration into flow cytometry, sorting, and quality control in cell manufacturing.

Adaptable pulsatile flow generated from stem cell-derived cardiomyocytes using quantitative imaging-based signal transduction.

Endothelial cells (EC) in vivo are continuously exposed to a mechanical microenvironment from blood flow, and fluidic shear stress plays an important role in EC behavior. New approaches to generate physiologically and pathologically relevant pulsatile flows are needed to understand EC behavior under different shear stress regimes. Here, we demonstrate an adaptable pump (Adapt-Pump) platform for generating pulsatile flows from human pluripotent stem cell-derived cardiac spheroids (CS) via quantitative imaging-based signal transduction. Pulsatile flows generated from the Adapt-Pump system can recapitulate unique CS contraction characteristics, accurately model responses to clinically relevant drugs, and simulate CS contraction changes in response to fluidic mechanical stimulation. We discovered that ECs differentiated under a long QT syndrome derived pathological pulsatile flow exhibit abnormal EC monolayer organization. This Adapt-Pump platform provides a powerful tool for modeling the cardiovascular system and improving our understanding of EC behavior under different mechanical microenvironments.