Fluorescence intravital microscopy captures large data sets of dynamic multicellular interactions within various organs such as the lungs, liver, and brain of living subjects. In medical imaging, edge detection is used to accurately identify and delineate important structures and boundaries inside the images. To improve edge sharpness, edge detection frequently requires the inclusion of low-level features. Herein, a machine learning approach is needed to automate the edge detection of multicellular aggregates of distinctly labeled blood cells within the microcirculation. In this work, the Structured Adaptive Boosting Trees algorithm (AdaBoost.S) is proposed as a contribution to overcome some of the edge detection challenges related to medical images. Algorithm design is based on the observation that edges over an image mask often exhibit special structures and are interdependent. Such structures can be predicted using the features extracted from a bigger image patch that covers the image edge mask. The proposed AdaBoost.S is applied to detect multicellular aggregates within blood vessels from the fluorescence lung intravital images of mice exposed to e-cigarette vapor. The predictive capabilities of this approach for detecting platelet-neutrophil aggregates within the lung blood vessels are evaluated against three conventional machine learning algorithms: Random Forest, XGBoost and Decision Tree. AdaBoost.S exhibits a mean recall, F-score, and precision of 0.81, 0.79, and 0.78, respectively. Compared to all three existing algorithms, AdaBoost.S has statistically better performance for recall and F-score. Although AdaBoost.S does not outperform Random Forest in precision, it remains superior to the XGBoost and Decision Tree algorithms. The proposed AdaBoost.S is widely applicable to analysis of other fluorescence intravital microscopy applications including cancer, infection, and cardiovascular disease.

Neutrophils are the body’s front-line defenders against foreign insult and are key players in a variety of inflammatory conditions. This body of work examines the role of neutrophils in promoting pathology in three distinct inflammatory contexts. In the pro-inflammatory state provoked by breast cancer, neutrophils decondense their nuclei and release cytotoxic web-like structures known as neutrophil extracellular traps (NETs). NETs form most commonly via histone modifications facilitated by the enzyme PAD4. NETs are known to be a harbinger of disease progression and promote metastasis through capture of circulating tumor cells. It was hypothesized that breast tumors release small particles known as extracellular vesicles (EVs) into the circulation which interact with peripheral neutrophils to cause NET release. The first group of studies herein examined for the first time whether tumor-released EVs promote NET release in a PAD4- dependent manner, as well as whether PAD4 expression by the parent cell influences the capacity of EVs to promote NET formation.

Despite the perception that e-cigarettes are safer than conventional cigarettes, numerous findings demonstrated that e-cigarette aerosol (EC) exposure induced compromised immune functionality, vascular changes even after acute exposure, and lung injury. Notably, altered neutrophil functionality and platelet hemodynamics have been observed post-EC exposure. It was hypothesized that EC exposure initiates an inflammatory response resulting in altered neutrophil behavior and increased neutrophil-platelet interaction in the pulmonary microvasculature. Neutrophil and platelet responses were examined up to 48 hrs following whole-body, short-term EC exposure without flavorants or nicotine in a murine model, which most closely modeled secondhand exposure. This study is the first to investigate the impact of EC exposure through lung intravital imaging. Compared to room air-exposed mice, EC-exposed mice displayed significantly increased 1.7‒1.9-fold number of neutrophils in the pulmonary microvasculature associated with no marked change in neutrophils within whole blood or bronchoalveolar lavage fluid (BALF). Neutrophil-platelet interactions were also significantly elevated 1.9‒2.5-fold in exposed mice. Plasma concentration of myeloperoxidase was markedly reduced 1.5-fold 48 hr following exposure cessation, suggesting suppressed neutrophil antimicrobial activity. Cytokine expression exhibited changes indicating vascular damage. Effects persisted for 48 hr post-EC exposure. Data demonstrated that EC exposure repeated for 3 consecutive days in 2.5 hr intervals in the absence of flavorants or nicotine resulted in modified pulmonary vasculature hemodynamics, altered immune functionality, and a pro-inflammatory state in female BALB/cJ mice.

