Epigenetic and transcriptional responses in circulating leukocytes are associated with future decompensation during SARS-CoV-2 infection.

To elucidate host response elements that define impending decompensation during SARS-CoV-2 infection, we enrolled subjects hospitalized with COVID-19 who were matched for disease severity and comorbidities at the time of admission. We performed combined single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on peripheral blood mononuclear cells (PBMCs) at admission and compared subjects who improved from their moderate disease with those who later clinically decompensated and required invasive mechanical ventilation or died. Chromatin accessibility and transcriptomic immune profiles were markedly altered between the two groups, with strong signals in CD4+ T cells, inflammatory T cells, dendritic cells, and NK cells. Multiomic signature scores at admission were tightly associated with future clinical deterioration (auROC 1.0). Epigenetic and transcriptional changes in PBMCs reveal early, broad immune dysregulation before typical clinical signs of decompensation are apparent and thus may act as biomarkers to predict future severity in COVID-19.

Human Immune Cell Epigenomic Signatures in Response to Infectious Diseases and Chemical Exposures.

Variations in DNA methylation patterns in human tissues have been linked to various environmental exposures and infections. Here, we identified the DNA methylation signatures associated with multiple exposures in nine major immune cell types derived from peripheral blood mononuclear cells (PBMCs) at single-cell resolution. We performed methylome sequencing on 111,180 immune cells obtained from 112 individuals who were exposed to different viruses, bacteria, or chemicals. Our analysis revealed 790,662 differentially methylated regions (DMRs) associated with these exposures, which are mostly individual CpG sites. Additionally, we integrated methylation and ATAC-seq data from same samples and found strong correlations between the two modalities. However, the epigenomic remodeling in these two modalities are complementary. Finally, we identified the minimum set of DMRs that can predict exposures. Overall, our study provides the first comprehensive dataset of single immune cell methylation profiles, along with unique methylation biomarkers for various biological and chemical exposures.

Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.

SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.

Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.

SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associate with mild or moderate symptoms are already robust and include severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity is marked by upregulation classical antiviral pathways including those regulating IRF1 and IRF7, whereas in moderate disease these classical antiviral signals diminish suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.

Quantitative phase microscopy spatial signatures of cancer cells.

We present cytometric classification of live healthy and cancerous cells by using the spatial morphological and textural information found in the label-free quantitative phase images of the cells. We compare both healthy cells to primary tumor cells and primary tumor cells to metastatic cancer cells, where tumor biopsies and normal tissues were isolated from the same individuals. To mimic analysis of liquid biopsies by flow cytometry, the cells were imaged while unattached to the substrate. We used low-coherence off-axis interferometric phase microscopy setup, which allows a single-exposure acquisition mode, and thus is suitable for quantitative imaging of dynamic cells during flow. After acquisition, the optical path delay maps of the cells were extracted and then used to calculate 15 parameters derived from the cellular 3D morphology and texture. Upon analyzing tens of cells in each group, we found high statistical significance in the difference between the groups in most of the parameters calculated, with the same trends for all statistically significant parameters. Furthermore, a specially designed machine learning algorithm, implemented on the phase map extracted features, classified the correct cell type (healthy/cancer/metastatic) with 81-93% sensitivity and 81-99% specificity. The quantitative phase imaging approach for liquid biopsies presented in this paper could be the basis for advanced techniques of staging freshly isolated live cancer cells in imaging flow cytometers. © 2017 International Society for Advancement of Cytometry.

Late-outgrowth endothelial progenitors from patients with coronary artery disease: endothelialization of confluent stromal cell layers.

Patients with coronary artery disease (CAD) are the primary candidates to receive small-diameter tissue-engineered blood vessels (TEBVs). Peripheral blood derived endothelial progenitor cells (EPCs) from CAD patients (CAD EPCs) represent a minimally invasive source of autologous cells for TEBV endothelialization. We have previously shown that human CAD EPCs are highly proliferative and express many of the hallmarks of mature and healthy endothelial cells; however, their behavior on stromal cells that comprise the media of TEBVs has not yet been evaluated. Primary CAD EPCs or control human aortic endothelial cells (HAECs) were seeded over confluent, quiescent layers of human smooth muscle cells (SMCs) using a direct co-culture model. The percent coverage, adhesion strength, alignment under flow and generation of flow-induced nitric oxide of the seeded CAD EPCs were compared to that of HAECs. The integrin-binding profile of CAD EPCs was also evaluated over a layer of confluent, quiescent SMCs. Direct comparison of our CAD EPC results to analogous co-culture studies with cord blood EPCs show that both types of blood-derived EPCs are viable options for endothelialization of TEBVs.

Nonlinear phase dispersion spectroscopy.

Nonlinear phase dispersion spectroscopy is introduced as a means to retrieve wideband, high spectral resolution profiles of the wavelength-dependent real part of the refractive index. The method is based on detecting dispersion effects imparted to a light field with low coherence transmitted through a thin sample and detected interferometrically in the spectral domain. The same sampled signal is also processed to yield quantitative phase maps and spectral information regarding the total attenuation coefficient using spectral-domain phase microscopy and spectroscopic optical coherence tomography (SOCT), respectively. Proof-of-concept experiments using fluorescent and nonfluorescent polystyrene beads and another using a red blood cell demonstrate the ability of the method to quantify various absorptive/dispersive features. The increased sensitivity of this method, novel to our knowledge, is compared to intensity-based spectroscopy (e.g., SOCT), and potential applications are discussed.

Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry.

We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

Spectral-domain differential interference contrast microscopy.

We present a fiber-optic low-coherence imaging technique, termed spectral-domain differential interference contrast microscopy (SD-DIC), for quantitative DIC imaging of both reflective surfaces and transparent biological specimens. SD-DIC combines the common-path nature of a Nomarski DIC interferometer with the high sensitivity of spectral-domain low-coherence interferometry to obtain high-resolution, quantitative measurements of optical pathlength gradients from a single point on the sample. Full-field imaging can be achieved by scanning the sample. A reflected-light SD-DIC system was demonstrated using a USAF resolution target as the phase object. Live cardiomyocytes were also imaged, achieving a resolution of 36 pm for pathlength gradient measurements. The dynamics of cardiomyocyte contraction were recorded with high sensitivity at selected sites on the cells.

Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress.

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α(5)ß(1) integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α(5)ß(1) with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.

Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis.

OBJECTIVE: To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. METHODS AND RESULTS: Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface α(5)ß(1) integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdc(scid)/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. CONCLUSIONS: The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.

Whole-cell-analysis of live cardiomyocytes using wide-field interferometric phase microscopy.

We apply wide-field interferometric microscopy techniques to acquire quantitative phase profiles of ventricular cardiomyocytes in vitro during their rapid contraction with high temporal and spatial resolution. The whole-cell phase profiles are analyzed to yield valuable quantitative parameters characterizing the cell dynamics, without the need to decouple thickness from refractive index differences. Our experimental results verify that these new parameters can be used with wide field interferometric microscopy to discriminate the modulation of cardiomyocyte contraction dynamics due to temperature variation. To demonstrate the necessity of the proposed numerical analysis for cardiomyocytes, we present confocal dual-fluorescence-channel microscopy results which show that the rapid motion of the cell organelles during contraction preclude assuming a homogenous refractive index over the entire cell contents, or using multiple-exposure or scanning microscopy.

ApoA-I induced CD31 in bone marrow-derived vascular progenitor cells increases adhesion: implications for vascular repair.

Transgenic over expression of apolipoprotein A-I (ApoA-I) the major structural apolipoprotein of HDL appears to convey the most consistent and strongest anti atherogenic effect observed in animal models so far. We tested the hypothesis that ApoA-I mediates its cardio protective effects additionally through ApoA-I induced differentiation of bone marrow-derived progenitor cells in vitro. This study demonstrates that lineage negative bone marrow cells (lin(-) BMCs) alter and differentiate in response to free ApoA-I. We find that lin(-) BMCs in culture treated with recombinant free ApoA-I at a concentration of 0.4 microM are twice as large in size and have altered cell morphology compared to untreated cells; untreated cells retain the original spheroid morphology. Further, the total number of CD31 positive cells in the ApoA-I treated population consistently increased by two fold. This phenotype was significantly reduced in untreated cells and points towards a novel ApoA-I dependent differentiation. A protein lacking its best lipid-binding region (ApoA-I Delta 10) did not stimulate any changes in the lin(-)BMCs indicating that ApoA-I may mediate its effects by regulating cholesterol efflux. The increased CD31 correlates with an increased ability of the lin(-) BMCs to adhere to both fibronectin and mouse brain endothelial cells. Our results provide the first evidence that exogenous free ApoA-I has the capacity to change the characteristics of progenitor cell populations and suggests a novel mechanism by which HDL may mediate its cardiovascular benefits.

Increased activity of phosphatase PP2A in the presence of the PlA2 polymorphism of alphaIIbbeta3.

Polymorphisms in alphaIIbbeta3 are important genetic factors that alter platelet biology and have been associated with susceptibility to thromboembolic disorders. To define the molecular mechanisms that lead to variance in thrombotic diathesis dictated by the beta3 polymorphism, we examined regulation of intracellular signaling by alphaIIbbeta3, and studied the effects of a common beta subunit PlA2 polymorphism. We found that PP2A regulates alphaIIbbeta3 control of the ERK signaling in a polymorphism specific fashion. In CHO cells, exogenous expression of alphaIIbbeta3 reduced ATP-stimulated ERK phosphorylation and more so for PlA2 than PlA1. Interestingly, reduced level of ERK phosphorylation correlated with an increase in PP2A activity, with higher activity associated with PlA2 than PlA1. We tested the effect of PP2A on alphaIIbbeta3-dependent adhesion, and found that PP2A overexpression increased cell adhesion, while phosphatase inhibitors decreased cell adhesion. We propose that PlA2 alters cell signaling at least in part by increasing beta3-associated PP2A activity.

Reactive oxygen species in vascular endothelial cell motility. Roles of NAD(P)H oxidase and Rac1.

Reactive oxygen species (ROS) are acknowledged generally to be multi-faceted regulators of cellular functions that trigger various pathological states when present chronically or transiently at non-physiologically high levels. Here we focus on the physiological involvement of ROS in cellular motility, with special emphasis on endothelial cells (EC). An important source of ROS within EC is the non-phagocytic NAD(P)H oxidase, and the small GTPase Rac1 plays a central role in activating this complex. Rac1 is one of the three Rho-family molecules (Rac, Rho and Cdc42) involved in the control of the actin cytoskeleton in response to various signals. In this review we examine the evidence linking ROS production, Rac1 activation and actin organization to EC motility, considering mechanisms for direct interaction of ROS and actin and the effects of ROS on proteins that regulate the actin cytoskeleton. The accumulated evidence suggests that ROS are important regulators of the actin cytoskeletal dynamics and cellular motility, and more in-depth studies are needed to understand the underlying mechanisms.

Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3.

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.