Projects

Characterizing inflammatory neutrophils in sepsis using single-cell sequencing and other approaches

Antigen-presenting aged neutrophils induce CD4⁺ T cells to exacerbate inflammation in sepsis
https://doi.org/10.1172/JCI164585
Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow–derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture–induced sepsis but not in CIRP^–/– mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non–APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4⁺ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4⁺ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4⁺ T cell activation, Th1 polarization, and IFN-γ–mediated hyper-NETosis.

Investigating the Role of Inflammatory Mediators in Overweight Individuals

Increased plasma lipopolysaccharide-binding protein and altered inflammatory mediators in overweight women suggest a state of subclinical endotoxemia
https://doi.org/10.1101/2023.05.18.540879
Background Over 65% of American women are overweight or obese. Obesity and the closely related metabolic syndrome increase the probability for developing several diseases, including cardiovascular disease (CVD). Chronic low-grade inflammation has been recognized as an underlying event linking obesity to CVD. However, inflammatory alterations in individuals who are overweight remain understudied. To provide insight, we performed a pilot study to determine the levels of key circulating biomarkers of endotoxemia and inflammation in overweight vs. lean women with high cholesterol and/or high blood pressure – two important conventional risk factors for CVD.
Methods Plasma samples from adult female subjects who were lean (n=20, BMI=22.4±1.6 kg/m²) or overweight (n=20, BMI=27.0±1.5 kg/m²) with similar ages (55.65±9.1 years and 59.7±6.1 years), and race/ethnicity, and self-reported high cholesterol and/or high blood pressure were analyzed and compared. Samples were obtained through the Northwell Health “Genotype and Phenotype, GaP” registry. Plasma levels of lipopolysaccharide-binding protein (LBP), CRP, IL-6, leptin, and adiponectin were analyzed using commercially available assay kits.
Results Plasma levels of LBP (a recognized marker of metabolic endotoxemia in obesity) were significantly higher in the overweight group compared with the lean group (p=0.005). The levels of CRP, a general marker of inflammation, were also significantly higher in overweight subjects (p=0.01), as were those of the cytokine IL-6 (p=0.02) and the adipokine leptin (p=0.002), pro-inflammatory mediators associated with cardiovascular risk. Levels of adiponectin, an adipokine with anti-inflammatory and anti-atherogenic functions, were significantly lower in the overweight group (p=0.002). The leptin/adiponectin ratio, a preferential atherogenic marker was significantly increased in women who are overweight (p=0.02). Alterations in LBP, CRP, leptin, and adiponectin significantly correlated with BMI, but not with age. The absolute levels of these analytes were within the ranges reported for healthy subjects evaluated in larger clinical trials and thus can be classified as consistent with subclinical endotoxemia.
Conclusion These results document the presence of a pro-inflammatory state in overweight compared with lean women and are of interest for further evaluation of evidence of inflammation in overweight individuals as an additional risk factor for cardiometabolic disease.

Analyzing Menstrual Blood using Single-Cell RNA Sequencing, for Development of a Non-Invasive Screening Tool

Single cell analysis of menstrual endometrial tissues defines phenotypes associated with endometriosis
https://doi.org/10.1016/j.mvr.2021.104205
Background. Endometriosis is a common, complex disorder which is under-recognized and subject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic endometrial lining.
Methods. We have undertaken the first single cell RNA-sequencing (scRNA-Seq) comparison of endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects, including confirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who have chronic symptoms suggestive of endometriosis but have not been diagnosed).
Results. We identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-tissues from controls that is almost absent from endometriosis cases, along with a striking reduction of total uNK cells in the ME of cases (p<10^-16). In addition, an IGFBP1⁺ decidualized subset of endometrial stromal cells are abundant in the shed endometrium of controls when compared to cases (p<10^-16) confirming findings of compromised decidualization of cultured stromal cells from cases. By contrast, endometrial stromal cells from cases are enriched in cells expressing pro-inflammatory and senescent phenotypes. An enrichment of B cells in the cases (p=5.8 x 10^-6) raises the possibility that some may have chronic endometritis, a disorder which predisposes to endometriosis.
Conclusions. We propose that characterization of endometrial tissues in ME will provide an effective screening tool for identifying endometriosis in patients with chronic symptoms suggestive of this disorder. This constitutes a major advance, since delayed diagnosis for many years is a major clinical problem in the evaluation of these patients. Comprehensive analysis of ME is expected to lead to new diagnostic and therapeutic approaches to endometriosis and other associated reproductive disorders such as female infertility.

Characterizing Vasculature using Fractal Geometry and Atomic Force Microscopy

Morphological Characterization of Etv2 Explants Using Fractal Analysis and Atomic Force Microscopy
https://doi.org/10.1016/j.mvr.2021.104205
The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2⁺ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2⁺ HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2⁺ HUVECs have decreased fractal dimension and lacunarity compared to Etv2⁺ HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction
https://doi.org/10.1016/j.stem.2022.03.002
The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Whole Genome Sequencing Quality Control Pipeline

Empirical design of a variant quality control pipeline for whole genome sequencing data using replicate discordance
https://doi.org/10.1038/s41598-019-52614-7
The success of next-generation sequencing depends on the accuracy of variant calls. Few objective protocols exist for QC following variant calling from whole genome sequencing (WGS) data. After applying QC filtering based on Genome Analysis Tool Kit (GATK) best practices, we used genotype discordance of eight samples that were sequenced twice each to evaluate the proportion of potentially inaccurate variant calls. We designed a QC pipeline involving hard filters to improve replicate genotype concordance, which indicates improved accuracy of genotype calls. Our pipeline analyzes the efficacy of each filtering step. We initially applied this strategy to well-characterized variants from the ClinVar database, and subsequently to the full WGS dataset. The genome-wide biallelic pipeline removed 82.11% of discordant and 14.89% of concordant genotypes, and improved the concordance rate from 98.53% to 99.69%. The variant-level read depth filter most improved the genome-wide biallelic concordance rate. We also adapted this pipeline for triallelic sites, given the increasing proportion of multiallelic sites as sample sizes increase. For triallelic sites containing only SNVs, the concordance rate improved from 97.68% to 99.80%. Our QC pipeline removes many potentially false positive calls that pass in GATK, and may inform future WGS studies prior to variant effect analysis.

Polygenic Risk Score in Alzheimer’s Disease

Greater effect of polygenic risk score for Alzheimer’s disease among younger cases who are apolipoprotein E-ε4 carriers
https://doi.org/10.1016/j.neurobiolaging.2020.09.014
To evaluate how age and apolipoprotein E-ε4 (APOE4) status interact with APOE-independent polygenic risk score (PRS_non-APOE), we estimated PRS_non-APOE in superagers (age ≥ 90 years, N = 346), 89–controls (age 60-89, N = 2930), and Alzheimer’s disease (AD) cases (N = 1760). Using superagers, we see a nearly 5 times greater odds ratio (OR) for AD comparing the top PRS_non-APOE decile to the lowest decile (OR = 4.82, p = 2.5 × 10⁶), which is twice the OR as using 89– controls (OR = 2.38, p = 4.6 ×10⁹). Thus PRS_non-APOE is correlated with age, which in turn is associated with APOE. Further exploring these relationships, we find that PRS_non-APOE modifies age at onset among APOE4 carriers, but not among non-carriers. More specifically, PRS_non-APOE in the top decile predicts an age at onset 5 years earlier compared with the lowest decile (70.1 vs. 75.0 years; t-test p = 2.4 × 10⁵) among APOE4 carriers. This disproportionally large PRS_non-APOE among younger APOE4-positive cases is reflected in a significant statistical interaction between APOE4 status and age at onset (β =–0.02, p = 4.8 × 10³) as a predictor of PRS_non-APOE. Thus, the known AD risk variants are particularly detrimental in young APOE4 carriers.

Fitts’s Law in Diagnosing Fine Motor Disorders

Fitts’s Law: Application as a Diagnostic Tool for Fine Motor Disorders
Motor neuron diseases affect 7 to 11 million people worldwide, a number that is increasing every day. The most common of these diseases is the neurodegenerative disorder, Parkinson’s disease. There is currently no clear-cut procedure to successfully diagnose specific fine motor disorders, because many have similar symptoms. To solve this problem, a computer-based diagnostic tool was designed utilizing Fitts’s law, a theory that relates the distance between two targets, and their sizes, to the time it takes to travel between them. The results from the experiment were confirmed using an applied theoretical model based on the optimal stationary control theory. An unaffected user’s movement follows a bell-shaped curve and is fairly straight. Someone with a fine motor disorder has several smaller bell-shaped curves that vary in intensity, representing the tremors and pauses in their movements. Four different graphs were created using the data gathered – a movement path, one with scaled and averaged trajectories, a representation of Fitts’s law, and a bell-shaped curve showing the changing velocity of the subject’s movement between two targets. By simulating motor disorders through mouse cursor vibrations and random pauses in motion, these graphs could also be created for a specific motor disorder. By averaging these results, a model is created, both for an unaffected person and for each individual fine motor disorder. In the future, experiments and data-fitting will be performed to test the validity of the theory that weighing matrices play a key role when planning arm movement trajectories in the CNS.