Novel metal oxide nanoparticle (NP) contrast agents may offer safety and functionality advantages over conventional gadolinium-based contrast agents (GBCAs) for cancer diagnosis by magnetic resonance imaging. However, little is known about the behavior of metal oxide NPs, or of their effect, upon coming into contact with the innate immune system. As neutrophils are the body's first line of defense, we sought to understand how manganese oxide and iron oxide NPs impact leukocyte functionality. Specifically, we evaluated whether contrast agents caused neutrophils to release web-like fibers of DNA known as neutrophil extracellular traps (NETs), which are known to enhance metastasis and thrombosis in cancer patients. Murine neutrophils were treated with GBCA, bare manganese oxide or iron oxide NPs, or poly(lactic-co-glycolic acid) (PLGA)-coated metal oxide NPs with different incorporated levels of poly(ethylene glycol) (PEG). Manganese oxide NPs elicited the highest NETosis rates and had enhanced neutrophil uptake properties compared to iron oxide NPs. Interestingly, NPs with low levels of PEGylation produced more NETs than those with higher PEGylation. Despite generating a low rate of NETosis, GBCA altered neutrophil cytokine expression more than NP treatments. This study is the first to investigate whether manganese oxide NPs and GBCAs modulate NETosis and reveals that contrast agents may have unintended off-target effects which warrant further investigation.

More than 1 in 4 high school students use electronic nicotine delivery systems (ENDS) in the United States, with usage rising over 130% since 2017. It is well known that smoking can cause cardiovascular disease and thrombosis; however, few studies have investigated the adverse effects of ENDS use on the cardiovascular system, especially in the lungs. Neutrophils and platelets are known to undergo activation and proinflammatory phenotypic changes when exposed to ENDS vapor, and their aggregation could promote vascular occlusion and further inflammation. We hypothesize that platelet and neutrophil recruitment will be increased in mice exposed to ENDS vapor, and that higher rates of aggregate formation will be observed in the pulmonary vasculature of these subjects. To investigate lung vascular pathology following ENDS use, we have applied an innovative spinning disk confocal microscopy approach to visualize real-time interactions of blood cells in the lungs of live mice. Identifying the presence of platelet-neutrophil aggregates in the lung due to ENDS vapor exposure would suggest that ENDS users may be at higher risk of atherosclerotic and thrombotic complications that with chronic use could eventually result in respiratory disease. Mice were subjected to 3 consecutive days of ENDS vapor inhalation using a Juul device with Virginia Classic Tobacco e-liquid (5% nicotine) for 3 hours each day. Mice subjected to room air sham inhalation constituted the control group. 24 hours after completion of the inhalation course, mice were anesthetized to facilitate microsurgery enabling fluorescence microscopy of the living lung. Neutrophils and platelets were labeled with fluorescently tagged antibodies to compare overall presence and aggregation within the lung microvasculature (Fig. 1). Preliminary data suggest significant (p<0.01) increases (Fig. 2) in platelet (2.59-fold), neutrophil (2.06-fold), and platelet-neutrophil aggregate (2.02-fold) presence in mice exposed to ENDS vapor compared to untreated mice. Collectively, this illustrates that ENDS vapor induces a pro-inflammatory state in the pulmonary vasculature in as little as one day after limited exposure. These findings will prove useful in future studies seeking to model the consequences of long-term ENDS use, which will ultimately enhance both clinician monitoring of patients at risk of such complications and public awareness of the potential consequences of ENDS use.

Electronic cigarettes are frequently viewed as a safer alternative to conventional cigarettes; however, evidence to support this perspective has not materialized. Indeed, the current literature reports that electronic cigarette use is associated with both acute lung injury and subclinical dysfunction to the lung and vasculature that may result in pathology following chronic use. E-cigarettes can alter vascular dynamics, polarize innate immune populations towards a proinflammatory state, compromise barrier function in the pulmonary endothelium and epithelium, and promote pre-oncogenic phenomena. This review will summarize the variety of e-cigarette products available to users, discuss current challenges in e-cigarette study design, outline the range of pathologies occurring in cases of e-cigarette associated acute lung injury, highlight disease supporting tissue- and cellular-level changes resulting from e-cigarette exposure, and briefly examine how these changes may promote tumorigenesis. Continued research of the mechanisms by which e-cigarettes induce pathology benefit users and clinicians by resulting in increased regulation of vaping devices, informing treatments for emerging diseases e-cigarettes produce, and increasing public awareness to reduce e-cigarette use and the onset of preventable disease.

The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer.